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Current Pharmaceutical Analysis

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ISSN (Print): 1573-4129
ISSN (Online): 1875-676X

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Research Article

Determination of Thrombin Activity by Reversed-phase High Performance Liquid Chromatography

Author(s): Liang Zhang, Dehui Yang and Ye Liu*

Volume 19, Issue 3, 2023

Published on: 30 December, 2022

Page: [216 - 221] Pages: 6

DOI: 10.2174/1573412919666221124110031

Price: $65

Abstract

Background: A reversed-phase high-performance liquid chromatographic method for determining thrombin activity is described. The improved method can provide a high resolution between substrate Nα-P-toluenesulfonyl-L-arginine methyl ester (TAME) and the product of enzyme reaction, Nα-P-toluenesulfonyl-L-Arginine (TA).

Methods: When the substrate concentration was 5 mg/mL, the Michaelis equation approached the zero-order reaction, and the thrombin enzyme activity must have a good linear relationship with the consumption of TAME or the yield of TA.

Results: The method is suitable for quantitatively analyzing thrombin activity in formulation or serum.

Conclusion: Three batches of thrombin lyophilized preparations were determined for activity.

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Graphical Abstract

[1]
Wells, C.M.; Di Cera, E. Thrombin is a sodium ion activated enzyme. Biochemistry, 1992, 31(47), 11721-11730.
[http://dx.doi.org/10.1021/bi00162a008] [PMID: 1445907]
[2]
Chase, T.; Shaw, E.; Chase, T.; Shaw, E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. [J]. Biochemistry, 1969, 8(5), 2212-2224.
[http://dx.doi.org/10.1021/bi00833a063] [PMID: 4239491]
[3]
Stocker, K.; Barlow, G.H. The coagulant enzyme from Bothrops atrox venom (batroxobin). Methods Enzymol., 1976, 45, 214-223.
[http://dx.doi.org/10.1016/S0076-6879(76)45021-8] [PMID: 1011993]
[4]
Gaffney, P.J.; Lord, K.; Brasher, M.; Kirkwood, T.B.L. Problems in the assay of thrombin using synthetic peptides as substrates. Thromb. Res., 1977, 10(4), 549-556.
[http://dx.doi.org/10.1016/0049-3848(77)90210-9] [PMID: 854887]
[5]
Eric, D. Determination of the specific activity of recombinant hirudin. Thromb. Res., 1990, 60(6), 433-443.
[http://dx.doi.org/10.1016/0049-3848(90)90228-5] [PMID: 2082477]
[6]
Dang, Q.D.; Di Cera, E. A simple activity assay for thrombin and hirudin. J. Protein Chem., 1994, 13(4), 367-373.
[http://dx.doi.org/10.1007/BF01901692] [PMID: 7986342]
[7]
Hopfner, K.P.; Ayala, Y.; Szewczuk, Z.; Konishi, Y.; Di Cera, E. Chemical compensation in macromolecular bridge-binding to thrombin. Biochemistry, 1993, 32(12), 2947-2953.
[http://dx.doi.org/10.1021/bi00063a004] [PMID: 8457559]
[8]
Wu, Q.Y.; Sheehan, J.P.; Tsiang, M.; Lentz, S.R.; Birktoft, J.J.; Sadler, J.E. Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin. Proc. Natl. Acad. Sci. USA, 1991, 88(15), 6775-6779.
[http://dx.doi.org/10.1073/pnas.88.15.6775] [PMID: 1650482]
[9]
Siddiqui, M.R.; AlOthman, Z.A.; Rahman, N. Analytical techniques in pharmaceutical analysis: a review. Arab. J. Chem., 2017, 2017(10), S1409-S1421.
[http://dx.doi.org/10.1016/j.arabjc.2013.04.016]
[10]
Rahman, N.; Azmi, S.; Wu, H.F. The importance of impurity analysis in pharmaceutical products: an integrated approach. Accreditation and Quality Assurance, 2006, 11(1-2), 69-74.
[11]
Husain, A.; Iram, F.; Siddiqui, A.A.; Almutairi, S.M.; Mohammed, O.B.; Khan, S.A.; Azmi, S.N.H.; Rahman, N. Identification of metabolic pathways involved in the biotransformation of eslicarbazepine acetate using UPLC-MS/MS, human microsomal enzymes and in silico studies. J. King Saud Univ. Sci., 2021, 33(2), 101281.
[http://dx.doi.org/10.1016/j.jksus.2020.101281]
[12]
Salbego, P.R.S.; Bender, C.R.; Orlando, T.; Moraes, G.A.; Copetti, J.P.P.; Weimer, G.H.; Bonacorso, H.G.; Zanatta, N.; Hoerner, M.; Martins, M.A.P. Supramolecular similarity in polymorphs: Use of similarity indices (I X). ACS Omega, 2019, 4(6), 9697-9709.
[http://dx.doi.org/10.1021/acsomega.8b03660] [PMID: 31460060]
[13]
Rahman, N.; Sameen, S.; Kashif, M. Spectroscopic study on the interaction of haloperidol and 2,4-dinitrophenylhydrazine and its application for the quantification in drug formulations. Anal. Chem. Lett., 2016, 6(6), 874-885.
[http://dx.doi.org/10.1080/22297928.2016.1265898]

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