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

Abstract

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 VAMS_MPA to PL_MPA 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 VAMS_MPA to PL_MPA 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.

Keywords: Clinical validation; Cross-validation; LC–MS/MS; Mycophenolic acid; Renal transplantation; VAMS.

Fig. 1

Phases of cross and clinical validation are presented as the flowchart with the procedure that should be followed for each of the compared methods and paired samples. The algorithm is presented by the example of the data presented study. WB whole blood, PL plasma, VAMS volumetric absorptive microsampling, LC–MS/MS liquid chromatography-tandem mass spectrometry, HPLC–DAD high-performance liquid chromatography with diode array detection, PB Passing-Bablok regression, Vams_hi mycophenolic acid concentration corrected with hematocrit, VAMS_c mycophenolic acid concentration corrected with regression, VAMS_{c_DAD} mycophenolic acid concentration corrected with regression, MPE median prediction error, MPPE median percentage prediction error, RMSE root mean squared prediction error, MAPE median absolute percentage prediction error, R² Pearson’s correlation, SRCC Spearman rank correlation coefficient, ICC Intraclass correlation coefficient

Fig. 2

Evaluation of VAMS stability under different conditions [n = 4]. The stability was evaluated using two calibration levels of mycophenolic acid—LQC and HQC (0.35 µg/mL and 12.50 µg/mL, respectively) in different storage conditions (at RT with access to light and in the dark, and at 4 ℃ and – 20 ℃, both in the dark). The analyte in the VAMS sampler was stable during the six weeks in all storage places at both calibration levels. Stability is expressed as a percentage ratio to initial stability (100%). RT room temperature, RT (dark) room temperature in the dark condition, LQC lower quality control concentration, HQC higher quality control concentration, VAMS volumetric absorptive microsampling

Fig. 3

Scatter diagram showing the effect of the hematocrit (presented as cold/hot map of correlation between WB_{MPA(LC–MS/MS)} and VAMS_{MPA(LC–MS/MS)} concentrations differences). The correlation coefficient of the presented data was lower than zero (− 0.02596, − 0.31717 to 02696 for 95% CI), confirming that HT level does not influence analyte recovery from the VAMS sampler. WB_{MPA(LC–MS/MS)} MPA concentration in whole blood measured using tandem-mass liquid-chromatography analytical method (without correction), VAMS _{MPA(LC–MS/MS)} MPA concentration in volumetric absorptive micro sample measured using tandem-mass liquid-chromatography analytical method (without correction), HT hematocrit level, VAMS volumetric absorptive microsampling

Fig. 4

Passing-Bablok regression curves (a, c, e, g) and Bland–Altman plots (b, d, f, h) for cross-validation of the methods before conversion using hematocrit and regression formulas. Compared methods are described on the axis in each presented case. Data are presented as red circle points for all paired samples. The regression line, diagonal line, and confidence interval curve are presented as blue, red dotted, and blue area, respectively, on Passing-Bablok regression curves (a, c, e, g). The limit of agreement and the average difference are presented in red dotted lines and blue line, respectively, on Bland–Altman plots (b, d, f, h). In contrast, average bias is presented as a percent value with ± 1.96SD range. VAMS volumetric absorptive microsampling, LC–MS/MS liquid chromatography-tandem mass spectrometry, HPLC–DAD high-performance liquid chromatography with diode array detection, MPA_{VAMS-LC–MS/MS} mycophenolic acid concentration in VAMS sample determined LC–MS/MS, MPA_{WB-LC–MS/MS} mycophenolic acid concentration in whole blood sample determined LC–MS/MS, MPA_{PL-LC–MS/MS} mycophenolic acid concentration in plasma sample determined LC–MS/MS, MPA_{PL-HPLC–DAD} mycophenolic acid concentration in plasma sample determined HPLC–DAD

Fig. 5

Passing-Bablok regression curves (a, c, e) and Bland–Altman plots (b, d, f) for clinical validation of the methods after conversion before conversion using hematocrit and regression formulas. Compared methods are described on the axis in each presented case. Data are presented as red circle points for all paired samples. The regression line, diagonal line, and confidence interval curve are presented as blue, red dotted, and blue area, respectively, on Passing-Bablok regression curves (a, c, e). The limit of agreement and the average difference are presented in red dotted lines and blue line, respectively, on Bland–Altman plots (b, d, f). In contrast, average bias is presented as a percent value with ± 1.96SD range. VAMS volumetric absorptive microsampling, LC–MS/MS liquid chromatography-tandem mass spectrometry, HPLC–DAD high-performance liquid chromatography with diode array detection, MPA_{VAMS-LC–MS/MS} mycophenolic acid concentration in VAMS sample determined LC–MS/MS, MPA_{WB-LC–MS/MS} mycophenolic acid concentration in whole blood sample determined LC–MS/MS, MPA_{PL-LC–MS/MS} mycophenolic acid concentration in plasma sample determined LC–MS/MS, MPA_{PL-HPLC–DAD} mycophenolic acid concentration in plasma sample determined HPLC–DAD, MPA_{VAMSHt-LC–MS/MS} mycophenolic acid concentration corrected with hematocrit, MPA_{VAMSc-LC–MS/MS} mycophenolic acid concentration corrected with regression, MPA_{VAMSc_DAD-LC–MS/MS} mycophenolic acid concentration corrected with regression

Fig. 6

MPA determination results using different methods for each pediatric patient after renal transplantation—presented diagram included concentrations before and after conversion using formulas based on hematocrit value and Passing-Bablok regression model [n = 50]. The patient’s blinded numbers were assigned randomly. The meaning of using points, shapes, and colors is described in the legend on the graph. MPA_{VAMSHt-LC–MS/MS} mycophenolic acid concentration corrected with hematocrit, MPA_{PL-HPLC–DAD} mycophenolic acid concentration in plasma sample determined HPLC–DAD, MPA_{PL-LC–MS/MS} mycophenolic acid concentration in plasma sample determined LC–MS/MS, MPA_{VAMSc-LC–MS/MS} mycophenolic acid concentration corrected with regression, MPA_{VAMS-LC–MS/MS} mycophenolic acid concentration in VAMS sample determined LC–MS/MS, MPA_{WB-LC–MS/MS} mycophenolic acid concentration in whole blood sample determined LC–MS/MS, VAMS volumetric absorptive microsampling