Therapeutic drug monitoring of mycophenolic acid (MPA) using volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients: ultra-high-performance liquid chromatography-tandem mass spectrometry analytical method development, cross-validation, and clinical application

Background Mycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results. Methods This study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10–15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMSMPA to PLMPA concentration was performed using formulas based on hematocrit values and a regression model. Results LC–MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland–Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMSMPA to PLMPA concentration and confirmed on an independent group of samples. Conclusions This study is the first development of a triple matrix-based LC–MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC–DAD protocol. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s43440-023-00509-w.

Fresh whole blood (WB) and plasma (PL) for method validation were systematically obtained from healthy donors untreated with immunosuppressive drugs (TAC and MPA) at the Regional Centre of Blood Donation and Hemotherapy (Warsaw, Poland). Blood was stored at 4°C, while plasma was frozen at -20°C and used to prepare calibration curves for one or four weeks, respectively.
The above reference and internal standards were stored at −20°C in a freezer to maintain appropriate stability. Other chemical substances and reagents, such as the liquids used for mobile phase preparation, analyte extraction, and protein precipitation, were stored at room temperature or 4°C when prepared in the experimental mixtures.
Vacutainer test tubes (1.6 mL) containing K3-EDTA (tripotassium salt of ethylenediamine tetraacetic acid) as an anticoagulant for whole-blood collection, and 4 mL test tubes with clot activator, as well as lancets, and blood collection sets, were obtained from Becton Dickinson (Warsaw, Poland) or Sarstedt (Nümbrecht, Germany). Simple laboratory materials such as tips, test tubes, and falcon tubes were purchased from Sarstedt (Nümbrecht, Germany) or GenoPlast Biotech (Rokocin, Poland). Chromatographic vials integrated with 300 μL inserts and complementary screw caps were obtained from Thermo Scientific (Waltham, MA, USA) or Agilent (Santa Clara, CA, USA).

Calibrators
Primary stock solutions were prepared in glass ampules using solid standards for MPA (1 mg/mL), MPA-d3 (0.1 mg/mL), and MPAG (0.1 mg/mL) via dissolving in methanol: water mixture (50:50, v/v). The above solutions were appropriately diluted to obtain the estimated concentration of working solutions and finally to generate the calibration curve. The SIL-IS working solution was prepared at a 25µL/mL concentration level and, in the next step, diluted during calibration curve preparation. The MPAG solution has diluted a hundredfold and was used only as a control for chromatographic separation of MPA and MPAG in patients' samples.
Notable, the QC working solutions were prepared from other primary solutions of MPA than calibration curve points (but the same primary solution concentration-1 mg/mL). Working solutions were stored at -20 o C in the freezer.
During the investigation, three types of matrices were used for calibration methods: plasma, whole blood, and capillary blood in VAMS samplers. The WB and PL samples were fortified abreast by spiking 50µL of each matrix with 10µL of calibration working solution or QC working solutions. The VAMS calibration sample was prepared by spiking the 50µL of WB with 10µL of the appropriate working solution after mixing by self-automated vortex (Chemland, Stargard, Poland) gently absorbing by VAMS tip. The calibration levels and quality controls (QC) for MPA were prepared at levels: 0.25, 0.35 (lower quality control, LQC), 0.5, 1.0, 2.5, 3.50 (medium quality control, MQC), 5.0, 10.0, 12.5 (higher quality control, HQC), and 15.0 µg/mL. The WB and PL without immunosuppressants were obtained from healthy volunteers.

Sample preparation protocol
The 50μL of WB or PL was diluted with 90 μL pure water, and 10 μL of internal standard (d3-MPA) was added. In the next step, 400 μL of the precipitation mixture (0.1M zinc sulfate aqueous solution and acetonitrile, 50:50 (v/v)) was added. After that, the sample was shaken for 10 min at RT using an automatic shaker (ThermoScientific, Waltham, MA, USA) and