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International Journal of Analytical and Bioanalytical Chemistry

Universal Research Publications. All rights reserved

ISSN-2231-5012
Review Article
A Review: GC Method Development and validation
Santosh Kumar Bhardwaja,b, K. Dwivedia and D. D. Agarwala
a
School of Studies in Chemistry, Jiwaji University, Gwalior-474011, Madhya Pradesh, India
b
Shimadzu Analytical India Pvt Ltd, Delhi, India.
Email: sbhardwaj81@yahoo.com
Received 16 January 2016; accepted 26 January 2016
Abstract
The intension of this paper is to review and discuss the various steps involved in GC method development and validation.
Gas chromatography is a sensitive, accurate, reproducible, quantitative and versatile tool well adapted for the analysis of
complex mixtures. This techniques plays an important role in analysis of drugs and pharmaceutical products. However the
use of GC is limited to volatile thermally stable compounds or the molecules that may undergo derivatization reactions to
thermally stable products. Method development and validation play important role in the discovery, development and
manufacture of pharmaceuticals. Method development is the process of proving that an analytical method is acceptable for
use to measure the concentration of an API in a specific compound dosage form which allow simplified procedures to be
employed to verify that an analysis procedure, accurately and consistently will deliver a reliable measurement of an active
ingredient in a compound’s preparation. The method validation is essential for analytical method development and tested
extensively for specificity, linearity, accuracy, precision, range, detection limit, quantitation limit, robustness and system
suitability.
© 2016 Universal Research Publications. All rights reserved
Keywords: GC, Method development, Validation

1.0 Introduction: component takes place, and to this must be added the
Gas chromatography is a unique and versatile technique. In source and control of the carrier gas flow through the
its initial stages of development it was applied to the column, a mean of sample introduction and a means of
analysis of gases and vapors from very volatile detection of the components as they elute from the end of
components. The work of Martin and Synge and then the column. Since temperature will influence the
James and Martin in gas–liquid chromatography (GLC) volatility of the analytes, the column is placed in a
opened the door for an analytical technique that has thermostatically controlled oven. 2Unlike most other types
revolutionized chemical separations and analyses. As an of chromatography, the mobile phase does not interact with
analytical tool, GC can be used for the direct separation and molecules of analyte but only transport the analyte through
analysis of gaseous samples, liquid solutions, and volatile the column3. Faster gas chromatographic separation is a
solids. If the sample to be analyzed is nonvolatile, the generally beneficial option. Since the decrease time of
techniques of derivatization or pyrolysis GC can be analysis results in the increased sample throughput and
utilized. Gas chromatography is the analytical technique consequently, the laboratory operation costs can be reduced
used for product identification (under very controlled significantly. Reduction of analysis time can be achieved
conditions) and must be directly coupled to a by changing column parameters (shorter length, smaller
massspectrometer when information other than a column inner diameter, thinner film of stationary phase) or
comparative fingerprint (program) is required, such as operational parameters (faster temperature program rate,
positive identification of peaks on the chromatogram. 1The isothermal analysis. Different carrier gas, higher carrier gas
basic principal of gas chromatography is that greater the flow rate or a combination of both approaches can be
affinity of the compound for the stationary phase, more the applied.4A basic gas chromatograph is represented
compound will be retained by the column and longer it will diagrammatically in figure -1.
be before it is eluted and detected. Thus the heart of the gas 2.0 GC Method Development:
chromatograph is the column in which separation of the Methods are developed for new products when no official
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 Schematic Diagram of Gas Chromatography

Steps involved in GC Method development

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 Gas Chromatographic Method Validation
methods are available. Alternate method for existing (Non- considerations must be given to the number of samples to
Pharmacopoeial) products are to reduce the cost and time be analysed. 8Proper selection of the methods depends upon
for better precision and ruggedness. When alternate method the nature of the sample (volatile or nonvolatile molecule),
proposed is intended to replace the existing procedure its molecular weight, solubility and melting point. 9As
comparative laboratory data including merit/demerits are much as possible informations should be gathered about the
made available. 5Several steps are being considered for GC samples including aggregation state, sample composition
method development like column selection (stationary (analytes, matrix, solvent), information on GC relevant
phase and dimensions: column id, length, and film properties, such as boiling point range, polarity, functional
thickness), carrier gas selection (Nitrogen, Helium, flow groups, solubility, reactivity, stability at room temperature
rate), temperature programing (Initial temperature, initial in the presence of air. It should need to be checked that,
hold, ramp rate, final temperature, and final hold), injector does sample contain thermally or chemically labile
tempreture and detector tempreture.6 components, aggressive substances (acid, bases), water or
Steps involved in Method development are.7 nonvolatile residues, does the sample contain hazardous
 Understanding the Physicochemical properties of substances, any special handling required.10If the sample to
sample. be analyzed is nonvolatile, the techniques of derivatization
 Selection of chromatographic conditions. or pyrolysis GC can be utilized. This latter technique is a
modification where in a nonvolatile sample is pyrolyzed
 Developing the approach of analysis.
before it enters the column.8
 Sample preparation
2.2 Selection of chromatographic conditions
 Method optimization The initial choices of column and supporting
 Method validation (figure-2) instrumentation have a profound influence on the
2.1 Understanding the physicochemical properties of possibilities for, and ultimate results of, separation
sample: optimization. Manipulation of column parameters
Gas chromatography is a unique and versatile technique. In (stationary phase, inner diameter, length, and film
its initial stages of development it was applied to the thickness) gives chromatographers control over column
analysis of gases and vapors from very volatile efficiency, resolution and speed of analysis.1 In gas
components. The mixture to be separated and analyzed by chromatography, the elution order of analytes is governed
GC may be either a gas, a liquid, or a solid in some by several factors such as latent vapour pressures,
instances.1Before beginning the GC method development, solubilities in stationary phase, and propensities for
it is important to review what is known about the sample. molecular interaction in the stationary phase. All of these
The goal of the analysis should be defined at this point and effects change with temperature and their concerted effect

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ultimately determine the equilibrium distribution of solute analysis times are substantially increased. The resolution of
molecules between the mobile and stationary phase.11 peaks eluting in the middle of the chromatogram can be
2.2.1 Selection of Column: altered by change in ramp rate. If there is excessive peak
A column is of course, the starting and central piece of a resolution, the ramp rate can be increased to reduce
chromatograph. An appropriately selected column resolution and the analysis time. If there is insufficient
can produce a good chromatographic separation which resolution, decrease the ramp rate, but there will be an
provides an accurate and reliable analysis. An improperly increase in the analysis time. Better resolution of later
used column can often generate confusion, inadequate, and eluting peaks often occurs when decreasing the ramp rate.
poor separations which can lead to results that are invalid Another option to alter resolution of peaks in the middle of
or complex to interpret. There are over 10,000 compounds a chromatogram is to use a mid-ramp hold. A mid ramp
that can be analysed by GC and over 400 GC capillary hold is a several minute isothermal portion somewhere
columns. It is a challenge for a column manufacturer to during a temperature ramp. Stop the temperature program
give detailed column selection guidelines to meet such a shortly after last peak has eluted from the column. If
wide variety of applications.12An optimized column’s isothermal temperature limit is reached and peaks
chromatographic separation begins with the column. The are still eluting, a final hold time is necessary. Only use a
selection of the proper capillary column for any application final hold time if the temperature limit is reached.12,13
should be based on four significant factors which are 2.2.4 Optimization of Injector type, temperature &
stationary phase, column internal diameter, film thickness, injection volume
and column length. The differences in the chemical and Introduction of the sample into GC system is a critical step
physical properties of injected organic compounds and their in separation. The reproducibility of the amount of sample
interactions with stationary phase are the basis of injected is important to ensure the reproducibility of results.
separation process. When strength of the analyte-phase A sample can be injected manually into the system or by
interactions differs significantly for two compounds, one is using an auto sampler system. A major errors in GC is poor
retained longer than the other. How long they are retained injection technique. The injector temperature for
in the column (retention time) is a measure of these separation. The temperature of injector is used to rapidly
analyte-phase interactions. Changing the chemical features vaporize the liquid sample into gaseous phase that can be
of the stationary phase alters its physical properties. Two carried to the column for separation. In capillary and micro
compounds that co-elute (do not separate) on a particular packed gas chromatography (GC), there are four primary
stationary phase might separate on another phase of techniques for vaporizing a sample and transferring it onto
different chemistry.12 the inlet of the analytical column: split, splitless, direct, and
2.2.2 Selection of Carrier Gas: Several inert gases can be on-column injections. Of these, split and splitless injections
used as the carrier gas or mobile phase of GC. Hydrogen, are the most commonly used techniques. Split Injector was
helium and nitrogen are all common carrier gases. Each selected for analysis of sample with high concentration
carrier gas has its benefits and systems for which it is best levels. In the split injection mode, only a fraction of the
suited.13The choice of gas to be used as mobile phase in gas vaporized sample is transferred onto the head of the
chromatography is influenced by the following column. The remainder of the vaporized sample is removed
requirements and considerations.2 from the injection port via the split vent line. Split
Inertness injections should be used only when sample concentrations
Dryness are high enough to allow a portion of the sample to be
Freedom from oxygen discarded during the injection process, while still
Safety maintaining a sufficient concentration of analytes at the
Cost detector to produce a signal.13,14
Availability 2.2.5 Optimization of detector type & detector
2.2.3 Optimization of Column oven temperature temperature.
program A variety of detector is commercially available to be used
The column resides in an oven, and temperature, which with GC, each having its own limitations and advantages.
greatly affects the effectiveness of the chromatographic The most commonly used detector in GC is flame
separation, is an extremely important factor used in ionization detector. FID is typically used for organic
controlling GC. In many cases, isothermal is not the most compounds and used in quality control analysis of
effective temperature mode for sample separation; in such pharmaceutical compounds. Detector temperatures and the
cases, a temperature program can be used. Most GC relative flow rates of carrier gas, hydrogen and air into the
temperature program have initial temperature, a ramp detector are the key operating parameters. 13a series of
(degree increase per minute) and a final temperature.Using standards is defined for evaluation of detector parameters
a linear temperature program as a starting point if previous such as drift, noise, sensitivity, linear range, dynamic range
analysis information is not available to use as aguide, the etc. The variation in detector response with flow rate
first program development step is to try a simple, linear depends on whether the detector is concentration or mass
temperature program. To improve the resolution of earlier flow dependent. For concentration dependent detectors
eluting peaks, decrease the initial temperature or increase (e.g., thermal conductivity detector, photo ionisation
the initial hold time. Decreasing the initial temperature detector) a decrease in the flow-rate does not affect the
usually results in the largest resolution improvement, but peak height, which remains approximately constant.

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However the peak width, and consequently the peak area, concentrate sample analytes. The sample pre-treatment
increase. In contrast, for mass flow detection systems (e.g., development can at times be more complex than the
flame ionization detection, flame photometric detection, separation itself. The goals of the separation should be
nitrogen phosphorus detection) the response is inversely specified at the beginning of the method development.
proportional to the retention time. Therefore, any change in Moreover, before the sample is injected during the GC
chromatographic conditions which cause a change in the method development, the detector should be selected to be
retention time will also affect the peak height. It follows sensitive to all sample components of interest. The final
that a decrease in the flow-rate results in reduced peak procedure should meet all the goals that have been defined
heights, however the peak area remains approximately at the beginning of the method development and when the
constant.15 method for quantitative use is finalized it should be
2.3. Developing the approach for analysis: validated.8
Two general approaches to GC method development can be 2.5 Method optimization :
followed, either start from scratch or scale a current Several steps are involved in method development and
method. The path that is taken depends on the status of the validation. In the case of analytical procedures like GC ,
current method. Method translation yields a scaled version these steps might include review of information on samples
of the current method. If the current method meets all of the to be analyzed, definition of separation goals; assurance of
analytical needs except speed, then translation is the best special procedure requirements, sample pretreatment if any;
way to go (especially if the analysis involves many detector selection and setting, separation conditions
components). If there are deficiencies with the current optimization, check for problems or special procedure
method, It might be better to redevelop the method from requirements, recovery of purified material, quantitative
scratch in orderto better meet the overall analytical calibration and qualitative method development.8The
requirements.14 Proper selection of the methods depends experimental conditions should be optimized to get desired
upon the nature of the sample (volatile or nonvolatile separations and sensitivity after getting appropriate
molecule), its molecular weight, solubility and melting separations. This will be achieved through
point.9factors that affect gas chromatographic analysis are planned/systemic examination of parameters.16 During
column temperature, carrier gas flow-rate, injection optimization one parameter is changed at a time and set of
temperature, split ratio, detector temperature and sample conditions are isolated rather than using a trial and error
size.15All of these parameters are selected on the basis of approach.17
trials and followed by considering the system suitability 2.6 Method Validation :
parameters. Typical parameters of system suitability are Validation is derived from Latin from which means “strong
e.g. retention time should be more than 5 min, the ness”. Validation is the strength or strongness of a
theoretical plates should be more than 2000, the tailing procedure, process, and capability of an equipment to
factor should be less than 2, resolution between 2 peaks operate and is proved for its acceptability with confirmation
should be more than 5, % R.S.D. of the area of analyte and documented legally on the basis of scientific
peaks in standard chromatograms should not be more than data.17Validation of an analytical method is the process by
2.0 % like other.16 which it is established by laboratory studies, that the
2.4 Samplepreparation: performance characteristics of the method meet the
The sample that is injected into the gas chromatograph requirements for the intended analytical application.
following sample preparation must be either a liquid or a Validation is required for any new or amended method to
gas, the analytes must be volatile enough under the ensure that it is capable of giving reproducible and reliable
conditions of the inlet and column to traverse the results, when used by different operators employing the
instrument, and, ideally, the matrix interferences must also same equipment in the same or different laboratories. The
be volatile, so as not to contaminate the instrument or type of validation program required depends entirely on the
column. In most cases, liquid samples must be dissolved in particular method and its proposed applications.13Results
a volatile organic solvent. The basic goal of sample from method validation can be used to judge the quality,
preparation is to ensure that these conditions are met, with reliability and consistency of analytical results; it is an
additional goals that the preparation be reproducible to integral part of any good analytical practice. Use of
meet quantitative analysis requirements and straight equipment that is within specification, working correctly
forward to perform, if the analysis is to be performed and adequately calibrated is fundamental to the method
routinely, as in quality assurance and in other routine validation process. Analytical methods need to be validated
testing laboratories.1Before beginning the GC method or revalidated.18 (figure-3)
development, it is important to review what is known about • Before their introduction into routine use;
the sample. The goal of the analysis should be defined at • Whenever the conditions change for which the method
this point and considerations must be given to the number has been validated
of samples to be analyzed, available equipment, etc. The • Whenever the method is changed
nature of the sample (e.g. whether it is hydrophilic or Typical parameters recommended by FDA, USP, and ICH
hydrophorbic, whether it contains protolytic functions, etc) are as follow.18-20
determines the best approach to method development. 1. Specificity
Some samples require a pre-treatment prior to analysis 2. Linearity & Range
because of the need to remove interferences or to 3. Precision

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  Method precision (Repeatability) that no interference exists. The accuracy is then calculated
 Intermediate precision (Reproducibility) from the test results as a percentage of the analyte
4. Accuracy (Recovery) recovered by the assay. It may often be expressed as the
5. Solution stability recovery by the assay of known, added amounts of
6. Limit of Detection (LOD) analyte.19-21
7. Limit of Quantification (LOQ) Solution stability: During validation the stability of
8. Robustness standards and samples is established under normal
9. System suitability conditions, normal storage conditions, and sometimes in
Specificity:specificity of an analytical method as its ability the instrument to determine if special storage conditions are
to assess unequivocally the analyte in the presence of necessary, for instance, refrigeration or protection from
components, which may be expected to be present. It can light.19
also be defined as ability of method to measure accurately Limit of Detection (LOD) : Limit of detection (LOD) of
an analyte in the presence of interference, such as synthetic an individual procedure is the lowest amount of analyte in a
precursors, excipients, enantiomers, and known (or likely) sample that can be detected but not necessarily quantitated
degradation products that may be expected to be present in as an exact value. In analytical procedures that exhibit
the sample matrix.19 baseline noise, the LOD can be based on a signal-to-noise
Linearity and range:The linearity of an analytical (S/N) ratio (3:1), which is usually expressed as the
procedure is its ability (within a given range) to obtain test concentration of analyte in the sample. The signal-to-noise
results, which are directly proportional to the concentration ratio is determined by: s = H/h Where H = height of the
of analyte in the sample. A linear relationship should be peak corresponding to the component. h = absolute value of
evaluated across the range of the analytical procedure. It is the largest noise fluctuation from the baseline of the
demonstrated directly on the drug substance by dilution of chromatogram of a blank solution.20-22
a standard stock solution of the drug product components, Limit of Quantification (LOQ) : The limit of Quantitation
using the proposed procedure. Linearity is usually (LOQ) or Quantitation limit of an individual analytical
expressed as the confidence limit around the slope of the procedure is the lowest amount of analyte in a sample that
regression line.20 For the establishment of linearity, can be quantitatively determined with suitable precision
minimum of five concentrations are recommended by ICH and accuracy. For analytical procedures such as HPLC that
guideline. The range of an analytical method is the interval exhibit baseline noise, the LOQ is generally estimated from
between the upper and lower levels that have been a determination of S/N ratio (10:1) and is usually
demonstrated to be determined with precision, accuracy confirmed by injecting standards which give this S/N ratio
and linearity using the method.21,22 and have an acceptable percent relative standard deviation
Precision: The precision of an analytical procedure as well.19-22
expresses the closeness of agreement (degree of scatter) Robustness: is defined as the measure of the ability of an
between a series of measurements obtained from multiple analytical method to remain unaffected by small but
sampling of the same homogeneous sample under the deliberate variations in method parameters (e.g. pH, mobile
prescribed conditions. Precision may be considered at three phase composition, temperature and instrumental settings)
levels: repeatability, intermediate precision and and provides an indication of its reliability during normal
reproducibility. It can also be defined as degree of usage. Determination of robustness is a systematic process
agreement among individual test results when method is of varying a parameter and measuring the effect on the
applied repeatedly to multiple sampling of a homogeneous method by monitoring system suitability and/or the analysis
sample. 20The precision of an analytical procedure is of samples.19,20
usually expressed as the standard deviation or relative System Suitability: System suitability tests are an integral
standard deviation of series of measurements. Precision part of liquid chromatographic methods. They are used to
may be either the degree of reproducibility or of the verify that the detection sensitivity, resolution and
repeatability of the analytical procedure under normal reproducibility of the chromatographic system are adequate
conditions. Intermediate precision (also known as for the analysis to be done. The tests are based on the
ruggedness) expresses within laboratories variations, as on concept that the equipment, electronics, analytical
different days, or with different analysts or equipment operations and samples to be analyzed constitute an integral
within same laboratory. Precision of an analytical system that can be evaluated as such. Factors, such as the
procedure is determined by assaying a sufficient number of peak resolution, number of theoretical plates, peak tailing
aliquots of a homogeneous sample to be able to calculate and capacity have been measured to determine the
statistically valid estimates of standard deviation or relative suitability of the used method.19-22
standard deviation.21 3. Conclusion
Accuracy (Recovery) : The accuracy of an analytical In recent years development of the analytical methods for
procedure expresses the closeness of agreement between identification, purity evaluation and quantification of drugs
the value which is accepted either as a conventional true has received a great deal of attention in the field of
value or an accepted reference value and the value found. It separation science. This review describes GC method
is determined by applying the method to samples to which development and validation in general way. A general and
known amounts of analyte have been added. These should very simple approach for the GC method development for
be analysed against standard and blank solutions to ensure the separation of compounds was discussed. Knowledge of

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6
the physiochemical properties of the primary compound is FID Method Development and Validation Parameters
of utmost importance prior to the any GC method for Analysis of Palm Oil (Elaeis guineensis Jacq.)
development. Severa lsteps are being considered for GC Fatty Acids Composition, Research in Plant Sciences,
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Source of support: Nil; Conflict of interest: None declared

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