actabiomedicaActa Biomedica ScientificaActa Biomedica Scientifica2541-94202587-9596Scientific Centre for Family Health and Human Reproduction Problems10.29413/ABS.2018-3.5.16actabiomedica-715Research ArticleФАРМАКОЛОГИЯ И ФАРМАЦИЯPHARMACOLOGY AND PHARMACEUTICSПОДХОДЫ К РАЗРАБОТКЕ БИОАНАЛИТИЧЕСКИХ МЕТОДИК ДЛЯ ОПРЕДЕЛЕНИЯ НЕСТАБИЛЬНЫХ СОЕДИНЕНИЙ В БИОЛОГИЧЕСКИХ ЖИДКОСТЯХAPPROACHES TO THE DEVELOPMENT OF BIOANALYTICAL METHODS FOR DETERMINATION OF UNSTABLE SUBSTANCES IN BIOLOGICAL FLUIDSХохловА. Л.KhokhlovA. L.

150000, Ярославль, ул. Революционная, 5.

доктор медицинских наук, профессор, член-корреспондент РАН, заведующий кафедрой клинической фармакологии.

ul. Revolyutsionnaya 5, Yaroslavl 150000.

Doctor of Medical Sciences, Professor, Corresponding Member of RAS, Head of the Department of Clinical Pharmacology.

al460935@yandex.ruЯичковИ. И.YaichkovI. I.

150000, Ярославль, ул. Революционная, 5.

150000, г. Ярославль, ул. Республиканская, 108/1.

аспирант кафедры клинической фармакологии; младший научный сотрудник Центра трансфера фармацевтических технологий.

ul. Revolyutsionnaya 5, Yaroslavl 150000.

ul. Respuplikanskaya 108/1, Yaroslavl 150000.

Postgraduate at the Department of Clinical Pharmacology.

lya_1993_08@mail.ruДжуркоЮ. А.DzhurkoYu. A.

150045, г. Ярославль, Ленинградский пр., 52г.

кандидат фармацевтических наук, старший аналитик.

Lenyngradsky pr. 52g, Yaroslavl 150045.

Candidate of Pharmaceutical Sciences, Senior Analyst.

y.dzhurko@qayar.ruРыскаМ.RyskaM.

Pražská 1486/18c, 102 00 Praha 15.

доктор естественных наук, RNDr, экс-президент.

Pražská 1486/18c, 102 00 Praha 15.

Doctor of Natural Sciences, RNDr, ex-president.

miroslav.ryska@quinta.czКубешВ.KubešV.

Pražská 1486/18c, 102 00 Praha 15.

заведующий лабораторией.

Pražská 1486/18c, 102 00 Praha 15.

Head of the Laboratory.

vladimir.kubes@quinta.czШитовЛ. И.ShitovL. N.

150000, Ярославль, ул. Революционная, 5.

150045, г. Ярославль, Ленинградский пр., 52г.

кандидат биологических наук, заведующий биоаналитической лабораторией; ассистент кафедры поликлинической терапии и клинической лабораторной диагностики.

ul. Revolyutsionnaya 5, Yaroslavl 150000.

Lenyngradsky pr. 52g, Yaroslavl 150045.

Candidate of Biological Sciences, Head of the Bioanalytical Laboratory.

schitov@inbox.ruФГБОУ ВО «Ярославский государственный медицинский университет» Минздрава России.Yaroslavl State Medical University.ФГБОУ ВО «Ярославский государственный медицинский университет» Минздрава России; ФГБОУ ВО «Ярославский государственный педагогический университет им. К.Д. Ушинского».Yaroslavl State Medical University; K.D. Ushinsky Yaroslavl State Pedagogical University.Биоаналитическая лаборатория «Квинта-Аналитика Ярославль».Quinta-Analytica Yaroslavl LLC.Quinta-Analytica s.r.o.Quinta-Analytica s.r.o.ФГБОУ ВО «Ярославский государственный медицинский университет» Минздрава России; Биоаналитическая лаборатория «Квинта-Аналитика Ярославль».Yaroslavl State Medical University; Quinta-Analytica Yaroslavl LLC.20182809201835106115Copyright © Хохлов А.Л., Яичков И.И., Джурко Ю.А., Рыска М., Кубеш В., Шитов Л.И., 20182018Хохлов А.Л., Яичков И.И., Джурко Ю.А., Рыска М., Кубеш В., Шитов Л.И.Khokhlov A.L., Yaichkov I.I., Dzhurko Y.A., Ryska M., Kubeš V., Shitov L.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.actabiomedica.ru/jour/article/view/715

В статье приведены подходы к разработке биоаналитических методик для определения веществ, содержащих в структуре нестабильные функциональные группы. Процессы окисления и гидролиза являются основными причинами разложения веществ в биологических жидкостях. В качестве примера легкоокисляющихся соединений были выбраны молекулы, содержащие в структуре фенольные гидроксилы, в качестве примера легкоокисляющихся соединений – глюкурониды лекарственных веществ. Для измерения концентраций микофеноловой кислоты, содержащей в структуре один фенольный гидроксил и в процессе метаболизма образующей глюкурониды, в плазме крови использовались методы высокоэффективной жидкостной хроматографии с масс-спектрометрическим и тандемным масс-спектрометрическим детектированием. Концентрации метилдопы, содержащей в структуре два фенольных гидроксила, в плазме измерялись с помощью высокоэффективной жидкостной хроматографии с тандемным масс-спектрометрическим детектированием в диапазоне 0,02–3,00 мкг/мл. Определение деметилированной мебевериновой кислоты, содержащей в структуре один фенольный гидроксил и метаболизирующейся с образованием фенольного глюкуронида, осуществляли в диапазоне 10–2000 нг/мл совместно с мебевериновой кислотой. В начале разработки методик была изучено влияние фрагментации глюкуронидов в источнике ионов на количественное определение изучаемых веществ. Затем был произведён выбор антикоагулянта на основании изучения краткосрочной стабильности и стабильности при замораживании/размораживании аналитов, а также обратной конверсии их глюкуроновых коньюгатов. Для стабилизации метилдопы была использована комбинация антикоагулянта К3ЭДТА и раствора антиоксиданта, содержащего смесь аскорбиновой кислоты, натрия сульфита и натрия гидрокарбоната в концентрациях 5,0 %, 0,2 % и 2,4 % соответственно.

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.

нестабильностьобратная конверсияплазмамикофеноловая кислотаметилдопадеметилированая мебевериновая кислотаinstabilityback conversionplasmamycophenolic acidmethyldopadesmethyl mebeverine acidReferencesОб утверждении Правил проведения исследований биоэквивалентности лекарственных препаратов в рамках Евразийского экономического союза: Решение Совета Евразийской экономической комиссии от 3 ноября 2016 г. № 85. – 2016. – Режим доступа: http://docs.cntd.ru/document/456026107.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.Руководство по экспертизе лекарственных средств / Под ред. А.Н. Миронова. – М.: ПолиграфПлюс, 2014. – Т. 1. – С. 328.Mironov АN. (ed.). (2014). Guidelines on the expertise of medicines [Rukovodstvo po ekspertize lekarstvennykh sredstv]. Мoskva, 328 p.Хохлов А.Л., Рыска М., Кукес В.Г., Писачкова М., Печена М., Яворский А.Н., Шитов Л.Н., Джурко Ю.А., Ромадоновский Д.П., Шитова А.М., Чудова Н.В., Цызман Л.Г., Лилеева Е.Г., Хохлов А.А., Поздняков Н.О., Мирошников А.Е., Воронина Е.А., Рыбачкова Ю.В., Мануилов Д.М., Зимина Н.В., Яичков И.И. Теоретические и практические основы проведения исследований воспроизведённых лекарственных препаратов. – Москва – Ярославль – Прага, 2017. – 227 с.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.Хохлов А.Л., Джурко Ю.А., Kubes V., Шитов Л.Н., Яичков И.И., Шитова А.М. Методика количественного определения метилдопы в плазме крови человека // Вестник Волгоградского государственного медицинского университета. – 2017. – Т. 63, № 3. – С. 105–108.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.Хохлов А.Л., Яичков И.И., Шитова А.М., Шитов Л.Н., Джурко Ю.А. Исследование сравнительной фармакокинетики таблетированных форм микофеноловой кислоты // Фармакокинетика и фармакодинамика. – 2016. – № 4. – С. 3–8.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.Яичков И.И., Хохлов А.Л., Джурко Ю.А., Шитов Л.Н., Трубников А.А. Способы стабилизации лекарственных веществ и их метаболитов в биологических жидкостях при биоаналитических исследованиях (обзор) // Разработка и регистрация лекарственных средств. – 2017. – № 2. – С. 160–164.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.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.2010.03.008Almeida 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.2010.03.008Benoit-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.2007.08.023Benoit-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.2007.08.023Brandhorst 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.2006.074336Brandhorst 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.2006.074336Dell D. (2004). Labile metabolites. Chromatogr Suppl, 59, 139-148. DOI: 10.1365/s10337-003-0169-5Dell D. (2004). Labile metabolites. Chromatogr Suppl, 59, 139-148. DOI: 10.1365/s10337-003-0169-5Elbarty 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.2007.09.036Elbarty 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.2007.09.036Elliott S, Burgess V. (2006). Investigative implications of the instability and metabolism of mebeverine. J Anal Toxicol, 30 (2), 91-97.Elliott S, Burgess V. (2006). Investigative implications of the instability and metabolism of mebeverine. J Anal Toxicol, 30 (2), 91-97.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.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.Food and Drug administration. (2013). Guidance for industry. Bioanalytical method validation. Available at: http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/ guidances/ucm368107.Food and Drug administration. (2013). Guidance for industry. Bioanalytical method validation. Available at: http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/ guidances/ucm368107.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.10.99Heinig 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.10.99Hilhorst 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.14.290Hilhorst 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.14.290Khatri 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.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.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.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.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.2017.12088Khokhlov 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.2017.12088Kuhn 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.2009.10.040Kuhn 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.2009.10.040Kuhn 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.2008.10.004Kuhn 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.2008.10.004Kristinsson 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.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.Li W, Zhang J, Tse F. (2013). Handbook of LC-MS bioanalysis. New Jersey, 675 p.Li W, Zhang J, Tse F. (2013). Handbook of LC-MS bioanalysis. New Jersey, 675 p.Li W, Zhang J, Tse F. (2011). Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices. Biomed Chromatogr,Li W, Zhang J, Tse F. (2011). Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices. Biomed Chromatogr,(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(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: 10.3109/00498254.2015.1103386Maddela 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.2017.9.20Maddela 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.2017.9.20Moskaleva 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.2017.02.006Moskaleva 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.2017.02.006Ohyama 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.200700952Ohyama 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.200700952Ohyama 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.2007.12.028Ohyama 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.2007.12.028Oliveira 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)00612-2Oliveira 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)00612-2Rissling 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: 10.1080/00365513.2016.1230775Rissling 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: 10.1080/00365513.2016.1230775Róna 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.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.Shihabi ZK. (2009). Enhanced detection in capillary electrophoresis: Example determination of serum mycophenolic acid. Electrophoresis, 30 (9), 1516-1521.Shihabi ZK. (2009). Enhanced detection in capillary electrophoresis: Example determination of serum mycophenolic acid. Electrophoresis, 30 (9), 1516-1521.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.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.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-0031-1296333Valizadeh 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-0031-1296333Vlase 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.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.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.11.170Van 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.11.170Wang 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.3958Wang 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

The authors declare that there are no conflicts of interest present.