Abstract
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.
Highlights
This article describes the method development approaches for bioassay of substances containing un stable functional groups and forming unstable metabolites using the example of mycophenolic acid, methyldopa and mebeverine metabolites
The selected chromatographic parameters for both methods allow separating the analyte and its main meta bolite (Figs. 2 and 3); that is the reason why its fragmen tation in the ionisation process does not affect the accu racy of measuring mycophenolic acid (MPA). This experiment was not carried out when using the GC-MS method, because MPAG is not recovered from the plasma under the selected extrac tion conditions
A preliminary evaluation of the stability of mycophenolic acid was made by applying the HPLC-MS/MS method in plasma samples at a concentration of 25.0 pg/ml using K3EDTA and lithium heparinate as anticoagulants
Summary
This article describes the method development approaches for bioassay of substances containing un stable functional groups and forming unstable metabolites using the example of mycophenolic acid, methyldopa and mebeverine metabolites. If an un acceptable 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. Oxidation and hydrolysis are the main causes for the decomposition of molecules of drugs and their metaboli tes in biological fluids
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