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©2015, Indian Pharmacopoeia Commission
All rights reserved
No part of this publication may be reproduced, stored in a
retrieval system/transmitted in any form by any means such
as electronic, mechanical including photocopying, recording
or otherwise without the prior permission of Indian Pharmacopoeia Commission.
ISBN:
Guidance Manual for Monographs Development
of Herbs and Herbal products.
On behalf of : Government of India
Ministry of Health and Family welfare
Produced and published by Indian Pharmacopoeia Commission
Government of India
Ministry of Health and Family Welfare
Sector 23, Raj Nagar,
Ghaziabad-201002
Tel: (91-120)- 2783401
Fax: (91-120)-2783401
Website: www.ipc.gov.in
Email: [email protected]
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Disclaimer This manual is prepared as a guidance document for the stakeholders of IP who intend to submit Herbs and Herbal Products monographs,
candidate material for Phytochemicals Reference Substances and Botanical Reference Substances to IPC. They have been verified and
reviewed by experts before incorporation in this manual. Clarification on any matter presented in the manual will be issued with due
verification only. For any clarifications please consult IPC. The stakeholders are advised to keep abreast of the changes in the content if any,
from time to time through the technical secretariat of Indian Pharmacopoeia Commission. We also welcome suggestions for updating this
manual.
Copy right: The contents of this Manual are not to be copied in full or part or reproduced in any manner without the authority of the Indian
Pharmacopoeia Commission.
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OBJECTIVES
1. The objective of the “Guidance Manual for Monographs Development for Herbs and
Herbal Products” is to facilitate the concerned manufacturers and other stakeholders
for understanding the process and development of monographs of herbs and herbal
products.
2. The Manual also aims to encourage and promote research and development in the
quality of herbals in India and overseas.
3. The Manual also gives current Standard Operating Procedures (SOPs) for
development of monographs as well as Botanical Reference Substances (BRS) and
Phytochemicals Reference Substances (PRS) and invites suggestions for further
updating.
4. Indian Pharmacopoeia Commission will be glad to consider submission of data and
draft monographs adopting this Manual for inclusion in IP subject to such data
meeting the criteria laid down
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CONTENT
S. No. TITLE PAGE No.
Core Expert Committee
Acknowledgements
Abbreviations
4
6
7
1. Introduction 8
2. List of Monographs on Herbals in IP 2014 including those in Addendum 2015 10
3. Inclusion/ Exclusion Criteria of a Herbal Monograph in IP 11
4. Process of IP Herbal Monograph Development and Content of a Herbal
Monograph 13
5. General Monograph for Herbs, Processed Herbs and Herbal Products 20
6. Standard Operating Procedure for Preparation of Monographs of Herbs,
Processed Herbs and Herbal Products In IP
25
7. Standard Operating Procedure for Herbal Extracts Monographs 31
8. Standard Operating Procedure for Preparation, Qualification, Certification &
Supply of Botanicals Reference Substance (BRS)
35
9. Standard Operating Procedure for Preparation, Qualification, Certification &
Supply of Phytochemicals Reference Substance (PRS)
39
10. Identification of Herbal Materials 43
11. Determination of Ash 52
12. Determination of Extractive Values 54
13. Loss on Drying 56
14. Contaminants 57
15. Thin Layer Chromatography 60
16. High Performance Thin Layer Chromatography 66
17. High Performance Liquid Chromatography 74
18. Analysis of Essential Oils 82
19. Examples of Some Herbal Monographs 87
20. Herbal Drugs Manufacturers/Suppliers in India 93
21. Bibliography 95
22. Books for Further Reading on Herbs 96
23. Feedback Form 98
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CORE EXPERT COMMITTEE
Chairman
Dr. G. N. Singh
Secretary-cum-Scientific Director,
Indian Pharmacopoeia Commission,
Ghaziabad
and
DCG (I),
Central Drugs Standard Control Organization (CDSCO),
FDA Bhawan, Kotla Road, New Delhi
Members
Sub-Group on Essential oils
Dr. D.B. Anantha Narayana
Chairman,
Herbal Products Expert Group (HPEG)
Indian Pharmacopoeia Commission,
Sec 23, Raj Nagar,
Ghaziabad.
Mr. B. Murali,
Sr. Manager - Quality Control
M/s Natural Remedies,
Bangalore
Prof S.S. Handa
Former Director,
Indian Institute of Integrative Medicines
(formerly RRL),
Jammu
Dr. C.K. Katiyar
Chief Executive Officer,
M/s Emami Healthcare,
Kolkata
Dr. Amit Agarwal,
Director- R&D
M/s Natural Remedies
Bangalore
Dr. George Patani,
Director- R&D
M/s Inga Pharmaceuticals Pvt. Ltd.
Mumbai
Mr. Ramakant Harlal Kha,
M/s Nishant Aromatics
424/425, Milan Industrial Estate, Off T J Road,
Cotton Green, Mumbai,
Maharashtra 400033.
Mr. B. Murali
(Affiliation - as above)
Dr. Rahul Singh,
M/s Emami (I) Pvt. Ltd.
687 Anandapur, EM Bypass, Kolkatta 700107
Dr. Hema Lohani
Centre for Aromatic Plants, Selaqui Industrial Area,
Dehradun, Uttarakhand
Ms. Bhuvana Nageswaran
M/s Ultra International Ltd.
64/1, Site 4, Sahibabad Industrial Area,
Ghaziabad, Uttar Pradesh 201010
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IPC Scientific Team
Other Participants
Scientists other than those mentioned above who contributed in the work related to development of
monographs included in IP on Herbs and Herbal Products till today:
Dr. Jai Prakash,
Senior Principal Scientific Officer
Mr Alok Sharma
Scientific Officer
Dr. Manoj Kumar Pandey
Scientific Officer
Dr. M. Kalaivani
Scientific Assistant
Ms. Shruti Rastogi,
Pharmacopoeial Associate
Mr. Hariom Singh,
Pharmacopoeial Associate
Mr.Ashish Kumar Kushwaha
Pharmacopoeial Associate
Dr. Divya Kaushik
Pharmacopoeial Associate
Ms. Neha Singh,
Pharmacopoeial Associate
Ms. Amandeep Bhatia,
Pharmacopoeial Associate
Ms. B. Gayatri Dr. Anil Thakan
Dr. Anil Kanaujia Dr. G. Trimurtulu
Mr. M. J. Saxena Mr. K. Ravikant
Dr. S.P.S. Khanuja Dr. Beena Bhatt
Mr. Praful Lahorkar Dr. Dharmendra Kushwah
Dr. B. Murali Dr. Vijay Chauhan
Dr. S. Natarajan Dr. Hemant Kumar Sharma
Dr. Abraham Patani Mr. Rajendra M Dobriyal
Dr. George Patani Dr. Ravi Jain
Prof. K. Satyamoorthy Mr. D.K. Ved
Dr. M. Rajani (Late) Dr. Rahul Singh
Dr. Y. K. S. Rathore Mr. Ashish Suthar
Dr. Nancy Pandita Mr. Hariharan
Dr. Padmalatha S. Rai Dr. Ravishankara Bellampalli
Prof. M. N. Nanjan Dr. Deepak
Dr. Ashish Suthar Dr. A.K. Rawat
Dr. Amit Agarwal Dr. Pulok Mukherjee
Dr. C.K.Katiyar Mr. Prakash Itankar
Dr. Ramakant Dr. Ramakant Harlal Kha
Ms. Bhuvana Nageswaran Dr. Hema Lohani
Dr. Vandita Shrivastava
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Acknowledgements
The Indian Pharmacopoeia Commission (IPC) wishes to acknowledge the contributions of all experts,
officials, scientists and academicians involved in bringing out this Herbal Guidance Manual. The
encouragement and support given by Dr. G. N. Singh, Secretary-cum-Scientific Director of the IPC
and DCG (I), is noteworthy. The preliminary draft of this Guidance Manual was prepared by
Dr. D.B.A. Narayana, Chairman, Herbal Products Expert Group and Member, Scientific Body of IPC.
Modifications were done by the IPC scientific team namely Dr. Manoj Kumar Pandey,
Dr. M. Kalaivani, Mr. Hariom Singh, Ms. Shruti Rastogi, Mr Ashish Kushwaha, Dr. Divya Kaushik,
Ms. Neha Singh and Ms. Amandeep Bhatia. Special thanks to Herbal Products Expert Group and the
Chairman who reviewed the draft critically and also to other experts who provided comments on
uploaded draft displayed on the website of IPC www.ipc.gov.in.
The Scientific staff of the IPC provided their valuable scientific inputs including arrangement, fine
editing and formatting of content of this Manual. In particular Dr. D.B.A. Narayana, Dr. Jai Prakash
and Dr. Manoj Kumar Pandey have played a key role in overall review, editing, compilation and
bringing it to present shape.
IPC also places on record the contribution of other participants listed in this Guidance Manual.
Secretarial assistance provided by Ms. Reena Tripathi is thankfully acknowledged. We are thankful to
the printing services officials for their kind support.
The feedback on this Manual will help in further improvement of this document.
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ABBREVIATIONS
BRS : Botanical Reference Substance
CDSCO : Central Drugs Standard Control Organization
CoA : Certificate of Analysis
CV : Coefficient of Variation
FD : Fluorescence Detector
GC : Gas Chromatography
HPEG : Herbal Products Expert Group
HPLC : High Performance Liquid Chromatography
IP : Indian Pharmacopoeia
IPC : Indian Pharmacopoeia Commission
LoD : Loss on Drying
NRA : National Regulatory Authority
PI : Participating Institution
PPM : Parts per million
PRS : Phytochemical Reference Substance
RID : Refractive Index Detector
RS : Reference Substance
SOPs : Standard Operating Procedures
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1. INTRODUCTION
1.1 IP 2014
The seventh edition of Indian Pharmacopoeia (IP 2014) is published by the Indian Pharmacopoeia
Commission (IPC) on behalf of the Government of India, Ministry of Health & Family Welfare. IP
2014 has come into effect from 01.04.2014. As per the provisions of the Drugs & Cosmetics Act 1940
& Rules 1945, IP prescribes the standards for the drugs that are manufactured, imported and stocked,
marketed/distributed in India. The standards of identity, purity and strength prescribed in IP ensure
quality control and quality assurance of the medicines. The standard prescribed in the book applies for
the drugs which have not been included in IP 2014, but included in IP 2010 and its Addendum. The
drugs are to conform the standards prescribed in the IP for the time being in force and its immediately
previous edition.
Herbal medicines have been used by the humanity since time immemorial for various healthcare
needs. Around 80% of the population of developing countries uses herbal medicines for treatment of
various diseases. India is one of the largest producers of medicinal herbs and is called as botanical
garden of the world. With the ever-increasing use of herbal medicines and the global expansion of the
herbal medicines market, quality and safety has become a major concern for the health of the people.
The quality of herbal medicines has a direct impact on their safety and efficacy. Thus, to regulate the
quality standards, various identification and quantification procedures are mentioned in IP 2014.
IP 2014 contains 136 herbal monographs out of which 68 monographs are of raw herbs, 29
monographs of herbal extracts, 39 monographs of processed herbs/ pharmaceutical aids/ formulations.
1.2 IP Addendum 2015
IP Addendum 2015 to IP 2014 is also published by the IPC which has the same authority as IP 2014
and includes the changes and new monographs. It contains 13 new herbal monographs. The effective
date of IP addendum 2015 is 1st April 2015.
1.3 Objective of the Herbal Guidance Manual
This herbal manual is designed to disseminate the practical information and promote the proper use
and development of herbs and herbal products for the benefit of mankind. It is to facilitate the
stakeholders to provide the monographs for inclusion in IP. The details regarding the identification
and testing of the herbs or herbal preparations are provided in the Manual to ensure its quality. This
manual has been developed with a goal of providing assistance to stakeholders i.e. industry, health
care professionals, analysts and researchers on how they can assure high quality procedures for
successful development of the monographs. However, the test methods described in this Herbal
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Guidance Manual are presented as examples of suitable methods for herbal materials. The analysis of
herbal medicines is, however, not restricted to those methods discussed or recommended here. Other
validated techniques may be used in exceptional cases.
Appearance of herbal drug monograph does not mean its approval as a drug under the law; it is to
provide qualitative and quantitative standards of quality for the herbs, its use either as a food item or
as supplement or ingredient, as a drug and/or as an ingredient in cosmetics.
1.4 Scope
Unless otherwise stated, the quality expectations outlined in this document apply to all the herbs and
herbal preparations. This document should be read in conjunction with the IP 2014 and Addendum
2015.
This document can be utilized as a starting point for the development of new monographs by the
various stakeholders for IP. The inclusion and exclusion criteria are mentioned in Chapter 3 of this
Manual.
The guidance provided in this manual will help ensuring quality of the herbal products by the
manufacturers, analysts etc.
1.5 Notes to Readers
The compiled draft of this Guidance Manual for IP was examined by the Herbal Experts Committee
and corrections incorporated as suggested by them. The manual was uploaded on the website of the
Commission www.ipc.gov.in for 45 days for public comments. The comments received from the
stakeholders were examined and then incorporated.
IPC requests all stakeholders to send their feedback on this Guidance Manual. A format for the
feedback is presented at the end of this Manual. Views can be communicated through e-mail also on
the following address:
Email: [email protected]
Website: www.ipc.gov.in
Telephone and fax numbers: +91-120-2783401, 2783392
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2. LIST OF MONOGRAPHS ON HERBALS IN IP 2014 INCLUDING
THOSE IN ADDENDUM 2015
RAW HERBS
(A)
HERBAL EXTRACTS
(B)
PROCESSED HERBS/
PHARMACEUTICAL AIDS/
FORMULATIONS
(C)
1. Acacia
2. Ajwain
3. Amalaki
4. Amaltas
5. Amra
6. Anantmula
7. Arjuna
8. Artemisia
9. Ashwagandha
10. Asthisamhrta
11. Bakuci
12. Bala
13. Bassant
14. Belladona Leaf
15. Bhibhitaki
16. Bhringraj
17. Bhuiamla
18. Birmi
19. Brahmi
20. Coleus
21. Daruharidra Roots
22. Daruharidra Stems
23. Draksha
24. Ergot
25. Garcinia
26. Ginseng
27. Gokhru
28. Gudmar
29. Guduchi
30. Guggul resin
31. Haridra
32. Haritaki
33. Hingu
34. Ispaghula Husk
35. Ivy Leaf
36. Kalmegh
37. Kaunch
38. Kundru
39. Kutki
40. Lasuna
41. Lavang
42. Lodhra
43. Mandukaparni
44. Manjistha
45. Maricha
46. Methi
47. Mirch
48. Nagakesar
49. Neem
50. Pippali Large
51. Pippali Small
52. Punarnava
53. Sahajana Leaf
54. Sahajana Stick
55. Shankhpushpi
56. Sarpagandha
57. Saunf
58. Senna Leaf
59. Senna Pods
60. Shatavari
61. Shati
62. Sunthi
63. Tulasi
64. Valerian Root
65. Vasaka
66. Vidanga
67. Vijayasara
68. Yasti
1. Amla Juice Powder
2. Arjuna Dry Extract
3. Ashwagandha Dry
Extract
4. Bassant Dry Extract
5. Belladonna Dry Extract
6. Belladonna Soft Extract
7. Bhibhitaki Aqueous
extract
8. Brahmi Extract
9. Coleus Dry Extract
10. Garcinia Aqueous
extract
11. Ginseng Dry Extract
12. Gugulipid
13. Haridra Dry extract
14. Haritaki Aqueous extract
15. Haritaki Extract
16. Ivy Leaf Dry Extract
17. Kalmegh Dry Extract
18. Senna Dry Extract
19. Sunthi Extract
20. Tulasi Dry Extract
21. Valerian Dry Extract
22. Vasaka Extarct
23. Yasti Dry Extract
24. Opium
25. Bhuiamla Dry Extract
26. Gudmar Dry Extract
27. Kunduru Dry Extract
28. Mandukaparani Dry
Extract
29. Malt Extract
1. Arachis oil
2. Basil Oil
3. Belladonna Tincture
4. Black pepper oil
5. Caraway Oil
6. Cardamom Oil
7. Castor Oil
8. Clove Bud Oil
9. Clove Leaf Oil
10. Clove Stem Oil
11. Coconut Oil
12. Coriander Oil
13. Cumin Oil
14. Dill Seed Oil
15. Prepared Ergot
16. Eucalyptus oil
17. Guar Gum
18. Hydrogenated Castor Oil
19. Lavender Oil
20. Lemon Grass Oil
21. Lemon Oil
22. Lime Oil
23. Mentha Oil
24. Mentha Arvensis Oil
25. Nutmeg Oil
26. Papain
27. Peppermint Oil
28. Rosemary Oil
29. Sarpagandha Powder
30. Sarpagandha Tablets
31. Shellac
32. Starch
33. Thyme Oil
34. Tolu Balsam
35. Tragacanth
36. Opium powder
37. Ipecac Tincture
38. Gugulipid Tablets
39. Senna Tablets
Gross Total: A+ B+ C =
68 + 29+ 39 =136
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3. INCLUSION/ EXCLUSION CRITERIA OF A HERBAL
MONOGRAPH IN IP Pharmacopoeial herbal monographs contain information on the definition followed by specifications.
The specifications cover the various qualitative and quantitative tests, procedures and acceptance
criteria. The monographs shall employ various validated analytical procedures for the tests that are
feasible to be performed by a trained and experienced analyst without any repetition or development
of new procedure.
3.1 Inclusion Criteria
The following criteria were agreed upon by the Expert Committee:
The herb should be commercially available.
Should be of public interest.
Of clear and defined botany (in case of more than one species/varieties, cultivars of
same species/variety, there could be more than one monograph in IP).
Of known phytochemistry and reported even if not fully.
Some amount of information and /or method for analysis should be available, which
can be used as basis for development and validation. For this knowledge of
“markers”, if available is an advantage.
Sustainable (if the herb is in any regulated list, but knowledge of its sustainability
improvement is ongoing, and is of importance for use, a monograph would be
considered for inclusion).
Knowledge of its safety profiles through known history of its use.
For inclusion of an extract, either the extracts are made using traditional
methods/processes on a commercial scale or standardized extracts that are available
commercially using solvents are selected. For the latter cases, such extracts should
have been in commercial productions and use for at least 15 years, and their safety
profiles are known to the stakeholders.
Herbs should have therapeutic/ prophylactic value.
For finished products offered for sale in market, the products should have been
approved as a drug under Drugs & Cosmetics Act 1940 by CDSCO or by State
Regulatory Authority, as the case may be.
3.2 Exclusion Criteria
Drugs banned in India
Obsolete Drugs
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Drugs considered inappropriate by Indian Pharmacopoeia Commission and
Regulatory Authority.
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4. PROCESS OF IP HERBAL MONOGRAPH DEVELOPMENT AND
CONTENT OF A HERBAL MONOGRAPH
The principles of ‘openness, justice and fairness’ are kept in mind during compiling,
verifying/validating and editing the contents of the monographs in IP.
The herbal drug monographs in IP include crude herbs, processed herbs, herbal materials, herbal
preparations and finished herbal products that contain as active ingredients, the parts of plants, or
plant materials, or combinations thereof. The specific monograph of IP of a herb details the Title and
Synonym if any, Definition, Limits of active ingredient/Marker compounds, Description, Category,
Identification, Chemical Tests, Assay of the marker constituents, Contaminants, Specific Tests,
Storage conditions etc. Monographs are included in IP based on documented quality specifications
and suggestions/feedback from the stakeholders.
The general test methods which are common in nature are cited in the Appendix and are covered in
Volume I of IP. It involves rigorous consultation with experts, verification and validation of data,
identification of monographs and updation on continuous basis as well as responding to the
stakeholder’s queries on the existing monographs.
The flow diagram for herbal monographs development is given below.
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Herbal Monograph Development
Feedback from Stakeholders
Herbal monograph published in IP
Incorporation of valid comments for adoption in Indian Pharmacopoeia
Examination of public comments
Draft monograph displayed on IPC website for the public comments for a period of 45 days
Draft circulated to HPEG expert committee members, editing and formatting of draft monograph is done and forwarded to HPEG review
The participating institution verifies/validates the data and sends the draft monographs to the technical liaison staff of Indian Pharmacopoeia Commission
Sample authenticated and comparison made with reference substance for monograph development
Methods to be used for the Identification and Assay with literature reference are searched for analysis.
Efforts made to obtain 3 samples from different sources
Concurrence of Chairman of HPEG for the items for the monograph development
based on laid down criteria
Screening of proposal / request from stakeholder in the Department of Phytopharmaceuticals for herbal monograph inclusion in Indian Pharmacopoeia
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Content of a Herbal Monograph
The following concepts are important in the development and setting of specifications and should be
provided for each herbal monograph. The monograph should include: Title, Definition, Limits of
active ingredients, Marker compounds, Description, Category, Identification, Chemical Tests, Assay
of the marker constituents, Contaminants, Specific Tests, and Additional Requirements if any.
4.1 Monograph Title
For monographs intended for inclusion in pharmacopoeias, the title of the monograph should include
the Latin binomial nomenclature or Synonym or Common Name whichever is appropriate and is
followed by the name of plant part(s) or plant product (e.g., resin, gum-resin), and where applicable
the processed form.
4.2 Definition
Some or all of the following are usually included in the definition:
the state of the drug: whole, fragmented, peeled, cut, fresh or dried;
the complete scientific name of the plant (genus, species, subspecies, variety, author);
commonly used synonyms may be mentioned
the part or parts of the plant used
where appropriate, the stage in the growth cycle when harvesting takes place, or other
necessary information
wherever possible, the minimum content of quantifiable constituents (either responsible for
the biological activity of the herb (bio-marker) or a chemical compound known to be present
in the herb even if not responsible for biological activity (chemical/ analytical marker)
Herbal drugs very often contain a mixture of related substances, in which case the total
content of quantifiable constituents is determined and expressed as one of the constituents,
usually the major constituent; separate limits may be given for different forms of the drug
(whole/cut)
4.3 Characters
This section contains a brief description of the organoleptic characters of the drug such as colour,
odour, taste etc.
4.4 Category
It includes the therapeutic/prophylactic category of the drug.
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4.5 Identification. The purpose of the Identification in a monograph is to ensure that the article under
examination is in agreement with what is stated in the Definition of the article. All the identifications
mentioned below are not necessarily included: some may be absent when they are not feasible or are
not significant for the purpose of identification. Macroscopic and microscopic requirements of an
herbal monograph should be provided in detail with colour photographs. The detail about
identification is mentioned in Chapter 10.
4.5.1 Macroscopic. The important macroscopic botanical characters of the drug are specified to
permit a clear identification. When two species/subspecies of the same plant are included in
the definition, the individual differences between them are indicated.
4.5.2 Microscopic. It involves gross microscopic examination of the drug and it can be used to
identify the organized/ unorganized drugs by their known histological characters. It is mostly
used for qualitative evaluation of organized crude drugs in entire and powder forms with help
of microscope. It involves using microscope for detecting various cellular tissues and their
arrangements such as trichomes, stomata, starch granules and calcium oxalate crystals etc.
Crude drug can also be identified microscopically by cutting the thin TS (transverse section)/
LS (Longitudinal section) especially in case of wood. Quantitative aspects of microscopy
include study of stomatal number and index, palisade ratio, vein-islet number, size of starch
grains and length of fibers etc.
4.5.3 Fingerprinting. Chromatographic or spectroscopic patterns, sometimes referred to as
“fingerprints”, may be used as standards for identification. These fingerprints can be obtained
by HPLC, UHPLC, capillary electrophoresis, GC, TLC/HPTLC, IR, and Mass Spectroscopy.
The fingerprints must be able to distinguish these materials from other materials with
potential for species substitution and suspected adulteration. The acceptance criteria for
identification tests using chromatographic methods such as HPLC, UHPLC, capillary
electrophoresis or GC methodology must contain a description of the critical features of the
fingerprint chromatograms such as the presence of specified peaks, retention time, their order
of elution, and where possible, their relative abundance. For methods of TLC/HPTLC,
description must include colour and position of the characteristic bands. A colour image of a
typical TLC/HPTLC chromatogram should be provided. A critical aspect of the identification
of herbal materials by separation techniques is the use of reference standards for comparison.
In addition to the Sample solution, a Standard solution containing the reference standard is
chromatographed concomitantly. The reference material used in the Standard solution may be
an Authenticated Botanical Reference Substances (BRS), a reference standard extract, a single
chemical entity, or a standardized mixture of substances.
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4.6 Tests
4.6.1 Physicochemical Evaluation
It is an important parameter in detecting adulteration or improper handling of drugs. It can serve as a
valuable source of information and provide appropriate standard to establish the quality of herbs.
These are:
Extractable matter :
It is considered useful to determine extractable matter only in herbal drugs where no
constituent suitable for an assay is known or where the material is used to produce a
preparation with a dry residue.
Total ash:
This test is always included unless otherwise justified. It is to be carried out on the powder
drug.
Acid- insoluble ash :
This test may be carried out depending on the nature of the particular herbal drug and is used
to detect unacceptable quantities of certain minerals.
4.6.2 Loss on Drying
Herbal drugs are dried for preservation purposes. If they are insufficiently dried, growth of yeasts or
moulds may occur. It is the loss of weight expressed as percentage w/w resulting from water and
volatile matter of any kind that can be driven off under specified conditions. The limit is specified on
the basis of the results obtained on a reasonable number of varied samples of acceptable quality.
4.6.3 Swelling Index
Applicable to certain hydrocolloid-containing herbal drugs.
4.6.4 Bitterness values
Applicable to herbal drugs containing bitter principles.
4.7 Contaminants- General
4.7.1 Foreign Organic Matter. It is the material consisting of any or all of the following:
Parts of the organs from which the drug is derived other than the parts named in the definition
and description or for the limit are prescribed in the individual monograph.
Any part of organs other than those named in the definition and description.
Matter not coming from the source plant and
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Moulds, insects or other animal contamination.
Generally a limit of 2% of foreign matter is imposed, unless otherwise prescribed in a specific
monograph. Where a limit for foreign matter greater than 2% is to be prescribed, it is stated in the
specific monograph with an indication of the type of foreign matter. Where necessary, the monograph
should indicate how the foreign matter is identified.
4.7.2 Heavy Metals
The test is prescribed where there is the potential for contamination by heavy metals. The limit of
heavy metals is indicated in the individual monograph in terms of ppm.
4.7.3 Microbial contamination
The Pharmacopoeial monographs should specify the total count of aerobic microorganisms, the total
count of yeasts and molds, and the absence of specific pathogenic bacteria (e.g., Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella and Salmonella species)
4.8 Contaminants- Specific
An individual herbal monograph may require certain specifications that are peculiar to that
monograph, especially when safety is an issue. Limits may be set in certain specific monographs for
the characters that are undesirable or have negative botanic characteristics. When one desires a limit
for harmful substances that are present either naturally in the substance or formed as a result of post-
harvest processing practices, such submissions must be accompanied by toxicity data.
4.9 Assay
Wherever possible, an assay is included. Assay is carried out using suitable instruments such as UV-
Visible spectrophotometer, LC, GC or by HPTLC system etc.
4.10 Additional Information
4.10.1 Storage
Storage conditions are applicable unless otherwise specified: store protected from light. Where
applicable, additional specific conditions are given in the individual monograph.
4.10.2 Labelling
Labelling of herbal products includes the label both upon the immediate container and other
associated labeling and written, printed or graphic materials. The label states the Latin binomial
followed by the authorized name; the plant part(s), plant product, or processed form contained in the
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container or from which the article was derived. Content in percentage of active principles or marker
compounds should be stated. Labelling should be in accordance with the applicable drug laws.
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5. GENERAL MONOGRAPH FOR HERBS, PROCESSED HERBS AND
HERBAL PRODUCTS
5.1 Herbs and Herbal Products
Herbs and herbal products have been in trade and commerce and are currently used for a variety of
purposes. India has a rich history of use of herbs, processed herbs and formulations containing herbs
both from traditional wisdom as well as cultural usage. Herbs and herbal products are also regulated
by various laws. For the purposes of pharmacopoeial standards various considerations have been
given. This chapter provides a general outline towards the definitions of crude herbs, processed herbs
and herbal formulations.
5.2 Crude Herbs
This term means, unless specified otherwise, mainly whole, fragmented or cut plants, parts of plants,
algae, fungi, and lichen in a form which is not processed. Herbs are usually in dried form, but
sometimes, when specified, may also be in a fresh form. In specific cases exudates which have not
been processed further also are covered under the term herbs. Processing, does not include, normally
expected value addition steps like grading, sizing, removal of weeds or parts of plants other than those
specified herb and removal of adulterants. The term herbs, though botanically generally refer to plants
of specified height and nature, for the purposes of pharmacopoeial reference, shall mean and include
plants and parts of plants not necessarily from herbs and shrubs, but cover the entire range namely
creepers, climbers, trees etc. Each monograph of a herb in the pharmacopoeia shall specify the
scientific name according to binomial system specifying the genus, species, variety and author. In
cases where there are controversial botanical identity, as is seen with mainly herbs known in the
Indian traditional system, the monograph shall specify the official name of the herb along with its
botanical scientific name and guidance is taken from Ayurvedic Pharmacopoeia of India* to decide
the same. In cases where, the same herb is available in different grades or sizes, if found appropriate
and necessary, separate monographs may be introduced in the pharmacopoeia to cover each of them
with appropriate standards. For example -Pippali (large) and Pippali (small).
*Ayurvedic Pharmacopoeia of India, Deptt. of Ayush, Ministry of Health and Family Welfare,
Govt. of India.
In order to improve authentication of botanical identity, especially in those cases where there are
controversial botanical identity or chances of substitution/adulteration is expected, IP has provided
general test method adopting DNA-based identification test. This method has been provided in
appendix 2.2.17 under General Tests. A detailed test method adopting this has been introduced in the
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monograph of Shatavari to begin with. Testing by this method has been made mandatory only in case
of a dispute.
While deciding to introduce a monograph for a herb in the pharmacopoeia, the criteria that would be
kept in mind, but not limited to are - herbs with specific name and a definitive botanical identity up to
species, availability and usage in trade and commerce, public interest, knowledge of and availability
of a specific chemical compound of well characterized structure [either responsible for the biological
activity of the herb (bio-marker) or a chemical compound known to be present in the herb even if not
responsible for biological activity (chemical/analytical marker)], availability of a quantitative method
for estimation of such a compound, knowledge of safety of the herb, and its sustainability. Herbs
which may figure in a regulated list under appropriate forest and other laws, may still be taken up for
a monograph for inclusion in pharmacopoeia, if there is knowledge of efforts to cultivate or take care
of sustainability issues and /or specific permission is available under law for use of the herb. As
already specified under “General Notices” in the pharmacopoeia, appearance of a monograph does not
mean its approval as a drug under the law. Monograph of a herb in the pharmacopoeia is to provide
qualitative and quantitative standards of quality for the herb for its use either as a food item or food
ingredient or food supplement/ nutraceuticals, as a drug, and/or as an ingredient in cosmetics. Each
such use would need to comply with applicable regulations. Each herb is regarded as one active
substance, irrespective of the knowledge about the active constituents of the herb is available or not.
Herbs may be exposed to low dose gamma radiation for purpose of reducing their microbial
contamination. Herbs treated with low dose gamma radiation shall meet national laws related to such
treatment and shall be labeled as per law.
5.3 Processed Herbs
Processed herbs means preparations obtained by subjecting herbs to treatment such as extraction,
distillation, expression, fractionation, purification, concentration and partial or full fermentation.
Processing may also be done by way of powdering herbs, preparing tincture, preparing extract,
distilling to get essential oils, fatty oils (either expressed or solvent extracted or a blend of both)
expressed juices, extracted exudates, gums and oleo resins, liquid extract where the solvent is
evaporated to yield concentrated semi solid mass or dried mass. Extraction may be performed by
means of appropriate technology such as infusion, maceration, soxhleting, boiling under ambient or
higher pressure, with or without specified enzymes, with or without agitation and combination
thereof. Drying of liquid extracts for removal of the solvent may be done by using various appropriate
technologies like air drying, sun drying, drying under vacuum or with forced air circulation, drying at
low temperature with air circulation, by way of lyophilization or freeze drying. Extracts of herbs may
also be prepared by using carbon dioxide as a solvent-super critical fluid extraction.
Usage of distillates of herbs especially adopting steam distillation is increasing – for use to provide
health benefits/pharmacological actions when used as products for external use as well as in some
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cases internally used, and also for flavour and fragrance purposes. Such distillates may be referred as
distilled oils. Distilled oils may be used with or without partial fractionation, purification, selective
removal of identified components (De-mentholized Mentha oil etc.) and blending of natural distilled
oil with fractionated oils. Distilled oils are normally packed in tightly closable glass containers or
glass-lined aluminum container or aluminum containers properly labelled and are to be stored and
transported at temperature below 25°. Exposure to heat and light are to be prevented recognizing their
high flammability, and potential oxidation.
Extracts may be liquid extracts and tinctures, semi solid (soft extracts) or solid dry extracts of known
consistency obtained from herbs. Standardized extract, a term commonly employed, would for
pharmacopoeial purposes, mean an extract adjusted within an acceptable tolerance to a given content
of bio-marker or chemical/analytical marker. Standardization may be achieved by adjusting the
extracts with approved inert material or by blending one or more batches of extracts. Wherever
possible, extracts shall specify the defined range of the constituents (bio-marker or chemical/
analytical marker). Extracts not covered in the above description would be defined by the process of
production of the herb to the extract, solvent used and technology applied. The difference between
extracts and tinctures would be, in the type of solvent used for extracting an herb, and tincture would
normally mean an extract where aqueous-ethanol is used as a solvent for extraction. Dry extracts
usually have a loss on drying or water content not greater than 5 per cent w/w, unless specified
otherwise in any monograph. It is normal to extrapolate safety aspects and history of use information
for extracts as long as the process, solvents, extraction ratios are comparable to the processes used in
documented traditional knowledge. Additionally in cases of standardized extracts the inert
excipients(s), if any used for standardization or adjustment of the content of constituents should also
are declared on the label of such extracts. Extracts shall be free from solvent used for extraction and
shall comply with the respective limits as given in Appendix 5.4 (Residual Solvents) of IP Volume I
Harmful and carcinogenic solvents shall not be used for extraction purposes. Solvents and solvent
systems may include use of propylene glycol, glycerin, sorbitol and such other polyhydroxy alcohols,
as long as the content of such polyhydroxy alcohol are within safe limit in the final product.
In cases where extraction and fractionation process leads to preparation of an extract, which consists
of a single chemical compound of more than at least 70 per cent purity, such extracts shall be treated
as an active pharmaceutical ingredient or a food additive or a cosmetic ingredient and would be
required to meet relevant laws.
Extracts may also be offered as purified or enriched extracts. Such extract of a herb is processed in
such a way to provide higher than normal proportion of the active constituent (s) of the herb as long as
the active constituent (s) is/are known. Such purified or enriched extracts may contain additional
valuable components which may provide specific properties like enhanced efficacy or stability or
solubility and availability of the active constituent (s). Purified and enriched extracts may also be
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prepared to reduce or remove other specific compound or group of compounds that is scientifically
considered undesirable in the herb extracts. Pharmacological, toxicological, pharmaceutical
considerations need to be applied while preparing such purified or enriched extracts. Mixed extracts
may also be offered which would cover combination of more than one herb extract for purposes of
providing simplification or economical way to manufacture herbal formulations.
Herbs may also be extracted using vegetable oils (approved by Food Law) for extraction purposes and
such extracts shall specify the oil used for processing.
Approved preservatives or preservatives system may be used during preparation of extracts. The
names of such preservatives used which would remain in the final extract shall be listed on the label
of such extract, and the proportion of preservatives used shall not exceed normally accepted safe
limits of their usage as per relevant laws or pharmacopoeial standards. No artificial colours may be
used in extracts of herbs unless and otherwise specified in the specific monograph. Only approved
colours shall be used.
Extracts may be exposed to ethylene oxide fumigation or low dose gamma radiation for purposes of
reducing their microbial contamination. In cases where they are fumigated, the final extracts exposed
shall meet residual levels of ethylene oxide limits as applicable. Herbs treated with low dose gamma
radiation shall meet national laws related to such treatment and shall be labelled as per law.
5.4 Herbal Formulations
Herbal formulation shall mean a dosage form consisting of one or more herbs or processed herb(s) in
specified quantities to provide specific nutritional, cosmetic benefits, and/or other benefits meant for
use to diagnose treat, mitigate diseases of human beings or animals and/or to alter the structure or
physiology of human beings or animals. Dosage forms commonly employed for food or cosmetic or
pharmaceuticals may be employed to formulate one or more herb or processed herbs. Dosage forms
known in traditional medicines may also be adopted for preparing herbal formulations, either for
external use or for internal administration. Adequate consideration for uniform distribution of herb or
processed herbs as well as stability of the same in the dosage form shall be provided during
formulation development.
Herbal formulation shall be labelled to comply with relevant labelling requirements under food or
drug or cosmetics laws as applicable. Additionally, adequate information shall be provided on label of
such formulations to include the name of the herb, parts used, nature and type of extract or processed
herb used, extraction ratios, quantity per unit dose or per serving, name (s) of inert excipients used
and any preservatives added shall be provided on the label.
Appearance of a monograph of a herbal formulation in the pharmacopoeia does not mean its approval
as a drug under the law. Monograph of a herbal formulation in the pharmacopoeia is to provide
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qualitative and quantitative standards of quality for the formulation for its use either as a food item or
food ingredient or food supplement/ nutraceuticals, as a drug and / or as a cosmetic. Each such use
would need to comply with applicable regulations. Each extract is regarded as one active substance
irrespective of the knowledge about the active constituents of the herb is available or not.
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6. STANDARD OPERATING PROCEDURE FOR PREPARATION OF
MONOGRAPHS OF HERBS, PROCESSED HERBS AND HERBAL
PRODUCTS IN IP
Standard Operating Procedure (SOP) of herbs, processed herbs and herbal products monographs for
submission to IPC is described below:
6.1 Requirements for inclusion of photograph of an herb /part of the herb
a) An herbal monograph in IP provides photograph of the herb. Such a photograph shall provide a
clear visual depiction of the herb, part of the herb.
b) A photograph of the herb shall appear immediately after the title/synonym in the monograph.
c) An authentic sample of herb/part of a herb, properly cleaned, kept within a grid printed on a
paper which gives it the size denotation as illustrated in Table 1 shall be photographed using an
appropriate camera, with a minimum of 3 megapixels capacity. The pieces should be clearly
visible.
d) Alternatively, place such a sample on a glass plate which can be illuminated from below using
a suitable lamp and photograph it from a suitable distance from the top with proper focus.
While doing so depending on the colour of the backgrounds like butter paper, white paper,
black paper etc. may be used suitably.
e) The photograph shall be saved and reproduced in the IP as a composite photograph occupying a
size of 8 x 6 cm.
f) Alternatively, the same may also be reproduced in such a way to cover the requisite units
occupying 5 x 6 cm and a photograph of 1 or maximum 2 single units in a “close up” mode
occupying 3 x 6 cm size. In no case any photograph shall exceed 8 x 6 cm size. Table 1.
Describe the number of units of each material to be taken for the photograph.
While taking the photograph, name of the botanical as given in the title of the monograph (this shall
be printed in “Times New Roman” Font, with a size of 14) shall be kept along with the units of
botanicals
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Table 1: Description of number of units of each herbal material
Category No. of
Units
Category No. of
Units
Woody and available in large pieces –
stem, wood, and heartwood, woody roots
(Eg. Deodar, Erandmool)
4-6 Stems and roots with smaller
diameters
(Eg. Ephedra, Manjistha, Kutaki)
8-14
Leafy and creepers cut into parts
(Eg. Bhringraj, Neem)
10 to 12 Stigma, Style, Anthers, Small Petals,
Buds
(Eg.Keshar, Lavang)
20-40
Fleshy Dried Rhizomes
(Eg.Vidarikand, Varahi)
4-8 Minute seeds and parts of seeds
(Eg.Vakuchi, Isabgol)
More than
40
Flowers, Larger Petals, Small Fruits
(Eg.Japakusum, Kusumphool)
10-20 Resins, Gums in dried form
(Eg. Heeng, Babool)
4-8
Bark cut into pieces
(Eg. Arjuna, Kutaz)
3-8 Minute parts like epidermal hair
(Eg. Kamela)
5-10
Example: Photograph of Arjuna
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Grid to be used to place the herbs for photograph (Each block is of 1 cm2)
Empty Grid
6.2 SOP for TLC/HPTLC profile
a) A “Typical TLC/HPTLC profile” depicts the results of the test for identification/assay used.
A photograph of such TLC profile should be submitted on a separate sheet.
b) Identification tests by TLC/HPTLC shall be performed as per specification given in the
respective monograph
c) As a common practice, the plate shall be of at least 5 cm width and 10 cm height. In this
dimension 2 bands each of 10 mm width would be spotted. As a rule the extreme left track
(track 1) shall always be a Botanical Reference Substance (BRS)/ Phytochemical Reference
Substance (PRS). The track 2 of 10 mm width band shall be of a solution of material under
examination. All the bands shall be applied at a height of 20 mm from the base of the plate.
During development, the solvent front shall be allowed to move to at 80% of the plate height.
d) A photo-documentation of the plate developed as above, after visualization under UV 254 nm
and 366 nm, and/or by any derivitizing or by spraying reagents shall be photographed.
e) On top of all pages where such typical TLC/HPTLC profiles are reproduced following text
should be written.
“Reproduce below is Typical TLC Profiles of herbs for which a monograph appear in IP. Each
typical profile bears the name of the monograph below the profile.”
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Example: Pippali (Small)
Reproduce below is Typical TLC Profiles of herbs for which a monograph appear in IP. Each typical
profile bears the name of the monograph below the profile.
(Under UV light at 254 nm) (Under UV light at 366 nm)
6.3 SOP for A typical “HPLC / GC chromatogram”
a) A HPLC/GC Chromatograms should be submitted on a separate sheet.
b) Separate chromatograms should be submitted for respective PRS and sample under
examination. The peak of the PRS/ compound under examination shall be labelled
accordingly in the respective chromatograms, suffixed by a small arrow pointing to the peak.
Other peaks that may appear in the chromatogram, whose chemical identity is not known,
need not be labeled.
c) While supplying such HPLC/GC chromatograms please ensure that the chromatogram
should;
Contain appropriate scale in X and Y axis with respective units.
Not contain any notations given by the equipments like date, sample details, annotation
and all such other matter.
Not contain names of analyst, firms that may appear as a routine part due to settings. On
top of all pages where such typical chromatograms are reproduced, following text should
be written:
“Reproduce below is Typical chromatograms of herbs for which a monograph appear in IP. Each
typical chromatogram bears the name of the monograph below it. Methods adopted and the
interpretations of the chromatogram are detailed in the monograph.”
(Under day light after spraying with
vanillin sulphuric acid reagent)
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Example: Typical HPLC Chromatogram of Shankhpushpi
Reproduce below is Typical chromatograms of herbs for which a monograph appear in IP. Each
typical chromatogram bears the name of the monograph below it. Methods adopted and the
interpretations of the chromatogram are detailed in the monograph
HPLC chromatogram of PRS
HPLC chromatogram of Shankhpushpi
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Example: Typical GC chromatogram of Nigella sativa”
“Reproduced below are Typical chromatograms of herbs for which a monograph appear in this
edition of IP”.
GC chromatogram of Nigella sativa
p-C
ym
ene
Res
pon
se (
mV
)
α-
Pin
ene
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7. STANDARD OPERATING PROCEDURE FOR HERBAL EXTRACTS
MONOGRAPHS
Requirements for Developing Monographs for Inclusion in IP
7.1 Purpose
This document provides guidance for preparation of monographs for herbal extracts (one of the types
of processed herbs) for inclusion in the Indian Pharmacopoeia”.
7.2 Definition
a) Processed herbs have been explained in detail in the General Requirements for “Herbs and
Herbal Products” in IP 2014, Vol 3; page 3169. “Extracts” may refer to either an extract of a
herb prepared by using water or organic solvent as a solvent for extraction.
b) As per IP 2014’s General Requirements, in the extract of the herb prepared using water as a
solvent for extraction, it is appropriate to extrapolate the history of safe use, if the herb is also
known in Ayurveda or other traditional systems of medicines.
c) For purposes of IP, extract of the herb prepared using an organic solvent for extraction is
referred as “Solvent extract”. As per IP 2014’s General Requirements, for such extracts, it
may not be appropriate to extrapolate the history of safe use from Ayurveda or other
traditional systems of medicines, as the processing methods /usage methods differ from that
known in tradition.
For such extracts the following additional requirements need to be met for inclusion in IP.
The solvents used should be safe, avoid chloroform and such solvents. The final product
(extracts) needs to conform to residual solvent levels requirements of the pharmacopoeia.
The extracts being included in IP, should have been in commercial usage for at least a
period of 10 years. The stakeholder who shares the data and provides information to help
IP include the monograph need to certify that the particular extract/process has been
followed by them. Additionally the stakeholders need to inform IP committee regarding
any safety/toxicology data.
In addition to steps of solvent extraction, if the processing of the herb involves other
steps like salting out, (as sodium/potassium/calcium salts) or other esterification, such
extracts may also be considered for inclusion in IP, subject to them meeting requirements
under section above.
Stakeholders should not suggest an extract whose process has undergone changes and
have not been in market for at least 10 years. This ten years commercial usage
requirement means that the particular candidate extract has been manufactured on a
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32
commercial scale and has been supplied to industry in India, or abroad for manufacture
of any category of products for human or animal consumption.
The decision of inclusion of any monograph for any extract monograph however is the
prerogative of Scientific Body of IPC. The stakeholders would need to make available
the safety information available with them for inspection to herbal of IPC. They need not
provide copies to herbal expert group as it would be the property of the firm. However,
the herbal expert group would need to see the data before agreeing for inclusion or
otherwise of the proposed extracts monograph. IPC’s herbal expert group may also
request for any additional information to substantiate the long years of usage of the
extract under consideration.
It may also be appreciated that the mere inclusion of a monograph for any extracts in IP
does not necessarily mean its approval as a drug. (Please refer to General Monograph on
Herbs, processed herbs and herbal products, IP 2014, Vol 3, pg. 3170).
The process of developing a monograph shall be the same as adopted by IP for raw herbs or for
finished herbal products. For this purposes samples to be tested should be from three commercial
batches and not from small batches made in the laboratory. Batches made in pilot plants can be used
provided the batch sizes are at least 15% of the normal commercial batch sizes intended or adopted
for commercial purposes. Samples of three such batches shall be involved in development of draft
monographs and theses samples will also undergo “RING TESTING”, with two more labs including
Indian Pharmacopoeia Laboratory. The final monograph, test methods and the tolerance limits, shall
be decided based on application of scientific thinking to the results of ring testing.
For each monograph on extracts there shall be a mandatory testing for quantitative assay of at least
one or more marker(s) compounds which are well characterized chemical substance(s).
At this stage testing for at least one marker is to be relied upon, and in cases where scientific
knowledge exists /or is developed for more than one marker that can be tested, such multiple marker
testing may be adopted and prescribed by IP:
a) The monograph of extract shall at the beginning prescribe the quantitative contents (minimum
or limits) for such marker(s).
b) These marker(s) may be either those that are linked to biological activity or just analytical
marker(s).
c) The marker(s) used for testing need not be the same, and can be different basis the presence of
the compounds and the scientific knowledge.
d) Test(s) for absence of any marker may also be considered & included, if felt necessary to
control quality or safety through such testing.
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e) Other physicochemical tests that can give information for objective assessment of quality of
the extract would be included in the Standard tests to be performed.
f) Other requirements like microbial contamination and heavy metals as per IP shall also be
applicable.
g) The monographs shall also mention and include any tests that may be required to check for
the excipients added, if any, if there is a need felt.
The final monograph developer shall also provide to IP, the chromatograms for inclusion in IP under
the relevant section as being done for herbs, as guide to users.
Example of Extract Monograph in IP 2014
Arjuna Dry Extract
Arjuna Dry Extract is obtained by extracting Arjuna
(Terminalia arjuna Wight and Arrn,
Fam. Combretaceae) bark with methanol or any other
suitable solvent and evaporation of solvent.
Arjuna Dry Extract contains not less than 90.0 per
cent w/w and not more than 110.0 per cent w/w of
stated amount of the arjunolic acid on the dried basis.
It may contain suitable added substances.
Category. Antihyperlipidaemic, antihypertensive,
astringent, cardioprotective, hrdroga.
Usual strength. 60 per cent w/w.
Description. A light brown to beige powder with or
without red tinge.
Identification
A. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase. A mixture of 92 volumes of chloroform
and 8 volumes of methanol.
Test solution. Dissolve 50 mg of the extract under
examination with 50.0 ml of methanol and filter.
Reference solution. A 0.1 per cent w/w solution of
arjunolic acid RS in the methanol.
Apply to the plate 5 µl of each solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 8 cm.
Dry the plate in air and spray with anisaldehyde
sulphuric acid reagent. Heat the plate at 110° for 10
minutes and examine the plate under ultraviolet light
at 365 nm and day light. The chromatographic profile
of the test solution is similar to that of the reference
solution.
B. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase. A mixture of 35 volumes of
chloroform, 10 volumes of methanol and 2 volumes of
water.
Test solution. Dissolve 50 mg of the extract under
examination with 10.0 ml of methanol and filter.
Reference solution. A 0.05 per cent w/w solution of
arjugenin RS in the methanol.
Apply to the plate 5 µl of each solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 8 cm.
Dry the plate in air and spray with vanilline sulphuric
acid reagent. Heat the plate at 110° for 10 minutes
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34
and examine the plate under ultraviolet light at 365
nm and day light. The chromatographic profile of the
test solution is similar to that of the reference solution.
Tests
Ethanol-soluble extractive (2.6.2). Not less than
80.0 per cent.
Total ash (2.3.19). Not more than 5.0 per cent.
Heavy metals (2.3.13). 1.0 g complies with the limit
test for heavy metals, Method B (20 ppm).
Loss on drying (2.4.19). Not more than 5.0 per cent,
determined on 1 g by drying in an oven at 105°.
Microbial contamination (2.2.9). Complies with the
microbial contamination tests.
Assay. Determine by liquid chromatography (2.4.14).
Test solution. Shake a quantity of the extract under
examination containing about 50 mg of arjunolic acid
in 50.0 ml of the methanol, filter.
Reference solution. A 0.1 per cent w/v solution of
arjunolic acid RS in methanol.
Chromatographic system
– a stainless steel column 25 cm x 4.6 mm
packed with octadecylsilane bonded to porous
silica (5 µm),
– mobile phase: a mixture of 35 volumes of 5 mM
α- cyclodextrin and 65 volumes of methanol,
– flow rate: 1 ml per minute,
– spectrophotometer set at 205 nm,
– injection volume: 20 µl.
Inject the reference solution. The test is not valid
unless the relative standard deviation for replicate
injections is not more than 2.0 per cent.
Inject the reference solution and the test solution.
Calculate the content of the arjunolic acid in the
extract.
Storage. Store protected from heat and moisture.
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8. STANDARD OPERATING PROCEDURE FOR PREPARATION,
QUALIFICATION, CERTIFICATION & SUPPLY OF BOTANICAL
REFERENCE SUBSTANCES (BRS)
8.1 Purpose
This SOP provides the procedure for preparation, qualification and supply of “Botanical Reference
Substance” (hereinafter referred to as BRS) to be used for identification of commercial supplies of
raw material, of botanical origin, as per the procedures mentioned in Indian Pharmacopoeia.
8.2 Definition
Botanical Reference Substance is a standard whose botanical identity and genuineness has been well
established to both genus and species level.
8.3 Need
It is to be used as a reference material for comparison and confirming the identity of the commercial
supplies of the respective botanical as prescribed under tests for identity in the monograph.
Compliance to identity test using microscopic, chromatographic (TLC / HPTLC fingerprint) and other
specified tests will involve use of BRS. The BRS generally has a shelf life of 2 years unless otherwise
stated. Each BRS should be supplied with documentation describing the above characteristic features.
8.4 Scope
Applicable to all the laboratories who are involved in generating ‘BRS’, who will prepare them for
Indian Pharmacopoeia Commission (IPC), who will certify the same prior to issue.
8.5 Responsibility
IPC: To appoint and authorize one or more laboratories to prepare specific (one or more)
BRS.
Appointed Laboratory: To prepare specific (one or more) BRS as per the procedure described
below and supply the same to IPC from time to time.
IPC to certify the quality of BRS and make them available to the users along with COA
(Certificate of analysis).
IPC to periodically upgrade or revalidate the available BRS.
IPC to make public, the availability of a list of all BRS and their current lot along with price.
8.6 Procedure for appointed Laboratory
a) Collect about 4.0 Kg of raw material after botanical authentication. Botanical authentication will
be done to confirm the genus and species of the botanical raw material. Botanical authentication is
to be done through a qualified and experienced Botanist/Pharmacognosist of any recognized
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36
Institutions who would issue a certificate of verification on his Institution’s letter head. Such
authentication should be done at least from two crude drug verification laboratories like
CSIR-National Institute of Science Communication and Information Resources, Dr. KS
Krishnan Marg, Pusa IARI Campus, New Delhi-110 012
CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow – 226001, UP
Agharkar Research Institute, Gopal Ganesh Agharkar Road, Pune – 411004
Jawaharlal Nehru Ayurvedic Medicinal Plants Garden, Near Gandhi Bhavan,
Kothrud, Pune- 411 029, Maharashtra.
Bombay Natural History Society, Hornbill House, Dr. Sálim Ali Chowk, Shaheed Bhagat
Singh Road, Mumbai - 400 023.
b) Prepare at least two specimen of the said plant material out of the 2.0 kg raw material
obtained, label them properly and preserve for future reference.
c) Dry the material in an appropriate manner to approximately 5% w/w moisture content.
Document the passport data including processing details. In case the dried material is
procured from a supplier, the passport data will not involve any processing details.
d) Perform the test(s) for identity as prescribed in the monograph of particular botanical in
Indian Pharmacopoeia. Material which meets the requirement(s) will only be taken up for
processing further. Document all the tests performed, their results and the inference properly
and generate the certificate of analysis (COA) duly signed by the analyst(s) who performed
the analysis and authenticated by the Head of the Laboratory.
e) Powder the raw material to fine size (to pass through 22#sieve) using previously cleaned and
dried suitable grinder free from contamination.
f) Distribute approximately 2 g depending upon the bulk density of the material, into either
manually or using suitable filling equipment such as in a cleaned, dried vial free from any
contamination. For this purpose amber colored USP type 1 Glass vials generally used for
injectables may be used. Vials of size from 2, 5, 10 and 30 ml as required may be used to
hold 2 g of the material without leaving too much of air head space. The mouth of the vial
should be plugged with the suitable size rubber plug previously cleaned and the mouth sealed
with aluminum “Tear Down Seals”. Tear down Seal can be easily applied and sealed on the
mouth of the vials either with a hand sealer or with mechanical sealing machine. Each such
vial containing the powdered material shall be affixed with a label containing the following
information.
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37
BOTANICAL REFERENCE SUBSTANCE
Name of BRS, IP (as given in IP Monograph):
Lot No: BM IL DDMMYY Date of Packing: June 15
Storage: Cool and dry place
========================================
Indian Pharmacopoeia Commission, Ghaziabad
Self-adhesive sticker label may be used on which information given below printed using a PC
or otherwise.
g) The Lot No should consist of 4 letters and 6 digits. The first two letters representing the Name
of the botanical, next two letters representing the name of the laboratory, followed by 2 digits
of date, 2 digits representing the month and last 2 digits representing the year of packaging
the BRS.
Eg. Lot No. “BM IL DDMMYY represents “Bacopa monniera, Indian Laboratory” and
the BRS has been packed on the respective date in format DDMMYY, where DD
represents Date, MM represents Month and YY represents Year.
h) Pack the sealed vials in suitable shipper with honeycomb partition as per the requirement of
size and shape of the radiation lab. Most of the existing radiation agencies accept 60 x 43 x 34
cm cartons. Affix copies of above label, in suitable size, at least on two panels of each
Shipper.
i) Send the entire shipper properly sealed and labeled as above to a center for gamma radiation
at 6- 14 k Gy along with suitable dispatch documents.
j) Collect the shippers after irradiation along with certificate of irradiation from the irradiation
centers.
k) Collect 10 irradiated BRS samples and preserve them as Control sample. Dispatch the rest of
the irradiated samples to IPC along with following documents.
Botanical Authentication certificates
Certificate of analysis
Certificate of irradiation issued by irradiating agency
Packing Chalan and in-voice
Certificate for transportation (certifying that material is non-hazardous etc)
Labelling requirement for candidate Botanical Reference Substance
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7.7 Procedure for IPC
a) On receipt of the consignment cross check the material received against
documents/certificates received.
b) Take adequate samples randomly from the shippers and perform identity test(s) in duplicate
as per standards given in Indian Pharmacopoeia.
c) Upon the samples meeting the requirements of IP, the consignment is ready for certification
as BRS. The COA is prepared as discussed earlier.
d) Upon conforming the materials as BRS, each vial is to be affixed a prior printed “SEAL”
which reads as below-
e) “CERTIFIED BRS – IPC”, such seals need to be affixed in such a way that part of seal
overlaps the label affixed by the appointed laboratory. A suggestive specimen of such seal is
given below. A hologram is also to be added to make sure duplication does not take place.
BOTANICAL REFERENCE SUBSTANCE
Name of BRS, IP (as given in IP Monograph):
Lot No: BOOL/210706 Date of Packing: July 06
Storage: Cool and dry place
=======================================
Indian Pharmacopoeia Commission, Ghaziabad
f) IPC will store the certified BRS samples consignment between “Temp 2-15 0C and 60% RH”.
g) IPC shall supply the requisite no of BRS packs as per order along with a copy of the COA.
7.8 Relevant documents to be maintained by the Appointed Laboratory.
Two reference samples of initial crude botanical.
Authentication certificates
Passport data
Irradiation certificate
COA
IPC
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9. STANDARD OPERATING PROCEDURE FOR PREPARATION,
QUALIFICATION, CERTIFICATION AND SUPPLY OF
PHYTOCHEMICAL REFERENCE SUBSTANCES (PRS)
9.1 Procedure for appointed Laboratory:
9.1.1 Perform a literature survey, identify and shortlist one or more relevant publications
which can form the basis for a procedure to be adopted to isolate the unique chemical
compound with known structure identified to be used as a PRS. Write down an
approach note/ block diagram of the steps involved in isolating the compound. If
available, note down the percentage of the compound reported in the literature from
that particular plant material.
9.1.2 Collect requisite quantity of fresh raw material (adequate to provide about 1000 mg
of PRS) after botanical authentication. Botanical authentication will be done to
confirm the genus and species of the botanical raw material. Botanical authentication
is to be done through a qualified and experienced Botanist/Pharmacognosist of any
recognized Institutions who would issue a certificate of verification on his
Institution’s letter head. Such authentication should be done at least from two crude
drug verification laboratories like –
CSIR-National Institute of Science Communication and Information Resources, Dr. KS
Krishnan Marg, Pusa IARI Campus, New Delhi-110 012
Agharkar Research Institute, Gopal Ganesh Agharkar Road, Pune – 411004
Jawaharlal Nehru Ayurvedic Medicinal Plants Garden, Near Gandhi Bhavan, Kothrud,
Pune-411 029, Maharashtra.
9.1.3 Process the raw material adopting the procedure as mentioned. Use solvents/solvents
mixtures and relevant chromatographic techniques which may involve column
chromatography or flash chromatography or semi-prep HPLC or GC. Collect the
relevant fractions which are expected to contain the compound to be isolated and
process them to obtain a mixture which is predominantly rich in the desired PRS.
Perform TLC and/or any other test at different stages of such separation to guide that
the process is moving in the right direction.
9.1.4 Purify the above fraction containing the desired PRS using column chromatography
/crystallization technique /preparative HPLC depending on the quantity of the
compound present in the fraction.
9.1.5 Assess the purity of the isolated compound using different chromatographic
techniques like TLC / HPLC / GC etc. If the purity of the compound is less than 98%
w/w, repeat the process as in 9.1.3 including any simple re-crystallization steps
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40
involving the relevant solvents of analytical reagents grade. Assess the purity again
to confirm greater than 98% purity before proceeding to the next stage.
9.1.6 Determine the structure of the compound adopting all necessary spectroscopic and
other measurements techniques including, but not limiting to CHN analysis, MS, IR,
UV, NMR, Specific rotation, chiral testing etc.
9.1.7 Prepare a report (COA) documenting the purity testing as well as all the relevant
structure determination testing along with the results obtained, interpretation and
attaching copies of purity and structure determination data.
9.1.8 In those cases, where the PRS compound is being prepared by a synthetic process
(and not isolated from the plant material as above), document the steps involved in
the synthetic process, purification of the final compound obtained as well as its purity
and structure confirmation.
9.1.9 Distribute either manually or using suitable filling equipment (previously cleaned and
dried, free from any contamination) approximately 10 mg (or a quantity as specified
by IPC) into previously cleaned and dried glass vials of suitable size depending upon
the bulk density of the material. Flush the vials with slow stream of nitrogen to
replace the air in the Vials and close with rubber bungs. For this purpose amber
colored USP type 1 Glass vials generally used for injectables may be used. Vials
from size of 2, 5, 10 ml as required may be used to hold the material without leaving
too much of air head space. The mouth of the vial should be plugged with the suitable
size previously cleaned rubber plug and the mouth sealed with aluminum “Tear
Down Seals”. Tear Down Seal can be easily applied and sealed on the mouth of the
vials either with a hand sealer or with mechanical sealing machine. Each such
container containing the PRS shall be affixed with a label containing the following
information.
Self-adhesive sticker label may be used on which information given above printed using a PC or
otherwise.
Labelling requirement for candidate Phytochemical Reference Substance
PHYTOCHEMICAL REFERENCE SUBSTANCE ___mg
Name of PRS, IP (as given in IP Monograph):
Lot No: BM IL DDMMYY Date of Packing: June 15
Storage: Cool and dry place
========================================
Indian Pharmacopoeia Commission, Ghaziabad
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41
9.1.10 The Lot no should consist of 4 letters and 6 digits. The first two letters representing
the Name of the botanical, next two letters representing the name of the laboratory,
followed by 2 digits of date, 2 digits representing the month and last 2 digits
representing the year of packaging the PRS.
Eg. Lot no. “BM IL DDMMYY represents “Bacopa monniera, Indian Laboratory” and
the PRS has been packed on the respective date in format DDMMYY, where DD
represents Date, MM represents Month and YY represents Year.
9.1.11 Pack the sealed vials in suitable shipper with honeycomb partition as per the
requirement. Affix copies of above label, in suitable size, at least on two panels of
each Shipper.
9.1.12 Preserve 5 PRS samples as Control sample. Dispatch the rest of the PRS vials to IPC
along with following documents.
Certificate of analysis – Original (Purity and structure confirmation test results along
with copies of all confirmatory test records like spectra and chromatograms)
Packing Chalan and in-voice (original two copies)
Certificate for transportation (certifying that material is non-hazardous etc)
9.2 Procedure for IPC
9.2.1 On receipt of the consignment cross check the material received against
documents/certificates received.
9.2.2 Take adequate samples randomly from the shippers and perform pre-decided test(s) so
as to confirm the purity/structure of the PRS tubes supplied by the appointed
laboratories in duplicate.
9.2.3 Upon the samples meeting the requirements or purity and identity, the consignment is
ready for certification as PRS. Document all the tests performed, their results and the
inference properly and generate the certificate of analysis (COA) duly signed by the
analyst(s) who performed the analysis and authenticated by the Head of the
Laboratory.
9.2.4 Upon conforming the materials as PRS, each tube is to be affixed a prior printed
“SEAL” which reads as below-
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42
“CERTIFIED PRS – IPC”, such seals need to be affixed in such a way that part of seal
overlaps the label affixed by the appointed laboratory. A suggestive specimen of such seal is
given below. A hologram is also to be added to make sure duplication does not take place.
9.2.5 IPC will store the certified PRS samples consignment between “Temp 2-15oC and
60% RH” OR at Room Temperature if the PRS would be stable at RT.
9.2.6
9.2.7 IPC shall supply the requisite no of PRS packs as per order along with a copy of the
COA.
9.3 Relevant documents to be maintained by the Appointed Laboratory
a) Spectra and Chromatograms containing data regarding purity and structural conformation
b) COA
c) Relevant literature data and the return down procedure for isolation and purification and/or
the procedure for preparing the PRS by a synthetic process respectively.
PHYTOCHEMICAL REFERENCE SUBSTANCE
Name of PRS, IP (as given in IP Monograph):
Lot No: BM IP 04062015 Date of Packing: June 15
Storage: Cool and dry place
========================================
Indian Pharmacopoeia Commission, Ghaziabad
IPC
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43
10. IDENTIFICATION OF HERBAL MATERIALS
The identification of herbal materials is done with respect to macroscopic, microscopic characteristics
and chromatographic procedures etc. An examination to determine these characteristics are the first
step towards establishing the identity and purity of such materials, and should be carried out prior to
any other tests. Wherever possible, authentic specimens of the materials and samples should be
available to serve as a reference.
These tests not only serve to ensure the accurate identification of the herbal material, but must be able
to distinguish it from related species that may pose potential for species substitution or adulteration.
For development of a monograph it is necessary to perform a comparative analysis between the herbal
material and the literature reference. The morphological testing must be done by a person suitably
qualified and experienced.
Macroscopic identity of herbal materials is based on shape, size, colour, surface characteristics,
texture, fracture characteristics and appearance of the cut surface.
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IDENTIFICATION OF HERBAL MATERIAL
Monograph available in other Pharmacopoeias
Yes No
Compliance with all
identification tests (macroscopic,
microscopic and
chromatographic method) in
available pharmacopoeial
references
ant Pharmacopoeia
Options available
Comparison against a
literature source (articles
in journals, in-house
generated data)
Comparison against an
Authenticated reference
Material
Analyst must perform a
comparative analysis
between the herbal
material and literature
reference
The comparison must
include three or more
of the following:
Macroscopical
characters
Microscopical
characters
Chromatographic
procedures
Chemical tests
Analyst must perform a
comparative analysis
between the herbal
material and literature
reference
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10.1 MACROSCOPIC EVALUATION
Macroscopic evaluation on the basis of following parameters.
Size
A graduated ruler in millimeters is adequate for the measurement of the length, width and thickness
of crude materials. Small seeds and fruits may be measured by aligning 10 of them on a sheet of
calibrated paper, with 1 mm spacing between lines, and dividing the result by 10.
Colour
Visually examine the untreated sample under diffuse daylight. If necessary, an artificial light source
with wavelengths similar to those of daylight may be used. The colored the sample should be
compared with that of a reference sample.
Surface characteristics, texture and fracture characteristics
Examine the untreated sample. If necessary, a magnifying lens (6x to 10x) may be used. Wetting with
water or reagents, as required, may be necessary to observe the characteristics of a cut surface. Touch
the material to determine if it is soft or hard; bend and rupture it to obtain information on brittleness
and the appearance of the fracture plane — whether it is fibrous, smooth, rough, granular, etc.
Based upon the anatomical structure of the plant, the parts having therapeutic action commonly used
are leaves, barks, flowers, fruits, seeds, wood, aerial parts etc. The characteristics of the individual
group are described below:
10.1.1 Aerial Parts: Aerial parts consist of stems and leaves and sometimes associated with flowers
and fruits. These materials can be detected based on the following description:
A) Description of Aerial Stem. The size, shape, colour, texture - smooth or rigid, hair present or
not etc.
B) Position and arrangement of Leaves.
Radical Arising from the crown of the root
Cauline Arising from the aerial stem
Adnation Fusion of part of leaf with stem
Alternate Leaves arise from stem in alternate manner e.g. Lobelia
species
Opposite Leaves arise in pairs alternately at right angles to the
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stem.
10.1.2 Leaves or Leaflets
A) Leaf base: Stipulate or estipulate
B) Surface appearance and texture:
Glabrous Surface is free from hair
Pubescent Surface is hairy
Glandular Where long and distinct hairs or glandular hairs are present
Punctuate When surface is dotted with oil glands
The texture can be brittle, cariaceous, papery, fleshy etc.
C) Lamina Structure:
Lamina could be described in terms of margin, incision, composition, apex, base, and venation.
10.1.3 Inflorescence and Flowers
A) Type of Inflorescence. Racemose, cymose, mixed or any other type.
B) Axis or Receptacle of Inflorescence. The main axis of inflorescence is called the rachis while
branches bearing flower clusters and individual flowers are termed peduncles and pedicles
respectively.
C) Type of flower.
1. Monocotyledon or dicotyledon.
2. Unisexual or hermaphrodite.
3. Regular or zygomorphic.
4. Hypogynous, perigynous or epigynous.
D) Calyx and Corolla: It may be polysepalous or gamosepalous; persistent or caduceus; the
description about colour, shape, absence or presence of hair etc.
10.1.4 Subterranean Organs
Crude drugs from subterranean organs include stem structures such as corms, bulbs, stem-tubers,
rhizomes and root structures such as root tubers or adventitious roots.
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A) Type of Roots, Rhizomes etc: either fresh or dry; whole or sliced; peeled or unpeeled.
B) Shape of the drug: straight, branching, tortuous, cylindrical, conical etc.
C) Surface characteristics: presence of scale leaves, cracks, scars, lenticels etc.
10.1.5 Fruits
The description of fruits shall include the class (Simple, Dry, Indehiscent fruits; Simple, Dry,
Dehiscent fruits; Schizocarpic or splitting fruits; Succulent fruits); shape and dimension; adhesion
(superior or inferior); Dehiscence (Dehiscent or indehiscent). Number of seeds and description about
organoleptic characteristics.
10.1.6 Seeds
Seeds may be produced from orthotropous, campylotropous or anatropous ovules. Care must be taken
to distinguish seeds from fruits or parts of fruits containing a single seed.
Size, Shape and Colour
Funicle
Hilum and Micropyle: Size and positions
Nature of Seed Coats
Presence or absence of perisperm and endosperm.
10.2 MICROSCOPIC EVALUATION
It involves gross microscopic examination of the drug and can be used to identify the organized/
unorganized drugs by their known histological characters. Before examination through microscope,
the material must be prepared by powdering, cutting the sections of the drug or preparing a macerate.
Crude drug can also be identified microscopically by cutting the thin TS (transverse section) / LS
(Longitudinal section). Staining reactions done on very fine sections with various staining reagents
for studies of various cellular tissues and their arrangements such as trichomes, stomata, starch
granules and calcium oxalate crystals etc. Quantitative aspects of microscopy also include study of
stomatal index, palisade ratio, vein-islet number, size of starch grains and length of fibers etc.
Examples of some of the staining reagents are:
Ferric chloride for tannin
Phloroglucinol and hydrochloric acid for lignified tissues
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Ruthenium red for gums and mucilage containing cells
Chlor-zinc iodide reagent for cellulosic tissues
Acetic acid and hydrochloric acid for calcium carbonate crystals
Potassium hydroxide for hydroxyl anthraquinone.
Equipments and reagents required:
A microscope equipped with lenses with a magnification of 4x, 10x and 40x. and100
x.
A set of polarizing filters.
A stage micrometer and ocular micrometer
A heating device for preparation of sample slide
Slides and cover slip/ watch glasses.
A set of dissecting instruments.
Common Stains and Reagents for Microscopic evaluation
For the purpose of identification and characterization of materials expected to be included in the
prescribed standards, the following stains and reagents are recommended for use wherever relevant, in
addition to those mentioned in the monograph.
Acetic Acid: Dilute 6 ml of glacial acetic acid with 100 ml of distilled water; used for identification
of cystoliths, which dissolve with effervescence.
Aniline Chloride Solution: Dissolve 2 g in a mixture of 65 ml of 30 per cent ethyl alcohol and 15 ml
distilled water and add 2 ml of conc. hydrochloric acid. Lignified tissues are stained bright yellow.
Bismarck Brown: Dissolve 1 g in 100 ml of 95 per cent of ethyl alcohol; used as a general stain for
macerated material (With Schultze’s).
Chlorinated Soda Solution (Bleaching Solution): Dissolve 75 g of sodium carbonate in 125 ml of
distilled water; triturate 50 g of chlorinated lime (bleaching powder) in a mortar with 75 ml of
distilled water, adding it little by little. Mix the two liquids and shake occasionally for three or four
hours. Filter and store, protected from light. Used for lightening highly coloured material, by warming
in it and washing the tissues thoroughly.
Breamer’s Reagent: Dissolve 1 g of sodium tungstate and 2 g of sodium acetate in sufficient
quantity of water to make 10 ml. Yellowish to brown precipitates; indicate the presence of tannins.
Canada Balsam (as a Mountant): Heat Canada balsam on a water bath until volatile matter is
removed and the residue sets to a hard mass on cooling. Dissolve residue in xylene to from a thin
syrupy liquid. Used for making permanent mounts of reference slides of selected debris.
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Choral Hydrate Solution: Dissolve 50 g of chloral hydrate in 20 ml of distilled water. A valuable
clarifying agent for rendering tissues transparent and clear, by freeing them from most of the ergastic
substances, but leaving calcium oxalate crystals unaffected.
Chloral Iodine: Saturate chloral hydrate solution with iodine, leaving a few crystals undissolved;
useful for detecting minute grains of starch otherwise undetectable.
Chlorziniciodine (Iodinated Zinc Chloride Solution): Dissolve 20 g of zinc chloride and 6.5 g of
potassium iodide in 10 ml of distilled water. Add 0.5 g of iodine and shake for about fifteen minutes
before filtering. Dilute, if needed, prior to use. Renders cellulosic walls bluish violet and lignified
walls yellowish brown to brown.
Chromic Acid Solution: 10 g of potassium chromate dissolved in 90 ml of dilute sulphuric acid: A
macerating agent similar to Schultze’s.
Corallin Soda: Dissolve 5 g of corallin in 100 ml of 90 per cent ethyl alcohol. Dissolve 25 g of
sodium carbonate in 100 ml distilled water, keep the solutions separate and mix when required, by 1
ml of the corallin solution to 20 ml of the aqueous sodium carbonate solution. Prepare fresh each
time, as the mixture will not keep for long. Used for staining sieve plates and callus bright pink and
imparts a reddish tinge to starch grains and lignidied tissues.
Ammonical Solution of Copper Oxide (Cuoxam): Triturate 0.5 g of copper carbonate in a mortar
with 10 ml of distilled water and gradually add 10 mlof strong solution of ammonia (ap.gr. 0.880)
with continued stirring; used for dissolving cellulosic materials.
Eosin: 1 per cent solution in 90 per cent ethyl alcohol; Stains cellulose and aleurone grains red.
Ferric Chloride Solution: A 5 per cent solution of ferric chloride in distilled water. Tannins
containing tissues coloured bluish or greenish black.
Glycerin: Pure or diluted as required with one or two volumes of distilled water. Used as a general
mountant.
Haematoxylin, Delafield’s: Prepare a saturated solution of ammonia alum. To 100 ml of this add a
solution of one g of Haematoxylin in 6 ml of ethyl alcohol (97 per cent). Leave the mixed solution
exposed to air and light in an unstopped bottle for three or four days. Filter and add to the filtrate 20
ml of glycerine and 25 ml of methyl alcohol. Allow the solution to stand exposed to light, till it
acquires a dark colour (about two months). Refilter and store as a stock solution. Dilute it 3 or 4 times
volumes with distilled water. Stains cellulosic fibers blue; used only on water washed material.
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Iodine Water: Mix one volume of decinormal iodine with 4 volumes of distilled water. Stains starch
blue, and reveals crystalloids and globoids when present in aleurone grains.
Iodine in Potassium Iodide Solution: Dissolve 1 g of potassium iodide in 200 ml of distilled water,
add 2 g of iodine to the solution and dissolve it; stains lignified walls yellow and cellulosic walls blue.
Lactophenol (Amman’s Fluid): Phenol 20 g, lactic acid 20 g, glycerine 40 g, dissolved in distilled
water 20 ml; reveals starch grains in polarized light with a well marked cross at hilum, and also
minute crystals of calcium oxalate as brightly polarizing points of light.
Methylene Blue: A solution of 0.1 g of methylene blue in 25 ml of ethyl alcohol (95 per cent). A
general stain for nucleus and bacteria.
Millon’s Reagent: Dissolve one volume of mercury in 9 volumes of fuming nitric acid (Sp. gr. 1.52),
keeping the mixture well cooled during reaction. Add equal volume distilled water when cool. Stains
proteins red.
Naphthol Solution: Dissolve 10 gm of naphthol in 100 ml of ethyl alcohol; a specific stain for
detection of inulin; cells containing inulin turn deep reddish violet.
Phloroglucinol: 1 gm of phloroglucinol dissolved in 100 ml of 90 per cent ethyl alcohol; mount
debris in a few drop, allow to react for a minute, draw off excess of reagent with a filter paper strip
and add a drop of conc. hydrochloric acid to the slide; lignified tissues acquire a deep purplish red
colour; very effective on water washed material but not in chloral hydrate washed debris, for which
alcoholic solution of safranin is more effective (See safranin).
Picric Acid Solution (Trinitrophenol Solution): A saturated aqueous solution made by dissolving 1
g of picric acid in 95 ml of distilled water; stains animal and insect tissue, a light to deep yellow; in a
solution with ethy alcohol, aleurone grains and fungal hyphae are stained yellow.
Potash, Caustic: A 5 per cent aqueous solution; used to separate tenacious tissues of epidermis and
also laticiferous elements and vittae, both of which are stained brown.
Ruthenium Red: Dissolve 0.008 g of ruthenium red in 10 ml of a 10 per cent solution of lead acetate;
(to be freshly prepared) used for identification of most kinds of mucilage containing tissues, which
turn pink. A 0.0008 g ruthenium red dissolved in 10 ml of distilled water and used immediately stains
cuticular tissues in debris to a light pink.
Safranin: A one per cent solution in 50 per cent ethyl alcohol; used to stain lignified cell walls deep
red, even after clearing with choral hydrate.
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51
Schultze’s Maceration Fluid: Add isolated debris to 50 per cent conc. nitric acid in a test tube and
warm over water bath: add a few crystals of potassium chlorate while warming, till tissues soften;
cool, wash with water thoroughly and tease out for mounting hard tissues; isolated cell structures are
clearly revealed, but the structures are not useful for measurement of dimensions.
Sehweitzer’s Reagent: Same as Ammoniacal Copper Oxide Solution (Cuoxam).
Sudan Red III: Dissolve 0.01 g of sudan red III in 5 ml of ethyl alcohol (90 per cent) and 5 ml of
pure glycerine; suberised walls of cork cells, and fatty material in cells are stained bright red.
Sulphovanadic Acid (Mandelin’s Reagent): Triturate one g of ammonium vandate with 100 ml
conc. Sulphuric acid. Allow the deposit to subside and use the clear liquid. This is to be prepared
fresh; useful for identification of alkaloids, particularly strychnine which turns violet in the cells
containing it.
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11. DETERMINATION OF ASH
The total ash method is designed to measure the total amount of material remaining after ignition.
This includes both “physiological ash”, which is derived from the plant tissue itself, and “non-
physiological” ash, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the
plant surface.
Acid-insoluble ash is the residue obtained after boiling the total ash with dilute hydrochloric acid, and
igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand
and siliceous earth.
Water-soluble ash is the difference in weight between the total ash and the residue after treatment of
the total ash with water.
11.1 Total Ash (IP, Appendix 2.3.19)
Method A. For crude vegetable drugs
Unless otherwise stated in the individual monograph, weigh accurately 2 to 3 g of the air-dried drug in
a tared platinum or silica dish and incinerate at a temperature not exceeding 450° until free from
carbon, cool and weigh. If a carbon-free ash is not obtained, wash the charred mass with hot water,
collect the residue on as ashless filter paper, incinerate the residue and filter paper until the ash is
white or nearly white, add the filtrate to the dish, evaporate to dryness and ignite at a temperature not
exceeding 450°. Calculate the percentage of ash on the dried drug basis.
Percentage of ash with reference to air dried drug =
11.2 Acid-insoluble ash
Use Method C unless otherwise directed.
Method C. Boil the ash (Method A) with 25 ml of 2 M hydrochloric acid for 5 minutes, collect the
insoluble matter in a Gooch crucible or on an ashless filter paper, wash with hot water, ignite, cool in
a desiccator and weigh. Calculate the percentage of acid-insoluble ash on the dried drug basis.
Method D. Place the ash (Method A), or the sulphated ash (2.3.18), as directed in the individual
monograph, in a crucible, add 15 ml of water and 10 ml of hydrochloric acid, cover with a watch
glass, boil for 10 minutes, and allow to cool. Collect the insoluble matter on an ashless filter paper,
Weight of ash (g)
Weight of the drug (g)
X 100
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wash with hot water until the filtrate is neutral, ignite to dull redness, cool in a desiccator and weigh.
Calculate the percentage of acid-insoluble ash on the dried basis.
11.3 WATER-SOLUBLE ASH
Boil the ash (Method A) for 5 minutes, with 25ml of water. Collect the insoluble matter in a gooch
crucible or on an ashless filter paper, wash with hot water and ignite for 15 minutes at a temperature
not exceeding 450°C. Subtract the weight of the insoluble matter from the weight of the ash; the
difference in weight represents the water soluble ash. Calculate the percentage of water soluble ash on
the dried basis.
Percentage of acid insoluble ash
with reference to air dried drug =
Weight of acid insoluble ash
Weight of the drug X 100
Weight of acid soluble ash
Weight of the drug X 100
Percentage of acid insoluble ash
with reference to air dried drug =
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12. DETERMINATION OF EXTRACTIVE VALUES
This method determines the amount of active constituents extracted with solvents from a given
amount of herbal material. The extractive values by different solvents are used to assess quality,
purity and to detect adulteration due to exhausted and incorrectly processed drugs.
12.1 ETHANOL – SOLUBLE EXTRACTIVE (IP Appendix 2.6.2)
Macerate 5g of the air-dried drug, coarsely powdered into a 250ml conical flask with
stopper.
Add 100 ml of ethanol of the specified strength.
Shake the flask frequently during the first 6 hours.
Keep it aside without disturbing for 18 hours and then filter rapidly taking
precautions against loss of ethanol.
Pipette out 25ml of the filtrate and evaporate to dryness in a tared flat-bottomed
shallow dish.
Then dry at 105° and weigh.
Calculate the percentage of ethanol soluble extractive with reference to air-dried
material by the given formula:
Weight of residue
Weight of the drug
It is expressed as per cent w/w of the ‘air-dried drug’.
12.2 WATER SOLUBLE EXTRACTIVE (IP Appendix 2.6.3)
Method I-
Proceed as directed for determination of Ethanol–Soluble Extractive, using chloroform water (2.5 ml
chloroform in purified water to produce 1000 ml) instead of ethanol.
Method II-
Add 5g to 50ml of water at 80° in a stoppered flask.
Shake well and allow standing for 10 minutes.
Cool and add 2g of kieselguhr and filter.
Transfer 5ml of the filtrate to a tared evaporating dish, 7.5cm in diameter.
X 100 Percentage of ethanol soluble extractive =
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Evaporate the solvent on a water-bath, continue drying for 30min
Finally dry in a steam oven for 2 hours and weigh the residue.
Calculate the percentage of water-soluble extractive with reference to air-dried
material by the given formula:
Weight of residue
Weight of the drug
X 100 Percentage of water soluble extractive =
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13. LOSS ON DRYING
Loss on drying is the loss of weight expressed as percentage w/w resulting from water and volatile
matter of any kind that can be driven off under specified conditions. The test is carried out on a well-
mixed sample of the substance. If the substance is in the form of large crystals, reduce the size by
rapid crushing to a powder form.
LOD is determined by drying the sample for specified period usually 3 hours at a specified
temperature (105oC). The limit is specified on the basis of the results obtained on a reasonable
number of samples of acceptable quality. When the drying temperature is indicated by a single value
other than a range, drying is carried out at the prescribed temperature ±2°.
Method (IP Appendix 2.4.19)
Unless otherwise specified in the individual monograph, use this method.
Weigh a glass-stoppered, shallow weighing bottle that has been dried under the same
conditions to be employed in the determination.
Transfer to the bottle the quantity of sample specified in the individual monograph.
Cover it and accurately weigh the bottle and the contents.
Distribute the sample as evenly as practicable by gently sidewise shaking to a depth
not exceeding 10mm.
Dry the substance by placing the loaded bottle in the drying chamber as directed in
the monograph, remove the stopper and leave it in the chamber.
Dry the sample to constant weight or for the specified time and at a temperature
indicated in the monograph.
After drying is completed, open the drying chamber, close the bottle promptly and
allow it to cool to room temperature (where applicable) in a desiccators before
weighing.
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14. CONTAMINANTS
Determination of foreign organic matter
Herbal materials should be free, as much as possible, of mould, insects and other animal contaminants
(animal excreta/other noxious foreign matter). Abnormal odour, discoloration, slimeness or signs of
deterioration should be absent. Amount of foreign organic matter should not be higher than the
pharmacopoeial limit.
Macroscopic examination can conveniently be employed for determining the presence of foreign
matter in whole or cut plant materials. However, microscopy is indispensable for powdered materials.
Any soil, stones, sand, dust and other foreign inorganic matter must be removed before herbal
materials are cut or ground for testing.
14.1 Definition
Foreign organic matter according to IP is material consisting of any or all of the following:
Parts of the organs or organs from which the drug is derived other than the parts named in the
definition and description or for which the limit is prescribed in the individual monograph.
Any organs other than those named in the definition and description.
Matter not coming from the source plant and
Moulds, insects or other animal contamination.
14.2 Method (IP, Appendix 2.6.1)
Weigh 100 to 500g, or quantity specified in the individual monograph, of the sample.
Spread it on a thin layer.
Inspect the sample with the unaided eye or with the use of a 6x lens.
Separate the foreign organic matter manually as completely as possible.
Weigh and determine the percentage of foreign organic matter from the weight of drug taken.
14.3 Determination of Heavy metals (IP Appendix 2.3.13). Heavy metals may be present in herbs
as a consequence of natural occurrence or from human activities such as industrial waste in the
soil; irrigation with contaminated water or airborne pollution. A general chapter on determination
of Heavy metals in herbal drugs ( Appendix 2.3.13) is included in the Indian Pharmacopoeia.
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Example: Ashwagandha Dry Extract Monograph, IP
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B
(20 ppm).
Methods of heavy metal determination is given below:
Method A
Standard solution. Into a 50-ml Nessler cylinder pipette 1.0 ml of lead standard solution (20 ppm
Pb) and dilute with water to 25 ml. Adjust with dilute acetic acid or dilute ammonia solution to a pH
between 3.0 and 4.0, dilute with water to about 35 ml and mix.
Test solution. Into a 50-ml Nessler cylinder place 25 ml of the solution prepared for the test as
directed in the individual monograph or dissolve the specified quantity of the substance under
examination in sufficient water to produce 25 ml. Adjust with dilute acetic acid or dilute ammonia
solution to a pH between 3.0 and 4.0, dilute with water to about 35 ml and mix.
Method B
Standard solution. Proceed as directed under Method A.
Test solution. Weigh in a suitable crucible the quantity of the substance specified in the individual
monograph, add sufficient sulphuric acid to wet the sample, ignite carefully at a low temperature until
thoroughly charred. Add to the charred mass 2 ml of nitric acid and 5 drops of sulphuric acid and heat
cautiously until white fumes are no longer evolved. Ignite, preferably in a muffle furnace, at 500° to
600°, until the carbon is completely burnt off. Cool, add 4 ml of hydrochloric acid, cover, digest on a
water-bath for 15 minutes, uncover and slowly evaporate to dryness on a water-bath. Moisten the
residue with 1 drop of hydrochloric acid, add 10 ml of hot water and digest for 2 minutes. Add
ammonia solution drop wise until the solution is just alkaline to litmus paper, dilute to 25 ml with
water and adjust with dilute acetic acid to a pH between 3.0 and 4.0. Filter, if necessary, rinse the
crucible and filter with 10 ml of water, combine the filtrate and washings in a 50-ml Nessler cylinder,
dilute with water to about 35 ml and mix.
14.4 Microbial Contamination (IP, Appendix 2.2.9)
Herbal plants and drugs are generally contaminated with a great number of bacteria and molds arising
from the soil and surrounding environment. In addition, further contamination results from harvesting
practices, handling, and processing.
A general chapter on Microbial Contamination on Non-sterile products designed for the estimation of
number of viable microorganisms present, for detecting the presence of designated microbial species
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and acceptance criteria for herbs, processed herbs and herbal products is included in the Indian
Pharmacopoeia.
Acceptance criteria for microbiological quality are based on the Total Aerobic Viable Counts
(TAC), Total Fungal Counts (TFC) and specified pathogens.
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15. THIN LAYER CHROMATOGRAPHY
15.1 INTRODUCTION
Thin layer chromatography (TLC) is a chromatographic technique applicable for quick screening of
herbs and herbal products by means of fingerprints. This technique is used as
1. The identity, purity, adulterations or substitutions of herbal drugs can be monitored by
comparison with the chromatogram of the standard drugs.
2. It enables entire chromatographic separation at a glance, along with semi-quantitative
information of active chemical constituents present, thus enabling assessment of quality of the
herb.
3. Optimization of TLC is fast and at a low cost by changing mobile and stationary phase.
4. TLC can be used to analyze combination of drugs with aid of appropriate separation
procedures.
15.2 BASIC PRINCIPLES OF TLC
TLC is based on the principle of adsorption, depending on the particular type of support, its
preparation and its use with different solvent. The components of the sample having more affinity
towards stationary phase migrate slowly as compared to the components having affinity towards
mobile phase. Separation in TLC is achieved by application of a solution of the sample containing a
mixture of compounds to the layer of sorbent (stationary phase), near one edge, as a spot/band on a
TLC plates of thickness 0.1-0.2 mm for analytical purposes and around 0.5-2mm for preparative TLC
are commercially available. The plate is then placed into a closed developing chamber. The solvent
front then migrates up the plate through the absorbent by capillary action.
The Rƒ is calculated by dividing the distance the compound travelled from the original position by the
distance the solvent travelled from the original position (the solvent front)
Distance travelled by sample from starting point
Distance travelled by the solvent front from starting point
15.3 Application of samples
Samples can be applied as spots or bands. Small amount of sample should be used and the sample and
reference substances should be dissolved in same solvent or solvent mixture. If the sample solution is
very dilute, several small applications in the same place should be done to allow the solvent to
evaporate between additions. Sample volume of 5-10µl should be applied on the plate, about one
Rƒ =
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centimeter above the base level. The starting position can be indicated by making a small mark near
the edge of the plate. The plate is then dipped in to a saturated TLC chamber containing suitable
solvent. The solvent front is allowed to rise up to 80% of the plate. Plate is then dried on a suitable
heating device and then visualized and inference is drawn.
15.4 Choice of Solvent system
The solvent system should be chosen to match the nature of analytes and sorbent being used. Because
the mobile phase competes with the sample for sorbent sites, polar substances require a polar solvent
to migrate on a silica gel or alumina adsorbent layer. In reverse-phase TLC, non-polar substances are
strongly attracted to the layer, and non-polar mobile phases are required to effect migration.
Some of solvent systems used for separation of herbs and herbal drugs have been shown in Table 1.
Table 1. Solvent systems used for development of TLC chromatogram of phytoconstituents
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SOLVENT SYSTEMS SOLVENT RATIO SORBENTS CHEMICAL
CONSTITUENT
Essential Oils
Toluene: Ethyl acetate 93:7 Silica gel 60F254 Anethole/ methylchavicol
or safrole, eugenol
Toluene - Silica gel 60F254 Eugenol
Toluene:Ethyl acetate 93:7 Silica gel 60F254
Carvone, Linalool,
Cineole, α – terpineol,
thymol, carvacrol
Toluene: Ethyl acetate 93:7 Silica gel 60F254
Menthol, menthone,
methyl acetate,
menthofuran
Alkaloidal Drugs
Toluene: Ethyl acetate :
Diethylamine 70:20:10 Silica gel 60F254
Suitable for major
alkaloids of most drugs
Ethyl acetate: Methanol:Water 100:13.5:10 Silica gel 60F254
Xanthine derivatives,
colchicum and Rauwolfia
alkaloids
Acetone: Water: conc.
Ammonia 90:7:3 Silica gel 60F254
Solanaceous, Atropine and
Hyoscyamine alkaloids
Chloroform: Diethylamine 90:10 Silica gel 60F254 Cinchona alkaloids
Toluene: Acetone: Ethanol :
conc. Ammonia 40:40:6:2 Silica gel 60F254
Opium alkaloids
n-Heptane: Ethylmethyl
ketone: Methanol 58:34:8 Silica gel 60F254
Rauwolfia alkaloids
Cyclohexane: Chloroform :
Glacial acetic acid 45:45:10 Silica gel 60F254
Berberine and
protoberberine type
alkaloids
Bitter Drugs
Ethyl acetate: Methanol:
Water 77:13.5:8 Silica gel 60F254
General system for
screening
Acetone: Choloroform: Water 70:30:2 Silica gel 60F254 Amarogentin
Chloroform: Methanol 95:5 Silica gel 60F254 Quassin
Iso-octane: Isopropanol:
Formic acid 83.5:16.5:0.5 Silica gel 60F254
Humulone and lupulone
n- heptanes: Isopropanol:
Formic acid 90:15:0.5 Silica gel 60F254
Bitter acids
Chloroform: Ethanol 95:5 Silica gel 60F254 Cucurbitacins
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Anthracene derivatives
Ethyl acetate: Methanol:
Water 100:13.5:10 Silica gel 60F254
All anthracene drug
extracts
n-propanol: Ethyl acetate:
Water: Glacial acetic acid 40:40:29:1 Silica gel 60F254
Senna
Light petroleum: Ethyl acetate
:Formic acid 75:25:1 Silica gel 60F254
Anthraquinone aglycones
Toluene: Ethylformate:
Formic acid 50:40:10 Silica gel 60F254
Hypericin
Cardiac Glycosides
Ethyl acetate: Methanol:
Water
100:13.5:10
Or
81:11:8
Silica gel 60F254 Applicable system for
cardiac glycosides
Ethyl acetate: Methanol:
Ethanol: Water 81:11:4:8 Silica gel 60F254
Strophanthoside. Addition
of ethanol increases Rf
value of strongly polar
compound
Chloroform: Methanol: Water 64:50:10 Silica gel 60F254 Separation of all saponin
mixtures from plants.
Flavonoids
Ethyl acetate: Formic acid:
Glacial acetic acid: Water 100:11:11:26 Silica gel 60F254
Screening system for
flavonoid glycosides
Ethyl acetate: Formic acid:
Glacial acetic acid:
Ethylmethyl ketone: Water
50:7:3:30:10 Silica gel 60F254 Screening system for
flavonoid glycosides
Benzene: Pyridine: Formic
acid 72:18:10 Silica gel 60F254 Flavonoid aglycones
Chloroform: Acetone: Formic
acid 75:16.5:8.5 Silica gel 60F254 Flavonoids
Toluene: Ethylformate:
Formic acid 50:40:10 Silica gel 60F254 Flavonoid aglycones
Coumarins
Toluene: Ether (1:1) saturated
With 10% acetic
Acid
Silica gel 60F254 Universally accepted for
coumarin aglycones
Ethyl acetate - Silica gel 60F254 Used for higher Rf
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Source: In Mukherjee PK (2002). Thin Layer Chromatography. Quality Control of Herbal Drugs. Business Horizons publisher. 1st edition.
15.5 Derivatization and Detection of TLC plate
Zone detection in TLC is based on natural colour, fluorescence, or UV absorption or on the use of
various universal or selective chemical detection reagents applied by spraying or dipping. Detection
with UV radiation at wavelengths 254 nm and/or 366 nm is fast and straight forward detection for
visualization of separated substances. After the plate is developed, it is sprayed with various reagents
for the development of colour. Often the colour reaction is not confined to a single compound but is
produced by several compounds belonging to a particular group. Some of the colouring reagents
commonly used for analysis of herbal drugs is listed in Table 2 wherein the correlations of colour
response with particular aspect of drug structure are reviewed.
Table 2: Most common derivatization reagents
Vanillin/Sulfuric acid A universal spray, many terpenoids, sterols and
saponins give red and blue colours.
Ammonia vapour Alkaloids, Flavonoids, mycotoxins, anthracene
Aniline-diphenylamine- phosphoric acid
Phenols, Flavonoids, tannins, plant acids, ergot
alkaloids, hypericin.
Anisaldehyde/ vanillin with sulphuric or
phosphoric acid
Steroids, higher alcohols, phenols, and
essential oils.
Bromocresol Green Solution Detection of flavonoids.
Dragendorff’s reagent Alkaloids, heterocyclic nitrogen compounds
and lipids. Alkaloids give dark orange or red
Ethyl acetate: Formic acid:
Glacial acetic acid: Water 100:11:11:27 Silica gel 60F254 For polar coumarins
Lignans
Chloroform: Methanol (6cm)
followed by toluene-acetone
90:10 followed by
65:35 Silica gel 60F254 Podophyllins
Pungent principles from plants
Toluene: Ethylacetate 70:30 Silica gel 60F254 Piper
Toluene: Ethyl ether: Dioxan 62.5:21.5:16 Silica gel 60F254
General for pungent
principles
Diethyl ether (saturated
chamber) - Silica gel 60F254
Capsaicin and related
compounds
Triterpenes
Ethyl acetate: Glacial acetic
acid: Formic acid: Water 100:11:11:26 Silica gel 60F254
Polar compounds
(isoflavone)
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coloration.
Fast blue salt Tannins, coumarins, cannabinoids, amines.
Ferric (III) chloride Phenolic compounds for example, coumarins,
flavonoids, tannins. Blue or red coloration will
be observed.
Iodine vapour For many organic compounds
Ninhydrin Amino-acids, Primary amines, secondary
amines and peptides.
Phosphomolybdic acid solution For detection of alkaloids
15.6 Troubleshooting in TLC analysis
When compound runs as a streak rather than a spot, due to overloading. Run the TLC again
after diluting your sample. Or, sample might contain many components, creating many spots
which run together and appear as a streak. Perhaps, the experiment did not go as well as
expected.
When the sample runs as a smear or an upward crescent. Compounds which possess strongly
acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with
this behaviour. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic
acids) to the eluting solvent to obtain clearer plates.
If the sample runs as a downward crescent, the adsorbent may be disturbed during the
spotting, causing the crescent shape.
When the plate solvent front runs crookedly. Either because of the adsorbent has flaked off
the sides of the plate or the sides of the plate are touching the sides of the container (or the
paper used to saturate the container) as the plate develops. Crookedly run plates make it
harder to measure Rf values accurately.
Many random spots are seen on the plate, indicating analyst has accidentally dropped any
organic compound on the plate.
No spots are seen indicating solvent level is above baseline.
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16. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
16.1 INTRODUCTION
High Performance Thin Layer Chromatography (HPTLC) is also known as planar chromatography.
This analytical technique is known for its high separation capability, performance and reproducibility
considered superior to classical TLC methods. HPTLC have been widely used for analysis of Herbs
and Herbal Products. Although, TLC is a simple, rapid method but it has many pitfalls. HPTLC came
into existence in 1975 with introduction of high efficiency, commercially precoated plates, that are
smaller (10 x 10 or 10 x 20 cm), have thinner (0.1- 0.2 mm) layer composed of sorbent with a finer
mean particle size (5-6µm) and a narrower particle size distribution or classification (4-8 µm) and are
developed over shorter distances (about 3-7 cm) compared to classical TLC plates, which are 20 x 20
cm with a 0.25 mm layer containing particles with an average size of 10-12 µm.
HPTLC is a flexible, versatile, economical process in which all the processes are independent. Since
herbs are complex mixtures and herbal extracts contain numerous compounds in different
concentrations, the analysis of herbs and herbal preparations is challenging. The advantages of
HPTLC include the following:
Non tedious sample preparation technique and multiple samples can be analyzed on one plate.
Great range of stationary phases available with unique selectivity for mixture components.
Ability to choose solvents for the mobile phase is not restricted by low UV transparency or the
need for ultra-high purity. Corrosive and UV-absorbing mobile phases can be employed.
No prior treatment for solvents like filtration and degassing.
Chromatographic conditions can be changed within a few minutes, and chromatographic chamber
requires little time for equilibration.
Multiple detection of the sample chromatogram is possible in order to improve selectivity and
confirm zone identity.
Automated sample application takes 0.5-2 min per sample depending on the size, application mode
and number of replicates.
Densitometry evaluation of plates can be accomplished within 10 minutes.
The results can be easily monitored, documented and the chromatograms of various herbs and
herbal preparations can be compared.
Amount of mobile phase required is small, minimizing the cost.
Accuracy and precision of quantification is high because samples and standards are
chromatographed and analyzed under same conditions on a single plate.
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HPTLC is a modern adaptation of TLC with better and advanced separation efficiency and detection
limits. The table below compares HPTLC and TLC (Table 1).
Table.1: Comparison of TLC and HPTLC
SNO. FEATURES TLC HPTLC
1 Technique Manual Semiautomatic
2 Layer Lab made/ Precoated Precoated
3 Sample
application Circular Rectangular or Circular
4 Solid support Silica gel , Alumina &
Kieselguhr etc..
Wide choice of stationary phases
like silica gel for normal phase and
C8 , C18 for reversed phase modes
etc
5 PC
connectivity No Yes
6 Method
storage No Yes
7 Validation No Yes
8 Quantitative
analysis Difficult Relatively easier
9 Sample holder Capillary/ Pipette Syringe
10 Wavelength
range 254 or 366 nm, visible 190 to 800nm, monochromatic
11 Analysis Time Slower Shorter migration distance and the
analysis time is greatly reduced
12 Scanning Not possible
Use of UV/ Visible/ Fluorescence
scanner scans the entire
chromatogram qualitatively and
quantitatively and the scanner is an
advanced type of densitometer
13 Accuracy Low High
16.2 HPTLC METHOD DEVELOPMENT
The method for analyzing herbals by HPTLC demands primary knowledge about the nature of
chemical constituents present in herbs. In combination with microscopic investigations, they provide a
means for identity check. Any adulterations in raw material can also be identified. Method
development and standardization involves trail and errors. The first step is the selection of appropriate
developing solvent. This can be done by, identifying the solvents that can have average separation
power for the desired constituent(s), employing a gradient system and use of modifiers like acids or
bases. Selection of a marker compound(s) for a particular herb or herbal preparation(s) for
quantification is also of critical importance. It is often impossible to separate all the components of the
plant/extract. Therefore, the method shall be designed such that the marker compound doesn’t co-
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elute with other substances. Procedure for HPTLC method development is outlined as follows
(Figure1) :
FIGURE 1 Schematic procedure for HPTLC method development
16.2.1 Stationary phase
HPTLC plates comprises of small particles with a narrow size distribution resulting in homogenous
layers with a smooth surface. HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly
decreased development distance (3-7cm) and analysis time (7–20 min). Commercial precoated plates
with glass or aluminium foil backing have been extensively used. Normal phase adsorption TLC on
silica gel, usually silica gel 60 (60 Å pore size), with a less polar mobile phase, such as chloroform–
methanol, has been used for more than 90% of reported analysis. Reversed-phase lipophilic C-18
chemically-modified silica gel phase; and hydrocarbon- impregnated silica gel plates developed with
a more polar aqueous mobile phase, such as methanol–water or dioxane-water, are used for reversed-
phase LC.
Other precoated layers that are used include aluminum oxide, magnesium silicate, magnesium oxide,
polyamide, cellulose, kieselguhr, ion exchangers, and polar modified silica gel layers that contain
bonded amino, cyano, diol, and thiol groups. HPTLC plates need to be stored under appropriate
conditions. Before use, plates should be inspected under white and UV light to detect damage and
impurities in the adsorbent. It is advisable to prewash the plates to improve the reproducibility and
robustness of the results.
Test solution and Reference solution Selection of chromatographic plate and mobile phase
Plater pre-washing if required
Saturation of chromatographic chamber with mobile
phase
Application of test and reference solution followed by drying of spotted plate
Chromatographic development followed by drying of spotted plate
Detection of spots
Scanning & Documentation of developed plate
Archiving
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16.2.2 Mobile Phase
The selection of mobile phase is based on adsorbent material used as stationary phase and physical
and chemical properties of analyte. Generally diethyl ether, methylene chloride, and chloroform
combined individually or together with hexane as the strength-adjusting solvent for normal-phase
TLC and methanol, acetonitrile, and tetrahydrofuran mixed with water for strength adjustments in
reverse-phase TLC are being used. Accurate volumetric measurements of the components of the
mobile phase must be performed separately and precisely in adequate volumetric glassware and
shaken to ensure proper mixing of the components.
16.2.3 Sample preparation and application of Spot
After preparation of mobile phase, the test solution is prepared by Infusion, Decoction, Maceration,
Soxhletion and Percolation technique. The amount of Test sample and Reference sample to be taken
for testing should be as specified in the monographs concerned..
A good solvent system is one that moves all the components of the mixture off the baseline, but does
not move them near to the solvent front. The spots /peaks of interest should be resolved between Rf
0.15 and 0.75. The more non-polar the compound, the faster it will elute (or the less time it will
remain on the stationary phase) and the more polar the compound the slower it will elute (or more
time on the stationary phase). The volume of reference solutions and test material may be applied to
the plate in the range of 5µl- 20µl so that when the same is applied as a band of width of
6mm-8mm, it provides reasonable amounts of the phytochemicals being aimed for separation-neither
too low nor too high proportions. . The narrow, homogenous sample bands lengths are usually applied
by use of an automated applicator. These ensure high-resolution separations, accurate and precise
quantitative results by scanning densitometry.
16.2.4 Chromatogram Development
A classical method of linear, ascending development in a mobile phase vapour-saturated, covered
glass chamber or tank is widely used for herbal analysis. The twin trough chamber is also used; it is a
normal chamber with inverted V-shaped ridge on the bottom dividing the chamber into two sections.
This allows use of low volume of mobile phase and easy conditioning of the layers. Complex
separations can be performed by use of two–dimensional development, in which the sample mixture is
applied to one corner of HPTLC plate. The plate is then developed with first mobile phase, dried and
then developed with the second mobile phase in the perpendicular direction. In addition to the usual
capillary flow development of plates, forced flow overpressure layer chromatography (OPLC) in
which the mobile phase is mechanically pumped through the layer covered with a membrane that is
under pressure have been used.
16.2.5 Developing the Plate
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After the plate is developed, it is dried in fume hood or oven to completely evaporate the mobile
phase. The separated compounds are detected by their natural colour, natural fluorescence under 366
nm UV light, quenching of fluorescence under 254 nm UV light on an “F” layer containing a
phosphor termed as UV indicator sprayed with various reagents for the development of colour often
the colour reaction is not confined to a single compound but is produce by several compounds
belonging to a particular group. Therefore along with using migration rates, various constituents of a
sample are also identified their response to chemical treatment. Universal or selective chromogenic
and fluorogenic detection reagents are applied by spraying onto the layer, dipping the layer into
reagent, or exposing the layer to reagent vapours. An important advantage of offline operation of
HPTLC is the flexibility to use multiple detection methods. For example, the layer can be viewed
under long wave, short wave UV light, followed by one or more chromogenic, fluorogenic or
biological detection methods. The most common derivatizing agents are enlisted in chapter of TLC.
16.2.6 Quantification
The quantitative evaluation is performed by measuring the predefined zones/spots obtained in the
samples and standards using a densitometer or scanner. The scanner and densitometers need to be set
to the desired UV wavelengths.
16.2.7 Documentation
Documentation to be done with the help of controlled automated system and help of software. While
doing so they need to be suitably numbered and a numbering system need to be developed and
adopted for proper storage of the documents/profiles/densitometry data and also for easy retrieval of
them.
16.3 Fingerprint of some medicinal plants by TLC/HPTLC method
SNO. HERBAL DRUGS (IP
2014)
CATEGORY MOBILE PHASE SPRAY
REAGENTS
1. Trachyspermum ammi
(Ajwain)
Antiasthmatic,
carminative,
germicidal,
antioxidant
Toluene : ethyl acetate
[93:7]
Anisaldehyde
sulphuric acid
reagent
2 Emblic myrobalan
(Amalaki)
Antiscorbutic, antacid,
carminative,
hepatoprotective
Toluene : ethyl acetate
: glacial acetic acid:
formic acid
[20:45:20:5]
Anisaldehyde
sulphuric acid
reagent
3 Cassia fistula
(Amaltas)
Purgative, diuretic,
antipruritic, febrifuge
Toluene : ethyl acetate
: formic acid :
methanol [3:3:0.8:0.2]
-
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4 Mangifera indica
(Amra)
Cardiotonic,
astringent, nourishing
tonic, in haemoptysis
Ethyl acetate : formic
acid : acetic acid :
water [100:11:11:25]
Vanillin glacial
acetic acid reagent
5 Terminalia arjuna
(Arjuna)
Antihyperlipidaemic,
antihypertensive,
astringent,
cardioprotective
Toluene : ethyl acetate
: acetic acid [5:5:0.5]
10% w/v sulphuric
acid in methanol
6 Atropa belladonna
(Belladona dry extract) Anticholinergic
Acetone : water :
strong ammonia
solution [90:7:3]
Modified potassium
iodobismuthate
solution
7 Asparagus racemosus
(Shatavari)
Anti-inflammatory,
immunomodulatory,
neuroprotective,
antidysentery
Chloroform : methanol
: water [13:10:2]
Vanillin sulphuric
acid reagent
8 Cassia angustifolia
(Senna)
Purgative,
anthelmintic,
febrifuge
n-propyl alcohol :
ethyl acetate : water :
glacial acetic acid
[40:40:29:1]
20% v/v of nitric
acid solution
9 Centella asiatica
(Mandukaparni)
Brain tonic, anabolic,
anxiolytic, alterative
Chloroform : glacial
acetic acid : methanol :
water [60:32:12:8]
Anisaldehyde
sulphuric acid
reagent
10 Adhatoda vasica
(Vasaka)
Expectorant,
bronchiodilator
Ethyl acetate :
methanol : strong
ammonia solution
[8:2:0.2]
Dragendorff’s
reagent
16.4 TROUBLESHOOTING IN HPTLC ANALYSIS
A poor band quality is observed due to variation in the gas flow, wrong distance between needle
tip and TLC layers or because of clogged needle. Check gas pressure and adjust to 2-5 bars,
check distance and adjust to 1mm, washing of the syringe.
Bad reproducibility due to sample overloading. Dilute the sample and inject.
Poor accuracy may be due to destroyed glass barrel of the syringe or damaged spray head due to
wrong sample dosage syringe. Replace the spray head.
The quality of bands can also be affected because of clogging in the nozzle, this requires cleaning
of the nozzle.
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16.5 VALIDATION
The suitability of any analytical procedure for its intended use in pharmaceutical analysis must be
based on objective validation data. Guidelines on the validation of analytical procedures are available
on the website of International Conference on Harmonization (ICH) www.ich.org
TLC/HPTLC, is one of the predominant techniques for identity, quantification and standard limit
tests for impurities, especially for those that are difficult or impossible to detect with liquid
chromatography. The process of validating a procedure cannot be separated from its development as
the analyst will not know whether the procedure and its performance are acceptable until validation
has been performed. Before outlining the validation protocol and the experimental design, it is
necessary to qualify all instrumentation and equipment. All chemicals, TLC plates, and reference
standards must be defined, specified, and tested. The analytical procedure must be developed,
optimised, and documented. A validation protocol, which includes the acceptance criteria and the
specified statistical approaches, must be agreed upon and signed.
A quantitative TLC purity test requires a comprehensive validation study - including specificity,
linearity and range, precision, accuracy, detection limits, quantitation limits etc.
16.5.1 Specificity
To demonstrate specificity, the sample, standard substance(s), pure and spiked placebos are
chromatographed simultaneously. The chromatographic method selectively separates the active
constituent from the impurities and excipients.
16.5.2 Linearity and Range
Linearity is the functional relationship between the concentration of the substance and the measured
value. Ideally the calibration curve should be linear that should pass through the origin when tested by
regression analysis. Unfortunately, numerous analytical techniques indicate/demonstrate or lead to a
nonlinear relationship between the concentration of analytes in samples and the corresponding
measurement signals. In TLC/HPTLC especially in the case of evaluation performed by scanning in
the UV/VIS reflection mode, most calibration functions are nonlinear. The absorption measurement
always yields 2nd degree functions. Therefore, for routine analytical procedures, a three-point
calibration model must be used.
16.5.3 Precision
Precision is the closeness of agreement between the values obtained in an assay. It is expressed as the
coefficient of variation (% CV). CV is the standard deviation of the assay values divided by the
concentration of the analyte. Several types of precision can be measured: intra-assay precision
(repeatability) is the % CV of multiple determinations of a single sample in a single test run.
Repeatability provides information about the variation caused by sample preparation, sample
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application, and evaluation within one analytical run and within a short period of time. The same
sample is determined on the same plate by applying at least six analytical solutions from individual
weighings. Inter-assay precision (also called intermediate precision) measures the % CV for multiple
determinations of asingle sample, controls and reagents analyzed in several assay runs in the same
laboratory. Reproducibility is the precision between different laboratories and is done on the same
sample with different apparatus.
16.5.4 Accuracy
Accuracy is the closeness of agreement between the actual value of the drug and the measured value.
Spike and recovery studies are performed to measure accuracy. The analyte is spiked with three
different concentrations of the impurities present. The recovery is calculated as a percentage value of
the added amount of impurity.
% Recovery = (Amount found/ Amount added)* 100
16.5.5 Limit of Detection/ Limit of Quantification
Limit of Detection (LOD) is the lowest amount of the analyte in a sample that can be detected but not
necessarily be quantified as an exact concentration or amount. Limit of Quantitation (LOQ) is the
lowest amount of an analyte that can be measured quantitatively in a sample with acceptable accuracy
and precision. For determination of LOD and LOQ the impurities and the analyte are run in triplicate.
A blank is also run simultaneously. Thus, LOD and LOQ are calculated based on the signal-to-noise
ratio.
Conclusion
Thus HPTLC methods are employed to analyze herbs and herbal preparations for the qualitative
(fingerprint) and quantitative determination, demonstrating their feasibility in the quality control of
phytoconstituents.
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17. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
17.1 Introduction
HPLC is a chromatographic technique widely used for qualitative and quantitative analysis of organic
compounds present in multi-component mixtures, such as herbal plants extracts. It has a broad range
of application as it can be applied to herbal products that contain components such as alkaloids,
glycosides, flavanones, organic acids, phenols and lignans. The main HPLC conditions include the
optimal choice of column, mobile phase and detector. The HPLC conditions should be optimized, so
that the components of the herbals can be resolved as much as possible. In other words, the more
chromatographic peaks obtained, the better it is. This allows the internal characteristics of the herbal
plants to be fully manifested, providing sufficient information for the fingerprint evaluation and for its
quality assessment.
For the separation of crude extracts, either raw mixtures or samples enriched by extraction via simple
solid-phase extraction or liquid-liquid extraction are injected into HPLC after passing through 0.45µm
filter. The separations are performed mostly in reverse-phase chromatography on C18 material with
the ACN-H2O or MeOH-H2O solvent system in gradient elution mode.
17.2 Normal and Reverse Phase Chromatography
In normal phase the polarity of the stationary phase is higher than that of the mobile phase. In Reverse
phase, the polarity of stationary phase is less than that of mobile phase. For ionizable solutes, the pH
of the mobile phase is an important factor in control of retention and selectivity. Figure 1 describes
the methods for choice of HPLC methods.
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75
17.3 HPLC COLUMN
MW
>20
00
M
W<2
00
0
Ionic (including
metal
chelates and
transition
metals)
Normal
phase(bonded
)
Water
Soluble
Organic
Soluble
Organic
Soluble
Chiral Sample
Hexane
Soluble
Normal
phase(adsorpt
ion)
Unmodified
silica packing
CN, NH2 bonded
phases
Non-ionic
MeOH,
MeOH/H2O
Soluble
THF
Soluble
Reverse
phase bonded
C18, C8, phenyl,
C4 , CN
bonded phases
Small
molecule GPC
Hydrophobic
small pore
packing’s
Reverse
phase(bonded)
C18, C8, phenyl,
C4 , CN
bonded phases
Reverse phase (with
ionizationcontrol)
C18, C8, phenyl, C4 , CN
bonded phases
(with pH control)
Reverse phase
(paired ion)
C18, C8, phenyl, C4 , CN
bonded phases
(with pH control)
Ion exchange Anion and cation
exchange columns
Water
Soluble
Chiral
chromatography
Chiral phases (eg. Pirkle
columns)
Reverse phase
(with
ionizationcontrol)
C18, C8, phenyl, C4 , CN
bonded phases
(with pH control)
Reverse phase
(paired ion)
C18, C8, phenyl, C4 , CN
bonded phases
(with pH control)
Ion exchange Anion and cation
exchange columns
Chiral
chromatography
Chiral phases (eg. Pirkle
columns)
Chiral
chromatography
Chiral phases (eg. Pirkle
columns)
Fig 1. Choice of HPLC method
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HPLC column consists of a metal cylinder packed with tiny silica or modified silica particles to
separate compounds in a mixture. The mobile phase passes through the column using high pressure,
resulting in each component in the mixture coming out at different times, and the elution is based on
the polarity (Table 1 ).
Table1 : Properties of solvents commonly used in HPLC.
S.No. Solvent Polarity Miscible with
water
UV cut off
1 Hexane Nonpolar No 200
2 Carbon
tetrachloride
Nonpolar No 263
3 Chloroform Nonpolar No 245
4 Methylene chloride Nonpolar No 235
5 Tetrahydrofuran Nonpolar Yes 215
6 Diethyl ether Nonpolar No 215
7 Acetone Nonpolar Yes 330
8 Ethyl acetate Nonpolar Poorly 260
9 Acetonitrile Nonpolar Yes 190
10 Isopropanol Nonpolar Yes 210
11 Methanol Nonpolar Yes 205
12 Water Polar Yes -
For the herbal products that contain alkaloidal compounds, the ion exchange column may be selected.
For those that contain mainly polysaccharides compounds, a gel column may be selected. For those
that contain mainly steroidal compounds, the C18 reverse-phase column may be selected. Currently
most commonly used columns are C18 bonded-phase type of reverse-phase ODS column. The normal-
phase column, which is rarely used, is for the separation of homologous or isomeric compounds. The
ion exchange column is used for the separation of water-soluble ionic compounds. Some compounds
may require the use of amino compounds.
17.3.1 Selection of HPLC Column
The selection of HPLC columns is based on following criteria:
a) Internal Diameter
An important parameter of a HPLC column is the internal diameter (ID) as this directly influences the
Detection, sensitivity, selectivity of separation and the quantity of analyte that can be loaded onto the
column
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Low ID columns have higher sensitivity and lower solvent consumption for analytical
purposes and require low loading of sample
Analytical scale columns (4 mm and 4.6 mm) are the most common type of columns and
often use a UV‐Vis absorbance detector.
Narrow‐bore columns with ID of ≤ 3mm are used for sensitive analytical applications and use
more advanced UV‐Vis, fluorescence or mass detectors
Large ID columns (≥ 7mm) have a high loading capacity and so are used to purify usable
amounts of sample material for preparative or semi‐preparative applications
Internal diameter of the column has vital role in resolution and is inversely proportional i.e.
lesser the internal diameter better the resolution.
b) Particle size
The size of the particles (beads) in the stationary phase of the column influences the separation of
the analyte. Smaller particles generally provide more surface area and better separations, but the
pressure required for optimum separation is greater.
Columns with 5and 3μm are commonly used. Columns with 5 μm particles provide high
resolution of peaks and are large enough to be suitable for analytical, preparative and
semi‐preparative applications and with 3μm particles are used when rapid analysis is needed and
are mostly suitable for analytical applications
c) Porosity
An ideal stationary phase will be porous enough to provide greater surface area so that the sample
material can fit into the column and be analysed. Small pores up to 120 Å provide greater surface
are a and lead to higher resolving ability and are typically used for smaller MW analytes. Larger
pores such as 300 Å are suitable for a wider range of samples especially for larger MW analytes
such as proteins and oligonucleotides.
d) Length. The length of a column determines the overall separation time and influences the
resolution of the peaks. For the same particle size:‐
3 ‐ 7.5cm length column provide faster analysis run times, however these may be relatively
costlier
10 – 25cm length columns provide better resolution and are commonly used.
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A compromise between the speed and resolution of analysis is normally needed to find the most
efficient column i.e. the shortest column with the best resolving capability.
e) Chemical nature of compounds
If compounds have many carbons, or differ in the number of carbons, choose a stationary
phase with carbon (C18, C8, and phenyl). This works well for molecules that are soluble in
solvents such as acetonitrile, methanol, etc.
Acids and bases can be difficult to separate. The "neutral" form is usually retained more on a
reversed phase (C18) column, while the "ionic" form is not retained as much. Control of pH
is required in such cases.
A phenyl column for aromatic compounds separation and amino and -Cyano columns can be
used for separating polar organic molecules such as sugars.
17.4 MOBILE PHASE
The choice of mobile phase depends on the nature of the drug and the solvent system with the most
optimized method should be used. The best separation should satisfy the following requirements:
All the components of the test sample can be analyzed or the sample can be analyzed into as
many components (peaks) as possible.
The peaks should be adequately resolved.
The analysis time is as short as possible.
Optimization of the separation conditions mainly involves optimization of chemical factors such as
mobile phase composition, pH value of the mobile phase and ion pair reagent concentration.
The separation may be achieved either in isocratic mode or gradient mode. As the herbal drugs
contain complex components, normal isocratic elution is unable to achieve the separation of
components of different natures, giving rise to fewer peaks in the chromatogram and insufficient
information required for fingerprint evaluation. Under most circumstances, gradient elution should be
used. Under suitable gradient conditions, compounds of widely differing natures are all separated.
17.5 DETECTORS
The detectors available for detection of herbal compounds include ultraviolet-visiblet (UV-Vis)
detector - diode array detector (DAD), fluorescence detector (FLD), electrochemical detector (ECD)
and refractive Index detector (RID).
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17.5.1 UV DETECTOR
It is suitable for detection of compounds with ᴫ -ᴫ conjugated or ᴩ -ᴫ conjugated structure, while a
large number of compounds that are without double bonds are not detected. Three types of UV
detectors are available: fixed wavelength, multiple wavelength or photodiode array (DAD). The fixed
wavelength detector is the least expensive and has higher intrinsic sensitivity because the light is
emitted at specific wavelengths with given lamps. However, the multiple-wavelength detector is more
versatile and can compensate for its lower sensitivity when a wavelength with the higher extinction
coefficient for the solutes is chosen. UV-visible detector has its application in detection of flavonoids,
terpenes, alkaloids, coumarins and alkamides.
17.5.2 FLUORESCENCE DETECTOR (FD)
Compared with HPLC-UV, fluorescence detector (FD) affords much greater sensitivity and
selectivity. In fluorescence, the molecular absorption of a photon triggers the emission of another
photon with a longer wavelength. This difference in wavelength provides more selectivity and the
fluorescent light is measured against a very low light background, thus improving the S/N ratio. Most
application of FD is related to detection of aflatoxins in food because they contain natural
fluorescence.
17.5.3 REFRACTIVE INDEX DETECTOR (RID)
RID is a simple and least expensive detector. The detector has been useful for detecting compounds
such as carbohydrates and polymers. However, RID lacks sensitivity and is susceptible to changes in
ambient temperature, pressure, and flow rate and cannot be used for gradient elution.
17.6 SAMPLE ANALYSIS
The herbal plants are prepared using optimized extraction and separation methods, and the analysis
should be carried out under the best chromatographic conditions for separation and analysis. Rigorous
method validation should be done to confirm the reliability of the test results, comprising mainly of
precision of the instrument and the reproducibility of the test method.
17.7 SYSTEM SUITABILITY
A system suitability test is required to ensure that a given operating system may be generally
applicable. System suitability also verifies that the resolution and reproducibility of the
chromatographic system are adequate for the analysis to be done.
Replicate injections of a standard solution used in the assay or other standard solution are compared to
determine whether the requirements for precision are met. Unless otherwise specified in the individual
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monograph, data from five replicate injections of the analyte are used to calculate the relative standard
deviation if the requirement is 2% or less; data from six replicate injections are used if the relative
standard deviation is more than 2%.
Replicate injections of the standard solution may be made before the injection of samples or may be
interspersed among sample injections. System suitability must be demonstrated throughout the run by
injection of an appropriate control preparation at appropriate intervals, including at the end of the
analysis. The control preparation can be the standard solution used in the test or a solution containing
a known amount of analyte.
17.8 TROUBLESHOOTING IN HPLC
Peak Problems
No peaks usually indicate instrumental problems. It could be due to a number of
causes including no sample injected, system not turned on properly, major leaks, a
dead detector, wrong mobile phase, or a particularly retentive or adsorptive column.
No flow usually indicates pump problems, flow system, blockages, or empty solvent
reservoirs.
Too many peaks occur from sample carryover, usually in the auto sampler or on the
column. Using a sample solvent stronger than the mobile phase may cause sample
precipitation in the column. The next blank run will show original chromatogram,
but at a lower concentration.
Too few peaks indicates poor resolution i.e. one or more peak overlap each other.
Increase the resolution or use “peak purity” software with a PDA to confirm
presence of unresolved peaks.
Fronting and tailing peaks: Fronting peaks are caused by column overload so dilute
the sample 1/10 and re-inject. Tailing peaks are caused by secondary interactions of
the analyte with the stationary phase or of excess void volume. All fittings and
tubing’s between the injector and the detector should be checked.
Broad Peaks and Split Peaks: These are indications of degraded column
performance caused by sample contamination, a partially blocked inlet frit, or
column voids caused by dissolution of silica particles usually at higher pH. Thus, the
column needs to be replaced.
Changes in Retention Time
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Changes in flow rate and mobile phase composition are the major factor responsible for changes in
retention time. Always prepare and measure pH in the aqueous portion of the mobile phase before
adding the organic solvent. Organic solvent changes pH.
Peak area problems
Peak area changes may be due to changes in volume injected, flow rate, wavelength of detector, pH,
leaks, sample stability, integration problems and partial loss of sample due to irreversible adsorption
on a dirty frit or inactive column.
Baseline drift
Column temperature fluctuation (even small changes cause cyclic baseline rise and
fall; most often affects refractive index and conductivity detectors, or UV detectors
at high sensitivity or in direct photometric mode). The column and mobile phase
temperature should be controlled.
Non-homogeneous mobile phase (drift usually to higher absorbance, rather than
cyclic pattern from temperature fluctuation). HPLC-grade solvents should be used
and the mobile phase should be degassed before use.
Contaminant or air buildup in detector cell. Flush the cell with methanol or other
strong solvent or if necessary clean cell with 1N HNO3 (never with HCl).
Mobile-phase mixing problem or change in flow rate. Composition/flow rate should
to correct.
Mobile phase contaminated deteriorated, or prepared from low-quality materials.
Detector (UV) is not set at absorbance maximum but at slope of the curve. The
wavelength should be set at UV absorbance maximum.
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18. ANALYSIS OF ESSENTIAL OILS
18.1 INTRODUCTION
An essential oil is primarily a substance comprising of volatile compounds which are formed through
the plant metabolism. They are isolated by physical means only (pressing and distillation) from a
whole plant or plant parts of known taxonomic origin, primarily composed of terpenes and their
oxygenated derivatives and are obtained by steam distillation or solvent extraction of different parts of
the aromatic plants including the buds, flowers, leaves, seeds, roots, stems, bark, wood and rhizomes
etc. Generally essential oils are immiscible with water, many of which are sufficiently soluble in
water to impart to its characteristics odor and taste. The two principal circumstances determine a plant
to be used as an essential oil plant:
A unique blend of volatiles like flower scents in rose. Such flowers produce and
immediately emit the volatiles by the epidermal layers of their petals.
Secretion and accumulation of volatiles in specialized anatomical structures such as
secretory idioblasts, cavities/ducts, or glandular trichomes. This leads to higher
concentration of the essential oil in the plant.
Essential oils may be classified using different criteria: consistency, origin, chemical
nature of main components.
The essential oil chemical profile is closely related to the extraction procedure employed, and thus,
choice of appropriate extraction method is crucial. On the basis of the properties of the plant material,
the following extraction techniques can be applied: steam distillation, solvent extraction, fractionation
of solvent extracts, maceration, etc.
The techniques commonly applied to assess essential oil physical properties are:
18.2 RELATIVE DENSITY
Relative density is defined as the ratio of the densities of given oil with respect to water at specified
temperature.
Unless otherwise stated, it is based on the ratio of the weight of a liquid in air at 25° to that of an
equal volume of water at the same temperature. Where a temperature is specified in an individual
monograph, the specific gravity is the ratio of the weight of the liquid in air at the specified
temperature to that of an equal volume of water at the same temperature.
18.3 OPTICAL ROTATION
Optical rotation, ‘α’ is the property shown by certain substances of rotating the plane of polarized
light. Such substances are said to be optically active in the sense that they cause incident polarized
light to emerge in a plane forming a measurable angle with a plane of the incident light.
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The optical rotation of a substance is the angle through which the plane of polarized light is rotated
when polarized light passes through the substance, if liquid, or a solution of the substance. Substances
are described as dextro-rotatory or laevo-rotatory according to whether [α]D25
the plane of polarization
is rotated clockwise or anticlockwise, respectively as determined by viewing towards the light source.
Dextro-rotation is designated (+) and laevo-rotation is designated (-).
The optical rotation unless otherwise specified, is measured at the wavelength of the D line of sodium
(λ= 589.3 nm) at 25°, on a layer 1dm length. It is expressed in degrees.
Optical activity is determined by using a polarimeter, with the angle of rotation depending on a series
of parameters, such as oil nature, the length of column through which the light passes, the applied
wavelength, and the temperature. The degree and direction of rotation are of great importance for
purity assessments, since they are related to the structures and the concentrations of the chiral
molecule in the sample. Each optically active substance has its own specific rotation.
Calculation
Calculate the specific optical rotation using the formulae, dextro-rotation and laevo-rotation being
designated by (+) and (-) respectively.
[α]D20
= α / ld20
Where, α = corrected observed rotation, in degress, at 20°
D = D line of sodium light (l = 589.3nm)
l = length of the polarimeter tube in dm
d20
= specific gravity of the liquid or solution at 20°
18.4 REFRACTIVE INDEX
The refractive index (n) of a substance with reference to air is the ratio of the sin of the angle of
incidence to the sin of the angle of refraction of a beam of light passing from air into the substance. It
varies with the wavelength of the light used in its measurement.
Unless otherwise specified in individual monograph, the refractive index, nD20
is measured at
20°±0.5° with reference to the wavelength of the d-line of sodium (λ=589.3 nm).
NOTE: The temperature should be carefully adjusted and maintained since the refractive index varies
significantly with temperature.
18.5 MONOGRAPH DEVELOPMENT OF ESSENTIAL OIL/ VOLATILE OIL
a) Naming the monograph
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The family of the plant along with its genus and species along with common name/ popularly
known name is mentioned as the name of the monograph.
b) Source of Essential oil
Essential oils are generally derived from one or more plant parts, such as flowers (e.g. rosemary,
lavender), leaves (e.g. mint, lemongrass), leaves and stems (e.g. Clove), bark (e.g. cinnamon,
cassia, canella), roots (valerian), seeds (e.g, castor, cumin, nutmeg), fruits (coriander, cumin),
rhizomes (e.g. ginger) and gums or oleoresin exudations.
c) Active constituents
The minimum content of quantifiable chief marker constituents shall be provided.
Eg:- Lemon Grass oil is the essential oil obtained by steam distillation of the leaves of
Cymbopogon flexuosus (Fam. Graminae). It contains not less than 70.0 per cent and not more
than 90.0 per cent of citral. Not less than 1.56 per cent and not more than 8.0 per cent of methyl
heptanoate.
d) Category
Therapeutic category and strength is specified wherever possible.
e) Description
This section contains a brief description of the organoleptic characters of the drug. The
information given is not to be regarded as representing mandatory requirements but is important
in preliminary evaluation. The colour of the drug, where this is characteristic.
The odour of the drug, no reference is made to taste of the drug. Eg:- A clear, pale yellow to
amber liquid, free from sediment, suspended matter and characteristic lemon like odour.
f) Identification
This section includes tests performed to identify the drug. It can be performed by GC, TLC and
flash point where applicable. All the identifications mentioned below are not necessarily
included: some may be absent when they are not feasible or are not significant for the purpose of
identification.
g) Gas Chromatography (2.4.13)
Where GC is used in a test or assay, it may also be referred to under Identification. Reference GC
chromatograms are mentioned in Volume I of IP. The chromatograms to be reproduced shall
consist of 2 parts:
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Part 1: Depicting the running of a gas chromatograph, as per the method given in the monograph
using the Phytochemical Reference Substance(s) (PRS)/ marker compound. Such chromatogram
shall be labeled clearly about X and Y axis, what they depict, clearly point out the “Retention
Time at which the peak of the PRS/ marker compound appears”. The peak shall be labeled
suitability, suffixed by a small arrow pointing to the peak. In such chromatogram, if any internal
standard used or any other compounds whose identity and retention time are known appear, they
may also be labeled.
Part 2: Depicting the running of a GC, as per the method given in the monograph using the
sample of the material under test. Such chromatogram shall be labeled clearly about X and Y
axis, what they depict, clearly point out the Retention Time at which the peak where the marker
compound under analysis is appearing. The peak shall be labeled with the name of the marker
compound, suffixed by a small arrow pointing to the peak. In such chromatogram, if any internal
standard if used or any other compounds whose identity and retention time are known, appear,
they may be also labeled. Other peaks that may appear in the chromatogram, whose chemical
identity is not known, need not be labeled.
On top of all pages where such typical chromatograms are reproduced, following text should be
written.
“Reproduced below is Typical chromatogram of an essential oil for which a monograph
appears in this edition of IP”. Each typical chromatogram bears the name of the monograph below it
Example: GC chromatogram of XYZ Seed Oil”:
h) Flash Point (2.4.44)
The flash point of a volatile material is the lowest temperature at which it can vaporize to form
an ignitable material in air. At the flash point, the vapor may cease to burn when the source of
ignition is removed. The flash point is often used as as a descriptive characteristic of liquid fuel,
Res
pon
se (
mV
)
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and it also used to help characterize the fire hazards of liquids. “Flash point” refers to both
flammable liquids and combustible liquids.
Flash point as per IP 2014 general chapter 2.4.44 is incorporated in identification.
Other identification test respective to each monograph as applicable are incorporated.
NOTE: THE TEST RESULTS ARE COMPARED WITH THAT OF REFERENCE
i) Tests
All the tests mentioned below are not necessarily included: i.e. may depend upon type of oil
(Essential oil/volatile oil).
Tests such as Relative density (2.4.29), Refractive index (2.4.27), Optical rotation (2.4.22), Acid
value (2.3.23), Light absorption (2.4.7), Peroxide value (2.3.35), Acetyl value (2.3.22), Hydroxyl
value (2.3.27), Saponification value (2.3.37), Iodine value (2.3.28), Foreign fatty substances,
Melting range (2.4.21) etc.
j) Assay
Wherever possible, an assay by chemical method or GC is carried out. Specific constituents of
the essential oil can be quantified by GC. The chromatographic system consisting of the details
of column, column temperature, mobile phase, detector, flow rate, injection volume and split
ratio.
k) Storage
Distilled oils are normally packed in tightly closable glass containers or glass-lined aluminum
container or aluminum containers and are to be stored and transported at temperature below 25°.
Exposure to heat and light are to be prevented recognizing their high flammability, and potential
oxidative properties.
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19. EXAMPLES OF SOME HERBAL MONOGRAPHS
RAW HERB
Arjuna
Terminalia arjuna Bark
Arjuna consists of dried stem bark of Terminalia
arjuna (Roxb) Wight &Arn (Fam. Combretaceae)
Arjuna contains not less than 0.02 per cent of
arjungenin calculated on the dried basis.
Category.Antihyperlipidaemic, antihypertensive,
astringent, cardioprotective, hrdroga.
Description.A flat or minutely curved thick pieces of
bark with reddish gray colour and astringent taste.
Identification
A. Macroscopic — Stem bark pieces, flat or minutely
curved, with reddish gray external surface and darker
inner surface. Internal surface has longitudinal minute
ridges. Fractures longitudinal.
B. Microscopic— Cork consisting of 6-10 layers of
elongated cells, phloem broad, medullary rays
uniseriate. Calcium oxalate clusters abundant. Few of
the parenchyma cells contain colouring matter.
C. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase.A mixture of 5 volumes of toluene, 5
volumes of ethyl acetate and 0.5 volume of acetic
acid.
Test solution. Reflux 2 g of coarsely powdered
substance under examination with 50 ml of
chloroform for 15 minutes, cool and filter. Reflux the
residue further with 50 ml of chloroform. Combine
the filtrate and concentrate under vacuum to dryness.
Dissolve the residue in 10 ml of ethanol at 50° for 10
minutes and filter.
Reference solution. Reflux 1 g of arjuna RS with 50
ml of chloroform for 15 minutes, cool and filter.
Reflux the residue further with 50 ml of chloroform.
Combine the filtrate and concentrate under vacuum to
dryness. Dissolve the residue in 5 ml of ethanol at 50°
for 10 minutes and filter.
Apply to the plate 10 µl of each solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 8 cm.
Dry the plate in air and examine under ultraviolet light
at 254 nm and 365 nm, spray with 10 per cent w/v
solution of sulphuric acid in methanol. Heat the plate
at 110º for 5 minutes and examine in day light. The
chromatographic profile of the test solution is similar
to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2.0
per cent.
Ethanol-soluble extractive (2.6.2). Not less than
20.0 per cent.
Water-soluble extractive (2.6.3). Not less than 20
per cent by method I.
Total ash (2.3.19). Not more than 30.0 per cent.
Acid-insoluble ash (2.3.19). Not more than 2.0 per
cent.
Heavy metals (2.3.13). 1.0 g complies with the limit
test for heavy metals, Method B (20 ppm).
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Loss on drying (2.4.19). Not more than 10.0 per cent,
determined on 5 g by drying in an oven at 105°.
Microbial contamination (2.2.9).Complies with the
microbial contamination tests.
Assay. Determine by liquid chromatography (2.4.14).
Test solution. Reflux 5 g of coarsely powdered
substance under examination with 50 ml of
chloroform for 15 minutes, cool and filter. Reflux the
residue further with 50 ml of chloroform, cool and
filter. Combine the filtrates and concentrate under
vacuum to dryness, then extract dried residue with 10
ml of ethanol at 50° for 10 minutes and filter.
Reference solution. A 0.1 per cent w/v solution of
arjungenin RS in ethanol.
Chromatographic system
– a stainless steel column 25 cm x 4.6 mm packed
with octadecylsilane bonded to porous silica (10
µm),
– mobile phase: A. acetonitrile (70 per cent) in
water,
B. acetonitrile (30 per cent) in
water,
– a gradient programme using the condition given
below,
– flow rate: 1.2 ml per minute,
– spectrophotometer set at 210 nm,
– injection volume: 20 µl
Time Mobile phase A Mobile phase B
(in min) (per cent v/v) (per cent v/v)
0 30 70
10 50 50
30 70 30
50 30 70
Inject the reference solution. The test is not valid
unless the relative standard deviation for the replicate
injections for the analyte peak corresponding to
arjungenin is not more than 2.0 per cent.
Inject the reference solution and the test solution.
Calculate the contents of arjungenin.
Storage. Store protected from light, heat, moisture
and against attack by insects and rodents.
EXTRACT
Arjuna Dry Extract
Arjuna Dry Extract is obtained by extracting Arjuna
(Terminalia arjunaWight and Arn,Fam. Combretaceae)
bark with methanol or any other suitable solvent and
evaporation of solvent.
Arjuna Dry Extract contains not less than 90.0 per cent
w/w and not more than 110.0 per cent w/w of stated
amount of the arjunolic acid on the dried basis. It may
contain suitable added substances.
Category. Antihyperlipidaemic, antihypertensive,
astringent, cardioprotective, hrdroga.
Usual strength. 60 per cent w/w.
Description. Alight brown to beige powder with or
without red tinge.
Identification
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A. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase. A mixture of 92 volumes of chloroform
and 8 volumes of methanol.
Test solution. Dissolve 50 mg of the extract under
examination with 50.0 ml of methanol and filter.
Reference solution. A 0.1 per cent w/w solution of
arjunolic acid RS in the methanol.
Apply to the plate 5 µl of each solution as bands 10 mm
by 2 mm. Allow the mobile phase to rise 8 cm. Dry the
plate in air and spray with anisaldehydesulphuric acid
reagent. Heat the plate at 110° for 10 minutes and
examine the plate under ultraviolet light at 365 nm and
day light. The chromatographic profile of the test
solution is similar to that of the reference solution.
B. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase.A mixture of 35 volumes of chlorofom,
10 volumes of methanol and 2 volumes of water.
Test solution. Dissolve 50 mg of the extract under
examination with 10.0 ml of methanol and filter.
Reference solution. A 0.05 per cent w/w solution of
arjugenin RS in the methanol.
Apply to the plate 5 µl of each solution as bands 10 mm
by 2 mm. Allow the mobile phase to rise 8 cm. Dry the
plate in air and spray with vanillinesulphuric acid
reagent. Heat the plate at 110° for 10 minutes and
examine the plate under ultraviolet light at 365 nm and
day light. The chromatographic profile of the test
solution is similar to that of the reference solution.
Tests
Ethanol-soluble extractive (2.6.2). Not less than 80.0
per cent.
Total ash (2.3.19). Not more than 5.0 per cent.
Heavy metals (2.3.13). 1.0 g complies with the limit
test for heavy metals, Method B (20 ppm).
Loss on drying (2.4.19). Not more than 5.0 per cent,
determined on 1 g by drying in an oven at 105°.
Microbial contamination (2.2.9).Complies with the
microbial contamination tests.
Assay. Determine by liquid chromatography (2.4.14).
Test solution. Shake a quantity of the extract under
examination containing about 50 mg of arjunolic acid in
50.0 ml of the methanol, filter.
Reference solution.A 0.1 per cent w/v solution of
arjunolic acidm RS in methanol.
Chromatographic system
– a stainless steel column 25 cm x 4.6 mm packed
with octadecylsilane bonded to porous silica (5
µm),
– mobile phase: a mixture of 35 volumes of 5
-cyclodextrinand 65 volumes of methanol,
– flow rate: 1 ml per minute,
– spectrophotometer set at 205 nm,
– injection volume: 20 µl.
Inject the reference solution. The test is not valid unless
the relative standard deviation for replicate injections is
not more than 2.0 per cent.
Inject the reference solution and the test solution.
Calculate the content of the arjunolic acid the extract.
Storage. Store protected from heat and moisture.
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ESSENTIAL OILS
Basil Oil
Tulsi ka tail
Basil Oil is the essential oil obtained by steam
distillation of part of plant (leaves and flowering tops)
Ocimum basilicum (Fam.Lamiaceae).
Basil Oil contains not less than 50.0 per cent and not
more than 75.0 per cent of methyl chavicol, and not less
than 15.0 per cent and not more than 25.0 per cent of
limonene.
Description.A clear, pale yellow to amber liquid, free
from sediment and suspended matter.
Identification
A. Determine by gas chromatography (2.4.13).
Test solution. A 2.0 per cent w/v solution of the oil
under examination in ethanol (95 per cent).
Reference solution.A 2.0 per cent w/v solution of basil
oil RS in ethanol (95 per cent).
Use chromatographic system described in the Assay.
The peaks in the chromatogram obtained with the test
solution corresponds to the peaks in the chromatogram
obtained with the reference solution.
B. Flash point (2.4.44). Not less than 100.0°.
Tests
Relative density (2.4.29).0.9300 to 0.960.
Refractive index (2.4.27). 1.488 to 1.500.
Optical rotation (2.4.22).– 6.0° to +7.5°.
Acid value (2.3.23). Not more than 1.0.
Assay. Determine by gas chromatography (2.4.13).
Test solution.A 2.0 per cent w/v solution of the oil
under examination in ethanol (95 per cent).
Reference solution(a).A 2.0 per cent w/v solution of
methyl chavicol RS in ethanol (95 per cent).
Reference solution (b).A 2.0 per cent w/v solution of
limonene RS in ethanol (95 per cent).
Chromatographic system
– a capillary column 30 m x 0.25 mm, coated with
methyl 5 per cent phenylpolysiloxane,
– temperature:
column. 50° for 1 minute, increase from 50° to
220° at a rate of 10° per minute and maintain at
this temperature for 13 minutes
– inlet port at 250º and detector at 280º,
– detection by flame ionization detector.
– flow rate 1 ml per minute using nitrogen as carrier
gas.
– injection volume: 1.0 µl.
– split ratio : 1 : 25
Inject reference solutions (a), (b) and the test solution.
Calculate the content of methyl chavicol and limonene
in the oil using area normalisation procedure.
Storage. Store protect from light and moisture.
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TINCTURE
Belladonna Tincture
Belladonna Tincture obtained from Belladonna leaf or
roots of one or more of the cultivated varieties of
Atropa belladonna Linn. or A. acuminata Royle ex
Lindley (Fam. Solanaceae) or a mixture of both species.
Belladonna Tincture contains not less than 90 per cent
w/w and not more than 110 per cent w/w of total
alkaloids, calculated as hyoscyamine, C17H23NO3.
Category. Anticholinergic.
Usual strength. 0.03 per cent w/w.
Description. A clear green or brownish green liquid.
Identification
A. Determine by thin-layer chromatography (2.4.17),
coating the plate with silica gel GF254.
Mobile phase. A mixture of 10 volumes of anhydrous
formic acid, 10 volumes of water, 30 volumes of
methyl ethyl ketone and 50 volumes of ethyl acetate.
Test solution. Evaporate 10 ml of the tincture under
examination in a water-bath at 40° under reduced
pressure. Dissolve the residue in 1.0 ml of the
methanol.
Reference solution. Dissolve 1 mg of chlorogenic acid
RS and 2.5 mg of rutin RS in 10 ml of the methanol.
Apply to the plate 40 µl of each solution as bands 10
mm by 2 mm. Allow the mobile phase to rise 15 cm.
Dry the plate in air, heat the plate at 110° for 10
minutes. Spray the warm plate with 1 per cent v/v
solution of diphenylboric acid aminoethyl ester in
methanol. Allow to cool and spray with 5 per cent v/v
solution of macrogol400 in methanol, allow the plate
to dry in air for 30 minutes and examine under
ultraviolet light at 365 nm. The chromatographic
profile of the test solution is similar to that of the
reference solution.
B. Atropine. Determine by thin-layer chromatography
(2.4.17), coating the plate with silica gel G.
Mobile phase. A mixture of 3 volumes of strong
ammonia solution, 7 volumes of water and 90 volumes
of acetone.
Test solution. To 15.0 ml of the tincture under
examination, add 15 ml of 0.05M sulphuric acid and
filter. Add 1 ml of strong ammonia solution to the
filtrate, with two quantities, each of 10 ml, of peroxide-
free ether, separate the ether layer by centrifugation if
necessary, dry the combined ether extracts over
anhydrous sodium sulphate, filter and evaporate to
dryness on a water-bath. Dissolve the residue in 0.5 ml
of methanol.
Reference solution. Dissolve 50 mg of hyoscyamine
sulphate in 9 ml of methanol and 15 mg of hyoscine
hydrobromide in 10 ml of methanol, separately. Mix 1.8
ml of the hyoscine hydrobromide solution and 8 ml of
the hyoscyamine sulphate solution.
Apply to the plate 20 µl and 40 µl of each solution as
bands 10 mm by 2 mm. Allow the mobile phase to rise
10 cm. Dry the plate at 105° for 15 minutes, spray with
potassium iodobismuthate solution, dry the plate and
spray with sodium nitrite solution until the plate is
transparent. Examine the plate after 15 minutes in day
light. The chromatogram obtained with the test solution
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corresponds to the chromatogram obtained with the
reference solution.
Tests
Ethanol (2.3.45). 64 to 69 per cent v/v by method III.
Assay. Evaporate 50.0 g of the tincture under
examination to a volume of about 10 ml. Transfer
quantitatively to a separating funnel, with the minimum
volume of ethanol (70 per cent v/v). Add 5 ml of strong
ammonia solution and 15 ml of water. Extract with
three quantities, each of 40 ml of a mixture of 1 volume
of dichloromethane and 3 volumes of peroxide-free
ether, carefully to avoid emulsion, until the alkaloids
are completely extracted. Combine the dichloromethane
and ether extracts; concentrate the solution to a volume
of about 50 ml by heating on a water-bath. Transfer the
resulting solution quantitatively to a separating funnel,
rinsing with peroxide-free ether. Add a quantity of
peroxide-free ether equal to at least 2.1 times the
volume of the solution to produce a layer having a
density well below that of water. Extract the resulting
solution with minimum of three quantities, each of 20
ml of 0.25M sulphuric acid until the alkaloids are
completely extracted. Separate the layers by
centrifugation if necessary and transfer the layers to a
separating funnel. Make the combined layers alkaline
with strong ammonia solution and extract with
minimum of three quantities, each of 30 ml of
dichloromethane until the alkaloids are completely
extracted. Combine the dichloromethane and ether
extracts, add 4 g of anhydrous sodium sulphate and
allow standing for 30 minutes with occasional shaking.
Decant the dichloromethane and filter. Wash the
sodium sulphate with three quantities, each of 10 ml of
dichloromethane. Combine the dichloromethane and
ether extracts, evaporate to dryness on a water-bath.
Heat the residue in an oven at 105° for 15 minutes.
Dissolve the residue in a few ml of dichloromethane,
evaporate to dryness on a water-bath and heat the
residue in an oven at 105° for 15 minutes again.
Dissolve the residue in a few ml of dichloromethane.
Add 20.0 ml of 0.01M sulphuric acid and remove the
dichloromethane by evaporation on a water-bath.
Titrate the excess of acid with 0.02M sodium hydroxide
using methyl red mixed solution as indicator.
1 ml of 0.01 M sulphuric acid is equivalent to 0.005788
g of total alkaloids calculated as hyoscyamine.
Calculate the content of total alkaloids with reference to
the dried material
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20. HERBAL DRUGS MANUFACTURERS/SUPPLIERS IN INDIA
A non-exhaustive source for extracts and marker compounds suppliers are given below:
S.No MANUFACTURER/
SUPPLIER
ADDRESS TELEPHONE / FAX /
1. Asthagiri Herbal Research
Foundation
7/1, Sundaram Colony, Thirumalesai St, Tambaram
East, Tambaram East, Chennai, Tamil Nadu 600059
044 2239 7645
2. Aayurmed Biotech Private
Limited
3, Satyam I-B, Raheja Complex, Nr Times of India
Press, Malad, Mumbai, Maharashtra 400 097,
022-32919302
3. Alchem International Private
Ltd.
201, Empire Plaza, Mehrauli - Gurgaon Road,
Sultanpur, New Delhi-110 030
0129-4266000
0129-2307192
4. Central Institute of Medicinal
and aromatic Plants
Central Institute of Medicinal and Aromatic Plants
P.O. - CIMAP, Near Kukrail Picnic Spot,
Lucknow - 226 015
91-522 - 2718524
5. Chromadex 1005 Muirlands Blvd, Suite G, Irvine, CA 92618 USA +1.949.419.0288
Fax: +1.949.419.0294
6. Cymbio Pharma Private
Limited
Cymbio Pharma Private Limited
No. 23/4, N. H. 7, Venkatala, Yelahanka Bengaluru-
560064
09900014078
7. Dabur India Ltd. Unit I & II Plot No.22, Site-IV Sahibabad ,Ghaziabad 0120 – 4378400
Fax : 0120 - 4376924
8. Exotic Naturals 118, Morya House,Off New Link Rd, Andheri (West),
Mumbai, Maharashtra 400053
022- 26733092
9. Govt. Opium and Alkaloids
Works
Mahuabagh, Ghazipur, Uttar Pradesh 233001 011-26417475
Fax: 011-26440667
10. Herbotech Pharmaceuticals VPO Pandori Waraich, Majitha Road,Amritsar, Punjab
143001
0183- 2221025
11. Indo Nacop Chemicals Ltd
23-579, Vishnukundina Nagar,Vinukonda Post -Guntur
District, Andhra Pradesh
08646-272954/ 273513
08646-272954, 09440968849
12. Inga Pharmaceuticals Inga House, Mahakali Road, Andheri East, Mumbai,
Maharashtra 400093,
022-28202932, 28202933
13. Indian Institute of Integrative
Medicine
Post Bag No. 3, Canal Road, Jammu-180001 0191-2584999, 2585222
14. Ivy Comm Systems 1405, Sector-14, Faridabad, Haryana 121 007, India 0129-4006805
15. Konark Herbal & Health Care 332, Adhyaru Industrial Estate Sun Mill Compound,
Lower Parel, Mumbai, Maharashtra-400 013
022-40914300
16. Kumaon Chemical Products Village Shivlalpur Ramnagar Distt., (Nainital),
Uttarakhand - 244715
05947-251243, 08045132916
Fax : 91-05947-251377
17. Min Chem India 117, Loha Bhawan, 93, P. D. Mello Road, Masjid East ,
Mumbai - 400009 Maharashtra
08049442137
18. M R Group Painth Parao, Ramnagar (Distt.) Nainital,
Uttarakhand- 244715
05947-251423
19. Kancor Ingredients Ltd No.VII/138, Kancor Road, Angamaly South,
Ernakulam, Kerala-683573
0484-3051000, 2456451
20. Kumaon Chemical Products Village Shivlalpur, Ramnagar Distut,
Uttarakhand-244715
05947-251716
21. Natural Remedies Pvt. Ltd.
Plot No. 5B, Veerasandra Industrial Area, 19th K.M.
Stone, Hosur Road, Electronic City (Post), Bangalore,
Karnataka-560 100
080-4020 9999
22. Nature & Nurture Healthcare
Pvt. Ltd.
305, Vardhman City-2, Plaza Commercial Centre, Asaf
Ali Road, New Delhi, Delhi-110002
043524267/ 43752230
23. Naturol BioEnergy Limited Plot No: 1056/1, Road No# 45, Jubilee Hills, 040-23556979, 23541829
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Hyderabad, Andhra Pradesh-500 033
24. Neeru Enterprises 6, Saleem Manzil, Civil Lines, Rampur, Uttar Pradesh
244 901
0595-2350829/ 2353744
25. Nishant Aromas 424/425, Milan Industrial Estate, Off T J Road, Cotton
Green, Cotton Green, Mumbai, Maharashtra-400033
022- 2471 0058
26. Phyto Concentrate
Phyto Concetrates
Factory Adress 1: Block No - 1145,Near Santej petrol
pump,
Santej, District: Gandhinagar
Factory Adress 2: Plot No 63/1, Industrial Estate,
Express Highway, Halol-389350,
Gujarat, India
02764-286568
02764-286409
27. Pioneer Enterprise
101, Raudat Tahera Street, Mumbai-400003
022-2347 2534
Fax: +9122 2347 0325
28. Plantmed Pharma Pvt. Ltd. No. 1187/4, Kaliamma Temple Road, T. Dasarahalli,
Bangalore, Karnataka- 560057
080-32212625
29. Quad Lifesciences Pvt.Ltd. Village Bhagwas, 6 K.M. Derabassi – Barwala Road,
Derabassi– 140 507 Distt, Mohali, Punjab
30. RYM Exports 23, Anuradha Society, Old Nagardas Rd. Andheri(E),
Mumbai, Maharashtra 400069
022-32956539, 28210025
31. Sami Labs Limited 19/1 & 19/2, I Main, II Phase, Peenya Industrial Area,
Bangalore, Karnataka 560 058,
080-28397973
32. Sanat Products 3rd Floor, Sagar Plaza, Distt Centre, Laxmi Nagar,
Vikas Marg Laxmi Nagar 110092, New Delhi, Delhi
011- 2251 8794
33. Shashi Phytochemical
Industries
1, Old Industrial Area, Alwar, Rajasthan 301001 0144-2373770, 72, 73
34. Sigma Aldrich
DSM-149,150,151, 1st Floor, DLF Towers, Shivaji
Marg, New Delhi-110015
011-6676 2800
Fax: 011-6676
35. Sonic Biochem Extractions 38, Patel Nagar, Indore, Madhya Pradesh 452001 0731-2466456, 2447099
36. Surya Pharmaceutical Ltd. S.C.O: 164-165, Sector 9-C, Chandigarh 160 009 0172-5005000
37. Ultra International 64/1, Site 4, Sahibabad Industrial Area, Ghaziabad,
Uttar Pradesh 201010
0120 438 8500
38. Venbiotech Pvt. Ltd. 10, Chokkanathar Street, Karthikeyan Nagar,
Maduravoyal, Chennai, Tamil Nadu 600095
044-42918171
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BIBLIOGRAPHY
1. Chandra H, Purohit VK. High Performance Thin Layer Chromatography (HPTLC): A
Modern Analytical Tool for Biological Analysis. Nature and Science 2010;8(10):58-61.
2. Kamboj A. Analytical evaluation of herbal drugs. Drugs Discovery Research in
Pharamocognosy/Analytical Evaluation of Herbal Drugs. (Eds): Vallisuta O, Olimat SM,
2012; 23-29.
3. Mukherjee PK. Quality Control of Herbal Drugs published by Business Horizons 2002; 1:
426-491.
4. WHO’s Quality Control Methods for Medicinal Plant Materials 1998. World Health
Organization, HQ, Geneva.
5. EMEA 1998, Quality of Herbal Medicinal Products Guidelines European Agency evaluation
of Medicinal Products (EMEA), London.
6. Wagna H, Bladt S. Plant Drug Analysis- A Thin Layer Chromatography Atlas from Springer
2009;2: 351-364.
7. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy, Nirali Prakashan 31st edition 2005.
8. Meier B, Sprianod. Moden HPTLC a perfect tool for quality control of herbals and their
preparations, Journal of AOAC International 2010; 95(5):1399-1409.
9. Sherma J. Biennial review of planar chromatography 2009-2011. Journal of AOAC
International 2011; 95(4):992-1008
10. Sherma J. Review of HPTLC in drug analysis: 1996-2009. Journal of AOAC International
2010; 93(3):754-764.
11. Sadek PC. Trouble shooting HPLC system: A Bench Manual, Wiley, New York 2000.
12. Wolfender JL. HPLC in natural product analysis: The petechan issue, Planta Medica 2009; 75
(7):719-734.
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BOOKS FOR FURTHER READING ON HERBS
1. Indian Pharmacopoeia 2014 and Addendum 2015. Ministry of Health and Family Welfare,
Government of India, Indian Pharmacopoeia Commission, Ghaziabad, U P.
2. The Ayurvedic Pharmacopoeia of India 2011. Government of India, Ministry of Health and
Family Welfare, Department of AYUSH, New Delhi. Part I, Volume I to IX.
3. The Ayurvedic Pharmacopoeia of India 2011. Government of India, Ministry of Health and
Family Welfare, Department of AYUSH, New Delhi. Part II, Volume I and II.
4. Ayurvedic Formulary of India1978. Government of India, Ministry of Health and Family
Welfare, Department of AYUSH, New Delhi. Part I and Part II.
5. Pulok K Mukherjee 2009. Peter J Houghton (Editors), Evaluation of Herbal Medicinal
Products, Perspectives on Quality, Safety and Efficacy, Pharmaceutical Press, London.
6. Indian Herbal Pharmacopoeia 2002. Indian Drugs Manufacturers Association, Mumbai, India.
7. Quality Standards of Indian Medicinal Plants 2008. Indian Council of Medical Research,
New Delhi, A K Gupta (Coordinator), Volumes I to XI.
8. Reviews on Indian Medicinal Plants 2011. Indian Council of Medical Research, New Delhi,
A K Gupta and Neeraj Tandon (Editors), Volumes I to VII.
9. Y K Sareen. Illustrated Manual of Herbal Drugs used in Ayurveda 1999. Council of Scientific
and Industrial Research and Indian Council of Medical Research.
10. Major Herbs of Ayurveda 2002. Churchil Livingstone – Elsevier Science, London, Elizabeth
M Williamson.
11. Thin Layer Chromatographic Atlas of Ayurvedic Pharmacopoeial Drugs 2009. Government
of India, Ministry of Health and Family Welfare, Department of AYUSH, New Delhi. Part I,
Volume I and II.
12. Macroscopic and Microscopic Atlas of Pharmacopoeial Drugs 2009. Government of India,
Ministry of Health and Family Welfare, Department of AYUSH, New Delhi. Part I,
Volume I to V.
13. British Herbal Pharmacopoeia 1996. British Herbal Medicine Association, London.
14. British Herbal Compendium Volume I 1992 and Volume II 2006. British Herbal Medicine
Association, London.
15. British Pharmacopoeia 2015. Department of Health Social Services and Public safety,
Medicines and Healthcare products Regulatory Agency (MHRA), London.
16. US Pharmacopoeia National Formulary 2015. United States Pharmacopoeial Convention,
Rockville, MD.
17. The Japanese Pharmacopoeia JP XVI 2011. Ministry of Health, Labour and Welfare.
Pharmaceutical and Medical Device Regulatory Science Society of Japan, Tokyo, Japan
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18. Pharmacopoeia of the People’s Republic of China. Vol III 2010. Chinese Pharmacopoeia
Commission. Beijing, China.
19. USP Dietary Supplements Compendium 2015 Vol IV. United States Pharmacopoeial
Convention, Rockville, MD.
20. Sukh Dev, Prime Ayurvedic Plant Drugs 2006. Anamaya Publishers (New Delhi) and Anshan
Tunbridge Wells, UK.
21. Malati G Chauhan and APG Pillai. Microscopic Profile of Powdered Drugs used in Indian
Systems of Medicine, Institute of Ayurvedic Medicinal Plant Sciences, Gujarat Ayurveda
University, Jamnagar, India, Volume I to V.
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