Abstract
The aim of the work was to prepare a simple but reliable HPLC-UV method for the routine monitoring of mycophenolic acid (MPA). Sample preparation was based on plasma protein precipitation with acetonitrile. The isocratic separation of MPA and internal standard (IS) fenbufen was made on Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) using a mobile phase: CH3CN:H2O:0.5M KH2PO4:H3PO4 (260:700:40:0.4, v/v). UV detection was set at 305 nm. The calibration covered the MPA concentration range: 0.1–40 µg/mL. The precision was satisfactory with RSD of 0.97–7.06% for intra-assay and of 1.92–5.15% for inter-assay. The inaccuracy was found between −5.72% and +2.96% (+15.40% at LLOQ) and between −8.82% and +5.31% (+19.00% at LLOQ) for intra- and inter-assay, respectively, fulfilling acceptance criteria. After a two-year period of successful application, the presented method has been retrospectively calibrated using the raw data disregarding the IS in the calculations. The validation and stability parameters were similar for both calculation methods. MPA concentrations were recalculated and compared in 1187 consecutive routine therapeutic drug monitoring (TDM) trough plasma samples from mycophenolate-treated patients. A high agreement (r2 = 0.9931, p < 0.0001) of the results was found. A Bland–Altman test revealed a mean bias of −0.011 μg/mL (95% CI: −0.017; −0.005) comprising −0.14% (95% Cl: −0.39; +0.11), whereas the Passing–Bablok regression was y = 0.986x + 0.014. The presented method can be recommended as an attractive analytical tool for medical (hospital) laboratories equipped with solely basic HPLC apparatus. The procedure can be further simplified by disapplying an internal standard while maintaining appropriate precision and accuracy of measurements.
Highlights
Mycophenolic acid (MPA) is an immunosuppressive agent presenting high inter- and intra-subject pharmacokinetic variability
The absolute recovery was analyzed by comparing the peak areas for extracted calibration standards with those obtained from direct injection of equivalent quantities of standards taking into account the volume ratio
The analysis of the results presented in the tables showed showed that
Summary
Mycophenolic acid (MPA) is an immunosuppressive agent presenting high inter- and intra-subject pharmacokinetic variability. It is available either as inactive pro-drug mycophenolate mofetil (MMF), or as enteric-coated mycophenolate sodium (EC-MPS) [1,2,3]. The AUC parameter (mainly: AUC0–12h ), better correlated with the clinical effect, has all the disadvantages of multiple drug determinations, the leading centers have been using a limited sampling strategy (LSS) and calculating AUC0–12h values based on the algorithms obtained for the therapeutic scheme used in the center [3,4,5]. The use of AUC-based monitoring requires the determination of MPA concentrations several times higher than with C0 monitoring. MPA is extensively biotransformed into the inactive glucuronide (MPAG) observed in concentrations many times exceeding MPA concentration in plasma, and into the active acyl-glucuronide (AcMPAG)
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