INTRODUCTION:

ANALYTICAL METHOD DEVELOPMENT:

Analytical chemistry deals with methods for identification, separation, and quantification of the chemical components of natural and artificial materials. The choice of analytical methodology is based on many considerations, such as: chemical properties of the analyte and its concentration sample matrix, the speed and cost of the analysis, type of measurements i.e., quantitative or qualitative and the number of samples. A qualitative method yields information of the chemical identity of the species in the sample.

A quantitative method provides numerical information regarding the relative amounts of one or more of the analytes in the sample. The steps of method development and method validation¹

· Method development plan definition

· Background information gathering

· Laboratory method development, it includes various stages namely sample preparation, specific analytical

· method, detection and data processing

· Generation of test procedure

· A well-developed method should be easy to validate.

A method should be developed with the goal to rapidly test preclinical samples, formulation prototypes, and commercial samples. There are five common types of analytical methods, each with its own set of validation requirements¹

· Identification tests

· Potency assays

· Quantitative tests for impurities

· Limit test for the control of impurities

· Specific tests

The first four tests are universal tests, but the specific tests such as particle-size analysis and X ray diffraction are used to control specific properties of the active pharmaceutical ingredient (API) or the drug product. The most widely used methods for quantitative determination of drugs and metabolites in biological matrices such as blood, serum, plasma, or urine includes Gas chromatography, (GC) High-performance liquid chromatography, (HPLC) Thin layer chromatography, (TLC) combined GC and LC mass spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GCMS, and GC-MSMS, techniques like NMR is used for structure identification. Chromatography in different forms is the leading analytical method for separation of components in a mixture. The chromatographic procedure for the separation of substances is based on differences in rates of migration through the column arising from different partition of the compounds between a stationary phase (column packing) and a mobile phase transported through the system. Chromatographic methods can be classified according to the physical state of the mobile phase into the following basic categories: gas chromatography, (GC) supercritical fluid chromatography (SFC) and liquid chromatography (LC). The technique was originally developed by the Russian botanist M.S. Tswett in 1903¹.

TERMINOLOGY²:

Validation:

It is accepted that during the course of a typical drug product development program, a defined analytical method will undergo many modifications because composition changes, lower strength may be added or percentage of coating material may change on the formulation. Because of the changes the analytical method may be modified and if modified it should be verified so it requires different levels of validation. Two different levels/types of method validations, complete validation and partial validation or mini, validation, are defined and characterized as follows. Complete validation complete validation is necessary before executing clinical batch or registration batch of drug product².

Mini validation:

Mini validations are required for all the test methods like Assay, Related substance, UOD and Blend Uniformity for analysing the routine samples prior starting the complete validation some parameters to be checked as per ICH Guidelines detailed and acceptance criteria discuss later².

VALIDATION PARAMETERS:

Linearity:

A linear relationship should establish across the range. It may be demonstrated directly on the active substance by linear dilution and percentage level of each parameter. The linear range of the method must be determined regardless of the all stage of the formulation during drug development. ICH guidelines recommend evaluating a minimum of five concentrations to assess linearity and wider range of concentration and other approaches should be justified. The correlation coefficient, Y-intercept, Slope of the regression line and residual sum of square shall be submitted by appropriate statistical method. The linearity solutions are prepared by performing serial dilutions of a single stock solution, for related substance, residual solvent impurity blend or solvent blend shall be used. The response of the interested peak is plotted against the, corresponding theoretical concentration. For related substance method, relative response factors and relative retention times for each impurity should establish with respect to the active compound. Response factors allow the end user to not using the impurity material for each analysis, and it is useful to correcting for response differences and final impurity calculation. To determine the relative response factors, linearity curve method or about 0.2 and 0.4 percent for each impurity and the active compound should be injected and find the relative response factor from the slope of the linearity curve or ratio between the response factor of impurity and response factor of active compound².

Selectivity (Specificity):

For clinical and before registration batch of the drug product, the analytical method must demonstrate specificity including degradation study. The method must have the ability to separate each known impurity and degradation product at the quantitation level and if any blank, placebo peaks are found it should be properly separated from impurity peak and interested peak. For identification tests, discrimination of the method should be demonstrated by obtaining positive results for samples containing the analyte and negative results for samples not containing the analyte. The method must be able to differentiate between the analyte of interest and compounds with a similar chemical structure that may be present. For a high-performance liquid chromatography (HPLC) identification test, peak purity evaluation should be used to assess the homogeneity of the peak corresponding to the analyte of interest. For assay/related substances methods, the active peak and each impurity should be adequately resolved from all impurity/degradant peaks, placebo peaks, and blank peaks. Resolution of each impurity peaks and impurity peak from interested peak should complice US Pharmacopeia. Blank, Placebo and sample matrix components should be analyzed without the active present in order to identify possible interferences. If filters are to be used to clarify sample solutions, an aliquot of filtered sample diluents should be analyzed for potential interferences and result should compared with centrifuged sample. If the impurities/degradants are unknown or unavailable, forced degradation studies should be performed. Forced degradation studies of the active pharmaceutical ingredient (API), placebo and finished product, using either peak purity analysis or a mass spectral evaluation, should be performed to identify and separate the potential degradation products. The forced degradation studies should consist of exposing the API, placebo and finished product to acid, base, peroxide, heat, light conditions and moisture or water until adequate degradation of the active has been achieved. An acceptable range of degradation may be 10-30% for assay and about 10% for related substance but may vary based on the active being degraded. Over degradation of the active or known impurity should be avoided to prevent the formation of secondary degradants. If placebo material is available, it should be stressed under the same conditions and for the same duration and as the API and finished product. The degraded placebo samples should be evaluated to ensure that any generated degradants are resolved from the analyte and impurity peak of interest. Forced degraded sample should pass the peak purity and does not show any purity flag by using a photodiode array detector for chromophoric compound. Non chromophoric compound or GC sample should confirm mass spectral evaluation of degradation product peak and each known impurity peak should not show any significant changes in the fragmentation pattern from the parent compound².

Precision:

Precision reflects the closeness of agreement of a series of measurements between the series measurement obtained from multiple sampling from the same sample under the same condition at the same time. Precision may be considered in three levels repeatability, intermediate precision and reproducibility².

Repeatability:

Repeatability expresses the precision under the same operating condition over a short interval of time. It is also termed intra-assay precision. A minimum of six replicate sample preparation of a same sample or homogenous sample prepared at the 100% test concentration².

Intermediate precision:

Intermediate precision reflects within-laboratory variations such as different days, different analysts, and different equipments. Intermediate precision testing can consist of two different analysts, each preparing six sample preparations, as per specified analytical method. The analysts execute their testing on different days using separate instruments and analytical columns².

Reproducibility:

Reproducibility expresses the precision of a method with in the laboratory variation like different days, different analyst and different equipments etc. Each testing site can prepare a total of six sample preparations, as per the analytical method. Results are evaluated to ensure statistical equivalence among various testing sites. Acceptance criteria similar to those applied to intermediate precision also apply to reproducibility².

Accuracy:

Accuracy should be performed at a minimum of five concentration levels, for LOQ and maximum concentration will be six replicate preparation and median concentration must be three replicates as per specified test method or it can be spiked synthetic mixture of product component and the acceptance criteria and number sample preparation².

Solution Stability:

The solution stability is stability of standard and extracted sample solution (ready to inject) from the sample or matrix and analyzed as per specified method, and it should be stored properly in room temperature and refrigerated condition depending upon the stability of the sample and standard solution. The stability of standard and sample solution should be established in room temperature and refrigerated, if refrigerated before analyzing it should be thawing to room temperature. A minimum two preparations of standard and sample solution should be prepared and analysed as per specified method. The analysed solutions stored in necessary condition and the stability can be established for two days or solution stability can be established by an hour basis depending upon the nature of the product².

Ruggedness (Robustness):

Robustness, of an analytical procedure measure of its capacity to remain unaffected by small but deliberate variation in method parameters and provides an indication of its reliability during normal usage. Robustness must not necessarily include in mini validation or in preclinical stage validation, but in complete validation and before transfer the analytical method to another laboratory it should be established, the procedure and acceptance criteria².

Limit of quantification and detection

(LOD and LOQ):

The LOQ is the lowest amount of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy. There are different approaches to the determination of LOQ. LOQ is a parameter to the quantitation of the sample at low level in the compound and is used particular for the impurities, degradation products and/or residual solvents. LOD and LOQ can be established from signal to noise ratio method and linearity curve method. LOQ based on signal to noise ratio (SIN): This approach can only be applied if there is no much baseline noise, LOQ can be calculated as per US Pharmacopoeia or it can be derived from the computer aided soft ware’s. For related substance and residual solvents, a least concentration of impurities can be spiked in to the test solution (if no impurities present in the test solution) or synthetic mixture of product component of placebo preparation. The low concentration solution gives signal to noise ratio is about 10, that could be the LOQ and the low concentration solution gives signal to noise ratio is about 3, that could be the LOD of the particular impurity and it should be established for each impurities and interested peak².

BASIC CRITERIA FOR NEW METHOD DEVELOPMENT OF DRUG ANALYSIS³:

• The drug or drug combination may not be official in any pharmacopoeias

• A proper analytical procedure for the drug may not be available in the literature due to patent Regulations

• Analytical methods may not be available for the drug in the form of a formulation due to the Interference caused by the formulation excipients

• Analytical methods for the quantization of the drug in biological fluids may not be available

• Analytical methods for a drug in combination with other drugs may not be available

• The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.

METHOD DEVELOPMENT LIFE CYCLES³:

Steps in method development:

The steps of method development and method validation

• Method development plan definition

• Background information gathering

• Laboratory method development, it includes various stages namely sample preparation, specific analytical method, detection and data processing.

Generation of test procedure³:

· Sample information, define separation goals

· Sample pre-treatment, need of special HPLC procedure

· Selection of detector and detector settings

· Selection of LC method; preliminary run; estimate best separation conditions

· Optimize separation conditions

· Check for problems or requirement for special procedure

· Method validation

Sample information³:

· Number of compounds present

· Chemical structure of compounds

· Chemical nature

· Molecular weight of compounds

· pKa Value(s) of compounds

· Sample solubility

· Sample stability and storage

· Concentration range of compounds in sample

· UV spectra of compounds or properties for detection of compounds.

A well-developed method should be easy to validate. A method should be developed with the goal to rapidly test preclinical samples, formulation prototypes, and commercial samples. There are five common types of analytical methods, each with its own set of validation requirements

· Identification tests

· Potency assays

· Quantitative tests for impurities

· Limit test for the control of impurities

· Specific tests

Analytical Method Development Is Required for:

· Herbal Products

· New process and reactions

· New molecules

· Active ingredients (Macro analysis)

· Residues (Microanalysis)

· Impurity Profiling

· Component of Interest in different matrices

Analytical methodology contents³:

An Analytical Methodology consists of the following

· Techniques

· Method

· Procedure

· Protocol

Importance of analytical methodology to an analyst³:

· The required data for a given analytical problem

· The required sensitivity

· The required accuracy

· The required range of analysis

· The required precision

REFERENCES:

1. Dr Rajesh Z. Mujoriya, Analytical Method Development and Validation of Pharmaceutical Technology: An Overview, Research Journal of Pharmaceutical Dosage Forms and Technology. 5(4): July-August, 2013, 213-220.

2. G. Geetha, Karanam Naga Ganika Raju, B. Vignesh Kumar and M. Gnana Raja, Analytical Method Validation: An Updated Review, international journal of advances in pharmacy, Biology and chemistry, 1(1): Jan-March, 2012, 64-71.

3. Bhagyasree T, Neelam I, Ajitha A, Uma Maheshwara Rao, A review on analytical method development and validation, International Journal of Pharmaceutical Research & Analysis, 498),2014; 444-448.

Received on 01.07.2017 Modified on 20.08.2017

Res. J. Pharm. Dosage Form. & Tech. 2017; 9(3):93-97.

DOI: 10.5958/0975-4377.2017.00016.7

Mrs Rupali H Tiple*

Mrs Rupali H Tiple^*