1 INTRODUCTION

1. INTRODUCTION

1.1 ANALYTICAL CHEMISTRY

Analytical Chemistry(1)

seeks ever improved means of measuring the chemical

composition of natural and artificial materials. The techniques of this science are used to

identify the substances which may be present in a material and to determine the exact

amounts of the identified substances. Analytical chemists work to improve the reliability

of existing techniques to meet the demands for better chemical measurements.

Analytical chemistry serves the needs of many fields in industry; analytical chemistry

provides the means of testing raw materials and for assuring the quality of finished

products. Chemical analysis may be qualitative or quantitative.(2)

a. Qualitative:

Qualitative analysis involves attempting to identify what materials are present in a

sample. Answering the question: “What is it?”

b. Quantitative:

Quantitative analysis involves determining how much of a material is present in a sample.

Answering the question: “How much is present?”

Modern analytical methods make it possible to identify hundreds of components in a

single sample and to detect specific substances present in less than one part per million.

Analytical chemistry is the science of making quantitative measurements. In practice,

quantifying an analyte in a complex sample becomes an exercise in problem solving. To

be efficient and effective, an analytical chemist must know the tools that are available to

tackle a wide variety of problems. To be effective and efficient, analyzing samples

requires expertise in:

a. The chemistry that can occur in a sample

b. Analysis and sample handling methods for a wide variety of problems (the tools-

of-the-trade)

c. Proper data analysis and record keeping to meet these needs, Analytical

Chemistry courses usually emphasize equilibrium, spectroscopic and

electrochemical analysis, separations and statistics.

2 INTRODUCTION

Specific Technologies And Instrumentation3

:

A) Spectrometric techniques

o Ultraviolet and visible Spectrophotometry

o Fluorescence and phosphorescence Spectrophotometry

o Atomic Spectrometry (emission and absorption)

o Infrared Spectrophotometry

o Raman Spectroscopy

o X-Ray Spectroscopy

o Radiochemical Techniques including activation analysis

o Nuclear Magnetic Resonance Spectroscopy

o Electron Spin Resonance Spectroscopy

B) Electrochemical Techniques

o Potentiometry

o Voltametry

o Voltametric Techniques

o Amperometric Techniques

o Colorimetry

o Electrogravimetry

o Conductance Techniques

C) Chromatographic Techniques

o Gas Chromatography

o High performance Liquid Chromatography

o Thin Layer Chromatography

D) Miscellaneous Techniques

o Thermal Analysis

o Mass Spectrometry

o Kinetic Techniques

E) Hyphenated Techniques

o GC-MS (Gas Chromatography – Mass Spectrometry)

o ICP-MS (Inductivity Coupled Plasma Mass Spectrometry)

3 INTRODUCTION

o GC-IR (Gas Chromatography – Infrared Spectroscopy)

o MS-MS (Mass Spectrometry – Mass Spectrometry)

1.2. ANALYTICAL METHOD DEVELOPMENT

Methods are developed for new products when no official methods are available.

Alternate methods for existing (non-pharmacopoeial) products are developed to reduce

the cost and time for better precision and ruggedness. Trial runs are conducted, method is

optimized and validated. When alternate method proposed is intended to replace the

existing procedure comparative laboratory data including merit / demerits are made

available.

1.2.1. Steps of method development4

Documentation starts at the very beginning of the development process, a system for full

documentation of the development studies must be established. All data relating to these

studies must be recorded in laboratory notebook or an electronic database.

1. Analyte standard characterization

a) All known information about the analyte and its structure is collected i.e., physical

and chemical properties, toxicity, purity, hygroscopic nature, solubility and

stability.

b) The standard analyte (100% purity) is obtained. Necessary arrangement is made

for the proper storage (refrigerator, desicators, freezer).

c) When multiple components are to be analyzed in the sample matrix, the number of

components is noted, data is assembled and the availability of standards for each

one is determined.

d) Only those methods (MS, GC, HPLC etc.,) that are compatible with sample

stability are considered.

2. Method requirements

The goals or requirements of the analytical method that need to be developed are

considered and the analytical figures of merit are defined. The required detection limits,

selectivity, linearity, range, accuracy and precision are defined.

4 INTRODUCTION

2. Literature search and prior methodology

The literature for all types of information related to the analyte is surveyed. For

synthesis, physical and chemical properties, solubility and relevant analytical

methods. Books, periodicals, chemical manufacturers and regulatory agency

compendia such as USP / NF, AOAC and ASTM publications are reviewed.

Chemical Abstracts Service (CAS) automated computerized literature searches are

convenient.

4. Choosing a method

a) Using the information in the literatures and prints, methodology is adapted. The

methods are modified wherever necessary. Sometimes it is necessary to acquire

additional instrumentation to reproduce, modify, improve or validate existing

methods for in-house analytes and samples.

b) If there are no prior methods for the analyte in the literature, from analogy, the

compounds that are similar in structure and chemical properties are investigated

and are worked out. There is usually one compound for which analytical method

already exist that is similar to the analyte of interest.

5. Instrumental setup and initial studies

The required instrumentation is setup. Installation, operational and performance

qualification of instrumentation using laboratory standard operating procedures (SOP’s)

are verified. Always new consumables (e.g. solvents, filters and gases) are used, for

example, method development is never started, on a HPLC column that has been used

earlier. The analyte standard in a suitable injection / introduction solution and in known

concentrations and solvents are prepared. It is important to start with an authentic,

known standard rather than with a complex sample matrix. If the sample is extremely

close to the standard (e.g., bulk drug), then it is possible to start work with the actual

sample. Analysis is done using analytical conditions described in the existing literature.

6. Optimization

During optimization one parameter is changed at a time, and set of conditions are

isolated, rather than using a trial and error approach. Work has been done from an

organized methodical plan, and every step is documented (in a lab notebook) in case of

dead ends.

5 INTRODUCTION

7. Documentation of analytical figures of merit

The originally determined analytical figures of merit limit of quantitation (LOQ), Limit

of detection (LOD), linearity, time per analysis, cost, sample preparation etc., are

documented.

8. Evaluation of method development with actual samples

The sample solution should lead to unequivocal, absolute identification of the analyte

peak of interest apart from all other matrix components.

9. Determination of percent recovery of actual sample and demonstration of

quantitative sample analysis:

Percent recovery of spiked, authentic standard analyte into a sample matrix that is shown

to contain no analyte is determined. Reproducibility of recovery (average +/- standard

deviation) from sample to sample and whether recovery has been optimized has been

shown. It is not necessary to obtain 100% recovery as long as the results are reproducible

and known with a high degree of certainty. The validity of analytical method can be

verified only by laboratory studies. Therefore documentation of the successful

completion of such studies is a basic requirement for determining whether a method is

suitable for its intended applications.

1.2.2. Introduction to validation

Validation of an analytical method is the process by which it is established, by laboratory

studies, that the performance characteristics of the method meet the requirements for the

intended analytical applications5.

Validation is defined as follows by different agencies:

Food and Drug administration (FDA)

Establishing documentation evidence, which provides a high degree of assurance that

specific process, will consistently produce a product meeting its predetermined

specification and quality attributes.

World Health Organization (WHO)

Action of providing that any procedure, process, equipment, material, activity, or system

actually leads to the expected results.

6 INTRODUCTION

European Committee (EC)

Action of providing in accordance with the principles of good manufacturing practice that

any procedure, process, equipment, material, activity or system actually leads to the

expected results. In brief validation is a key process for effective Quality Assurance6

1.2.3.Reasons For Validation7:

There are two important reasons for validating assays in the pharmaceutical industry. The

first, and by for the most important, is that assay validation is an integral part of the

quality-control system. The second is that current good manufacturing practice regulation

requires assay validation.

Steps followed for validation procedures

1. Proposed protocols or parameters for validations are established.

2. Experimental studies are conducted.

3. Analytical results are evaluated

4. Statistical evaluation is carried out.

5. Report is prepared documenting all the results.

1.3. Objective and Parameters of Analytical Method Validation

The objective of validation of an analytical procedure is to demonstrate that it is suitable

for its intended purpose. According to ICH guidelines, typical analytical performance

characteristics that should be considered in the validation of the types of methods are8.

1. Accuracy

2. Precision

3. Specificity

4. Detection Limit

5. Quantitation Limit

6. Linearity

7. Range.

8. Ruggedness

9. Robustness

7 INTRODUCTION

1) Accuracy

The accuracy is the closeness of the measured value to the true value for the sample.

The ICH documents recommended that accuracy should be assessed using a minimum of

nine determinations over a minimum of three concentrations levels the specified range (

i.e, three concentrations and three replicates of each concentration)

Accuracy was tested (% Recovery and % RSD of individual measurements) by analyzing

samples at least in triplicate, at each level (80,100 and 120 % of label claim) is

recommended. For each determination fresh samples were prepared and assay value is

calculated. Recovery was calculated from slope and y intercept of the calibration curve

obtained in linearity study. Accuracy was determined from the mean relative error for a

set of replicate analysis (i.e. the difference between measured and nominal concentration)

for spiked samples.

2) Precision

The precision of an analytical procedure expresses the closeness of agreement between

series of measurements obtained from multiple sampling of the same homogenous

sample under the prescribed conditions. Precision of an analytical method is usually

expressed as the standard deviation, realative standard deviation or coefficient of

variation of a series of measurement. The ICH documents recommend the repeatability

should be assessed using a minimum of nine determinations covering specified range of

procedure. Precision may be measure of either the degree of reproducibility or of

repeatability of the analytical method under normal operating conditions.

Repeatability: Repeatability expresses the precision under the same operating conditions

over a short interval of time. Repeatability is also termed intra –assay precision.

Intermediate Prescision: Intermediate precison expresses with in laboratories variations:

different days, different analyst, different equipment.

Reproduciblity: When the procedure is carried out by different analyst in different

laboratories using different equipment, regents and laboratories setting. Reproducibility

was determined by measuring repeatability and intermediate precision. Reproducibility is

assessed by means of an interlaboratory trial.

8 INTRODUCTION

Fig No.1.1: Difference between Accuracy and Precision illustrated by using target

diagram

3) Specificity

An investigation of specificity should be conducted during the validation of identification

tests, the determination of impurities.ICH documents defines specificity as the ability to

assess unequivocally the analyte in the presence compounds that may be expected to

products and matrix components.

Examples of the extraneous peaks are as followings:

For the drug substance or raw material, the related substances to consider are process

impurities (which include isomeric impurities) from the synthetic process, residual

pesticide, solvents and other extraneous components from extracts of natural orgin.

For the drug product, the related substance may be impurities present in an active drug,

degradation product, interaction of drug with the expients, extraneous components e.g

residual solvents from the expients or manufacturing process, leachable or extractables

from the container and closure system or from the manufacturing process.

The definition has the following implications:

Identification test: Suitable identification tests should be able to discriminate

compounds of closely related structure which are likely to be present .Ensure identity of

an analyte. The analyte should have no interference from other extraneous components

and be well resolved from them.

9 INTRODUCTION

Purity Test: To ensure that all the analytical procedures performed allow an accurate

statement of the content of impurity of the content of impurity of an analyte i.e related

substances test, heavy metals, residual solvents etc.

Assay: To provide an exact result this allows an accurate statement on the content or

potency of the analyte in a sample.

4) Detection Limit (LOD)

It is the lowest amount of analyte in a sample that can be deceted, but not necessarily

quantities as an exact value, under the stated experimental conditions. The detection limit

is usually expressed as the concentration of analyte (Percentage parts per million) in the

sample. The quantitative limit is generally determined by the analysis of samples with

known concentration of analyte and by establishing the minimum level can be accuracy

and precision.

Based on Signal-to-Noise Approach: This approach can only be applied to analytical

procedures that exhibits baseline noise. Determination of the signal-to-noise ratio is

performed by compairing measured signals from samples with known low concentration

of analyte with those of blank samples and by establishing the minimum concentration at

which the analyte can be really quantified. A typical signal-to-noise ratio is 10:1.

Based on the Standard deviation of the response and the slope:

LOD = 3 * SD / slope of calibration curve

SD = Standard deviation of intercepts

5) Limit of quantification (LOQ)

It is the lowest amount of analyte in a samples that can be determined with acceptable

precision and accuracy under the stated experimental conditions. Quantification limit is

expressed as the concentration of analyte (e.g- % ppm) in the sample.

LOQ = 10 * SD / slope of calibration curve

SD = Standard deviation of intercepts

Based on S.D of the blank: Measurement of the magnitude of analytical background

response is performed by analyzing an appropriate number of blank samples and

calculating the standard deviation of these responses.

10 INTRODUCTION

Based on the calibration curve: A specific calibration curve should be studied using

samples, containing an analyte in the range of QL. The residuals S.D of regression line or

the S.D of intercepts of regression lines, may be used as the S.D. The quantitative limit is

a parameter of quantitative assay for low levels of compounds in sample matrics, and is

use particularly for the determination of impurities or degradation products.

6) Linearity and Range

The linearity of an analytical procedure is its ability to obtain test results which are

directly proportional to concentration of the analyte in the sample. The range of an

analytical is the intervals between the upper and lower concentration (amounts) of analyte

in the sample for which it has been demonstrated which it has been demonstrated that the

analytical procedure has a suitable level of precision accuracy and linearity. For

determination of linearity standard were tested six times. A calibration curve was

constructed and the proposed method was evaluated by its correlation coefficient and

intercept value, calculated in the corresponding statistical study (ANOVA). Calibration

plots were constructed for drug and related substances

in either standard solution or synthetic mixtures of drug product components by plotting

the concentration of compounds versus peak area response using least square method.

The ICH recommends that for the establishment of linearity a minimum of five

concentrations normally used.

7) Ruggedness

Degree of reproducibility of test results obtained by the analysis of the same samples

under a variety of condition such as different laboratories, different analysts, different

instruments etc, normally expressed as the lack of influence on test results of operational

and environmental variable of the analytical method. Ruggedness is a measurement of

reproducibility of test results under the variation in condition normally expected from

laboratory to laboratory and from analyst to analyst. Degree of represenatative of test

results is then determined as a function of the assay variable. By analysis of aliquots from

homogenous lots in different laboratories, by different analyst, using operational and

environmental conditions that may differs but are still with in the specified parameter of

the assay variable.

11 INTRODUCTION

8) Robustness

Robustness of an analytical method is measure of its capacity to remain unaffected

small but deleribate variations in method parameters and provides an indication of its

reliability during normal usage.

Testing varing some or all condition:

Age of column

Column temperature

PH of buffer in mobile phase

Reagents and flow rate

9) System Suitability

System suitability tests are based on the concept that the equipment, electronics,

analytical operations and samples constitute an integral system that can be evaluated as a

whole. According to USP system suitability are an integral part of chromatographic

methods. These tests verify that the resolution and reproducibility of the system are

adequate for the analysis to be performed. One consequence of the evaluation of

robustness and ruggedness should be that a series of system suitability parameters is

established to ensure that the validity of the analytical method is maintained whenever

used. The parameters which are recommended by International Committee of

harmonization to be validated for different types of assays are shown in Table No. 1.2.

12 INTRODUCTION

Table No.1.2:

Validation Parameters Recommended by

International Committee on Harmonization (ICH)8

ASSAY TYPE VALIDATIONS

Identification tests are intended to ensure the identity of an

analyte in a sample. This is normally achieved by

comparison of a property of the sample to that of a

reference standard.

Specificity

Impurities quantitations are intended to accurately reflect

the purity characteristic of the sample. Different validation

characteristics are required for a quantitative test than for a

limit test.

Accuracy

Precision

Specificity

Detection limit

Quantitation

limit

Linearity

Range

Impurities Limit are intended to reflect the purity

characteristics of the sample.

Specificity

Detection limit

Content / Potency, Dissolution are intended to measure the

analyte present in a given sample. A quantitative

measurement of the major component (s) in the drug

substance.

Accuracy

Precision

Specificity

Linearity

Range

13 INTRODUCTION

Table No– 1.3

Validation Characteristics Versus Type of Analytical Procedures Test of

Impurities9:

Type

of Procedure Identification Quantitation Limit

Dissolution

Measurement

(Content /

Potency)

Accuracy No Yes No Yes

Precision

/ Repeatability

No Yes No Yes

Intermediate

Precision

No Yesa No Yes

a

Specificity Yes Yes Yes Yes

Detection Limit No Nob Yes No

Quantitation

Limit

No Yes No No

Linearity No Yes No Yes

Range No Yes No Yes

a. When reproducibility is performed, intermediate precision is not

needed.

b. May be needed in some cases.

The comparison of different official guidelines in case of parameters required to be

validated for different assays is shown in Table – 1.3

14 INTRODUCTION

Table – 1.4

Comparative Table Representing FDA, USP and

ICH Requirements

Criteria GMP FDA USP ICH

Accuracy x X x x

Reproducibility x x

Sensitivity x

Specificity x X x x

Linearity X x x

Precision X x x

Detection Limit x x

Quantitation Limit x x

Range x x

Recovery X

Ruggedness X x

1.3.1. Benefits Of Method Validation7:

A fully validated process may require less in-process control and end –product testing.

It deepens the understanding of processes, decrease the risks of processing problems, and

thus assure the smooth running of the process.

Quality: Customer – patient satisfaction. It has been built into the product.

1.3.2. Cost Reduction

Increased efficiency, shortening lead time resulting in lower inventories.

Fewer rejects and reworks.

15 INTRODUCTION

Longer equipment life by operating the equipment as per manufacturer’s

specifications and the establishing of cost effective preventive maintenance

schedules.

Possible reduced testing of raw materials bulk formulations and finished

products.

1.3.3. STATISTICAL ANALYSIS10

:

Statistical procedures and representative calculations:

The consistency and suitability of the developed method are substantiated through the

statistical analysis like standard deviation, relative standard deviation and theoretical

plates per meter.

For Accuracy: Standard deviation = =1

)xx(2

i

n

Where, x = sample, xi = mean value of samples, n = number of samples

Relative Standard Deviation = /xi × 100

Molar extinction coefficient (mol-1

cm-1

) = A/C × L

Where, A= Absorbance of drug, C= concentration of drug, L= Path length

Sandell, s sensitivity (µg/cm2/0.001 absorbance units) = C/A×0.001

Where, C= concentration of drug, A= Absorbance of drug

16 INTRODUCTION

1.5. HPLC system is composed of several components. These components are11

:

o Sample delivery system, which includes pump, associated pressure, and flow

controls and filters on the inlet side.

o Sample injection function.

o The column.

o The detector

o Data processor.

o Recorder.6

Fig. No.1.5: Diagram of a Typical HPLC

17 INTRODUCTION

1.4.1. HPLC method validation12

Everyday many chromatographers face the need to develop a HPLC separation whereas

individual approaches may exhibit considerable diversity; method development often

follows the series of steps summarized in the following fig.

Fig 1.6: - Steps involved in HPLC method validation

1. Introduction on sample

Define separation goals

2. Need for special HPLC

Procedure, sample, pretreatment, etc

3. Choose detector and

Detector settings

4. Choose LC method;

Preliminary run; estimate

best separation conditions

5. Optimize separation

condition

6. Check for problems or requirements

for special procedure

7a. Recover

Purified material

7c. Qualitative

Method

7b.Quantitative

Calibration

8. Validation method for release to

routine laboratory

18 INTRODUCTION

1.4.2. Selection of Column:

The HPLC column is the heart of the method, critical performing the separation. The

column must possess the selectivity, efficiency and reproducibility to provide good

separation. Commonly used reversed phase columns are C18 (octadecyl silane,), C8 (octyl

silane,) phenyl and cyano. They are chemically different bonded phases and demonstrate

significant changes in the selectivity using the same mobile phase. During method

development selection of column can be streamlined by starting with shorter columns

(150,100 or even 50mm long.). By selecting a shorter column with an appropriate phase

run time can be minimized so that an elution order and an optimum mobile phase can be

quickly determined. It is also advantageous to consider the column internal diameter,

many laboratories use 4.6mm ID as standard, but it is worth considering use of 4mm ID

column as an alternative. This requires only 75% of the solvent flow than that of 4.6mm

column.Selecting an appropriate stationary phase can also help to improve the efficiency

of method development. For example, a octyl phase (C8) can provide time saving over a

octadecyl (C18) as it doesn’t retain analytes as strongly as the C18 phase. For normal phase

applications Cyano phases are the most versatile. C18 (250*4.6mm) column are more

often used in the laboratory. These columns are able to resolve a wide variety of

compounds due to their selectivity and high plate counts.

1.4.3. Role of Flow rate

Flow rate, more for isocratic than gradient separation, can sometimes be useful and

readily utilized to increase the resolution, although its effect is very modest. The slower

flow rate will also decrease the column back pressure. The disadvantage is that when

flow rate is decreased, to increase the resolution slightly, there is a corresponding

increase in the run time. Method development involves considerable trial and error

procedures. Optimization can be started only after a reasonable chromatogram has been

obtained. A reasonable chromatogram means that all the compounds are detected by

more or less symmetrical peaks on the chromatogram.

19 INTRODUCTION

The parameters that are affected by the changes in chromatographic conditions are,

Capacity factor (k’),

Selectivity (),

Column efficiency (N) and

Peak asymmetry factor (As).

1. Capacity factor (k')

k' = (t R- t0) / t 0

The capacity factor is a measure of the degree of retention of an analyte relative to an

unretained peak, where tR is the retention time for the sample peak and to is the retention

time for an unretained peak.

Recommendations:

The peak should be well-resolved from other peaks and the void volume. Generally the

value of k' is > 2.

2. Backpressure

The pressure required to pump the mobile phase through the column. It is related to

mobile phase viscosity (η), flow rate (F), column length (L), and diameter (dc), and

particle size (dp) by the following equation:

ΔP α FLη / dp2

dc2

3. Resolution (Rs)

Ability of a column to separate chromatographic peaks, It can also be expressed in terms

of the separation of the apex of two peaks divided by the tangential width average of the

peaks.

Rs = ΔtR / W1/2 + W2/2;

Where ΔtR = t2 – t1

For reliable quantitation, well-separated peaks are essential for quantitation.

20 INTRODUCTION

Resolution can be improved by increasing column length, decreasing particle size,

increasing temperature, changing the eluent or stationary phase

Recommendations:

Rs > 2 between the peak of interest and the closest potential interfering peak (impurity,

excipient, degradation product, internal standard, etc.) are desirable.

4. Tailing factor (T)

A measure of the symmetry of a peak, given by the following equation where W0.05 is the

peak width at 5% height and f is the distance from peak front to apex point at 5% height.

Ideally, peaks should be Gaussian in shape or totally symmetrical.

T = W0.05 / 2f

The accuracy of quantitation decreases with increase in peak tailing because of the

difficulties encountered by the integrator in determining where/when the peak ends and

hence the calculation of the area under the peak. Integrator variables are preset by the

analyst for optimum calculation of the area for the peak of interest.

Recommendations:

T of ≤ 2

5. Theoretical plate number / Efficiency (N)

Theoretical plate number is a measure of column efficiency, that is, how many peaks can

be located per unit run-time of the chromatogram. A measure of peak band spreading

determined by various methods, some of which are sensitive to peak asymmetry. The

most common are shown here, with the ones most sensitive to peak shape shown first:

4-sigma / tangential: N = 16 (tR / W) 2

= L / H

Half height: N = 5.54 (tR / W) 2 = L / H

Where tR is the retention time for the sample peak and W is the peak width.

21 INTRODUCTION

N is fairly constant for each peak on a chromatogram with a fixed set of operating

conditions. H, or HETP, the height equivalent of a theoretical plate, measures the column

efficiency per unit length (L) of the column. Parameters which can affect N or H include

Peak position, particle size in column, flow-rate of mobile phase, column temperature,

viscosity of mobile phase, and molecular weight of the analyte

Recommendations:

The theoretical plate number depends on elution time but in general should be > 2000.

General Recommendation:

The amount of testing required will depend on the purpose of the test method. For

dissolution or release profile test methods using an external standard method, k', T and

RSD are minimum recommended system suitability tests. For acceptance, release,

stability, or impurities/degradation methods using external or internal standards, k', T, Rs

and RSD are recommended as minimum system suitability testing parameters. System

suitability testing is essential for the assurance of the quality performance of the

chromatographic system13

.

22 INTRODUCTION

1.5 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) (14-

19) :

1.5.1. INTRODUCTION:

The term “thin layer chromatography” was coined by Egon Stahl in Germany in the late

1950’s. Stahl’s greatest contribution to the field was standardization of materials,

procedures and nomenclature and description of selective solvent systems for the

resolution of many important compound classes. In present day HPTLC is without doubt

one of the most versatile and widely used separation methods in chromatography.

Commercially, many sorbents on a variety of backings and mobile phases are now

available and allows the handling of a large number of samples in one chromatographic

run. It has found use in a wide range of application areas as the concept of HPTLC is so

simple and samples usually require only minimal pretreatment, detecting components at

low nanogram sensitivities. HPTLC technique is much more reliable and reproducible

method for the standardization of both single and compound herbal and allopathic

formulations. HPTLC is a separation technique based on a stationary phase and a liquid

mobile phase. Separations are achieved by adsorption. The stationary phase is a flat thin

layer coated onto an inert support (glass, aluminum or polyester). In its simplest form,

HPTLC costs little, but even including the more sophisticated instrumentation, it still

remains less expensive per sample analysis than HPLC. The modern HPTLC has seen a

strong move in the direction of plate scanning and video imaging as a means of providing

sensitive and reliably accurate results and a more permanent record of chromatogram.

The HPTLC is the improved method of TLC which utilizes the conventional technique of

TLC in more optimized way. It is also known as planar chromatography or Flat-bed

chromatography.

Features of HPTLC

1. Simultaneous processing of sample and standard - better analytical precision and

accuracy less need for Internal Standard

2. Several analysts work simultaneously

3. Lower analysis time and less cost per analysis

4. Low maintenance cost

5. Simple sample preparation - handle samples of divergent nature

23 INTRODUCTION

6. No prior treatment for solvents like filtration and degassing

7. Low mobile phase consumption per sample

8. No interference from previous analysis - fresh stationary and mobile phases for

each analysis - no contamination

9. Visual detection possible - open system

10. Non UV absorbing compounds detected by post-chromatographic derivatization

Various steps involved in HPTLC:

1. Selection of HPTLC plates and adsorbent

2. Sample preparation

3. Application of sample

4. Development

5. Detection and post-chromatographic derivatisation

6. Quantification

7. Documentation

24 INTRODUCTION

Fig. No.1.7: Schematic procedure for HPTLC

Validation method for release to

routine laboratory

25 INTRODUCTION

1.5.2. Selection of HPTLC plates:

Pre coated plates:

The plates are used which have different support materials and sorbent layers with

different format and thickness.

HPTLC Plates:

HPTLC grew out of improvements in the quality of sorbents and consistency of plate

manufacture. HPTLC plates are characterized by smaller particles (<10 μm), thinner

layers (<150 μm) and smaller plates (<10 cm developing distance). Also, the particle size

distribution of the sorbent is narrower than for conventional TLC layers. HPTLC plates

provide more resolving power per unit distance, develop faster and consume less solvent.

However, sample diffusion in the direction of migration must be kept to a minimum to

prevent band broadening, and the thin sorbent layer dictates sample volume in HPTLC be

kept to a maximum of 1 µL per stroke.

Supports:

Table 1.8: List of supports materials for HPTLC plate

Materials Advantage Disadvantage

Glass 1. resistant to heat and chemicals.

2. easy to handle and offers superior

flat surface for work.

1. fragile.

2. costs more for

additional packaging.

Polyester sheets

(0.2 mm thick)

1. more economical as produced

even in roll forms.

2. unbreakable.

3. less packing material.

4. spots can be cut and eluted thus

eliminates dust from scrapping

1. charring reactions if

temperature exceeds

120oC as the plates are

dimensionally unstable

beyond this temperature.

Aluminum Sheets

(0.1 mm)

1. increasesed temperature

resistance.

1. eluents containing high

concentration of mineral

acids or ammonia can

attack chemically on

aluminum.

26 INTRODUCTION

Plate size:

10X20 cm, 10X10 cm, 5X10 cm and 5X7.5 cm

The plates should have good cut edges of sheets to obtain constant Rf values if not flaking

off the layer on one or both sides of cut edges which leads for capillary cracks between

chromatographic layer and the foil in which mobile phase will travel much more rapidly

forward than it does in the center of chromatogram. The cracks causes mobile phase to

migrate from edges of the layer to the center thus causing deformation of zones and

distortion of tracks.

Pre-washing of pre-coated plates:

Pre-washing is required because sorbents with large surface area absorb not only water

vapors and other impurities from atmosphere but also other volatile substances which

often condense particularly after the packing has been opened and exposed to laboratory

atmosphere for a long time. Such impurities include elutable components of the binder

usually give dirty zones and fail to give reproducible results. So all the times pre-coated

plates are always packed with glass or aluminum foil side upward.

Some common methods involved in pre-washing:

a) Ascending method: In this technique the chromatographic plates are run blank

(i.e. before application of the sample) with suitable solvent / mobile phase. The

solvent/mobile phase carries the impurities to the top of the plate. It takes longer

time but cleaning effect is superior. The disadvantage of this technique is active

dirt gets accumulated at the solvent front.

b) Dipping method: In this technique, the chromatographic plate is dipped in a

suitable solvent for specified period of time, removed from the chamber and finally

dried. Dipping method is quicker and yields uniform layer but cleaning effect is

often not as good as Ascending method.

c) Continuous method: In this technique, the plate to be washed is placed in

chamber having an entrance and exit slits. The solvent is made to flow

continuously through the chamber that carries the impurities from the plate. The

wanted plates should always be stored in a dust free atmosphere, under ambient

conditions. Usually desiccators of suitable size are used for storage of plates.

27 INTRODUCTION

Solvents used for pre-washing:

a. Methanol

b. Chloroform: methanol

c. Chloroform: Methanol: Ammonia

d. Methylene chloride: Methanol

e. Ammonia solution

The plates are then dried for 20 minutes in a clean drying oven at 120°C. The plates are

then removed and equilibrated with lab atmosphere (temperature, relative humidity) in a

suitable container providing protection from dust and fumes. Further then the plates are

handled either on both side edges or on the top edge. The pre-washed plates are kept in

desiccators without applying grease for sealing of edges.

1.5.4. Sample Preparation:

1. Sampling, mechanical crushing, extraction, filtration and enrichment of minor

compounds are the several steps for sample preparation.

2. Sample and reference substances should be dissolved in the same solvent to ensure

comparable distribution at starting zones.

Application of sample:

The selection of a sample application technique and the device to be used depends

primarily on:

Sample volume

Number of samples to be applied

Required precision and degree of automation.

In order to maximize the separation, it is important to restrict the size of the sample origin

in the direction of chromatography to a minimum. The maximum sample volume that can

be applied spot wise (in one stroke) are 5 µL L on conventional layers and 1 µL L on

HPTLC layers. Spot wise application of larger volumes requires a device with

controllable delivery speed.

Sample application in the form of bands or rectangles:

Spraying-on samples as narrow bands allows the application of larger volumes. Narrow

bands as starting zones always ensure the highest resolution. Very large sample volumes

28 INTRODUCTION

or samples with high matrix content can be sprayed-on in the form of rectangles. All

types of Thin-Layer Chromatography, whether qualitative, quantitative or preparative,

benefit from optimized resolution as a result of the appropriate sample application

technique. Sample application is the first step of instrumental Thin-Layer

Chromatography and thus determines the quality of the analysis. For application, the

sample should be dissolved in the solvent of lowest suitable solvent strength in order to

minimize the effects of spot broadening during application. For application by spraying,

the speed has to be adjusted to them properties of chosen solvent. In order to avoid

volume errors highly volatile solvents should not be used to prepare and apply standards

and samples for quantitative analyses. Samples are applied as bands by spray-on

technique or as spots using the following parameters:

Table 1.9: Parameters for HPTLC

Parameters HPTLC

Distance from lower edge of plate in for TLC 8 mm

Minimum distance from left and right edge of plate 10 mm

Minimum space in mm between bands / spots 4 mm

Band length 8 mm

Maximum diameter of application spot 5 mm

1.5.5. Chromatogram Development: Thin-layer chromatography differs from all other

chromatographic techniques in the fact that in addition to stationary and mobile phase a

gas phase is present. This gas phase can significantly influence the result of the

separation.

Processes in the Developing Chamber:

The classical way of developing a chromatogram is to place the plate in a chamber, which

contains a sufficient amount of developing solvent. The lower end of the plate should be

immersed several millimeters. Driven by capillary action the developing solvent moves

up the layer until the desired running distance is reached and chromatography is stopped.

The following considerations primarily concern silica gel as stationary phase, which can

29 INTRODUCTION

be described as adsorption chromatography. The process occurring within the

development chamber is as follows:

a. Between the components of the developing solvent and their vapor, equilibrium is

established eventually. This equilibrium is called chamber saturation. Depending

on the vapor pressure of the individual components the composition of the gas

phase can differ significantly from that of the developing solvent.

b. While still dry, the stationary phase adsorbs molecules from the gas phase. This

process of adsorptive saturation is also approaching an equilibrium in which the

polar components will be withdrawn from the gas phase and loaded onto the

surface of the stationary phase.

c. Simultaneously a part of the layer which is already wetted with mobile phase

interacts with the gas phase. Thereby especially the less polar components of the

liquid are released into in the gas phase. This process is not as much governed by

vapor pressure as by adsorption forces.

d. During migration, the components of the mobile phase can be separated by the

stationary phase under certain conditions, causing the formation of secondary

fronts.

The problems occurring due to this can be avoided by keeping increasing the chamber

saturation time and this can be achieved by as follows: keeping the chamber more or less

completely with filter paper soaked with developing solvent, waiting a certain time

between the introduction of developing solvent into the chamber and beginning of

chromatography. The advantages of chamber saturation is that the, components of the

developing solvent, which have been loaded onto the dry layer via the gas phase, the gas

phase are pushed ahead of the mobile phase with invisible solvent front. The exceptions

are very polar components such as water, methanol, acids, or bases. This results in Rf

values being lower in saturated chambers and particularly on pre-conditioned layers, than

in unsaturated chamber. The consequences of not allowing chamber saturation proceeds

in non-equilibrium between stationary, mobile, and gas phase. For this reason it is very

difficult to describe the conditions in a developing chamber. Reproducible

chromatographic results can only be expected when all parameters are kept as constant as

30 INTRODUCTION

possible. Chamber shape and saturation are playing a predominant role in this regard.

Unfortunately this means that the chromatographic result is different in each chamber.

Choosing a developing chamber:

The selection of the proper developing chamber is done during method development and

generally follows practical considerations such as which chamber is available, which one

must be used due to sop, or which one has been used in the past if a results comparison is

to be made. However, a focus should also be on economical aspects such as time

requirement and solvent consumption. A selection of glass chambers can be done by as

follows:

The Horizontal Developing Chambers: It is economical, flexible and reproducible

in operation. Although designed for applications where the plate is developed from two

sides, they are also suitable for single-sided developments in unsaturated, saturated and

sandwich configuration as well as for preconditioning of HPTLC plates.

Twin trough chamber: Twin trough chambers offer several ways to improve the

results of TLC/HPTLC developing techniques. The trough is divided into two divisions,

where the mobile phase is added to one division and allowed for the plate saturation. This

reduces the time for chamber saturation when compared to flat bottom chambers. The

development is started only when developing solvent is introduced into the trough with

the plate. The main advantage in this is the amount of mobile phase required is not more

than 20 mL for 20X20 cm plate. This not only saves solvent, but also reduces waste

disposal problem.

Automatic Developing Chamber (ADC): In this system, the chromatographic

development can be fully automated and can control the chamber saturation, pre-

conditioning of the layer and final drying. This instrument not only eliminate any effects

of the operator when introducing the plate into a saturated chamber, but also the activity

of the layer prior to start of chromatography can be set and drying of the

chromatographed plate is rapid and complete. The main advantage in this system is that

one can control the humidity required for the development of chromatographic plate.

Automated Multiple Development (AMD): This is used In case the sample contains

polar and non-polar components, which must be separated in the same analysis.

31 INTRODUCTION

Development is performed on the basis of a solvent gradient from polar to non-polar over

several steps with intermediate drying.

1.5.6. Mobile phase (M P):

The Mobile phase should be of high grade,

1. Chemical properties and analytes and sorbent layer factors should be considered

while selection of mobile phase.

2. Use of mobile phase containing more than three or four components should

normally be avoided as it is often difficult to get reproducible ratios of different to

get reproducible ratios of different components.

Properties of Mobile phase:

1. M P optimization is necessary while performing HPTLC.

2. Various components of M P should be measured separately and then placed in mixing

vessel. This prevents contamination of solvents and also error arising from volumes

expansion or contraction on mixing.

3. Trough chambers are used in which smaller volumes of M P usually 10-15 mL is

required. Usually volumetric pipettes are used.

4. Different components of M P are mixed first in mixing vessel and then transferred to

developing chambers.

5. Chambers containing multi component M P are not generally used for re-use for any

future development, due to differential evaporation and adsorption by layer and also

once the chamber is opened, solvents evaporate disproportionally depending on their

volatilities.

1.5.7. Post-chromatographic Derivatization:

After the development, it’s an inherent advantage of TLC that fractions remain stored on

the plate and can be derivatized after chromatography. By derivatization substances that

do not respond to visible or UV light can be rendered detectable. In many cases,

substances or classes of substances can be identified by specific reagents. Derivatization

can be achieved with gas, by liquid spraying or dipping (immersion). In any case the

reagent needs to be homogenously transferred to the chromatogram. By immersing a

TLC plate into the derivatizing reagent a very homogenous reagent transfer can be

achieved. Dipping and withdrawing has to be performed smoothly in order to avoid

32 INTRODUCTION

tidemarks. Using the suitable Chromatogram Immersion Device the reproducibility of the

derivatization step can be significantly improved when compared to spraying.

Furthermore, no fumes are generated during this derivatization technique and the

exposure to hazardous chemicals is limited. If the reagent is suitable, dipping should be

preferred over spraying. However, the fact is that spraying is most widely used for

reagent transfer onto the TLC plate because it is simple and quick. No expensive

equipment is necessary and only small volumes of reagent are needed. In addition

spraying is very flexible and indispensable when reagents have to be applied in sequence.

Also during method development, when searching for the most suitable reagent, spraying

is more frequently mentioned. The two principal heating devices are ovens and plate

heaters.

Chromatogram evaluation with classical densitometry or electronic image

acquisition: Quantitative evaluation of a TLC/HPTLC plate is always performed

densitometrically, either in absorption or fluorescence mode. The signal of each

substance zone is compared to the substance free plate background. For calibration and

result calculation the obtained peak data of the unknowns are compared against data

obtained for standards on the same plate. Quantitative evaluation can be performed with

data from classical densitometry or with those from electronic image acquisition.

Classical densitometry uses monochromatic light and a slit of selectable length and width

to scan the tracks of a chromatogram, measuring the diffusely reflected light. The latest

available instrument uses the entire spectral range from 190 to 800 nm with high spectral

selectivity for data acquisition. Absorption spectra for substance identification and for

selection of the most suitable measurement wavelength can be recorded within this range.

The strengths of classical densitometry are the spectral resolution of the light source and

the higher reproducibility of quantitative determinations. Currently electronic image

acquisition works only in the visible range. The UV region – exceptionally useful for

Thin-Layer Chromatography – is only indirectly accessible to image acquisition. In this

respect this technology exactly parallels the human eye.

The advantages of spectral selectivity are as follows: Spectral selectivity is only

accessible through the classical densitometer. UV-absorbing substances can be detected

by image acquisition technology. The quenching of a fluorescence indicator embedded

33 INTRODUCTION

in the layer, i.e. detection is shifted to the visible region. The more a substance to be

quantified absorbs at or near the excitation maximum of the fluorescence indicator (254

nm), the higher is sensitivity and accuracy of image processing but the lower the

absorbance at 254 nm, the less sensitive and less accurate it becomes. Recording of UV

spectra with their information about absorption maxima, identity and purity, is only

possible with classical densitometry.

Electronic image acquisition uses polychromatic light (white light, UV 254 or UV 366)

to illuminate the entire object and to capture an electronic image with a digital camera.

For the documentation of Thin-Layer Chromatograms, electronic image acquisition has

essentially replaced photography. Electronic images can easily be archived and can be

retrieved at any time, for review or to perform a quantitative evaluation. The strength of

electronic image acquisition is the view of the complete image of the chromatogram. This

possibility to get a visual impression of the chromatogram is one of the principal

advantages of Thin-Layer Chromatography over all other chromatographic techniques.

1.5.8. Applications of HPTLC:

1. Pharmaceutical industry: Quality control, content uniformity, uniformity test,

identity/purity check.

2. Food Analysis: Quality control, additives, pesticides, stability testing, analysis of

sub-micron levels of toxins etc

3. Clinical Applications: Metabolism studies, drug screening, stability testing etc

4. Industrial Applications: Process development and optimization, In-process check,

validation etc.

5. Forensic: Poisoning investigations.

34 INTRODUCTION

1.6 DRUG PROFILE (20-25)

1.6.1. PIOGLITAZONE ( PIO )

Structural formula

IUPAC name : 5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-

dione

Formula: C19H20N2O3S

Mol.Weight: 356.4390 g mol -1

Appearance: A crystalline solid

Approved Name: Pioglitazone

Category: Antidiabetic agent, Hypoglycemic Agents

Mechanism of action: Pioglitazone acts as an agonist at peroxisome proliferator activated

receptors (PPAR) in target tissues for insulin action such as adipose tissue, skeletal

muscle, and liver. Activation of PPAR-gamma receptors regulates the transcription of

insulin-responsive genes involved in the control of glucose production, transport, and

utilization. In this way, pioglitazone enhances tissue sensitivity to insulin.

Half life: 3-7 hour

Uses: Treatment of Type II diabetes mellitus

35 INTRODUCTION

1.6.2. ROSIGLITAZONE ( ROS)

Structural formula

IUPAC name: 5-[[4-[2-(methyl-pyridin-2-ylamino)ethoxy]phenyl]methyl]-1,3-

thiazolidine-2,4-dione

Formula: C18H19N3O3S

Mol. Weight: 357.42 g mol -1

Appearance: A crystalline solid

Approved Name: Rosigliazone maleate, Rosiglitazone maleate, Rosiglitazone

Category: Antidiabetic agent ,Hypoglycemic Agent

Mechanism of action: Rosiglitazone acts as an agonist at peroxisome proliferator

activated receptors (PPAR) in target tissues for insulin action such as adipose tissue,

skeletal muscle, and liver. Activation of PPAR-gamma receptors regulates the

transcription of insulin-responsive genes involved in the control of glucose production,

transport, and utilization. In this way, rosiglitazone enhances tissue sensitivity to insulin

Half life: 3-4 hours

Uses: For the treatment of Type II diabetes mellitus

36 INTRODUCTION

1.6.3. METFORMIN ( MET)

Structural formula

IUPAC name: 3-(diaminomethylidene)-1,1-dimethylguanidine

Formula: C4H11N5

Mol. weight: 129.16 g mol -1

Approved Name: Metformin hydrochloride

Appearance: A crystalline solid

Category: Antidiabetic agent , Hypoglycemic Agent

Mechanism of action: Metformin's pharmacologic mechanisms of action are different

from other classes of oral antihyperglycemic agents. Metformin decreases hepatic

glucose production, decreases intestinal absorption of glucose, and improves insulin

sensitivity by increasing peripheral glucose uptake and utilization

Half life: 6.2 hours

Uses: Metformin is an antihyperglycemic agent, which improves glucose tolerance in

patients with type 2 diabetes, lowering both basal and postprandial plasma glucose.

Metformin is not chemically or pharmacologically related to any other classes of oral

antihyperglycemic agents.

37 INTRODUCTION

1.6.4. GLIMEPIRIDE ( GLP )

Structural formula

Systematic(IUPAC)name:

3-ethyl-4-methyl-N-[2-[4[(4methylcyclohexyl)carbamoylsulfamoyl]phenyl]ethyl]-

2-oxo-5H-pyrrole-1-carboxamide

Formula: C24H34N4O5S

Mol. weight: 490.61 g mol -1

Approved Name: Glimepride

Appearance: A crystalline solid

Category: Antidiabetic agent , Hypoglycemic Agent

Mechanism of action: The mechanism of action of glimepiride in lowering blood glucose

appears to be dependent on stimulating the release of insulin from functioning pancreatic

beta cells, and increasing sensitivity of peripheral tissues to insulin. Glimepiride likely

binds to ATP-sensitive potassium channel receptors on the pancreatic cell surface,

reducing potassium conductance and causing depolarization of the membrane. Membrane

depolarization stimulates calcium ion influx through voltage-sensitive calcium channels.

This increase in intracellular calcium ion concentration induces the secretion of insulin.

Half life: 5 hours

Uses: Glimepiride is used with diet to lower blood glucose by increasing the secretion of

insulin from pancreas and increasing the sensitivity of peripheral tissue for concomitant

use with insulin for the treatment of noninsulin-dependent (type 2) diabetes mellitus..

Glimepiride, like glyburide and glipizide, is a "second-generation" sulfonylurea agents.

38 INTRODUCTION

1.6.5. REPGLINIDE ( REP )

Structural formula

Systematic(IUPAC)name:

2-ethoxy-4-[2-[[(1S)-3-methyl-1-(2-piperidin-1-ylphenyl)butyl]amino]-2-

oxoethyl]benzoic acid

Formula: C27H36N2O4

Mol. Weight: 452.58 g mol -1

Appearance: A crystalline solid

Approved Name: Repaglinide

Category: Antidiabetic agent, Hypoglycemic Agent

Mechanism of action: Repaglinide closes ATP-dependent potassium channels in the b-

cell membrane by binding at characterizable sites. This potassium channel blockade

depolarizes the b-cell, which leads to an opening of calcium channels. The resulting

increased calcium influx induces insulin secretion. The ion channel mechanism is highly

tissue selective with low affinity for heart and skeletal muscle.

Half life: 3-7 hours

Uses: For the treatment of Type II diabetes mellitus. Repaglinide is an oral blood

glucose-lowering drug of the meglitinide class used in the management of type 2 diabetes

mellitus (also known as non-insulin dependent diabetes mellitus or NIDDM).

39 INTRODUCTION

Repaglinide lowers blood glucose levels by stimulating the release of insulin from the

pancreas. This action is dependent upon functioning beta cells in the pancreatic islets.

Insulin release is glucose-dependent and diminishes at low glucose concentrations.