APPROACHES TO THE DEVELOPMENT OF BIOANALYTICAL METHODS FOR DETERMINATION OF UNSTABLE SUBSTANCES IN BIOLOGICAL FLUIDS

The approaches to bioanalytical method development for determination of substances which contain unstable functional groups in the structure are described. The oxidation and the hydrolysis are main causes of the decomposition of substances in biological fluids. Phenolic hydroxyls contain drugs were selected as examples of oxidable compounds, glucuronides of drugs were selected as examples of hydrolysable compounds. Determination of mycophenolic acid, which contains one phenolic hydroxyl and metabolized by formation of glucuronides, in plasma was performed using high performance liquid chromatography with mass-spectrometry and tandem mass-spectrometry detection. Methyldopa, which contains two phenolic hydroxyls, in stabilized plasma was assayed by high performance liquid chromatography – tandem mass-spectrometry in the range of 0.02–3.00 μg/ml. Concentrations of desmethyl mebeverine acid, which contains in the structure one phenolic hydroxyl and metabolized by formation of phenolic glucuronide, was measured simultaneously with mebeverine acid in the range of 10–2000 ng/ml. The influence of the ion source conversion of glucuronides on the quantitative determination of the substances was studied in the initial part of methods development. The next, selection of anticoagulants based on the study of short-term stability and freeze/thaw stability of the analytes and back conversion of their glucuronides was performed. The combination of anticoagulant K3EDTA and the antioxidant solution containing a mixture of ascorbic acid, sodium sulfite and sodium hydrogen carbonate in the concentrations of 5.0 %, 0.2 % and 2.4 %, respectively, was used to prevent degradation of methyldopa.

instability, back conversion, plasma, mycophenolic acid, methyldopa, desmethyl mebeverine acid

1. On the approval of the Rules for conducting bioequivalence studies of medicines in the Eurasian Economic Union. Decision of the Council of the Eurasian Economic Commission N 85 d.d. November 3, 2016 [Ob utverzhdenii Pravil provedeniya issledovaniy bioekvivalentnosti lekarstvennykh preparatov v ramkakh Evraziyskogo ekonomicheskogo soyuza: Reshenie Soveta Evraziyskoy ekonomicheskoy komissii ot 3 noyabrya 2016 g. № 85]. (2016). Available at: http://docs.cntd.ru/document/456026107.

2. Mironov АN. (ed.). (2014). Guidelines on the expertise of medicines [Rukovodstvo po ekspertize lekarstvennykh sredstv]. Мoskva, 328 p.

3. Khokhlov AL, Ryska M, Kukes VG, Pischakova M, Pechena M, Yavorskiy AN, Shitov LN, Dzhurko YuA, Romadonovskiy DP, Shitova AM, Chudova NV, Tsyzman LG, Lileeva EG, Khokhlov AA, Pozdnyakov NO, Miroshnikov AE, Voronina EA, Rybachkova YuV, Manuilov DM, Zimina NV, Yaichkov II. (2017). Theory and practice of conducting the researches of generic drugs [Teoreticheskie i prakticheskie osnovy provedeniya issledovaniy vosproizvedennykh lekarstvennykh preparatov]. Moskva, Yaroslavl, Praga, 227 p.

4. Khokhlov AL, Dzhurko YA, Kubeš V. Shitov LN, Yaichkov II, Shitova AM. (2017). Method for quantitative determination of methyldopa in human plasma [Metodika kolichestvennogo opredeleniya metildopy v plazme krovi cheloveka]. Vestnik Volgogradskogo gosudarstvennogo meditsinskogo universiteta, 63 (3), 105-108.

5. Khokhlov AL, Yaichkov II, Shitova AM, Shitov LN, Dzhurko YA, Miroshnikov AE. (2017). Study of comparative pharmacokinetics of coated tablets of mycophenolic acid [Issledovanie sravnitel’noy farmakokinetiki tabletirovannykh form mikofenolovoy kisloty]. Farmakokinetika i farmakodinamika, (4), 3-8.

6. Yaichkov II, Khokhlov AL, Dzhurko YA, Shitov LN, Trubnikov AA ( 2017). Methods of stabilizing drug substances and their metabolites in biological fluids in bioanalytical researches (review) [Sposoby stabilizatsii lekarstvennykh veshchestv i ikh metabolitov v biologicheskikh zhidkostyakh pri bioanaliticheskikh issledovaniyakh (obzor)]. Razrabotka i registratsiya lekarstvennykh sredstv, (2), 160-164.

7. Almeida S, Filipe A, Neves R, Spínola AC, Tanguay M, Ortuño J, Farré A, Torns A. (2010). Mycophenolate mofetil 500-mg tablet under fasting conditions: single-dose, randomized-sequence, open-label, four-way replicate crossover, bioequivalence study in healthy subjects. Clin Therap, 32 (3), 556-574. DOI: 10.1016/j. clinthera.2010.03.008

8. Benoit-Biancamano MO, Caron P, Levesque E, Delage R, Couture F, Guillemette C. (2007). Sensitive high-performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of mycophenolic acid and its glucuronide metabolites in human plasma and urine. J Chromatogr B Analyt Technol Biomed Life Sci, 858, 159-167. DOI: 10.1016/j. jchromb.2007.08.023

9. Brandhorst G, Streit F, Goetze S, Oellerich M, Armstrong VW. (2006). Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites. Clin Chem, 52 (10), 1962-1964. DOI: 10.1373/clinchem.2006.074336

10. Dell D. (2004). Labile metabolites. Chromatogr Suppl, 59, 139-148. DOI: 10.1365/s10337-003-0169-5

11. Elbarty FA, Shoker AS. (2007). Liquid chromatographic determination of mycophenolic acid and its metabolites in human kidney transplant plasma: Pharmacokinetic application. J Chromatogr B, 859 (2), 276-281. DOI: 10.1016/j.jchromb.2007.09.036

12. Elliott S, Burgess V. (2006). Investigative implications of the instability and metabolism of mebeverine. J Anal Toxicol, 30 (2), 91-97.

13. European Medicines Agency. Committee for Medical Products of Human Use. (2010). Guideline on bioanalytical method validation. – Available at: www.ema.europa.eu/docs/en_GB/document_library/Scientific_ guideline/2011/08/WC500109686.pdf.

14. Food and Drug administration. (2013). Guidance for industry. Bioanalytical method validation. Available at: http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/ guidances/ucm368107.

15. Heinig K, Bucheli F, Hartenbach R, Gajate-Perez A. (2010). Determination of mycophenolic acid and its phenyl glucuronide in human plasma, ultrafiltrate, blood, DBS and dried plasma spots. Bioanal, 2 (8), 1423-1435. DOI: 10.4155/bio.10.99

16. Hilhorst M, Van Amsterdam P, Heinig K, Swanziger E, Abbott R. (2015). Stabilization of clinical samples collected for quantitative bioanalysis – a reflection from the European Bioanalysis Forum. Bioanal, 7 (3), 333-343. DOI: 10.4155/bio.14.290

17. Khatri CA, Phanikumar ChV, Jayaveera K. (2012). Development and validation of bioanalytical method for simultaneous quantification of veratric acid, mebeverine acid and desmethyl mebeverine acid in human EDTA plasma by using LC-MS/MS. Pharm Chem J, 4 (6), 11-18.

18. Khokhlov AL, Dzhurko YA, Yaichkov II, Shitov LN, Shitova AM, Miroshnikov AE, Khozova LA. (2017). The rapid and sensitive HPLC-MS/MS-Method of determination of mebeverine metabolites in human plasma. Mathews J Pharm Sci, 1 (2), 010. Available at: mathewsopenaccess. com/PDF/pharmaceutical-science/M_J_Phar_2_1_010.pdf.

19. Khokhlov AL, Yaichkov II, Dzhurko YA, Shitov LN. (2017). Methodical approaches to bioassay of phenolic hydroxylenes contain substances. Med News North Cauc, 12 (3), 294-299. DOI: 10.14300/MNNC.2017.12088

20. Kuhn J, Götting C, Kleesiek K. (2009). Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography – electrospray ionization tandem mass spectrometry. Talanta, 80 (5), 1894-1898. DOI: 10.1016/j.talanta.2009.10.040

21. Kuhn J, Prante C, Kleesiek K, Götting C. (2009). Measurement of mycophenolic acid and its glucuronide using a novel rapid liquid chromatography – electrospray ionization tandem mass spectrometry assay. Clin Biochem, 42 (1-2), 83-90. doi: 10.1016/j.clinbiochem.2008.10.004

22. Kristinsson J, Snorradbttir I, Johannsson M. (1994). The metabolism of mebeverine in man: identification of urinary metabolites by gas chromatography/ mass spectrometry. Pharmacol Toxicol, 74 (3), 174-180.

23. Li W, Zhang J, Tse F. (2013). Handbook of LC-MS bioanalysis. New Jersey, 675 p.

24. Li W, Zhang J, Tse F. (2011). Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices. Biomed Chromatogr,

25. (1-2), 258277. DOI: 10.1002/bmc.1572 25. Liu Q, Jiao Z, Zhong M, Zhang M, Geng F, Zhao H. (2017). Effect of long-term co-administration of com pound glycyrrhizin tablets on the pharmacokinetics of mycophenolic acid in rats. Xenobiotica, 46 (7), 627-633. DOI: 10.3109/00498254.2015.1103386

26. Maddela R, Pilli NR, Maddela S. (2017). A novel and rapid LC–MS/MS assay for the determination of mycophenolate and mycophenolic acid in human plasma. J Young Pharm, 9 (1), 107-114. DOI: 10.5530/jyp.2017.9.20

27. Moskaleva NE, Baranov PA, Mesonzhnik NV, Appolonova SA. (2017). HPLC-MS/MS method for the simultaneous quantification of desmethylmebeverine acid, mebeverine acid and mebeverine alcohol in human plasma along with its application to a pharmacokinetics study. J Pharm Biomed Anal, 138, 118-125. DOI: 10.1016/j. jpba.2017.02.006

28. Ohyama K, Kinoshita N, Kishikawa N, Kuroda N. (2008). A simple and rapid CZE method for the analysis of mycophenolic acid and its phenol glucuronide metabolite in human. Electrophoresis, 29 (17), 3658-3664. DOI: 10.1002/elps.200700952

29. Ohyama K, Kishikawa N, Nakagawa H, Kuroda N, Nishikido M, Teshima M, To H, Kitahara T, Sasaki H. (2008). Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis. J Pharm Biomed Anal, 47 (1), 201-206. DOI: 10.1016/j.jpba.2007.12.028

30. Oliveira CH, Barrientos-Astigarraga RE, Sucupira M, Graudens GS, Muscará MN, Nuccia G. (2002). Quantification of methyldopa in human plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry. Application to a bioequivalence study. J Chromatogr B, 768 (2), 341-348. DOI: 10.1016/S15700232(01)00612-2

31. Rissling O, Bauer S, Shipkova M, Glander P, Mai M, Hambach P, Budde K. (2016). Simultaneous determination of mycophenolate and its metabolite mycophenolate-7-o-glucuronide with an isocratic HPLC-UV-based method in human plasma and stability evaluation. Scand J Clin Lab Invest, 76 (8), 612-619. DOI: 10.1080/00365513.2016.1230775

32. Róna K, Ary K, Renczes G, Gachályi B, Grézal GY, Drabant S, Klebovich I. (2001). Comparative bioavailability of alpha-methyldopa normal and film tablet formulations after single oral administration in healthy volunteers. Eur J Drug Metab Pharmacok, 26 (1-2), 25-30.

33. Shihabi ZK. (2009). Enhanced detection in capillary electrophoresis: Example determination of serum mycophenolic acid. Electrophoresis, 30 (9), 1516-1521.

34. Stockis A, Guelen PJM, De Vos D. (2002). Identification of mebeverine acid as the main circulating metabolite of mebeverine in man. J Pharm Biomed Anal, 29 (1-2), 335-340.

35. Valizadeh H, Nemati M, Hallaj-Nezhadi S, Ansarin M, Zakeri-Milani P. (2010). Single dose bioequivalence study of a-methyldopa tablet formulations using a modified HPLC method. Arzneimittelforschung, 60 (10), 607-611. DOI: 10.1055/s-0031-1296333

36. Vlase L, Mihu D, Popa D-S, Popa A, Briciu C, Loghin F, Ciortea R, Mihu C. (2013). Determination of methyldopa in human plasma by LC/MS-MS for therapeutic drug monitoring. Stud Univer Babes-Bolyai Chem, 58 (1), 31-41.

37. Van de Merbel NC, Hendriks G, Imbos R, Tuunainen J, Rouru J, Nikkanen H. (2011). Quantitative determination of free and total dopamine in human plasma by LC-MS/MS: the importance of sample preparation. Bioanal, 3 (17), 1949-1961. DOI: 10.4155/bio.11.170

38. Wang L, Qiang W, Li Y, Cheng Z, Xie M. (2017). A novel freeze-dried storage and preparation method for the determination of mycophenolic acid in plasma by high-performance liquid chromatography. Biomed Chromatogr, 31 (9), 3-27. DOI: 10.1002/bmc.3958