A Review on Bioanalytical Method Development and Validation of Pioglitazone by RP-HPLC

Diabetes mellitus, a chronic disease characterized by increased blood glucose levels, results from a deficiency in insulin production, insulin activity or both. It is a serious global health problem that affects millions of people worldwide. The International Diabetes Federation (IDF) estimates that there were 537 million adults aged 20–79 years with diabetes in 2021 and this is projected to increase to 783 million by 2045¹. Long-term harm, particularly to the eyes, kidneys, nerves, heart, and blood vessels, is caused by persistently high blood sugar levels in diabetes. Type 1 diabetes (T1D) and type 2 diabetes (T2D) are the two main types of diabetes. The primary cause of T1D is the autoimmune death of pancreatic beta cells, which leads in a complete lack of insulin. Type 2 diabetes, the more common form, occurs due to a combination of inadequate insulin production and insulin resistance². Microvascular problems such as diabetic retinopathy, nephropathy and neuropathy²⁰. Macrovascular problems such as cardiovascular problems like coronary artery disease, cerebrovascular disease and peripheral artery disease³.

Diabetes management requires a comprehensive approach, including medications, regular blood glucose monitoring, and lifestyle modifications. Antidiabetic medications are important for maintaining glycemic control and can prevent or delay complications of diabetes. These medications, which include insulin and several oral hypoglycemic agents (OHAs), are categorized based on how they work. OHAs are categorized as Biguanides (e.g., Metformin), Sulfonylureas (Glimepiride), Thiazolidinediones (Pioglitazone), Dipeptidyl Peptidase-4 (DPP-4) Inhibitors (Sitagliptin), Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists (Exenatide), Sodium-Glucose Co-Transporter-2 (SGLT2) Inhibitors (Empagliflozin), and Alpha-Glucosidase Inhibitors (Acarbose)^10,22,23.

Pioglitazone hydrochloride, a thiazolidinedione class antidiabetic drug used in the management of T2D acts as a selective agonist of peroxisome proliferator activated receptor-gamma (PPAR-γ), thereby enhancing insulin sensitivity in adipose, skeletal muscle and hepatic tissues³⁸. Owing to its widespread use, extensive clinical interest exists in understanding its pharmacokinetics and ensuring consistent therapeutic exposure in patients. Its concentration is the major measured variable in plasma for pharmacokinetic studies, therapeutic drug monitoring, drug interaction studies and bioequivalence testing.

Bioanalytical method development is the process of developing and optimizing an analytical technique for the accurate and reproducible measurement of a drug and/or its metabolites in biological matrices such as plasma, serum, urine, or tissue. Bioanalytical methods are a series of techniques for collection, processing, storage and analysis of biological matrices containing chemical compounds⁴. Bioanalytical method validation (BMV) is done to prove that a quantitative analytical procedure is appropriate and reliable for biochemical use. Systematic validation experiments with satisfactory and repeatable results ensure the quality and reliability of the method. Validation of a bioanalytical assay also includes the stability of analytes in biological samples collected in clinical studies, and the stability of critical assay reagents such as analyte stock solutions.

Bioanalytical method validation is the process required to demonstrate that an analytical method is suitable for the quantitation of analytes in biological matrices such as blood, plasma, serum or urine and that the method is accurate, reliable and consistent. Validation is a comprehensive laboratory procedure for establishing whether the performance characteristics of a method are appropriate for the planned analytical purpose.

The quantitative determination of medications and their metabolites in biological fluids, which supports the evaluation and interpretation of bioavailability, bioequivalency, pharmacokinetic, and toxicokinetic data, depends on the validation of bioanalytical methods. Completing regulatory reporting obligations is another reason why these investigations are crucial²⁸.

The quality of the data obtained is directly related to the reliability of bioanalytical investigations. It is therefore of utmost importance to establish and communicate clear validation criteria for analytical procedures in the pharmaceutical industry. RP-HPLC and LC-MS/MS are both widely used for plasma drug bioanalysis, each with their own set of advantages. Different chemicals are often evaluated using RP-HPLC with UV, PDA or fluorescence detection. These chromatographic procedures have advantages such as low detection limits, simple sample preparation requirements, possibility to obtain structural information and adaptation to analytes of different polarity²⁹. Bioanalytical method validation is a series of steps to ensure an analytical method is appropriate for the quantitative determination of analytes. The main validation criteria were accuracy, selectivity, precision, linearity, recovery, robustness, stability and range. The primary objective of the validation was to demonstrate the analytical process was fit for the intended purpose. The ICH Q2 (R1) guideline is one of the most extensively used international standards for validating analytical methods in the medical and pharmaceutical sciences³⁹. Regulatory bodies like the FDA and EMA have also released more thorough bioanalytical-specific guidelines under the titles "Guidance for Industry: Bioanalytical Method Validation" and "Guidelines on Bioanalytical Method Validation"^40,41. Parameters including matrix effects, carryover, and dilution integrity have been highlighted in recent advancements in validation procedures.

Another crucial component of bioanalytical research is the evaluation of analyte stability under various experimental settings. The preparation of investigational new drug applications (INDs), new drug applications (NDAs), abbreviated new drug applications (ANDAs), and related supplements incorporating clinical pharmacology, bioavailability, bioequivalency, and pharmacokinetic studies is aided by these regulatory recommendations.

Selection and Optimisation of Internal Standard

Internal standardization compensates for differences in matrix effects, extraction efficiency, and instrument responsiveness by adding a structurally comparable molecule at a predetermined concentration to every sample^24,25. To avoid detection issues in bioanalytical testing, ISs must be kept away from untreated samples. For easy identification and detector compatibility, the chosen ISs should have a steady and distinctive structure. To evaluate the quality of separation, they should ideally elute after the analyte⁵. To guarantee comparable extraction efficiency, retention time, stability, and detection responses, the IS and analyte should share structural similarities. Additionally, by decreasing the need for reanalysis and correcting both systematic and random fluctuations, ISs aid in the reduction of measurement errors. They also assist in maintaining peak shape, identifying analytical difficulties, and guaranteeing precise retention time measurements^26,27.

Pioglitazone is often measured using high-performance liquid chromatography (HPLC) because of its accuracy, reliability, and compatibility with various biological matrices²¹. Despite these advantages, aberrations including matrix interference, inconsistent sample preparation, and detector variability can impact HPLC experiments. In order to solve these problems, internal standards (ISs) are essential. Stable isotope-labelled ISs or structurally related compounds serve as surrogates that closely mimic the retention, extraction, and analyte detection properties. Early IS introduction during protein precipitation, liquid-liquid extraction, deproteinization, or solid phase extraction improves recovery and lowers variability. Complex treatments are further stabilized and signal suppression is reduced by advanced processes, such as automated ASTED dialysis combined with SPE. By correcting response variations and increasing signal-to-noise ratios, ISs also enhance the performance of detection systems, such as UV, PDA, fluorescence, and MS. In complicated matrices like plasma, urine, and tissue, where endogenous chemicals may obstruct drug quantification, their significance is especially clear. Linearity and repeatability are strengthened by careful IS selection, which guarantees constant retention and peak area ratios.

This section focuses on HPLC methods for the analysis of pioglitazone and emphasizes how ISs enhance assay performance. The selection criteria for ISs, their impact on chromatographic separation, and the most effective ways to integrate ISs across detection modes are all covered. It also outlines how optimized IS strategies improve the sensitivity, accuracy, and stability of calibration curve.

The most suitable internal standard for RP-HPLC bioanalysis of pioglitazone was rosiglitazone. It is frequently used because it is in the same thiazolidinedione class and thus has similar extraction, polarity, and chromatographic properties. The similarity in structure largely eliminates the variability due to the preparation of the sample and the instrumental analysis. Nonetheless, a number of structurally unrelated substances have also been used because they offer reliable recovery, suitable retention behavior, and sufficient UV absorbance while remaining well separated from the analyte peak. Availability, chromatographic compatibility, and the lack of endogenous plasma component interference are the criteria used to choose them. The HPLC methods, including sample preparation methods, chromatographic conditions, LLOQ, and IS applications across biological matrices, are compiled in Table:1.

Table:1 Internal standards reported for RP-HPLC based bioanalysis of Pioglitazone Hydrochloride

METHOD DEVELOPMENT

The establishment of an analytical method requires the development of a process that allows the identification and quantification of the target molecule in a sample matrix. Since a compound can be analysed by a number of techniques, the technique best suited to a particular application has to be selected according to a number of parameters such as chemical properties of the analyte, range of concentration, nature of the sample matrix, cost and speed of analysis, qualitative or quantitative measurement, required precision, and equipment available. The analytical chain is the process of developing a technique. It involves sampling, sample preparation, separation, detection and interpretation of the result.

Usually reversed phase C18 columns (e.g. Hypersil ODS, Inertsil C18, Apollo C18) or C8 columns (eg: Nova-Pak C8) and mobile phases combining phosphate or acetate buffers with MeOH or ACN or water pH of which was adjusted using glacial acetic acid, perchloric acid based on the pKa of the drug of choice³⁷. Flow rates were between 1.0 and 1.5 ml min−1. Detection depends on the required sensitivity. UV and PDA detectors are commonly used (220-269nm).

HPLC-UV techniques combined with protein precipitation or liquid-liquid extraction are dependable and useful methods that provide adequate sensitivity, precision and extraction recovery for high dose levels and regular treatment monitoring.  For more difficult bioanalytical applications, especially those involving low analyte concentration or complex biological matrices, solid phase extraction in conjunction with automated HPLC systems or mass spectrometric detection offers improved analytical performance.  Despite these benefits, HPLC is not widely used in tissue level studies and advanced metabolite characterization.  There is also a growing need for more integrated imaging based approaches with chromatographic procedures and standardized, high-throughput analytical workflows. Increasing the use of HPLC in antidiabetic medication bioanalysis would necessitate developing multianalyte analytical platforms, optimising matrix specific extraction techniques etc., Optimizing matrix-specific extraction techniques, creating multi-analyte analytical platforms, and integrating pediatric-focused approaches are all necessary to increase the importance of HPLC in antidiabetic medication bioanalysis. Continued advancements in sample preparation strategies and internal standard design are essential for preserving the clinical and regulatory significance of HPLC in the rapidly evolving field of diabetes pharmacotherapy.

Sample collection

Blood, plasma, urine, and serum are common biological samples utilized for analyte assays. Depending on the test sensitivity and study needs, blood is often drawn from human volunteers via venipuncture using a hypodermic syringe in amounts of roughly 5–7 ml. Tubes containing anticoagulants, like heparin or EDTA, are filled with the blood. After that, plasma was separated using centrifugation at 4000 rpm for 15 minutes, yielding between 30 and 50 percent of the initial sample volume⁶.

Sample preparation

The analytical procedure includes an important step, sample preparation. This includes the techniques used for the isolation and concentration of analytes from complex biological matrices such as blood, serum, and tissues. Common interfering chemicals present in biological samples may complicate the detection and quantification of the target analytes. Hence, suitable sample preparation methods are needed to remove contaminants, extract analytes, and enhance detectability to achieve reliable and consistent results^7,8. The demand for efficient, environmentally friendly sample preparation techniques is rising. The extraction methods using solvents such as protein precipitation extraction (PPE), solid-liquid extraction (SLE) and liquid-liquid extraction (LLE) were very advantageous for the bioanalytical applications of pioglitazone.. These techniques have reduced the cost of drug development and increased interest in pharmaceutical production⁹. Automation can lead to higher sample throughput, which improves accuracy and generates less hazardous waste. The main goal of sample preparation before analysis is to concentrate and purify the analyte³⁰. This reduces the likelihood of chemicals interfering with the analysis, the chromatographic column or the detector. The fastest and cheapest method for pioglitazone analysis is protein precipitation, but in terms of sample purification and analytical performance, liquid-liquid extraction and solid phase extraction are better. Due to the high protein binding (>99%) of pioglitazone, simple protein precipitation using acetonitrile (ACN) or methanol (MeOH) resulted in a strong matrix effect as compared to liquid-liquid extraction (LLE) and solid-phase extraction (SPE). However, due to these problems protein precipitation extraction (PPE) still remains the method of choice for routine analysis and for pioglitazone samples at high concentrations. It is low cost, easy to operate and requires minimum sample preparation. Due to its lipophilic nature, liquid-liquid extraction is also often used to efficiently isolate pioglitazone from the aqueous plasma phase into organic solvents. This method yields better chromatograms because it separates pioglitazone and several polar components of the matrix remain in the aqueous phase. Another consequence is the decrease of limits of detection (LOD) and quantification (LOQ), crucial for pharmacokinetic and bioequivalence evaluations. If lower levels of quantification and regulatory investigations are needed then SPE is the method of choice. SPE is a method that efficiently separates medicines from plasma components using a cartridge. Table :2 lists the various sample preparation strategies employed for the bioanalysis of pioglitazone using RP-HPLC.

Table no:2 Sample reparation strategies employed for bioanalytical estimation of Pioglitazone Hydrochloride – A Comparative overview

BIOANALYTICAL METHOD VALIDATION (BMV)

The validation of a bioanalytical procedure aims to demonstrate the performance characteristics and reliability of the method. This helps establish confidence in the results. As Shah et al have mentioned, all bioanalytical methods must be validated if the results are to be used to support the registration of a new drug or the reformulation of an existing drug. Remember that the first validation is just the beginning. When a method is in use, it is necessary to monitor it to verify

that it is functioning as originally validated^8,31. Validation is the demonstration of the suitability and robustness of the performance characteristics of a method for the intended analytical applications, by means of documentation supported by specific laboratory experiments.

Need for bioanalytical method validation

Bioanalytical method validation is conducted to demonstrate that an analytical method is reliable, accurate, precise and fit for the intended purpose for the quantification of pharmaceuticals and their metabolites in biological matrices such as plasma, serum, urine or tissues.

• To obtain dependable and interpretable results, it is vital to employ bioanalytical methods that are thoroughly characterized and fully validated.
• Bioanalytical techniques are constantly evolving and improving, representing the forefront of technological advancement.
• Each approach has unique characteristics and each is dependent on the analyte. Each molecule may require a different set of validation criteria.
• The applicability of the method also depends on the overall goal of the study. When the analyses are carried out in different sites the validation must be carried out in each site with proper documentation to assure consistency and inter laboratory dependability³².

Various important parameters that need to be validated includes selectivity, accuracy, precision, linearity and range, limit of detection, limit of quantification, recovery, robustness and stability.

Table: 3 Validation parameters¹⁰

The comparison of the published RP-HPLC methods for quantification of Pioglitazone in various biological samples shows that the validated methods fulfill the regulatory standards of bioanalytical method validation. Most of the methods exhibited good linearity (correlation coefficient > 0.99) over the individual calibration ranges indicating the accurate quantification of the analyte over wide range of concentration. The accuracy was within acceptable limits of nominal concentrations and the intra-day and inter-day precision values are typically < 15 indicating good reproducibility and reliability. The main variations in the reported procedures were in the sample preparation methods and detection systems. The stability experiments consistently demonstrated the stability of Pioglitazone in different biological samples under various bench top, freeze thaw, autosampler and long term storage conditions, suggesting the suitability of the methodologies for pharmacokinetic and bioequivalence studies. Overall, most of the reported RP-HPLC methods employed for the bioanalysis of Pioglitazone showed comparable validation performance, with variations mainly arising from extraction procedures, chromatographic conditions and analytical sensitivity rather than from fundamental differences in method reliability. Table: 4 depicts the summary of validation parameters reported for RP-HPLC based bioanalysis of Pioglitazone.

Table:4 Comparative summary of validation parameters of reported RP-HPLC methods for the bioanalysis of Pioglitazone in different biological samples