Recent Advances in Bioanalytical Techniques for the Analysis of Phytoconstituents: A Review

Rohini Pargonda Patil¹, Shailendra Sanjay Suryawanshi¹*, Mahesh Palled²

¹Department of Pharmaceutical Chemistry, KLE College of Pharmacy, Belagavi,

KLE Academy of Higher Education and Research, Belagavi, Karnataka, India.

²Department of Pharmaceutical Analysis, KLE College of Pharmacy, Belagavi,

KLE Academy of Higher Education and Research, Belagavi, Karnataka, India.

*Corresponding Author E-mail: shailendrasuryawanshi@klepharm.edu

ABSTRACT:

Medicinal plants have long been a valuable source of bioactive compounds with therapeutic potential. The bioactive compounds present in these plants continue to be a fundamental resource for new drug development. This article reviews various bioanalytical methods utilized for the analysis of phytochemicals. It highlights the significance of creating and validating reliable analytical methods for detecting and measuring phytochemicals across various biological samples, including plasma, blood, urine, and serum. The primary aim is to support researchers in applying these methods effectively for accurate quantification in biological samples. Bioanalytical method validation ensures that the techniques for quantifying phytochemicals are reliable, consistent, and reproducible. Various techniques, such as protein precipitation, solid-phase extraction, and liquid-liquid extraction, can be used for sample preparation to isolate and concentrate analytes from biological matrices. Following extraction techniques like high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gas chromatography (GC) are used to analyze and quantify phytochemicals. This review explores bioanalytical methods for detecting and measuring medicinal plant phytochemicals, offering guidance on developing precise and reliable techniques for various biological matrices that are applicable in pharmacokinetic studies.

KEYWORDS: Bioanalysis, Method development and validation, Phytochemicals.

INTRODUCTION:

BACKGROUND:

Bioanalysis, a branch of analytical chemistry, focuses on the quantitative measurement of drugs or analytes in biological systems. It is crucial in drug development, clinical trials, and therapeutic drug monitoring¹. Bioanalysis focuses on quantifying active analytes and their metabolites in biological matrices such as blood, plasma, tissue, serum, and cerebrospinal fluid. Bioanalytical methods in pharmaceuticals aid in evaluating pharmacokinetics, pharmacodynamics, toxicokinetics, bioavailability, and therapeutic monitoring². According to the USFDA, the purpose of bioanalytical method development and validation is essential for sample analysis³. Using validated bioanalytical methods is essential for obtaining reliable and interpretable results⁴. Biomarkers have been used in clinical medicine for decades. It is one of the most used methods in clinical settings due to its easy availability, affordable, effectiveness and low toxicity⁵. Herbs are naturally occurring plant-based substances that are used to treat ailments in local healing practices. These products are a complex mixture of organic composed of a variety of medicinal components that are beneficial to the human body. The chemical constituents present in the plant affect the effectiveness and efficiency of the herbal preparations^6-7. In analytical chemistry, drug analysis is useful for the isolation, estimation, and quantification of compounds derived from both natural and synthetic sources. Trends in analytical chemistry are necessary to overcome errors in analytical method development. Various analytical techniques such as spectroscopy and chromatography, are used⁸. The ultraviolet-visible spectrophotometer measures how a substance in solution absorbs UV light⁹. HPLC is a widely used technique in analytical chemistry for separating mixtures of substances. It facilitates the detection, measurement, and isolation of specific components within mixtures¹⁰. Analytical method development involves creating a procedure to identify and quantify a compound in a sample. The selection of a method is influenced by factors such as the chemical characteristics of the analytes, their concentration, cost, analysis speed, sample matrix, measurement type, and precision¹¹.

Sample collection and preparation:

The analyte content is commonly found in various biological fluids. The blood is drawn via venipuncture and collected in anticoagulant-treated tubes. Plasma is separated by centrifugation, yielding 30-50 % of the volume. Protein precipitation, Liquid-liquid extraction and solid-phase extraction are used for sample preparation aims to purify and concentrate the sample.

Liquid-Liquid Extraction (LLE):

It is based on the principles of differential solubility and analyte molecule partitioning equilibrium in the aqueous (original sample) and organic phases. Liquid extraction is the process of extracting a material from one liquid phase into another. Traditional LLE procedures have been replaced by more advanced and improved methods such as liquid phase micro extraction, single drop liquid phase micro extraction, and supported membrane extraction.

Solid Phase Extraction (SPE):

Solid-phase extraction is a targeted sample preparation technique where the analyte binds to a solid support, unwanted substances are removed, and the analyte is selectively isolated. The process consists of 4 steps: conditioning, sample loading, washing, and elution.

Protein Precipitation (PP):

Precipitation alters protein solubility using organic modifiers, salts, or pH changes. After centrifugation, the supernatant is injected into an HPLC or evaporated for concentration. It is faster than solid-phase extraction but less selective, with risks of protein residues and interference in RP-HPLC. Methanol is preferred for clarity, while salt-induced precipitation causes proteins to aggregate as salt levels increase.¹¹

Validation Parameters:

Table 1 indicates that the US Food and Drug Administration (USFDA) provides guidelines for validating bioanalytical methods.

Table 1: US FDA guidelines for bioanalytical method validation ¹²

Parameters	US FDA guidelines
Selectivity	Blank samples of relevant biological matrix should be gathered from at least six distinct sites before analysis. It is critical to assess the interference and selectivity of each blank at the LLOQ level.
Accuracy	To ensure accuracy, six different measurements are necessary per concentration. To determine accuracy, utilize at least three concentrations within the anticipated concentration range. LLOQ allows for up to 20% variance, however the mean must be within ±15% of the actual value. The degree of accuracy is assessed by how far the mean values differ from actual values.
Precision	To ensure accuracy, it is advised to do at least five determinations per concentration. Three concentrations within the expected concentration range should be considered at a minimum. Except the LLOQ, where the precision should not exceed 20% of the CV, the CV should not be greater than 15% at all concentration levels.
Recovery	Recovery experiments should be carried out at lower, medium, and higher concentrations, with unextracted standards standing in for 100% recovery.
LOQ	The reaction to the analyte should be five times that of the blank response. A recognizable, distinct, and reproducible analyte peak with 20% precision and 80-120% accuracy is required.
LOD	The smallest amount of analyte that can be detected but not measured. The determination of the LOD is subject to misinterpretation since some bioanalytical laboratories simply measure the lowest quantity of a reference solution that can be detected, while others assess the lowest concentration that can be identified in a biological sample34. There is general agreement that the LOD should represent the least detectable amount or concentration of the analyte of interest.
Freeze–thaw stability	Three freeze-thaw cycles must be used to assess analyte stability. Thaw at room temperature after 24 hours of storage at the prescribed temperature; divide into low and high concentration aliquots. Once fully thawed, refreeze for another 12 to 24 hours. Before analyzing the third cycle, this cycle must be repeated twice more. The third cycle should include an examination of the standard deviation of error after two additional rounds.
Short-term stability	After defrosting at room temperature, three separate portions of lower and higher concentrations must be kept at room temperature for four to twenty-four hours before analysis. The percentage variance should not be more than 15%.
Long-term stability	Under the same conditions as the research samples, at least three aliquots of each concentration were collected. Examine three separate cases. Make sure the storage time is longer than the time it takes to collect the first sample and analyze the last one.

LITERATURE REVIEW ON BIOANALYTICAL METHODS FOR PHYTOCHEMICALS:

Sandhu P et al.,¹³ have established a precise and sensitive QbD approach method for quercetin dehydrate by HPLC method. A C18 column was used for separation with a mobile phase of acetonitrile and ammonium acetate buffer (35:65 v/v, pH 3.5, 0.1% acetic acid), a flow rate of 0.7ml/min, and UV detection at 237nm.

Bhandari R et al.,¹⁴ have analyzed drug naringenin using a Waters HPLC system equipped with a photodetector, and the results were processed with the Waters Empower® system. This chromatographic separation was performed on a Waters®, Sunfire®, RP-18 filter unit using methanol and 0.5% OPA (70:30) as a mobile phase. The instrument utilizes a PDA detector with a 200-400nm wavelength range and records the chromatogram at 289nm.

Kazuo I et al.,¹⁵ have performed HPLC to determine of rutin in human plasma for pharmacokinetic studies. Oasis MAX catridges, possessing both reverse-phase and anion exchange properties, were used for solid-phase extraction. Ammonium acetate, EDTA, acetonitrile, and glacial acetic acid in a 16.5:82:0.5:1 (v/v) ratio at pH 3.8 was used, with a flow rate of 0.3ml/min. A preliminary study involving a healthy volunteer who received a 500 mg oral dose of rutin confirmed the method’s suitability for plasma rutin analysis.

Kumar R et al.,¹⁶ have carried out an HPLC method to determine fisetin in rat plasma utilizing a C-18 column. A 30:70 blend of acetonitrile and 0.2% OPA was used as the mobile phase, with detection set at 362nm and a flow rate of 1ml/min.

Singh G et al.,¹⁷ have developed a method for resveratrol quantification in human plasma. Phenomenex C18 column was used for separation in isocratic mode with UV detection at 306nm. The mobile phase, a methanol-phosphate buffer mix, flowed at 1.0ml/min, ensuring accuracy and precision.

Alvi S et al.,¹⁸ have designed an HPLC method to measure caffeine in human plasma using synthetic plasma for calibration. After deproteination with perchloric acid, caffeine and antipyrine were separated on a Waters Atlantis C18 column with a mobile phase of 15Mm potassium phosphate (pH3.5) and acetonitrile (83:17, v/v). Detection was performed at 274nm for accurate monitoring in healthy volunteers.

Sangkasat A et al.,¹⁹ have performed a method for determining melatonin in human plasma using HPLC-fluorescence detection. It involves liquid-phase extraction with dichloromethane, internal standard. Melatonin formulations in rats will be analyzed using HPLC separation on a C18 column with a mobile phase of 25% acetonitrile and phosphate buffer (pH 7.0) at a 1ml/min flow rate for pharmacokinetic studies.

Mane V et al.,²⁰ have performed and validated a cost-effective and reliable bioanalytical technique to quantify gallic acid in rat plasma using RP-HPLC with a gradient elution method. HPLC analysis was conducted on a Zorbax SB C18 column with a mobile phase of water containing 0.1% formic acid and acetonitrile with 0.08% formic acid. The method utilized a 1.0ml/min flow rate and PDA detection at 271nm. The method demonstrated accuracy, precision, and stability, with gallic acid remaining stable under various conditions.

Sharma P et al.,²¹ have developed an HPLC-UV method to quantify Asiatic acid in Wistar rat blood. Extraction used a solvent mixture, separation occurred on an RP-C18 column with a water-methanol gradient, and detection was at 205nm, ensures sensitive, reproducible, and stable for 14 days at -20°C.

Konam K et al.,²² have performed a specific method for Kaemferol in rat plasma used ibuprofen as an internal standard.  Extraction with acetonitrile ensured high recovery. Separation on a C18 column with a phosphate buffer-acetonitrile mobile phase enabled detection at 370 nm, with an 8.07min retention time. The method was applied to pharmacokinetic studies after a 50ng/kg oral dose.

Cen M et al.,²³ have developed an optimized HPLC-UV method for the sensitive quantification of isochlorogenic acid A in rat plasma. Shodex C18column with a mobile phase of 0.1% phosphoric acid in water and methanol (50:50, v/v) at a flow rate of 1.0ml/min was used for study. The method exhibited strong linearity within the 0.04-40 μg/ml range and was effectively used for pharmacokinetic studies in rats.

Kumar M et al.,²⁴ have developed a rapid RP-HPLC method using protein precipitation for morin estimation in human plasma. Separation on a C18 column used a phosphate buffer-acetonitrile mobile phase, with UV detection at 260nm. The validated method showed linearity (100-500ng/ml) and was applied to pharmacokinetic studies.

Mendes N et al.,²⁵ have analyzed UENF 1613 C. and validated the HPLC-based bioanalytical method to quantify chlorogenic acid. The results showed selectivity for chlorogenic acid in the extract of C. baccatum. The relative standard deviation and precision, including repeatability and intermediate precision, were within the acceptable limit of 15%. The phenolic compound showed a recovery between 77% and 93%. The validated method, employing external standardization, was effectively used to quantify chlorogenic acid in the UENF accession.

Harish V et al.,²⁶ have developed and validated an RP-HPLC method for XH quantification in rat plasma using curcumin as an internal standard. Isocratic elution with a 15:85 mobile phase at 0.8ml/min ensured a 10min run time. Protein precipitation was used for extraction, and stability studies confirmed accuracy, making the method suitable for plasma analysis.

Mane V et al.,²⁷ have developed a robust RP-HPLC method for quantifying Epigallocatechin 3- gallate in Wistar rat plasma. Using a Zorbax SB C18 column with a formic acid-water/acetonitrile mobile phase, the method showed stability and suitability for pharmacokinetic and bioequivalence studies.

Pozharitskaya O et al.,²⁸ have performed the RP-HPLC method for Taxifolin pharmacokinetic analysis in rabbit plasma using biochanin A as an internal standard. Taxifolin was extracted via liquid-liquid extraction after enzymatic hydrolysis. A Luna C18 column was used for separation with gradient elution and detection at 290nm. The method was applied to pharmacokinetic studies, showing an oral bioavailability of 36% for taxifolin in a liquid solution.

Satyavert G et al.,²⁹ have developed an RP-HPLC-UV method for quantifying hydrazinocurcumin in rat plasma and organs. An octadecylsilane column was used for separation with an isocratic mobile phase of methanol, acetonitrile, and water. The method indicates excellent linearity within the concentration range of 0.05 to 5 µg/ml, with detection observed at 332nm and 380nm. This method demonstrated stability and suitability for pharmacokinetic and organ distribution studies.

Jadhav V et al.,³⁰ have established a reliable HPLC-UV method for quantifying artemether in mouse plasma, employing artemisinin as the internal standard. The method exhibited strong linearity, with an RSD of 1.8% and dependable system suitability.

Mallu U et al.,³¹ have carried out a rapid and efficient RP-HPLC method for quantifying Vinorelbine (VNRB) in human plasma. VNRB was extracted using liquid-liquid extraction with methanol, reconstructed in the mobile phase, and analyzed with Paclitaxel. Validation was carried out as per USFDA bioanalytical guidelines, demonstrating simplicity, precision, accuracy, and no plasma matrix interference.

D’cruz D et al.,³² have established a fast and precise RP-HPLC method to quantify ticagrelor in human plasma using diethyl ether for protein precipitation, with separation achieved via a 60:40 acetonitrile-methanol mobile phase at 1ml/min and detection at 254nm.

Summary: Bioanalytical method validation is crucial to confirm the reliability of test results. A summary of bioanalytical methods for phytochemicals is shown in Table 2.

Table 2: Summary of Bioanalytical Methods for Phytochemicals

Title of paper	Type of matrix used	Phyto-constituent	Mobile Phase	Flow rate ml/min	Column
Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate	Rat plasma	Quercetin dihydrate	acetonitrile and ammonium acetate buffer (35:65) v/v	0.7 ml/min	C18
Development of a new, sensitive, and robust analytical and bio-analytical RP-HPLC method for in-vitro and in-vivo quantification of naringenin in polymeric nanocarriers	Rat plasma	Naringenin	methanol and 0.5% ortho-phosphoric acid (70:30)	1ml/min	C18
Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection	Human plasma	Rutin	ammonium acetate solution containing 10 mM acetonitrile and 0.3 mM EDTA, glacial acetic acid (16.5:82.5:1, v/v,)	0.3 ml/min	Luna ODS-2 column
Development and validation of RP-HPLC method for estimation of fisetin in rat plasma	Rat plasma	fisetin	Acetonitrile and orthophosphoric acid (30:70)	1ml/min	C18
Development and validation of a HPLC method for the determination of trans-resveratrol in spiked human plasma	Human plasma	Resveratrol	methanol: phosphate buffer	1ml/min	Phenomenex C18
Validated HPLC method for determination of caffeine level in human plasma using synthetic plasma: application to bioavailability studies	Human plasma	Caffeine	potassium phosphate and acetonitrile (83:17, v/v)	-	Waters Atlantis C18
Validation of method for determination of melatonin in human plasma by HPLC-fluorescence detector	Human plasma	Melatonin	25% acetonitrile/phosphate buffer	1ml/min	HiQ Sil C18 V
A Novel RP-HPLC Gradient Elution Technique For Bioanalytical Method Development And Validation For Estimating Gallic Acid In Wistar Rat Plasma	Rat plasma	Gallic Acid	0.1 % formic acid (A): acetonitrile (ACN) with 0.08 % formic acid (B).	1ml/min	Zorbax SB C18
Bioanalytical HPLC method development and validation for quantification of asiatic acid from centella asiatica linn	Rat plasma	Asiatic acid	water, methanol	-	C18
Bioanalytical method development and reaction rate study of kaempferol in albino rats plasma using RP-HPLC method	Rat plasma	Kaempferol	Potassium dihydrogen Phosphate buffer (0.05M, pH 2.1) and acetonitrile (65: 35 v/v) with 0.1% trimethylamine	1.5 ml/min	Lichrocart C18
Development and validation of a HPLC method for determination of isochlorogenic acid a in rat plasma and application to pharmacokinetic study	Rat plasma	Isochlorogenic acid	0.1% phosphoric acid aqueous solution (solvent A) and methanol (solvent B) (50:50, v/v)	1ml/min	Shodex C18
Method development and validation of Bio Flavanoid-Morin Hydrate by RP-HPLC in human plasma	Human plasma	Morin Hydrate	potassium dihydrogen phosphate (pH 5.0) and acetonitrile (60:40, v/v)	1ml/min	C18
Bioanalytical method validation for the quantification of the chlorogenic acid in Capsicum baccatum through High Performance Liquid Chromatography	-	Chlorogenic acid	phosphoric acid (pH 3.2) and acetonitrile	1ml/min	Macherey-Nagel RP-18
Bioanalytical Method Development, Validation and Stability Assessment of Xanthohumol in Rat Plasma	Rat Plasma	Xanthohumol	0.1% v/v OPA (A): Methanol (B)  15:85 v/v	0.8 ml/min	C18
Development and Validation of a Novel Bioanalytical Method for Estimating Epigallocatechin 3 Gallate in Wistar Rat Plasma by RP-HPLC Employing Gradient Elution Techniques	Rat Plasma	Epigallocatechin 3 Gallate	0.1 percent formic acid (A) and acetonitrile (ACN) with 0.08% formic acid (B)	1ml/min	Zorbax SB C18
Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography	Rabbit plasma	Taxifolin	Acetonitrile and 0.03% water solution of trifluoroacetic acid	1ml/min	Luna C18
Development and validation of bioanalytical method for the determination of hydrazinocurcumin in rat plasma and organs by HPLC-UV	Rat plasma	Hydrazinocurcumin	Methanol-acetonitrile- water (36:27:37 v/v)	1ml/min	Inertsil-ODS-3V
Development and validation of HPLC-UV method for quantification of artemether in plasma	Plasma	Artemether	ACN: water 70:30 v/v.	1ml/min	C18
Development and validation of a simple high-performance liquid chromatography–UV method for the determination of vinorelbine-a chemotherapeutic drug in spiked human plasma	Human plasma	Vinorelbine-a	Acetate buffer and methanol 85:15 (v/v)	1ml/min	Kromasil ® C18
Bioanalytical method development and validation of ticagrelor by RP-HPLC.	Human plasma	Ticagrelor	Acetonitrile and methanol (60:40) % v/v.	1ml/min	Phenomenex C18

CONCLUSION:

Developing and validating bioanalytical methods for phytoconstituents is essential to ensure the accuracy and reliability of analytical results, particularly in natural products and herbal medicine. These methods are vital for accurately quantifying and identifying active compounds within complex mixtures, which is necessary for quality assurance, pharmacokinetic analysis, and therapeutic monitoring. The development process involves choosing suitable techniques, optimizing extraction methods, and refining analytical conditions to maximize sensitivity and specificity. Validation, on the other hand, confirms that the method is consistent, accurate, and resilient across various conditions. As the research into phytoconstituents expands, the significance of robust bioanalytical methods will grow, playing a pivotal role in the progress of Natural product research and innovation.

CONFLICT OF INTEREST:

The authors confirm that they have no financial or personal conflicts of interest that could have influenced the work presented in this paper.

ACKNOWLEDGMENTS:

I thank KAHER KLE College of Pharmacy, Belagavi, for providing the necessary support to carry out this study

REFERENCES:

1. Tijare L, Rangari N, Mahajan U. A review on bioanalytical method development and validation. Asian J Pharm Clin Res. 2016; 9(3): 6-10.

2. Deshpande M, Kasture V, Mohan M, Chavan M. Bioanalytical Method Development and Validation: Recent Adv. Anal. Chem. 2019 Apr 10; 97.

3. Meesters R, Voswinkel S. Bioanalytical method development and validation: from the USFDA 2001 to the USFDA 2018 guidance for industry. J. Appl. Bioanal. 2018 Jul 19; 4(3): 67-73.

4. Saudagar R. B. and Thete P. G. Bioanalytical Method Validation: A Concise Review., Asian J. Res. Pharm. Sci. 2018; 8(2): 107–114

5. Bodaghi A, Fattahi N, Ramazani A. Biomarkers: Promising and valuable tools towards diagnosis, prognosis and treatment of Covid-19 and other diseases. Heliyon. 2023 Feb 1; 9(2).

6. Sam S. Importance and effectiveness of herbal medicines. J, Pharmacogn Phytochem, 2019, 8(2), 354–357.

7. U. Venkatesh, V. Ramana. A Review UV Method Development and Validation. Asian J. Pharm. Res. 2023; 364-73.

8. Sharma S, Singh N, Ankalgi A, Rana A, Ashawat M. Modern trends in analytical techniques for method development and validation of pharmaceuticals: a Review. J. Drug Deliv. Ther. 2021 Feb 15; 11(1-s):121-30.

9. Bhavyasri K, Surekha T, Rambabu D. Bioanalytical Method Development and Validation of Atorvastatin in Human Plasma by Using UV-Visible Spectrophotometry. Int. J. Pharm. Sci. Res. 2019 Jun 1; 11(6): 2243-6.

10. Kumar S, Kumar D. Importance of RP-HPLC in analytical method development: a review. Int. J. Pharm. Sci. 2012 Dec 1; 3(12): 4626.

11. Kirthi A, Shanmugam R, Prathyusha M, Basha D. A review on bioanalytical method development and validation by RP-HPLC. J. global trends Pharm. Sci. 2014; 5(4): 2265-71.

12. S. Shaharwale1, N. Shinde. A review on bioanalytical method development and validation by RP-HPLC. Eur. Chem. Bull, 2022: 1030-1040.

13. Sandhu P, Beg S, Kumar R, Katare O, Singh B. Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate. J. Liq. Chromatogr. Relat. Technol. 2017 Jun 15; 40(10): 506-16.

14. Bhandari R, Kuhad A, Paliwal J, Kuhad A. Development of a new, sensitive, and robust analytical and bio-analytical RP-HPLC method for in-vitro and in-vivo quantification of naringenin in polymeric nanocarriers. J. Chromatogr. B Biomed. Appl. 2019 Dec; 10: 1-4.

15. Ishii K, Furuta T, Kasuya Y. Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection. J. Chromatogr. B Biomed. Appl. 2001 Aug 5; 759(1): 161-8.

16. Kumar R, Kumar R, Khursheed R, Awasthi A, Khurana N, Singh SK, Khurana S, Sharma N, Gunjal P, Kaur J, Corrie L. Development and validation of RP-HPLC method for estimation of fisetin in rat plasma. S Afr J Bot. 2021 Aug 1; 140: 284-9.

17. Singh G, Pai R, Pandit V. Development and validation of a HPLC method for the determination of trans-resveratrol in spiked human plasma. J. adv. pharm. Technol Res. 2012 Apr 1; 3(2): 130-5.

18. Alvi SN, Hammami MM. Validated HPLC method for determination of caffeine level in human plasma using synthetic plasma: application to bioavailability studies. J Chromatogr Sci. 2011; 49(4): 292–296.

19. Sangkasat A, Johns N, Priprem A. Validation of method for determination of melatonin in human plasma by HPLC‑fluorescence detector. Laos J Sci. 2011; 2: 53–57.

20. Mane V, Killedar S, More H, Gaikwad A, Tare H. A novel RP‑HPLC gradient elution technique for bioanalytical method development and validation for estimating gallic acid in Wistar rat plasma. Int J Appl Pharm. 2023; 15(2): 153–160.

21. Sharma PD, Surana SJ, Jadav RB, Patel PH. Bioanalytical HPLC method development and validation for quantification of asiatic acid from centella asiatica linn. Int J Pharm Sci Rev Res. 2011; 10(2): 46-50.

22. Konam K, Chinta R, Lukaraju P, Bonthu M, Pottabathula H. Bioanalytical method development and reaction rate study of kaempferol in albino rats plasma using RP-HPLC method. Int. J. Biol. Res. 2012; 3(2): 197-203.

23. Cen M, Liang H, Xiong X, Zeng J, Cheng X, Wang S. Development and validation of a HPLC method for determination of isochlorogenic acid A in rat plasma and application to pharmacokinetic study. J Chromatogr Sci. 2017; 55(10): 1037–1042.

24. Kumar M, Muralidharan S. Bioanalytical method development and validation of griseofulvin nanoparticles using RP‑HPLC. J Young Pharm. 2015; 7(4 Suppl): 46–50.

25. Mendes N, Pereira S, Arantes M, Glória L, Nunes C, Passos M, Vieira I, de Moraes L, Rodrigues R, Oliveira D. Bioanalytical method validation for the quantification of the chlorogenic acid in Capsicum baccatum through High Performance Liquid Chromatography (HPLC-DAD). Food Chemistry. 2020 Sep 30; 325: 126929.

26. Harish V, Almalki WH, Alshehri A, Alzahrani A, Alzarea SI, Kazmi I, Gulati M, Tewari D, Chellappan DK, Gupta G, Dua K. Bioanalytical method development, validation and stability assessment of xanthohumol in rat plasma. Molecules. 2022 Oct 21; 27(20): 7117.

27. Mane V, Killedar S, More H, Salunkhe S, Tare H. Development and Validation of a Novel Bioanalytical Method for Estimating Epigallocatechin 3 Gallate in Wistar Rat Plasma by RP-HPLC Employing Gradient Elution Techniques. J Res Pharm. 2023 May 1; 27 (3): 1039-55.

28. Pozharitskaya O, Karlina M, Shikov A, Kosman V, Makarova M, Makarov V. Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography. Phytomedicine. 2009; 16 (3–4): 244–251.

29. Satyavert S, Nair A, Attimarad M. Development and validation of a bioanalytical method for the determination of hydrazinocurcumin in rat plasma and organs by HPLC‑UV. J Chromatogr B Analyt Technol Biomed Life Sci. 2020; 1156: 122310.

30. Jadhav V, Singh K. Development and validation of an HPLC‑UV method for quantification of artemether in mouse plasma using artemisinin as internal standard. J Pharmacogn Phytochem. 2018; 7 (SP6):75–78. doi:10.22271/phyto.2018.v7.isp6.1.17.

31. Mallu U, Anna V, Kasimala B. Development and validation of a simple high-performance liquid chromatography–UV method for the determination of vinorelbine-a chemotherapeutic drug in spiked human plasma. Development. 2019; 12(1).

32. D’cruz D, Babu A, Joshy E, Aneesh TP. Bioanalytical method development and validation of ticagrelor by RP-HPLC. Int J App Pharm. 2017 May 1; 9(3):51-4.

Received on 06.05.2025      Revised on 18.08.2025

Accepted on 24.10.2025 Published on 27.01.2026

Available online from February 02, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(1):51-56.

DOI: 10.52711/2231-5675.2026.00008

©Asian Pharma Press All Right Reserved

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.