Разработана экспрессная и чувствительная методика количественного определения метилдопы в плазме крови с применением ВЭЖХ-МС/МС. Динамический диапазон измерения концентраций составил 0,02-3,00 мкг/мл. Данная методика использована для проведения исследования фармакокинетики таблетированной формы метилдопы.Ключевые слова: метилдопа, ВЭЖХ-МС/МС, плазма, стабилизация, фармакокинетика. Quinta-Analytica, YaroslavlWe developed a rapid and sensitive method for quantifying plasma concentrations of methyldopa using HPLC-MS/MS. The range of concentration measurements was 0,02-3,00 g/ml. The method was applied for pharmacokinetic study of methyldopa tablet formulations.

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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.

Introduction: This article describes the method development approaches for bioassay of substances containing unstable functional groups and forming unstable metabolites using the example of mycophenolic acid, methyldopa and mebeverine metabolites. Materials and Methods: The concentration of mycophenolic acid, which contains one phenolic hydroxyl and forms glucuronides during metabolism, was measured in plasma using HPLC-MS/MS, HPLC-MS and GC-MS. The determination of methyldopa, containing two phenolic hydroxyls, in stabilised plasma was performed by HPLC-MS/MS in the range of 0.02-3.00 μg/ml. Desmethyl mebeverine acid, which contains one phenolic hydroxyl and is metabolised by forming phenolic glucuronide, was assayed simultaneously with mebeverine acid in the range of 10-2000 ng/ml. Results and Discussion: The selection of storage conditions of the samples containing unstable substances should begin with selecting an anticoagulant based on the study of its short-term stability and freeze/thaw stability. If an unacceptable result is obtained, a combination of the anticoagulant and a stabiliser solution, as well as a concentration of this solution and its volume ratio to the biological fluid should be titrated. After which, this method should be validated by using the selected anticoagulant or the combination of the anticoagulant and stabiliser solution. Conclusion: The application of this approach to developing a bioanalytical method for determination of unstable compounds makes it possible to avoid obtaining false assay results.