INTRODUCTION:

In the discovery, development, and production of pharmaceuticals, analytical methods, development and validation play crucial roles. To assure the identification, purity, potency, and effectiveness of drug goods, quality control laboratories use the official test procedures that come as a result of these processes^1,2 and all the processes demonstrating the reliability and reproducibility of a certain method employed for quantitative measurement of analytes in a specified biological matrix, such as blood, plasma, serum, or urine.

Recent studies have demonstrated the significance of sample throughput in the development of bioanalytical methods that involve effective sample preparation.^2,4By using specialised laboratory studies, validation entails proving that the method's performance characteristics are appropriate and dependable for the intended analytical applications. The pharmaceutical industry has been forced to review and redefine some aspects of the development and validation of bioanalytical methods for the quantification of these therapeutics in biological matrices in support of preclinical and clinical studies due to the rise in the use of biological agents as therapeutics.^5,6 Bioavailability, bioequivalence, pharmacokinetic, and toxicokinetic study data are all evaluated and interpreted using bioanalytical technique validation, which is used for the quantitative assessment of medicines and their metabolites in biological fluids. In general, these studies are in favour of regulatory submissions.⁷ The quality of the underlying bioanalytical data is directly correlated with the quality of these research. Therefore, it is crucial to define and communicate to the pharmaceutical industry guiding principles for the validation of these analytical procedures. Drug bioanalysis in plasma can be done using either RP-HPLC or LCMS-MS.⁸Each instrument has advantages of its own. Numerous chemicals can be estimated using RP-HPLC in conjunction with a UV, PDA, or fluorescence detector. Low detection limits, the ability to produce structural information, the need for little sample preparation, and the ability to cover a large range of analytes with varying polarity are some of these chromatographic principles' key benefits.⁹

BIOANALYSIS CONCEPT:

Identification and measurement of analytes in biological samples (blood, plasma, serum, saliva, urine, faeces, skin, hair, and organ tissue) is referred to as bioanalysis. In addition to analysing small compounds like medicines and metabolites, bioanalysis also identifies big molecules like proteins and peptides^.10 Pharmaceutical businesses have a long history of using bioanalysis to aid in medication development and discovery. The toxicokinetic (TK), pharmacokinetic (PK), and pharmacodynamics (PD) studies of novel medications all heavily rely on bioanalysis.¹¹Additionally, forensic, preclinical, and clinical toxicology laboratories all use bioanalysis. Thus, bioanalysis is a crucial field in many areas of study, including the creation of new medications, forensic investigation, doping management, and the discovery of biomarkers for the diagnosis of numerous diseases.¹² It is common knowledge that complex matrices like blood, plasma, and urine require careful sample preparation before being injected into an analytical device. Modern bioanalysis calls for high throughput sample preparation and hyphenated analytical equipment. Drug bioanalysis has long made use of tandem mass spectrometry (MS/MS) in conjunction with liquid chromatography (LC). The most critical component of regulated bioanalysis is method validation. To show the effectiveness of the bioanalytical approach, validation is required.¹³

METHOD DEVELOPMENT:

The goal of developing a bioanalytical technique is to specify the design, operating circumstances, constraints, and applicability of the method for the intended use, as well as to make sure that the method is prepared for validation. Prior to developing a bioanalytical method, the sponsor should be aware of the analyte of interest (e.g., ascertain the drug's physicochemical qualities, in vitro and in vivo metabolism, and protein binding) and take into account any potential applications of any previous analytical methods.¹⁴The creation of bioanalytical methods does not necessitate substantial note or record keeping. To justify any modifications made during the development of the technique, the sponsor should keep a record of the methods that were changed as well as any problems and how they were fixed¹⁵ The process of developing a method is scientific. It entails the assessment and improvement of the numerous chromatographic settings, detection, and quantification steps in the sample preparation process.¹⁶

Separation and Detection by RP-HPLC:

In bioanalysis, HPLC is a strong technology. Due to the wide range of selectivity of HPLC columns, several medicines and metabolites in various matrices were separated using HPLC. In bio-analytical method development various activities are carried out. The following are the stages involved in RP-HPLC method development¹⁷

● Literature search for drugs.

● Identification of analytical technique and optimization.

● Reference standard preparation.

● Selection and optimization of extraction method.

● Optimization of HPLC method.

METHOD VALIDATION:

Every GLP investigation includes technique validation, which serves to guarantee the calibre of the analytical procedure. To confirm the method's dependability, reproducibility, and quality, method validation is used. Processes, parameters, and data processing are all part of validating bioanalytical methods^.18,19

There are various forms of validation.

● Full validation

● Partial validation

● Cross validation

USFDA guidelines for bioanalytical method validation.

A concise, in-depth description of the bioanalytical procedure in writing should be determined before-hand. This could be a protocol, study plan, report, or even a standard operating procedure. The potential impact of environmental, matrix, or procedural variables on the estimation of analyte in the matrix from the time of sample collection to the time of analysis should be assessed at each stage of the process.²⁰

To ensure that there are no matrix effects during the implementation of the approach, the proper precautions should be performed.^21,22The parameters for bioanalytical technique validation in accordance with ICH and USFDA recommendations.

● Accuracy

● Precision

● selectivity

● Sensitivity

● Reproducibility

● Linearity

● Limit of detection

● Limit of quantification

● Robustness

● Ruggedness

● Range

● Recovery

● Stability

Types of Bioanalytical Method Validation:

● LLE: liquid-liquid extraction:

LLE is still a desirable sample preparation method despite significant advances in sample preparation methods.²³ It is frequently used to prepare biological and aqueous samples. To extract the analyte into the organic phase.²⁴for injection into an analytical device, an aqueous sample (such as plasma or urine) and an immiscible organic solvent are combined in LLE. This technique can deliver a clean sample and good recovery. With a high extraction recovery, LLE was employed to extract basic and acidic medicines from biological material^.25

● SPE: solid phase extraction:

SPE is currently the most widely used sample preparation technique thanks to its great efficiency, low cost, excellent reproducibility, relatively low environmental impact, and simplicity in use and automation.²⁶ SPE has benefits including concentrating and separating traces of analytes in biological samples. SPE is an easy approach for removing necessary analytes from a complicated matrix. It uses a sorbent of 50–200 mg ascartridge. Other SPE formats, like flat discs and SPE, have also recently been developed. The type and quantity of the The key performance indicators for SPE include sorbent, loaded sample volume (with sufficient recovery), composition, and volume of washing and elution solutions^27,28.

● Protein precipitation:

Compared to the LLE and SPE, protein precipitation is the most straightforward extraction method. This can be done by utilising the appropriate organic solvents, which have strong analyte solubility and protein precipitation capabilities.^29,30 Because it completely precipitates proteins, acetonitrile is the preferred solvent for protein precipitation, and methanol is the preferred organic.^31,32The analyte's solubility in various solvents was provided by the precipitant. Protein precipitation follows, and the The resulting supernatant can be injected into or it might be evaporated and reconstituted using the HPLC. as the mobile stage and additional cleaning of the Micro centrifugal devices can be used to process samples. at a very rapid rate^.33

● Solid phase micro extraction.(SPME):

Since its introduction in 1989, SPME has gained popularity as a potent solvent-free extraction method, particularly for GC. In environmental analysis and to a lesser extent in bioanalysis, SPME has been accepted as a simple, automated procedure.^34,35 A fused silica fibre is frequently coated with a stationary phase in SPME. By placing the analyte in the matrix and exposing the fibre, the equilibrium in SPME is created.³⁶

● Matrix solid phase dispersion. (MSPD):

Solid matrices are used to prepare samples for the solid matrix diffusion technique. It is referred to as a microscale extraction method since it requires less than 1 g of sample and a little amount of solvent^.37 The amount of solvent used is decreased by over 98%, and sample turnaround time is 90%. This technique has recently been used to extract organic molecules using acidic alumina. The length of the analysis and the limit of determination (LOD) are two crucial components of this procedure.³⁸

● Supercritical fluid extraction:

To extract non-polar to moderately polar analytes from matrices, supercritical fluid extraction is frequently used. It provides advantages for the environment and is required by regulations to be an alternative to organic solvents. Although supercritical fluids have the same density as liquids, they have gaseous and liquid values for viscosity and diffusivity.³⁹ SFE is typically finished in 10 to 60 minutes. Supercritical fluids' solvent power is susceptible to variations in temperature and pressure, but less so. At ambient temperatures, a large number of supercritical liquids are gases. Compared to bodily fluids, this makes analyte recovery easier.⁴⁰

CONCLUSION:

Pharmaceutical research and development heavily rely on bioanalysis and the generation of PK, toxicokinetic, and metabolic data; as a result, the data must be generated in accordance with recognised scientific standards. From the perspective of the quality assurance department, an effort has been made to comprehend and explain the development and validation of bioanalytical methods. This article reports on some of the methods and how validation is carried out in various scenarios found in the analysis of the study sample. The fundamental terms and definitions needed for bioanalytical technique validation were presented in this review and addressed. The most used sample preparation methods in bioanalysis are SPE and LLE. New SPE formats and selective sorbents are just two examples of the significant advancements made in SPE technology. The new ideas and recommendations discussed can be used to improve the development of RP-HPLC bioanalytical methods and the matrix effect brought on by the presence of unwanted analytes or other interfering chemicals in the sample. The major components of developing and validating a bioanalytical method are described in this review.

Sample preparation, chromatographic separation, and detection using the appropriate analytical method are the three fundamental, linked components of the creation of a bioanalytical method.

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Received on 14.01.2024 Modified on 25.05.2024

Asian J. Pharm. Res. 2024; 14(3):281-284.

DOI: 10.52711/2231-5691.2024.00043