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.


Figure 1. Graphic formulae of mycophenolic acid (А), methyldopa (B) and desmethyl mebeverine acid (C)

Figure 2. MRM-chromatogram of the mixture of MPA and MPAG when using HPLC-MS/MS method.

Figure 3. Chromatograms of MPA ( А ) and MPAG ( B ) solutions when using HPLC-MS method.

Table 1.  Study of Back-Conversion of Phenolic Glucuronide of Mycophenolic Acid During Storage.

Figure 4. Сhromatograms of DMAG (А) and DMA (B) solutions.

Table 2. Selection of Stabilisers to Prevent Oxidation of Methyldopa.

Table 3. Validation Results of Methods Developed.

Note: LLOQ - lower limit of quantification, LQC- low QC samples; HQC - high QC samples; NMF – normalised matrix factor; MF- matrix factor

Table 4.  Stability Study of Analytes in Blood Plasma.

Table 5.  Cross Validation of Methods for Determining Mycophenolic Acid in Plasma.

Table 6.  Main Pharmacokinetic Parameters of Test and Reference Methyldopa Drug.

Note : C max - maximum plasma concentration in blood; T max - time-to-peak concentration; AUC 0-t - area under the pharmacokinetic concentration-time curve from time zero to the last blood sampling procedure; AUC 0-∞ - area under the pharmacokinetic curve from time zero to infinity; C max /AUC 0-t - relative absorption rate; K el - terminal elimination rate constant; Т Ѕ - drug elimination half-life; MRT - mean residence time.

Figure 5. Averaged pharmacokinetic profiles of methyldopa concentrations in blood plasma after administration of test (T) and reference (R) drug.

Table 7. Results of Comparison of Bioequivalence of Methyldopa formulations.

Table 8.  Pharmacokinetic Parameters of Mebeverine Metabolites.

Figure 6. Averaged pharmacokinetic profiles of MA and DMA concentrations in blood plasma after administering a single dose of Duspatalin (200 mg).

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