Abstract
Introduction. Gestobutanoil is a synthetic pregnane steroid with gestagenic activity. Gestobutanoil has two pharmacologically active metabolites (AMOL and megestrol acetate). This implies the need for a detailed study of the kinetics of metabolites. It is rational to combine the study of the pharmacokinetics of gestobutanoil and its metabolites (AMOL and megestrol acetate). The simultaneous determination of several analytes in the rats’ serum can be carried out using chromatography-mass-spectrometry.Aim. Development of an analytical method for the simultaneous determination of gestobutanoil and two its metabolites in a biomatrix (rat serum).Materials and methods. The following methods were used to determine gestobutanoyl and two its metabolites in a biological matrix: GC-MS, HPLCESI-MS, HPLC-ESI-MS with derivatization, HPLC-APCI-MS.Results and discussion. When working with GC-MS, the chromatographic peaks of gestobutanoyl, AMOL, and megestrol acetate were strongly blurred and superimposed on each other, which is apparently due to the thermolability of the substances. The GC-MS method was abandoned in favor of HPLC. Analytes were separated by HPLC gradient elution on a C18 column. ESI ionization did not give typical protonated ions of gestobutanoyl and AMOL, and the intense signals of their cationized ions and fragment ions, which were observed in the spectra of AMOL and gestobutanoyl, could not ensure the reproducibility of the spectra, since the conditions of their formation are not suitable for routine analysis. Derivatization of analytes to form oximes and substituted hydrazones did not give the expected reaction products for HPLC-ESI-MS. APCI made it possible to remove intense cationized ions from the spectra of gestobutanoyl and AMOL and to increase the reliability of the method. The HPLCAPCI-MS technique was reproduced on model rat blood serum.Conclusion. An HPLC-MS method was developed for the simultaneous determination of gestobutanoyl, megestrol acetate, and AMOL. The technique was tested on a model rat blood serum containing all three analytes.
Highlights
Gestobutanoil is a synthetic pregnane steroid with gestagenic activity
It is rational to combine the study of the pharmacokinetics of gestobutanoil and its metabolites (AMOL and megestrol acetate)
When working with GC-MS, the chromatographic peaks of gestobutanoyl, AMOL, and megestrol acetate were strongly blurred and superimposed on each other, which is apparently due to the thermolability of the substances
Summary
Субстанция 17α-ацетокси-3β-бутаноилокси-6метилпрегна-4,6-диен-20-она (ГБ) серия 280917, чистота ≥ 99,0 % (ВЭЖХ) и субстанция 17α-ацетокси-3β-гидрокси-6-метилпрегна-4,6-диен-20-она (АМОЛ) – продукт промежуточного синтеза субстанции ГБ, чистота ≥ 99,0 % (ВЭЖХ) – предоставлены ФГАОУ ВО РНИМУ им. Для дериватизации готовили два раствора: раствор реактива Жирара Т и раствор гидроксиламина. Реактив Жирара Т готовили путем растворения 0,5 г реактива в смеси метанол : уксусная кислота (лед.) (9 : 1). Раствор гидроксиламина гидрохлорида готовили путем растворения 35 мг реактива в 3 мл воды, а затем к раствору добавляли 7 мл ацетонитрила. Матричные растворы аналитов ГБ, МА и АМОЛа готовили в ацетонитриле в концентрации 0,1 мг/мл. Далее готовили два стандартных раствора – смесь аналитов в ацетонитриле. Для приготовления модельного образца к 180 мкл сыворотки добавляли 20 мкл стандартного раствора 1 или 2 и перемешивали.
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