Methodology
HPLC Method Development 12
Sample preparation is a critical step during analysis. First consideration for developing
the analytical methods is to determine the solubility of sample under analysis.13 Selecting
specific solubility of active pharmaceutical ingredient in a general solvent like water, methanol,
Acetonitrile, Chloroform etc . Mobile phase composition plays a key role in separation of
different molecules. Mobile phase composition should be selected such way that it will not
degrade the column packaging material and HPLC contact parts. Hence strong acids, bases and
halide solution are avoided in mobile phase13. Viscosities to be maintain low i.e. less than 0.5
centipoises in order to avoid the higher backpressure. The chemicals and solvents used should be
high purity and easily available. 13
Detector: The selection of the detector is based on the chemical characteristic of the
molecules under investigation. Due to the easy availability and as most of the chemical
compounds shows absorbance in the UV Visible range, maximum method development starts
from UV Visible detector. In some cases the molecules are converted into different form where
the resulting compound shows absorbance in UV Visible range. When UV Visible response is
not possible than only other types of detectors are considered.
Column : Now a day’s intensions is to develop an analytical method where the
chromatograms should show maximum separation between different molecules within short run
time and to use minimum solvent and protect the environment and cost , short length columns
are preferred. The selection of Stationary phase to be selected based on molecule under
analysis, generally reverse phases are more preferred e.g. C8, C18.In case of normal phases ,
most preferred stationary phases is Cyano (Nitrile phase.) 14
Subsequently the method is fine tuned selecting proper chromatographic Parameters like Flow
rate, Column oven Temperature, Wave length, injection volume, tray temperature etc. Each
HPLC analytical method should have few of the system suitability parameters such as Column
efficiency, Resolution Factor, Tailing Factor, % RSD of peak area and % RSD of Retention
times of multiple injections, Relative Retention Time, Similarity Factor, Capacity Factor, signal
to noise ratio are complying to ensure the correctness of analysis .These parameters are based on
the outcome of the analytical method validation.
Quality by design Approach (QbD):15 A systematic approaches to development that begins with
predefined objectives and emphasizes product and process understanding and process control,
based on sound science and quality risk management. During the analytical method development
as per QbD approach the variability occurring due to HPLC column, Mobile phase composition,
pH etc are evaluated.
Method validation:
Background - Analytical Method Validation. 16
Analytical Methods validation is the process of demonstrating that analytical procedures
17.
are suitable for their intended use Validation is not only as a test of acceptability of using the
conditions (e.g. flow rate, sample size, column type etc) prescribed but the goal of validation
process is to challenge the method and determine limits of allowed variability for the conditions
needed to run the method.18
Analytical method is validated for the following parameters.
a) Specificity and selectivity:
The ability of a method to measure the analyte in the presence of components that may
be expected to be present , such as impurities , degradation products , and excipients interference
(if any ). Lack of specificity of an individual analytical procedure may be compensated by other
supporting analytical procedure(s).
Specificity in liquid chromatography is obtained by choosing optimal columns and
setting chromatographic conditions, such as mobile phase composition, column temperature and
detector wavelength. Besides chromatographic separation, the sample preparation step can also
be optimized for best selectivity.
b) Precision:
The precision of the analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of the homogeneous
samples. Precision is expressed as repeatability (Use of the analytical procedure by one analyst
in a short series of analysis), intermediate precision (Use of the analytical procedure by at least
two analyst on at least two separate occasions) or reproducibility (Use of the analytical procedure
by at least two labs) 19
The precision of an analytical procedure is usually expressed as the variance, standard
deviation or coefficient of variation of a series of measurements. 16
c) Linearity:
The linearity of an analytical method is its ability to obtain test results (within given
range) which are directly proportional to the concentration of analyte in the samples 16
d) Range:
The range of an analytical procedure is the range of concentrations (upper and lower) of
the analyte for which the accuracy, precision and linearity is acceptable 16
For assay tests, ICH requires the minimum specified range to be 80 to 120 percent of the test
concentration. It also requires the range for the determination of an impurity to extend from the
limit of quantitation or from 50 percent of the specification of each impurity, whichever is
greater, to 120 percent of the specification.
e) Accuracy:
The accuracy of an analytical procedure is the closeness of the test results obtained by
that procedure to the true value. The accuracy of an analytical procedure should be established
across its range.16
This test evaluates the specificity of the method in the presence of the excipients under
the chromatographic condition used for the analysis of drug product. It will pick up recovery
problems that could be encountered during the sample preparation and the chromatographic
procedures 17
f) Solution stability:
It is a process to establish time interval within which the solution will retain its integrity
and can be relied to give correct results, when stored at appropriate condition.
Solution stability of drug product after preparation according to the test method should be
evaluated according to respective test method. This is of concern especially for drugs that can
undergo degradation by hydrolysis, photolysis or adhesion to glassware 17
g) Limit of Detection (LOD):
The limit of detection is the lowest amount of analyte in a sample that can be detected,
but not necessarily quantitated, under the stated experimental conditions. 12
h) Limit of quantification (LOQ):
Limit of quantification is the lowest concentration of analyte in a sample that can be
quantitatively determined with acceptance precision and accuracy under the stated experimental
conditions. 12 Age of detector lamp, age of HPLC column, detector sensitivity of particular model
or manufacturer has impact on LOD and LOQ values
i) Robustness:
Robustness of an analytical method is a measure of its capacity to remain unaffected by
small but deliberate variations in method parameters and provides an indication of its reliability
during normal use .Typical variations are stability of analytical solutions, mobile phase pH ,
mobile phase composition , different lots or different suppliers of columns , column oven
temperature , flow rate, wavelength etc.16
Robustness is be partly assured by good system suitability specifications. Thus, it is
important to set tight, but realistic, system suitability specifications 17
j) System Suitability:
System suitability Tests are used to verify that the resolution and reproducibility of a
chromatographic system are adequate for the analysis to be done. The tests are based on the
concept that the equipment, electronics, analytical operations and samples to be analyzed
constitute an integral system that can be evaluated as such.
The tests are performed by collecting data from replicate injections of reference standard
or other solutions as specified in the individual test method. Unless otherwise in the directed,
system suitability parameters are determined from the analyte peak. The accuracy and precision
of HPLC data collected begin with a well behaved chromatographic system. The system
suitability specifications and tests are parameters that provide assistance in achieving this
purpose.17
k) Forced Degradation Study:
In this study the product under analytical method development is forcefully degraded by
creating the unfavorable conditions such as acid hydrolysis, base hydrolysis, oxidation,
reduction, thermal shocks etc and the analytical method s challenged by analyzing the degraded
sample and it is ensured that the impurities which are formed are not interfered with the peaks of
interest.
WORK PLAN:-
First Year: Literature study.
First six month - Literature Survey to find out the availability / non availability of High
Performance Liquid Chromatographic methods, potential problems in available method
Second six month - Availability of placebo, Sample, impurities, Column, chemicals etc.
Second Year: Development and validation of analytical Method
First six month - Development of analytical method.
Second six month - Validation of developed analytical method and reporting of the
results obtained.
