Development and validation of a method for isolating stavudine from biological fluids

Objective - study of isolating Stavudine from biological fluids (urine, blood plasma).

Materials and methods. Objects of research: substance and tablets of 30 mg Stavudine. Reagents: purified water, chloroform, ethyl acetate, dichloromethane, ether, heptane, toluene, acetone, 95% ethyl alcohol, 0.1 M hydrochloric acid solution, 10% ammonium hydroxide solution, 5%solutions of sodium sulfate, 20%sodium sulfate saturated, sodium chloride, sodium chloride saturated, 20%ammonium sulfate, ammonium sulfate saturated. Universal ionomer IT-1101. Stavudine was isolated by liquid-liquid extraction. To detect and quantify Stavudine, thin - layer sorbent chromatography (TLC) and UV spectrophotometry were used. Statistical data processing was performed using the software package for Windows XP (Microsoft Excel), and using the Student's t-test.

Results. When using thin-layer chromatography to identify Stavudine, it was found that the R_f value of the spots of Stavudine extracts from biological material corresponded to the R_f value range of 0.67-0.69. The R_f spot value of the standard Stavudine witness sample corresponded to (0.68±0.01). The stability study showed that the Stavudine solution is stable in a hydrochloric acid solution of 0.1 M, in which the absorption spectrum of Stavudine is characterized by absorption maxima at wavelengths of 209±1 and 267±1 nm. The wavelength of 267±1 nm was chosen as the analytical wavelength for the quantitative determination of Stavudine. In the course of the study, various factors affecting the degree of extraction of Stavudine from aqueous solutions were studied. The extractant is ethyl acetate, pH 3, the electrolyte is a saturated solution of ammonium sulfate. The extraction time is 7 minutes, twice.

Conclusion. Methods of identification and quantitative determination of Stavudine in extracts from urine and blood plasma by TLC and UV spectrophotometry have been developed. Methods of isolation of Stavudine from urine and blood plasma using the liquid-liquid extraction method have been developed (93.33±2.02%; 89.77±2.02% respectively).

1. Барсегян С.С., Саломатин Е.М., Плетенева Т.В., Максимова Т.В., Долинкин А.О. Методические рекомендации по валидации аналитических методик, используемых в судебно-химическом и химико-токсикологическом анализе биологического материала. Москва: Российский центр судебно-медицинской экспертизы, 2014. 75 с.

2. Государственная фармакопея Российской Феде-рации. XIV издание. Том I. Москва: Федеральная электронная медицинская библиотека. URL: http://femb.ru/femb/pharmacopea.php

3. Дарикулова Б.У., Мусабек Ш. Конструктивные подходы к проведению химико-токсикологического анализа и некоторые аспекты совершенствования химико-токсикологических исследований на примере Англии. Вестник КазНМУ. 2018;(3):101-103

4. Илларионова Е.А., Чмелевская Н.В., Гончикова Ю.А. Химико-токсикологическое определение ламивудина в биологических объектах. Судебно-медицинская экспертиза. 2020;63(1):42-46. DOI: 10.17116/sudmed20206301142

5. Илларионова Е.А., Чмелевская Н.В., Гончикова Ю.А., Скрипко А.А. Химико-токсикологический анализ зидовудина. Фармация. 2019;68(7):16-20. DOI: 10.29296/25419218-2019-07-03

6. Комаров Т.Н., Белова М.В., Столярова Д.Д., Шохин И.Е., Богданова Д.С., Мискив О.А., Медведев Ю.В., Коренская И.М. Химико-токсикологический анализ антиретровирусных препаратов. Разработка и регистрация лекарственных средств. 2019;8(4):53-60. DOI: 10.33380/2305-2066-2019-8-4-53-60

7. Мошкович Г.Ф., Минаева С.В., Варлова Л.В., Горяева М.П., Гуляева С.С., Тихонова Е.В. Сравнительное исследование применения нуклеозидных ингибиторов обратной транскриптазы в схемах антиретровирусной терапии у больных, получающих лечение хронического гепатита С. Вопросы вирусологии. 2016;61(1):34-39. DOI: 10.18821/0507-4088-2016-61-1-34-39

8. Нечаева О.Б. Мониторинг туберкулеза и ВИЧ-инфекции в Российской Федерации. Медицинский алфавит. 2017;3(30):24-33

9. Покровский В.В., под ред. Вич-инфекция и СПИД. Национальное руководство. Москва: ГЭОТАР-Медиа, 2013. 630 с.

10. Турсунов Р.А., Канестри В.Г., Симонова Е.Г., Раичич Р.Р. Антиретровирусная терапия - новая эпоха профилактики ВИЧ-инфекции. ВИЧ-инфекция и иммуносупрессии. 2018; 10(1):37-46. DOI: 10.22328/2077-9828-2018-10-1-37-46

11. Шалдина М.В., Пирогова И.А. Антиретровирусная терапия как основной метод лечения ВИЧ-инфекции. Вестник Совета молодых ученых и специалистов Челябинской области. 2017;2(4): 71-74

12. Bedor D.C.G., de Souza Filho J.H., Ramos V.L.S., Gonçalves T.M., de Sousa C.E.M., de Santana D.P. A sensitive and robust Lc-Ms/Ms method with monolithic column and electrospray ionization for the quantitation of efavirenz in human plasma: Application to a bioequivalence study. Química Nova. 2011;34(6):950-955. DOI: 10.1590/S0100-40422011000600007

13. Capparelli E.V., Letendre S.L., Ellis R.J., Patel P., Holland D., McCutchan J.A. Population pharmacokinetics of abacavir in plasma and cerebrospinal fluid. Antimicrob Agents Chemother. 2005;49(6): 2504-2506. DOI: 10.1128/AAC.49.6.2504-2506.2005

14. Kiran B.V., Rao B.S., Dubey S.S. Validation of Abacavir Sulfate in Pharmaceutical dosage by Reverse phase HPLC with Internal standard method. International Journal of pharmaceutical sciences and research. 2012;3(8):2590-2598. DOI: 10.13040/IJPSR.0975-8232.3(8).2590-98

15. Lea A.P., Faulds D. Stavudine: a review of its pharmacodynamic and pharmacokinetic properties and clinical potential in HIV infection. Drugs. 1996;51(5):846-864. DOI: 10.2165/00003495-199651050-00009

16. McDowell J.A., Lou Y., Symonds W.S., Stein D.S. Multiple-dose pharmacokinetics and pharmacodynamics of abacavir alone and in combination with zidovudine in human immunodeficiency virus-infected adults. Antimicrob Agents Chemother. 2000;44(8): 2061-2067. DOI: 10.1128/AAC.44.8.2061-2067.2000

17. Melroy J., Nair V. The antiviral activity, mechanism of action, clinical significance and resistance of abacavir in the treatment of pediatric AIDS. Curr Pharm Des. 2005;11(29):3847-3852. DOI: 10.2174/138161205774580642

18. Nayak D., Boxi A., Ashe S., Thathapudi N.C., Nayak B. Stavudine loaded gelatin liposomes for HIV therapy: Preparation, characterization and in vitro cytotoxic evaluation. Mater Sci Eng C Mater Biol Appl. 2017;(73):406-416. DOI: 10.1016/j.msec.2016.12.073

19. Riddler S.A., Anderson R.E., Mellors J.W. Antiretroviral activity of stavudine (2',3'-didehydro-3'-deoxythymidine, D4T). Antiviral Res. 1995;27(3):189-203. DOI: 10.1016/0166-3542(95)00016-f

20. Sanne I., Piliero P., Squires K., Thiry A., Schnittman S.; AI424-007 Clinical Trial Group. Results of a phase 2 clinical trial at 48 weeks (AI424-007): a dose-ranging, safety, and efficacy comparative trial of atazanavir at three doses in combination with didanosine and stavudine in antiretroviral-naive subjects. J Acquir Immune Defic Syndr. 2003;32(1):18-29. DOI: 10.1097/00126334-200301010-00004

21. Savaser A., Goraler S., Tasoz A., Uslu B., Lingeman H., Ozkan S.A. Determination of abacavir, lamivudine and zidovudine in pharmaceutical tablets, human serum and in drug dissolution studies by HPLC. Chroma. 2007;65(5-6):259-265. DOI: 10.1365/s10337-006-0166-6

22. Sungkanuparph S., Manosuthi W., Kiertiburanakul S., Piyavong B., Chumpathat N., Chantratita W. Options for a second-line antiretroviral regimen for HIV type 1-infected patients whose initial regimen of a fixed-dose combination of stavudine, lamivudine, and nevirapine fails. Clin Infect Dis. 2007;44(3):447-452. DOI: 10.1086/510745

23. Venter W.D.F., Kambugu A., Chersich M.F., Becker S., Hill A., Arulappan N., Moorhouse M., Majam M. et al. Efficacy and Safety of Tenofovir Disoproxil Fumarate Versus Low-Dose Stavudine Over 96 Weeks: A Multicountry Randomized, Noninferiority Trial. J Acquir Immune Defic Syndr. 2019;80(2):224-233. DOI: 10.1097/QAI.0000000000001908