An investigation into the bias between liquid chromatography-tandem mass spectrometry and an enzyme multiplied immunoassay technique for the measurement of mycophenolic acid.

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 microL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and gamma-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.

Low hematocrit and serum albumin concentrations underlie the overestimation of tacrolimus concentrations by microparticle enzyme immunoassay versus liquid chromatography-tandem mass spectrometry.

BACKGROUND: Rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are used increasingly for tacrolimus (TRL) monitoring but show a negative difference with respect to a microparticle immunoassay (MEIA). This report examines possible reasons for this difference between methods. METHODS: We collected 1156 blood samples from 277 adult and 121 pediatric recipients of liver, renal, and bone marrow grafts or hepatocyte or pancreatic islet cell implants. TRL was measured in whole blood by MEIA and LC-MS/MS, and hematologic and biochemical data were collected when available. RESULTS: LC-MS/MS was significantly more precise (P <0.02) than the MEIA with increased sensitivity. The MEIA had a median difference of 16.2% vs LC-MS/MS overall, and this was significantly affected by patient cohort (P <0.001). The difference was greater in adult or pediatric liver graft recipients while they were inpatients rather than outpatients (31.8% and 14.0% vs 7.5% and 6.5%, respectively). The difference was also greater in bone marrow than kidney graft recipients (32.8% vs 15.8%, respectively). Multiple linear regression analysis showed significant inverse relationships of this difference with hematocrit (packed cell volume) and plasma albumin (P <0.001) in the total cohort and a positive relationship with plasma bilirubin in a subgroup of pediatric liver graft recipients. CONCLUSIONS: Patients with a low packed cell volume and plasma albumin are likely to show artificially high concentrations of TRL when measured by MEIA. The increased risk of underimmunosuppression must be considered should doses be reduced to lower these seemingly high TRL concentrations.

Mycophenolic acid pharmacokinetics in pediatric liver transplant recipients.

The aim of this study is to study mycophenolic acid (MPA) pharmacokinetics in stable pediatric liver transplant recipients and determine which times best represent the area under the concentration versus time curve (AUC) of MPA plasma concentrations. MPA pharmacokinetic profiles were determined in 21 liver transplant recipients (age, 2 to 15 years; 12 boys) administered mycophenolate mofetil (MMF) for at least 6 months. Ten patients were coadministered cyclosporine A (CsA), and 11 patients were coadministered tacrolimus (Tac). Plasma MPA levels were analyzed by enzyme-multiplied immunoassay technique in blood samples at 0, 0.33, 0.67, 1.25, 2, 3.5, 5, and 7 hours after MMF administration. The AUC of plasma concentrations to 7 hours (AUC(0-7)) was calculated using the linear trapezoidal rule. MPA plasma trough concentration (C(0)), maximal concentration, and AUC(0-7) values ranged 9- to 14-fold at a median of 1.81 mg/L (range, 0.4 to 3.7 mg/L), 10.5 mg/L (range, 2.8 to 40.0 mg/L), and 30.2 mg/L.hr (range, 9.3 to 80.3 mg/L.hr), respectively. AUC(0-7) correlated significantly with MMF dose (r = 0.552; P =.010) and C(0) (r = 0.844; P <.001). Median AUC(0-7) (29.6 v 31.4 mg/L.hr; P =.918) was similar in children comedicated with CsA or Tac. Median MMF dose was greater in the CsA group (500 v 250 mg; P =.006). Consequently, median AUC(0-7) was significantly lower in the CsA group when equalized for dose and body weight (2.02 v 3.85 microg/L.hr per mg of MMF dose per kg of weight; P =.002). Variations of MPA pharmacokinetics in pediatric liver transplant recipients suggest that monitoring MPA plasma levels is required. C(0) correlates closely with AUC. Comedication with CsA increased MMF dosage requirements compared with children on Tac therapy.