LC-MS/MS-BASED QUANTITATIVE PROFILING OF PAPAIN ENZYME IN CARICA PAPAYA L.: METHOD DEVELOPMENT AND VALIDATION
DOI:
https://doi.org/10.22159/ijap.2024v16i2.49921Keywords:
Papain, C. Papaya, Matrix, LC-MS/MS, SANCO guidelineAbstract
Objective: To develop and validate a sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for quantifying the Papain enzyme in Carica papaya L. plant extracts.
Methods: As an internal standard (IS), Papain was procured. A gradient-based LC-MS/MS method was developed using the Quasar C18, 150 mm × 2.1 mm, 3 µm column; the drug separation was accomplished using a gradient elution mode with a mobile phase consisting of 0.2% formic acid in water (A) and a 98:2% ratio of formic acid in acetonitrile (B). The mobile phase was injected with a volume of 10 µl at a flow rate of 0.5 ml/min.
Results: Matrix-matched calibration adhering to SANCO guidelines was employed for accurate quantification, covering a concentration range of 1 to 2000 ng/ml. The method's selectivity and linearity were confirmed with an R2 correlation value of 0.990. Limits of detection (LOD) and quantification (LOQ) were determined as 170.5 ng/ml and 516.8 ng/ml, respectively. The developed LC-MS/MS method demonstrated robust analytical performance, providing a solid basis for quantifying Papain enzyme levels in Carica papaya L. plant extracts. The matrix-matched calibration approach, with a well-defined standard calibration curve, showcased the method's selectivity and linearity. The LOD and LOQ values further underscored the method's sensitivity.
Conclusion: This study successfully establishes and validates a gradient-based LC-MS/MS method for accurate quantification of Papain enzyme levels in Carica papaya L. plant extracts. The demonstrated precision, accuracy, and sensitivity support the reliability of this analytical approach, offering a valuable tool for future investigations into the biological effects of Papain.
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Kamphuis IG, Kalk KH, Swarte MB, Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984;179(2):233-56. doi: 10.1016/0022-2836(84)90467-4, PMID 6502713.
Cordara G, Van Eerde A, Grahn EM, Winter HC, Goldstein IJ, Krengel U. An unusual member of the papain superfamily: mapping the catalytic cleft of the Marasmius oreades agglutinin (MOA) with a caspase inhibitor. PLOS ONE. 2016;11(2):e0149407. doi: 10.1371/journal.pone.0149407, PMID 26901797.
Glazer AN, Emil EL, Boyer PD. Papain and other plant sulfhydryl proteolytic enzymes. Enzymes. 1971;8:501-46. doi: 10.1016/S1874-6047(08)60405-9.
Kushwaha K, Saini SS, Waghmode B, Gaid M, Agrawal PK, Roy P. Volatile components in papaya fruits are the non-invasive biomarkers to monitor the ripening stage and the nutritional value. Eur Food Res Technol. 2021;247(4):907-19. doi: 10.1007/s00217-020-03673-y.
Asanarong O, Minh Quan V, Boonrungsiman S, Sukyai P. Bioactive wound dressing using bacterial cellulose loaded with papain composite: Morphology, loading/release and antibacterial properties. Eur Polym J. 2021;143(8):110224. doi: 10.1016/j.eurpolymj.2020.110224.
Tsuge H, Nishimura T, Tada Y, Asao T, Turk D, Turk V. Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex. Biochem Biophys Res Commun. 1999;266(2):411-6. doi: 10.1006/bbrc.1999.1830, PMID 10600517.
Foroughi F, Keshavarz T, Evans CS. Specificities of proteases for use in leather manufacture. J Chem Technol Biotechnol. 2006;81(3):257-61. doi: 10.1002/jctb.1367.
Khanna N, Panda PC. The effect of papain on tenderization and functional properties of spending hen meat cuts. Indian J Anim Res. 2007;41(1):55-8.
Christopher N, Kumbalwar M. Enzymes used in food industry A. Syst Rev. 2007;4(10):7.
Dietrich RE. Oral proteolytic enzymes in the treatment of athletic injuries: a double-blind study. Pennsyl J. 1965;68(10):35-7.
Trickett P. Proteolytic enzymes in the treatment of athletic injuries. Appl Ther. 1964;6:647-52. PMID 14195254.
Piva E, Ogliari FA, Moraes RR, Cora F, Henn S, Correr Sobrinho L. Papain-based gel for biochemical caries removal: influence on microtensile bond strength to dentin. Braz Oral Res. 2008;22(4):364-70. doi: 10.1590/s1806-83242008000400014, PMID 19148394.
Anthoni C. Antifungal effects of pawpaw seed extracts and papain on postharvest Carica papaya L. fruit rot. Afr J Agric Res. 2010;5(12):1531-5. doi: 10.5897/AJAR.9000056.
Arvind G, Bhowmik D, Duraivel S, Harish G. Traditional and medicinal uses of Carica papaya. J Car Pap. 2013;1(1);3862:2320.
Chandran SP, Nachinmuthu KP, Natarajan SB, Inamdar MG, Shahimi MSBM. Papain loaded solid lipid nanoparticles for colorectal cancer therapy. Curr Can Ther Rev. 2018;14(1):75-87. doi: 10.2174/1573394713666170929160933.
Li T, Yan G, Bai Y, Wu M, Fang G, Zhang M. Papain bioinspired gold nanoparticles augmented the anticancer potency of 5-FU against lung cancer. Journal of Experimental Nanoscience. 2020;15(1):109-28. doi: 10.1080/17458080.2020.1746767.
Xu X, Man L. Papain mediated synthesized gold nanoparticles encore the potency of bioconjugated flutamide. Curr Pharm Biotechnol. 2021;22(4):557-68. doi: 10.2174/1389201021666200227121144, PMID 32106799.
Kruve A, Rebane R, Kipper K, Oldekop ML, Evard H, Herodes K. Tutorial review on validation of liquid chromatography-mass spectrometry methods: part II. Anal Chim Acta. 2015;870:8-28. doi: 10.1016/j.aca.2015.02.016, PMID 25819784.
SANCO. 12571/2013, Guidance document on analytical quality control and validation procedures for pesticide residue analysis in food and feed; 2013.
Nimmakayala MR, Kolli D, Durga Bhavani PN. Bioanalytical method development and validation of Maralixibat in rat plasma by LC-MS/MS detection and its application to a pharmacokinetic study. Int J App Pharm. 2023;15:166-72. doi: 10.22159/ijap.2023v15i4.47768.
Ferraz CC, Varca GH, Vila MM, Lopes PS. Validation of in vitro analytical method to measure papain activity in pharmaceutical formulations. Int J Pharm Pharm Sci. 2014;10(6):658-61.
Budama Kilinc Y, Cakir Koc R, Kecel Gunduz S, Zorlu T, Kokcu Y, Bicak B. Papain loaded poly(ε-Caprolactone) nanoparticles: in-silico and in vitro studies. J Fluoresc. 2018;28(5):1127-42. doi: 10.1007/s10895-018-2276-6, PMID 30097974.
Chankhampan C, Manosroi J, Yamamoto H, Tahara K, Manosroi W, Kawashima Y. Chemical stability enhancement and cytotoxicity reduction of papain loaded in PLGA nanospheres. Journal of Experimental Nanoscience. 2014;9(2):138-51. doi: 10.1080/17458080.2011.636385.
Manosroi A, Chankhampan C, Pattamapun K, Manosroi W, Manosroi J. Antioxidant and gelatinolytic activities of papain from papaya latex and bromelain from pineapple fruits. Chiang Mai J Sci. 2014;41(3):635-48.
Alara OR, Abdurahman NH, Ali HA, Zain NM. Microwave-assisted extraction of phenolic compounds from Carica papaya leaves: an optimization study and LC-QTOF-MS analysis. Future Foods. 2021;3:100035. doi: 10.1016/j.fufo.2021.100035.
International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human use, Validation of Analytical Procedures: Text and Methodology Q2. p. R1. (Complementary Guideline on Methodology; 2005.
Yin RF, Xiong K, Wen SM, Song ZM, Xu F. A simple LC-ESI-MS/MS method for quantification of bacopaside I in rat plasma and its application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1027:103-9. doi: 10.1016/j.jchromb.2016.05.031, PMID 27270262.
Li L, Feng R, Feng X, Chen Y, Liu X, Sun W. The development and validation of an HPLC-MS/MS method for the determination of eriocitrin in rat plasma and its application to a pharmacokinetic study. RSC Adv. 2020;10(18):10552-8. doi: 10.1039/C9RA10925K, PMID 35492908.
Shi R, Qiao S, Yu D, Shi X, Liu M, Jiang X. Simultaneous determination of five flavonoids from Scutellaria barbata extract in rat plasma by LC-MS/MS and its application to pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(19):1625-32. doi: 10.1016/j.jchromb.2011.03.058, PMID 21514902.
Roach JA, Musser SM, Morehouse K, Woo JY. Determination of usnic acid in lichen toxic to elk by liquid chromatography with ultraviolet and tandem mass spectrometry detection. J Agric Food Chem. 2006;54(7):2484-90. doi: 10.1021/jf052767m, PMID 16569032.
Alumuri T, Merugu K, Amarababu NLA, Kurnool A. Peramivir and related impurities in rat plasma and its applications in pharmacokinetic studies (bioanalytical method development and validation by LC-MS/MS). Int J App Pharm. 2022;14:53-61. doi: 10.22159/ijap.2022v14i5.45457.
Sveshnikova N, Yuan T, Warren JM, Piercey Normore MD. Development and validation of a reliable LC-MS/MS method for quantitative analysis of usnic acid in Cladonia uncialis. BMC Res Notes. 2019;12(1):550. doi: 10.1186/s13104-019-4580-x, PMID 31470895.
Zuo SJ, Cheng XL, Liu DZ, Feng WY, Cao YX, Zhang SQ. Quantitative analysis and pharmacokinetic study of a novel diarylurea EGFR inhibitor (ZCJ14) in rat plasma using a validated LC-MS/MS method. Acta Pharm. 2021;71(3):415-28. doi: 10.2478/acph-2021-0024, PMID 36654089.
Chandana OSS, Kumar DS, Babu RR. Method development and validation of eprosartan mesylate and its impurities using reverse phase high-performance liquid chromatography. Int J Curr Pharm Sci. 2016;8(4):49-53. doi: 10.22159/ijcpr.2016v8i4.15277.
Katolkar P, Jaiswal S. Analytical method development and validation for the estimation of cyamemazine tartrate in formulation by RP-HPLC with Stability indicating. Asian J Pharm Clin Res. 2022;2022:28-32. doi: 10.22159/ajpcr.2022.v15i9.45154.
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Copyright (c) 2024 CHANDAN C., RUSHIKESH SHIVAJI AGALAVE, TANIYA TYAGI, ADITYA KUMAR SINGH, RAMESH J., PHANI KUMAR G., SUSHMA B. V., JEYAPRAKASH M. R.
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