INTRODUCTION:

Analytical methods development and validation play important role in the drug discovery, development and manufacture of pharmaceuticals. Measurement of drug concentrations in biological matrix (such as serum, plasma, blood, urine, and saliva) is an essential aspect of medicinal product development. Such data may be required to support applications for new actives substances and generics as well as variation to authorised drug products. The analysis thus carried out must be verified for its alleged purpose and must be validated.

An investigation should be performed during each step to determine whether the external environment, matrix or procedural variables can affect the estimation of analyte in the matrix from the time of collection up to the time of analysis.^1,2

The bioanalysis procedure includes sampling, sample preparation, analysis, calibration and data evaluation and reporting. In modern bioanalysis a good sample preparation and a hyphenated instrumentation are required. In pharmaceutical research companies the development of comprehensive bioanalytical methods is very important during the process of drug discovery and development. In addition the method validation has an important role in regulatory bioanalysis to ensure the quality of the applied method. Bioanalytical method validation is very important for supporting of new drug applications or biologics license applications. Bioanalytical results of the efficacy trials make key decisions. Therefore, it is necessary to fully verify and documents. Various guidelines provide requirements for bioanalytical method validation and also address non-clinical requirements.^3,4

METHOD DEVELOPMENT:

The objective of the method development is too develop and validates highly specific, reliable and cost effective LC-MS/MS method for determination of drug in human plasma. The Scope of developing and validating the bioanalytical method is to get a suitable method which is more accurate and precise for the analyte of interest under given set of lab conditions by available resources.

Steps in Method Development (MD):

1. Literature search for drugs (Structure, Solubility, pKa etc.)

2. Identification of analytical techniques and optimization

3. Reference standard preparation

4. Selection of Internal Standard (ISTD)

5. Sample pre-treatment (Extraction Procedure)

6. Sample Storage

During the process of developing your drug, method development processes can also determine the multitude of constituents in your formulation. It's important to use method development to work out the product's purity, composition, and potency.

Sample collection and preparation:

The biological media that contain the analyte are usually blood, plasma, urine, serum etc. Blood is usually collected from human subjects by vein puncture with a hypodermic syringe up to 5 to 7ml (depending on the assay sensitivity and the total number of samples taken for a study being performed). The venous blood is withdrawn into tubes with an anticoagulant, e.g. EDTA, heparin etc. Plasma is obtained by centrifugation at 4000 rpm for 15 min. About 30 % to 50 % of the volume is collected. The purpose of sample preparation is to clean up the sample before analysis and to concentrate the sample. Material in biological samples that can interfere with analysis, the chromatographic column or the detector includes proteins, salts, endogenous macromolecules, small molecules and metabolic byproducts.

Liquid – Liquid extraction:

It is based on the principles of differential solubility and partitioning equilibrium of analyte molecules between aqueous (the original sample) and the organic phases. Liquid – Liquid extraction generally involves the extraction of a substance from one liquid phase to another liquid phase. Now a day’s traditional LLE has been replaced with advanced and improved techniques like liquid phase micro extraction, single drop liquid phase micro extraction and supported membrane extraction.^5,6

Solid Phase Extraction (SPE):

Solid phase extraction is selective method for sample preparation where the analyte is bound onto a solid support, interferences are washed off and the analyte is selectively eluted. Due to many different choices of sorbents, solid phase extraction is a very powerful technique.⁷

Conditioning:

The column is activated with an organic solvent that acts as a wetting agent on the packing material and solvates the functional groups of the sorbent. Water or aqueous buffer is added to activate the column for proper adsorption mechanisms.

Sample Loading:

After adjustment of pH, the sample is loaded on the column by gravity feed, pumping or aspirating by vacuum. Washing: Interferences from the matrix are removed while retaining the analyte.

Elution:

Distribution of analyte – sorbent interactions by appropriate solvent, removing as little of the remaining interferences as possible.

Protein Precipitation:

Protein precipitation is often used in routine analysis to remove proteins. Precipitation can be induced by the addition of an organic modifier, a salt or by changing the pH which influence the solubility of the proteins14. The samples are centrifuged and the supernatant can be injected into the HPLC system or be evaporated to dryness and thereafter dissolved in a suitable solvent. A concentration of the sample is then achieved. There are some benefits with precipitation method as clean-up technique compared to SPE.^8,9

Bioanalytical Method Validation (BMV):

The reason for validating a bioanalytical procedure is to demonstrate the performance and reliability of a method and hence the confidence that can be placed on the results.

Need of Bioanalytical Method Validation:

· It is essential to used well characterized and fully validated Bioanalytical methods to yield reliable results that can be satisfactorily interpreted.

· It is recognized that Bioanalytical methods and techniques are constantly undergoing changes and improvements; they are at the cutting edge of the technology.

· It is also important to emphasize that each Bioanalytical technique has its own characteristics, which will vary from analyte to analyte, specific validation criteria may need to be developed for each analyte. ¹⁰

· Moreover, the appropriateness of the technique may also be influenced by the ultimate objective of the study. When sample analysis for a given study is conducted at more than one site, it is necessary to validate the Bioanalytical method(s) at each site and provide appropriate validation information for different sites to establish inter-laboratory reliability.¹¹

Typical parameters to validate are include; selectivity, accuracy, precision, linearity and range, limit of detection, limit of quantification, recovery, robustness and stability.

Accuracy:

The degree of closeness of the observed concentrations to the nominal or known true concentration. It is typically measured as relative error (% RE). Accuracy should be measured using a minimum of five determinations per concentrations. A minimum of three concentrations in the range of expected study sample concentrations is recommended. The mean value should be within 15% of the nominal value except at LLOQ, where it should not deviate by more than 20%.¹²

Accuracy is best reported as % bias which is calculated from the expression:

Abso% Bias = measured value – true value/ true value X 100

Precision:

The precision of a bioanalytical method is a measure of the random error and is defined as the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogenous sample under the prescribed conditions.

It is typically measured as coefficient of variation (%CV) or relative standard deviation (R.S.D.) of the replicate measurement 20.

% C V = standard deviation/men X 100

Selectivity and specificity: The ability of the bioanalytical methods to measure and differentiate the analytes in the presence of components that may be expected to be present. These could include metabolites, impurities, degradants or matrix components

Linearity:

Calibration (standard) curve is the relationship between instrument response and known concentrations of the analytes. A calibration curve should be prepared in the same biological matrix as the samples in the proposed study by spiking the matrix with known concentrations of the analytes. The number of standards used in constructing a calibration Curve will be a function of the anticipated range of analytical values and the nature of the analytes/response relationship.¹³

System Suitability:

System Suitability is the ability of an analytical method to differentiate and quantify the analytes in the presence of other components in the sample. The selectivity of the method was evaluated by analyzing six replicates of plasma samples spiked at LLOQ (Lower Limit of Quantification).

Stability:

Drug stability in a biological fluid is a role of the storage conditions, the chemical properties of the drug, the matrix, and the container system. Stability evaluation is done to show that the concentration of analytes at the time of analysis corresponds to the concentration of the analytes at the time of sampling.

A) Stock Solution Stability The stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least six hours. If the stock solutions are refrigerated or frozen for the relevant period, the stability should be recognized. After completion of the desired storage time, the stability should be tested by comparing the instrument response of stored sample with that of freshly prepared solutions. ¹⁴

B) Bench Top Stability Bench top stability is to assess the stability of analytes in biological fluids over a period of time during which the sample are expected to be kept on bench while processing.

C) Freeze Thaw Stability Analytes stability should be determined after three freeze and thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at the intended storage temperature for 24 hours and thawed unaided at room temperature. When the sample completely thawed, the samples should be refrozen for 12 to 24 hours under the same conditions. The freeze–thaw cycle should be repeated two more times, and then analyzed on the third cycle. This Freeze-Thaw quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for the calculation of precision and accuracy.

D) Short Term Temperature Stability Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this temperature from 4 to 24 hours (based on the expected duration that samples will be maintained at room temperature in the intended study) and analyzed.

E) Long Term Temperature Stability The storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis. Long-term stability should be determined by storing at least three aliquots of each of the low and high concentrations under the same conditions as the study samples. The volume of samples should be sufficient for analysis on three separate occasions. The concentrations of all the stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stability testing.¹⁵

Recovery Study:

The recovery of analytes in an assay is the detector response obtained from an amount of the analytes added and extracted from the biological matrix, compared to the pure authentic standard. Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery of the analytes need not be 100%, but the extent of recovery of an analytes and of the internal standard should be consistent, precise, and reproducible.

Matrix Effect:

Matrix effect is defined as the effect of co-eluting residual matrix component of biological sample on the ionization of target. There are two types of the matrix effect e.g. absolute matrix effect and relative matrix effect. Absolute matrix effect is defined as difference between response of equally concentrated analyte in solvent and in matrix extracts.

CONCLUSION:

In this review, the basic required concepts and definitions in bioanalytical method validation were presented and discussed. SPE and LLE are most used sample preparation techniques in bioanalysis. Huge progress has been done in SPE technology such as new SPE format and new selective sorbents. In addition progress in the automated systems for sample preparation is expected to meet the need for high sample throughput. In addition progress in the automated systems for sample preparation is expected to meet the need for high sample throughput. Regulatory bioanalysis has an essential role in pharmaceutical research and development.

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Received on 20.07.2023 Modified on 17.10.2023

Asian J. Pharm. Ana. 2024; 14(1):47-50.

DOI: 10.52711/2231-5675.2024.00009