Title of the Thesis

Thesis submitted in partial fulfillment of the requirements for the degree ofMaster of ScienceinBIOTECHNOLOGYbyXXXXX(Roll No. : MRT12UGBBT23023)under the guidance ofXXXXXXAssociate ProfessorFaculty of Biological EngineeringShobhit University, Meerut

Department of BiotechnologyFaculty of Biological EngineeringSHOBHIT UNIVERSITY, MEERUT (June 2014)

DECLARATION

I hereby declare that the work which is being presented in the thesis titled as VALIDATION OF TETRACYCLINES IN HONEY BY UPLC-MS/MS in partial fulfillment of the requirement for the award of the degree of Bachelors of Technology in Biotechnology and submitted to the Departmen of Biotechnology, Faculty of Biological Engineering, Shobhit University, Meerut, is an authentic record of my own work carried out under the supervision of Mr. Aditya Pundhir, Assistant Professor Faculty of Biological Engineering, Shobhit University, MeerutThe work contained in this thesis has not been submitted by me for award of any degree or diploma or other similar title or recognition to any other University/institute.

XXXXXRoll No. : MRT12UGBBT23023Enrollment No.:000000000

CERTIFICATE FOR EXTERNAL WORK

CERTIFICATE

This is to certify that the present dissertation report entitled Method Validation, Glassware Calibration, Uncertainty & Testing Techniques used in Chemical Testing and Analysis of Food submitted to Shobhit University, Meerut in partial fulfillment of the requirement for the award of the degree of Bachelor of Technology in Biotechnology, is a bonafide work carried out by Mr. Manish Kumar Nirala, for the required period at Department of Biotechnology, Faculty of Biological Engineering, Shobhit University, Meerut.

It is further certified that the work has not been submitted in part or in full to any other university or institute for the award of any degree or diploma.

Mr. Aditya PundhirAssistant Professor Department of BiotechnologyFaculty of Biological Engineering Shobhit University, Meerut

ACKNOWLEDGEMENT

It is tough to keep a count of the host of helping hands and minds so benignant to me, over the time that I realized the inability inadequacy of mere words to convey the gratitude, so deeply felt. But amongst them all are some to whom I would do grave injustice if I dont account my sentiments and gratitude.While writing these lines first name come to in mind is of my reversed, esteemed honorable Vice Chancellor Dr. D.V Rai for his ever helping attitude and encouraging us to excel studies. I am sincerely indebted to Prof. (Dr.) Rekha Dixit, Head of the Department, Faculty of Biological Engineering, Shobhit University for her support, untiring efforts and constant help at every step in carrying out project work that made possible successful completion of this endeavor. In this project, I was greatly assisted and inspired by Mr. Aditya Pundhir, Assistant professor, Faculty of Biological Engineering, Shobhit University, Meerut, my mentor and supervisor. His able guidance and constructive counsel was the driving force for the pursuance of my research work.I would also like to pay my heartiest gratitude to the Director/Lab Manager Mr. Manoj Kumar Singh, and Quality Manager Aruna Singh of Intertek India Pvt. Ltd. for the support and exposure to industrial and laboratory that he provided us. My primary thanks goes to Assistant Manager Mr. Sachin Kumar Gupta, Senior Analyst Ms. Shashi Solanki, Senior Analyst Mr. David Antony, Analyst Dr. Subhash, Mr. Vikram Rana and Mr. Yatender Singh for her/his deep commitment and guidance during project. Words are inadequate in offering my thanks to the Technical Assistant and Project Assistants for their intense help, encouragement and cooperation in carrying out the project work. All the efforts would have been remain incomplete without the blessings of my father Sh. Bhagwat Singh does not only raise and nurture me but also introduce me to joy of reading from birth. He is a source of motivation and strength during moments of despair and discouragement and my mother always gives me endless love, support and encouragement and not only constantly inspired me but also provided me moral support, patience and affection throughout my research work. I would like to acknowledge invaluable help, love and constructive criticism from my brother which was the source of inspiration for me and made possible to achieve my objects. Above all, I remember the almighty who gave me courage, power, and enthusiasm and helped me in all my endeavors and for bringing me this far my educational career.

XXXXXX

ABSTRACT

Honey is now being used in a number of ways as food and food additives, drug and drug supplements and products with great prophylactic and therapeutically values, which contributes to human health maintaining and economically brings contribution to population welfare. Honey has been used by humans to treat in a number of ailments. Naturally a variety of honey found depends on nectar source i.e. blended, polyfloral, monofloral and honeydew honey etc. Typical honey analysis includes fructose (38.2%), glucose (31.3%), maltose (2.1%), sucrose (1.3%), water (17.2%), higher sugars (1.5%), ash (0.2%), other/undetermined (3.2%). Its glycemic index ranges from 31 to 78, depending on the variety. Honey has a density of about 1.36 kg/l (36% denser than water) and an acidic pH ranging from values 3 to 6.Other heavy metal amount ranges from g/kg to few mg/kg. Natural honey is free from contaminations and is usable for different purposes but a number of sources including natural and artificial, geographical position, bad apicultural practices, transporting hives over large geographic distances, potentially exposure of colonies to different pesticides, locality, industrial pollution and undetected routes of exposure affecting bees including through dust are causative agents of contamination. Antibiotic residues show a relatively long half-life and they may have direct toxic effects on consumers e. g., allergic reactions in hypersensitive individuals and disorder of the haemopoietic system, or cause problems indirectly through induction of resistant strains of bacteria. Tetracyclines, often complex molecules which may possess different functionalities within the same molecule inhibiting or abolishing the growth of microorganisms are of amphoteric nature with four natural variants i.e. OTC, TC, and CTC, 4epi-TC, 4epi-OTC, 4 epi-CTC and DC. A specific, sensitive and robust liquid chromatography tandem mass spectrometry for validating the method of tetracycline i.e. oxytetracycline, tetracycline, chlortetracycline, doxycycline, 4 epi-TC, 4epi-OTC, 4 epi-CTC in honey on UPLC-MSMS. Tetracycline analysis was performed using liquid chromatography electrospray ionization tandem mass spectrometry .We validate the method of tetracycline in Honey sample on UPLC-MSMS as per EIC .For the extraction of drug residues honey samples were dissolved with Na2EDTA, followed by a clean-up with filtration of 0.45m nylon filter and residues were extracted from the supernatant by solid-phase extraction (SPE), using OASIS HLB cartridges. The separation and determination was carried out by Ultra Pressure Liquid Chromatography coupled to tandem mass spectrometry (UPLC-MSMS), using an electrospay ionization source (ESI) in positive mode. Data acquisition under MS/MS was achieved by applying selective reaction monitoring (SRM) of two ion transitions per compound to provide a high degree of sensitivity and specificity. The method was validated, and mean recoveries were evaluated at five concentration levels (1, 2, 5, 10 and 15 g/kg).The samples spiked with different concentrations of , 2ppb, 5ppb, 10ppb, and 15ppb of standard tetracyclines analogues mixture were tested for the analysis of recoveries and preparation of samples.In India honey is regulated by the standards the Prevention of Food Adulteration (PFA) Act, the Bureau of Indian Standards (BIS) the Agricultural Produce Grading and Marking Act (Agmark) and the export is monitored by the Exports Inspection Council. The WHO, has recommended that antibiotics which are also licensed in human medicine should not be used any more as growth promoters in livestock as it is evident that residues of antibiotics in honey originate mostly not from the environment but from improper beekeeping practices.

Table of Contents Candidates Declaration ii

Certificate iii-iv

Acknowledgements v-vi

Abstractvii-viii

Table of Contentsix-xi

List of Abbreviations xii

List of Figures xiii

List of Tables xiv

Chapter 1 INTRODUCTION 1 1.1 Use and application 1 1.2 Production and consumption 2 1.3 Contamination of honey 3 1.3.1 Contamination due to environmental factor 4 1.3.2 Contamination due to apiculture practices 4 1.4 Objective 4

Chapter 2 LITERATURE SURVEY 5 2.1 Antibiotics 7 2.2 Parameter influencing the MS qunatafication 15 2.3 Instrumentation operation 17 2.3.1 Full scan analysis 18 2.3.2 Selected ion monitoring 20 2.3.3 Selected reaction monitoring 20

Chapter 3 MATERIAL AND METHODOLOGY 22 3.1 Instrumentation, chemical and material required 22 3.2 Preparation of reagents 23 3.3 Procedure 24 3.3.1 Preparation of working standard 24 3.3.2 Preparation of calibration curve standard 24 3.3.3 Sample Preparation 25 3.4 Instrumentation condition 26 3.4.1 LC condition 27 3.4.2 Mass spectrometer condition 27 3.5 Statics and calculation 30

Chapter 4 RESULTS AND DISCUSSION 31 4.1 Validation parameter 31 4.1.1 Specificity 31 4.1.2 System suitability 32 4.1.3 Linearity 33 4.1.4 Limit of quantification 34 4.1.5 Recovery 36 4.1.6 Repeatability 36 4.1.7 Within a Lab reproducibility 37 4.1.8 Range 37 4.1.9Decission Limit 38 4.1.10 Detection capability 39 4.1.11 Ruggedness 40 4.2 Performance criteria 40 4.2.1 Chromatographic separation 40 4.2.2 Mass spectropic detection 41 4.3 Data processing 42

Chapter 5 Summary and Future Scope 44

Reference 45

List of Abbreviations S.NOABBREVATION STAND FOR

1STPStandard Testing Procedure

2ACNAcetonitrile

3HPLCHigh Pressure Liquid Chromatography

4SRMSelective Reaction Monitoring

5SIMSelected Ion monitoring

6PpbParts Per Billion

7PpmParts Per Million

8LCMSMSLiquid chromatography Mass Spectroscopy

9UPLC MSMSUltra Pressure Liquid Chromatography

10EDTAEthylene Diamine Tetra acetic acid

11CTCChlorotetracycline

12OTCOxytetracycline

13TCTetracycline

14DC`Doxycycline

15DADDiode Array Detector

16EUEuropean Union

17EICExport Inspection Counseling

List of figures Fig. 1.1Contaminations as a result of malpractices3

Fig.2.1Structure of CTC And 4epi-CTC9

Fig.2.2Structures of DTC10

Fig.2.3Structures of TC And 4 epi TC10

Fig.2.4Structure of OTC and 4 epi-OTC11

Fig.2.5Total ion current19

Fig.2.6Triple quadrapole19

Fig.2.7Selected ion moinitering20

Fig.2.8Selected reaction monitering21

Fig.3.1Flow diagram of solid Phase extraction of sample 25

Fig.3.2Flow diagram of activation of OASIS HLB cartridge25

Fig.3.3Flow diagram of final preparation of sample26

List of tables Table 3.1Preparation of working standard24

Table 3.2Calibration Curve standard25

Table 3.3Selective Monitoring Reaction for the ion detect in positive mode ionization 29

Table 4.1Specificity of tetracycline in honey sample32

Table 4.2Selectivity and system suitability of tetracycline in honey sample32

Table 4.3Linearity of each analyte of tetracycline in honey sample34

Table 4.4LOQ of tetracycline in honey sample 35

Table 4.5Range of each analyte of tetracycline in honey sample 38

Table 4.6Decision limit of tetracycline in honey sample39

Table 4.7Detection limitof tetracycline in honey sample40

Table 4.8Relative intensity and tolerance41

Table 4.9Final report of validation42

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