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BACKGROUND: Tacrolimus (TAC) and mycophenolic acid (MPA) are commonly used immunosuppressive therapies after renal transplant. Our objective was to quantify TAC and MPA concentrations in peripheral blood mononuclear cells (PBMCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) and to evaluate and validate the performance of the methodology. A prospective follow-up cohort study was conducted to determine whether intracellular concentrations were associated with adverse outcomes in renal transplants. METHODS: PBMCs were prepared using the Ficoll separation technique and purified with erythrocyte lysis. The cells were counted using Sysmex XN-3100 and then packaged and frozen according to a 50 µL volume containing 1.0 × 106 cells. TAC and MPA were extracted using MagnaBeads and quantified using an LC-MS/MS platform. The chromatography was run on a reversed-phase Waters Acquity UPLC BEH C18 column (1.7 µm, 50 mm × 2.1 mm) for gradient elution separation with a total run time of 4.5 min and a flow rate of 0.3 mL/min. Mobile phases A and B were water and methanol, respectively, each containing 2 mM ammonium acetate and 0.1% formic acid. Renal transplant recipients receiving TAC and MPA in combination were selected for clinical validation and divided into two groups: a stable group and an adverse outcome group. The concentrations were dynamically monitored at 5, 7, 14, and 21 days (D5, D7, D14, and D21) and 1, 2, 3, and 6 months (M1, M2, M3, and M6) after operation. RESULTS: Method performance validation was performed according to Food and Drug Administration guidelines, showing high specificity and sensitivity. The TAC and MPA calibration curves were linear (r2 = 0.9988 and r2 = 0.9990, respectively). Both intra-day and inter-day imprecision and inaccuracy were less than 15%. Matrix effects and recoveries were satisfactory. The TAC and MPA concentrations in 304 "real" PBMC samples from 47 renal transplant recipients were within the calibration curve range (0.12 to 16.40 ng/mL and 0.20 to 4.72 ng/mL, respectively). There was a weak correlation between PBMC-C0TAC and WB-C0TAC (p < 0.05), but no correlation was found for MPA. The level of immunosuppressive intra-patient variation (IPV) was higher in PBMC at 77.47% (55.06, 97.76%) than in WB at 34.61% (21.90, 49.85%). During the dynamic change in C0TAC, PBMC-C0TAC was in a fluctuating state, and no stable period was found. PBMC-C0TAC did not show a significant difference between the stable and adverse outcome group, but the level of the adverse outcome group was generally higher than that of the stable group. CONCLUSIONS: Compared with conventional therapeutic drug monitoring, the proposed rapid and sensitive method can provide more clinically reliable information on drug concentration at an active site, which has the potential to be applied to the clinical monitoring of intracellular immunosuppressive concentration in organ transplantation. However, the application of PBMC-C0TAC in adverse outcomes of renal transplant should be studied further.
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(1) Background: Continuous monitoring of tacrolimus (TAC), mycophenolic acid (MPA), and creatinine (Cre) after renal transplantation is vitally important. In this study, we developed a new method based on volumetric absorptive microsampling (VAMS) combined with Ultra Performance Liquid Chromatography−Tandem Mass Spectrometry (UPLC-MS/MS) to simultaneously quantify three analytes including TAC, MPA, and Cre in whole blood. (2) Methods: The VAMS-based UPLC-MS/MS assay used a shared extraction and a single injection to simultaneously quantify the included TAC, MPA, and Cre. Development and validation were carried out following the Food and Drug Administration and European Medicines Agency guidelines for the validation of bioanalytical methods. Moreover, clinical validation for the three analytes was performed in both dried blood spot (DBS) and VAMS. Furthermore, a willingness survey was conducted using the system usability scale (SUS) for renal transplant recipients. (3) Results: The assay was successfully validated for all analytes. No interference, carryover, or matrix effects were observed, and extraction recoveries and process efficiencies were >90.00%. Analysis was unaffected by hematocrit (0.20~0.60, L/L) and anticoagulants (EDTA-2K). Dried VAMS samples were stable for 7 days at ambient temperature and stable for at least 1 month at −20 °C. During clinical validation, the measured TAC, corrected MPA, and Cre concentrations of VAMS samples met the analytical standards (95.00%, 88.57%, and 92.50%). When more stringent clinical acceptance criteria were set, the results obtained by VAMS (90.00%, 71.43%, and 85.00%) better than DBS (77.50%, 62.86%, and 70.00%). Compared with DBS, the survey found that renal transplant recipients are more inclined to use VAMS. (4) Conclusions: A robust extraction and UPLC-MS/MS analysis method in VAMS tips was developed and fully validated for the simultaneous quantification of TAC, MPA, and Cre concentrations. This method provides analytical support for the one-sample remote monitoring of both immunosuppressive drug concentrations and renal function in allo-renal recipients. Based on the good consistency between this method and the routine detection of venous blood samples and higher patient satisfaction than DBS, we believe that VAMS sampling can be a better alternative to venous whole-blood sampling.
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BACKGROUND Chronic allograft dysfunction (CAD) is the leading cause of graft loss among kidney transplant recipients (KTRs). Bile acids (BAs) play an important role in regulating inflammatory process, which is the major contributor to the development of CAD. The aim of this study was to evaluate the association between BAs metabolic dysregulation and CAD in KTRs. MATERIAL AND METHODS Fifteen serum BA species were determined in 43 healthy controls (HCs) and 131 KTRs by UPLC-MS/MS. KTRs were grouped into stable renal function (STA) and CAD1 and CAD2 groups based on eGFR levels. Circulating CYP7A1, CYP7B1, CYP27A1, and SLCO2B1 mRNA levels were determined by RT-PCR. RESULTS Total BA concentrations were comparable among the 4 groups. However, KTRs showed significantly different BAs profiling compared to HCs. KTRs with severe CAD (CAD2) had significantly lower unconjugated BAs and secondary BAs (SBAs) compared to the other 3 groups. KTRs had significantly lower SBAs/primary BAs (PBAs) ratios than HCs, which were comparable among the 3 KTR groups. Conjugated/unconjugated BAs ratios increased significantly with the deterioration of allograft function, which was further confirmed by correlation analysis. Differential correlation network analysis revealed that perturbations in intraclass and interclass BA coregulation existed during CAD progression. Moreover, relative gene expressions of CYP7B1 and CYP27A1 were positively correlated with eGFR. CONCLUSIONS BA species profiling, but not total BA concentrations, was significantly altered in KTRs with CAD. The shifts from unconjugated BAs toward conjugated BAs, SBAs toward PBAs, and distinct pairwise BAs coregulation patterns were the main characteristics of KTRs with CAD.
