Simultaneous quantification of quercetin and rifampicin in bronchoalveolar lavage fluid by HPLC-UV technique and its application to a pharmacokinetic study of investigational dry powder formulation

Year 2025, Volume: 29 Issue: 6, 2508 - 2518, 02.11.2025

Devang Tandel , Kalpana Patel , Vaishali Thakkar Tejal Gandhi , Jinal Tandel

https://doi.org/10.12991/jrespharm.1798001

https://izlik.org/JA96MA55UB

Abstract

A rapid and precise high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed for the quantitative determination of Rifampicin and Quercetin, in bronchoalveolar lavage fluid. The validation process was conducted in compliance with the USFDA guidelines. Separation was achieved using a C18-Luna Phenomenex column (5 μm, 4.60 × 150 mm) with a mobile phase comprising 20 mM disodium hydrogen orthophosphate (pH 4.5) and methanol in a 35:65 %v/v ratio. The retention times for Quercetin and Rifampicin were 5.42 and 8.60 minutes, respectively. Optimal protein precipitation was achieved using 500 μl of acetonitrile and 500 μl of methanol, yielding an extraction recovery exceeding 80 % for both drugs from bronchoalveolar lavage fluid. The extraction method yielded clean samples, rendering the process both straightforward and cost-effective. The method exhibited a strong linear correlation (r > 0.9995) between the peak area ratio and the concentrations for Rifampicin (50– 5000 ng/ml) and Quercetin (40–4000 ng/ml). Pharmacokinetic analysis revealed that poly(lactic-co-glycolic acid) nanoparticles sustained higher drug concentrations in the lung tissue for an extended period of up to 24 hours. In conclusion, the developed HPLC method, utilizing simple UV detection, offers several advantages, including costeffectiveness, high recovery rates, a broad linear range, robust quantification capability, and short analysis times.

Keywords

Rifampicin , Quercetin , Chromatography , Bioanalysis , Bronchoalveolar lavage fluid

References

• [1]Ughreja R, Bhatt V, Shah S, Boxa D. Prevalence of tuberculosis and drug-resistant tuberculosis in tertiary care rural hospital in Gujarat, India: a retrospective study. Eur J Transl Clin Med. 2023; 6(2): 36–44. https://doi.org/10.31373/ejtcm/175674
• [2]WHO Global tuberculosis report 2023. https://www.who.int/teams/global-tuberculosis-programme/tb-reports/globaltuberculosis-report-2023 (accessed on 10 September 2024).
• [3]Hari BNV, Chitra KP, Bhimavarapu R, Karunakaran P, Muthukrishnan N, Rani BS. Novel technologies: A weapon against tuberculosis. Indian J Pharmacol. 2010; 42(6): 338–344. https://doi.org/10.4103/0253-7613.71887
• [4]Das S, Tucker I, Stewart P. Inhaled dry powder formulations for treating tuberculosis. Curr Drug Deliv. 2015; 12(1): 26–39. https://doi.org/10.2174/1567201811666140716123050
• [5]Khan MF, Rita SA, Kayser MS, Islam MS, Asad S, Rashid R Bin, Bari MA, Rahman MM, Al Aman DAA, Setu NI, Banoo R, Rashid MA. Theoretically guided analytical method development and validation for the estimation of rifampicin in a mixture of isoniazid and pyrazinamide by UV spectrophotometer. Front Chem. 2017; 5:27. https://doi.org/10.3389/fchem.2017.00027
• [6]Campbell IA, Bah-Sow O. Pulmonary tuberculosis: diagnosis and treatment. BMJ. 2006; 332(7551): 1194–1197. https://doi.org/10.1136/bmj.332.7551.1194
• [7]El Khechine A, Drancourt M. Diagnosis of pulmonary tuberculosis in a microbiological laboratory. Med Mal Infect. 2011; 41(10): 509–517. https://doi.org/10.1016/j.medmal.2011.07.012
• [8]Butov D, Zaitseva S, Butova T, Stepanenko G, Pogorelova O, Zhelezniakova N. Efficacy and safety of quercetin and polyvinylpyrrolidone in treatment of patients with newly diagnosed destructive pulmonary tuberculosis in comparison with standard antimycobacterial therapy. Int J Mycobacteriol. 2016; 5(4): 446–453. https://doi.org/10.1016/j.ijmyco.2016.06.012
• [9]Prasad R, Singh A, Gupta N. Role of Bioenhancers in Tuberculosis. Int J Health Sci Res. 2016; 6(6): 307–313.
• [10]Javed S, Ahsan W, Kohli K. The concept of bioenhancers in bioavailability enhancement of drugs – a patent review. J Sci Lett. 2016; 1(3): 143–165.
• [11]Drabu S, Khatri S. Use of Herbal Bioenhancers to Increase the Bioavailability of Drugs. Res J Pharm Biol Chem Sci. 2011; 2(4): 107–119.
• [12]Kesarwani K, Gupta R, Mukerjee A. Bioavailability enhancers of herbal origin: an overview. Asian Pac J Trop Biomed. 2013; 3(4): 253–266. https://doi.org/10.1016/S2221-1691(13)60060-X
• [13]Randhawa GK, Kullar JS, Rajkumar. Bioenhancers from mother nature and their applicability in modern medicine. Int J Appl Basic Med Res. 2011; 1(1): 5–10. https://doi.org/10.4103/2229-516X.81972
• [14]Ajazuddin, Alexander A, Qureshi A, Kumari L, Vaishnav P, Sharma M, Saraf S, Saraf S. Role of herbal bioactives as a potential bioavailability enhancer for active pharmaceutical ıngredients. Fitoterapia. 2014; 97: 1–14. https://doi.org/10.1016/j.fitote.2014.05.005
• [15]Sasikumar K, Ghosh AR, Dusthackeer A. Antimycobacterial potentials of quercetin and rutin against Mycobacterium tuberculosis H37Rv. 3 Biotech. 2018; 8(10): 427–435. https://doi.org/10.1007/s13205-018-1450-5
• [16]Swain SS, Rout SS, Sahoo A, Oyedemi SO, Hussain T. Antituberculosis, antioxidant and cytotoxicity profiles of quercetin: a systematic and cost-effective in silico and in vitro approach. Nat Prod Res. 2022; 36(18): 4757–4761. https://doi.org/10.1080/14786419.2021.2008387
• [17]Vyavahare RD. Stability Indicating RP-HPLC Method for Rifampicin in Bulk and Pharmaceutical Dosage Form. International journal pharmacy of pharmaceutical research. 2017; 10(4): 265–282.
• [18]Shah P, Pandya T, Gohel M, Thakkar V. Development and Validation of HPLC method for simultaneous estimation of Rifampicin and Ofloxacin using experimental design. J Tai Uni Sci. 2019; 13(1): 146–154. https://doi.org/10.1080/16583655.2018.1548748
• [19]Khatak S, Khatak M, Ali F, Rathi A, Singh R, Singh GN, Dureja H. Development and validation of a RP-HPLC method for simultaneous estimation of antitubercular drugs in solid lipid nanoparticles. Indian J Pharm Sci. 2018; 80(6): 996–1002. https://doi.org/10.4172/pharmaceutical-sciences.1000449
• [20]Shah U, Patel S, Raval M. Stability Indicating Reverse Phase HPLC Method for Estimation of Rifampicin and Piperine in Pharmaceutical Dosage Form. Curr Drug Discov Technol. 2018; 15(1): 54–64. https://doi.org/10.2174/1570163814666170619092224
• [21]Strock J, Nguyen M, Sherma J. Transfer of Minilab TLC Screening Methods to Quantitative HPTLC-Densitometry for Pyrazinamide, Ethambutol, Isoniazid, and Rifampicin in a Combination Tablet. J Liq Chromatogr Relat Technol. 2015; 38(11): 1126–1130. https://doi.org/10.1080/10826076.2015.1028292
• [22]Jadhav S, Viswanathan V, Mukne AP. Validated HPTLC Method for Simultaneous Quantification of Isoniazid, Rifampicin and Glabridin. J Pharm Biomed Sci. 2016; 6(7): 453–459. http://dx.doi.org/10.20936/jpbms/160270
• [23]Luyen LT, Hung TM, Huyen LT, Tuan LA, Huong DTL, Duc H Van, Tung BT. Simultaneous determination of pyrazinamide, rifampicin, ethambutol, isoniazid and Acetyl Isoniazid in human plasma by LC-MS/MS method. J Appl Pharm Sci. 2018; 8(9): 61–73. https://doi.org/10.7324/JAPS.2018.8910
• [24]Temova Rakusa Z, Roskar R, Klancar Andrejc A, Trdan Lusin T, Faganeli N, Grabnar I, Mrhar A, Kristl A, Trontelj J. Fast and Simple LC-MS/MS Method for Rifampicin Quantification in Human Plasma. Int J Anal Chem. 2019; 2019(2): 1–8. https://doi.org/10.1155/2019/4848236
• [25]LLopis B, Funck-Brentano C, Tissot N, Bleibtreu A, Jaureguiberry S, Fourniols E, Aubry A, Zahr N. Development and validation of a UPLC-MS/MS method for simultaneous quantification of levofloxacin, ciprofloxacin, moxifloxacin and rifampicin in human plasma: Application to the therapeutic drug monitoring in osteoarticular infections. J Pharm Biomed Anal. 2020; 183: 113137. https://doi.org/10.1016/j.jpba.2020.113137
• [26]Burhan A, Vyas B. A rapid, sensitive and validated ultra performance liquid chromatography and tandem mass spectrometry method for determination of rifampicin in rat plasma: application to pharmacokinetic study. Int J Pharm Pharm Sci. 2017; 9(2): 222-230. https://doi.org/10.22159/ijpps.2017v9i2.15865
• [27]Yue-Ling M, Yu-Jie C, Ding-Rong W, Ping C, Ran X. HPLC determination of quercetin in three plant drugs from genus sedum and conjecture of the best harvest time. Pharmacog J. 2014; 9(6): 725–728. https://doi.org/10.5530/pj.2017.6.114
• [28]Sahani S, Jain V. A novel RP-HPLC method for simultaneous estimation of berberine, quercetin, and piperine in an ayurvedic formulation. Int J Appl Pharm. 2019; 11(1): 94–99. https://doi.org/10.22159/ijap.2019v11i1.29326
• [29]Baghel US, Nagar A, Pannu MS, Singh D, Yadav R. HPLC and HPTLC methods for simultaneous estimation of quercetin and curcumin in polyherbal formulation. Indian J Pharm Sci. 2017; 79(2): 197–203. https://doi.org/10.4172/pharmaceutical-sciences.1000217
• [30]Doshi G, Une H. Quantification of quercetin and rutin from Benincasa hispida seeds and Carissa congesta roots by high-performance thin layer chromatography and high-performance liquid chromatography. Pharmacog Res. 2016; 8(1): 37–42. https://doi.org/10.4103/0974-8490.171098
• [31]Patel AA, Amin AA, Patwari AH, Shah MB. Validated high performance thin layer chromatography method for simultaneous determination of quercetin and gallic acid in leea indica. Rev Brasil Farmacogn. 2017; 27(1): 50–53. https://doi.org/10.1016/j.bjp.2016.05.017
• [32]Tanaka S, Trakooncharoenvit A, Nishikawa M, Ikushiro S, Hara H. Comprehensive Analyses of Quercetin Conjugates by LC/MS/MS Revealed That Isorhamnetin-7- O-glucuronide-4′- O-sulfate Is a Major Metabolite in Plasma of Rats Fed with Quercetin Glucosides. J Agric Food Chem. 2019; 67(15): 4240–4249. https://doi.org/10.1021/acs.jafc.8b06929
• [33]Santos MC, Farias LS, Merlugo L, de Oliveira T V., Barbosa FS, Fuentefria AM, Henriques AT, Garcia C V., Mendez ASL. UPLC-MS for ıdentification of quercetin derivatives in Cuphea glutinosa Cham. & Schltdl (Lythraceae) and evaluation of antifungal potential. Curr Pharm Anal. 2017; 14(6): 586–594. https://doi.org/10.2174/1573412913666170918150924
• [34]Dou LL, Duan L, Gou L, Liu LL, Zhang YD, LI P, Liu EH. An UHPLC-MS/MS method for simultaneous determination of quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, isorhamnetin 3-O-rutinoside, bilobalide and ligustrazine in rat plasma, and its application to pharmacokinetic study of Xingxiong injection. Chin J Nat Med. 2017; 15(9): 710–720. https://doi.org/10.1016/S1875-5364(17)30101-2
• [35]Zupanets IA, Pidpruzhnykov Y V., Sabko VE, Bezugla NP, Shebeko SK. UPLC-MS/MS quantification of quercetin in plasma and urine following parenteral administration. Clin Phytosci 2019; 5(11): 1–12. https://doi.org/10.1186/s40816-019-0107-1
• [36]Ning ZW, Zhai LX, Peng J, Zhao L, Huang T, Lin CY, Chen WH, Luo Z, Xiao HT, Bian ZX. Simultaneous UPLC-TQ-MS/MS determination of six active components in rat plasma: Application in the pharmacokinetic study of Cyclocarya paliurus leaves. Chin Med. 2019; 14: 28. https://doi.org/10.1186/s13020-019-0248-7
• [37]Tandel DB, Patel KG, Thakkar VT, Sakure AA, Gandhi TR. Bioanalytical Method Development and Validation for Determination of Rifampicin and Quercetin in Rat Plasma by UHPLC-MS/MS: Applications to Pharmacokinetic Study. Anal Chem Lett. 2023; 13(1): 60–72. https://doi.org/10.1080/22297928.2022.2162830
• [38]Qin C, Feng W, Chu Y, Lee JB, Berton M, Bettonte S, Teo YY, Stocks MJ, Fischer PM, Gershkovich P. Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma. Biomed Chromatogr. 2020;34(11):e4934. https://doi.org/10.1002/bmc.4934
• [39]Sabale V, Jiwankar M, Sabale P. Bioanalytical method development, validation and quantification of flutamide in spiked rat plasma by using high-performance liquid chromatography. Futur J Pharm Sci. 2023; 9(1): 75–82. https://doi.org/10.1186/s43094-023-00528-7
• [40]Kharouba M, Mahmoud SH. Development and validation of a simple HPLC-UV Assay method for determination of levetiracetam concentrations in human plasma. Analytica. 2023; 4(1): 1–9. https://doi.org/10.3390/analytica4010001
• [41]Bioanalytical Method Validation Guidance for Industry. US Department of Health and Human Services Food and Drug Administration. 2018; 1–44.
• [42]Zar JH. Biostatistical analysis. fourth ed., Pearson education, New Jersey, USA 2004.