Paul Salm

A retrospective analysis of mycophenolic acid and cyclosporin concentrations with acute rejection in renal transplant recipients.

OBJECTIVES Although monitoring of cyclosporin (CsA) is standard clinical practice postrenal transplantation, mycophenolic acid (MPA) concentrations are not routinely measured. There is evidence that a relationship exists between MPA area under the concentration-time curve (AUC) and rejection. In this study, a retrospective analysis was undertaken of 27 adult renal transplant recipients. METHODS Patients received CsA and MPA therapy and had a four-point MPA AUC investigation. The relationship between MPA AUC performed in the first week after transplantation, as well as median trough cyclosporin concentrations, and clinical outcomes in the first month posttransplant were evaluated. RESULTS A total of 12 patients experienced biopsy proven rejection (44.4%) and 4 patients had gastrointestinal adverse events (14.8%). A statistically significant relationship was observed between the incidence of biopsy proven rejection and both MPA AUC (p = 0.02) and median trough CsA concentration (p = 0.008). No relationship between trough MPA concentration and rejection was observed (p = 0.21). Only 3 of 11 (27%) patients with an MPA AUC > 30 mg x h/L and a median trough CsA > 175 microg/L experienced acute rejection, compared with a 56% incidence of rejection for the remaining 16 patients. Patients who experienced adverse gastrointestinal events had significantly lower MPA AUC (p = 0.04), but median trough CsA concentrations were not significantly different (p = 0.24). Further, 3 of these 4 patients had rejection episodes. CONCLUSIONS In addition to standard CsA monitoring, we propose further investigation of the use of a 4-point sampling strategy to predict MPA AUC in the first week posttransplant, which may facilitate optimization of mycophenolate mofetil dose at a time when patients are most vulnerable to acute rejection.

Evaluation of Limited Sampling Strategies for Estimation of 12-hour Mycophenolic Acid Area Under the Plasma Concentration–time Curve in Adult Renal Transplant Patients

Mycophenolate mofetil, the oral prodrug of mycophenolic acid, is indicated as immunosuppressive therapy after renal transplantation. To aid in the investigation of pharmacokinetic–pharmacodynamic relationships of mycophenolic acid in the clinical setting, limited blood sampling strategies have been proposed, and models from these developed, for the estimation of mycophenolic acid area under the concentration–time curve (AUC). In the current study, the authors investigated the predictive performance of six published models to estimate AUC. A total of 49 profiles from 25 renal transplant patients were used to test each models performance against a full 14 time-point AUC. A wide range of agreement was found when predicted AUCs were compared with full AUCs using linear regression analysis (range:r2 = 0.499 to 0.836). Model 1, which uses 4 time-points over 6 hours, was found to be superior to all other models. The range of time-points used in this model takes into account patients with variable absorption. This model should be further tested on data sets from other centers. The relatively poor performance of the other models may be caused by their inability to describe the peak concentration in these patients. Caution is warranted when using limited sampling strategies on patients whose absorption of mycophenolic acid is altered, compared with those of the pharmacokinetic profiles from which the model was developed.

Simultaneous Quantification of Tacrolimus and Sirolimus, in Human Blood, by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

In this paper the authors present a validated method for the simultaneous analysis of tacrolimus and sirolimus in human blood by high-performance liquid chromatography–electrospray tandem mass spectrometry. Blood samples (500 &mgr;L) were prepared by C18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring. The assay was linear for both compounds over the range 0.25–100 &mgr;g/L (r2 > 0.996, n = 7). At the limit of quantification (0.25 &mgr;g/L), for both sirolimus and tacrolimus, the interday imprecision was <3% and the analytical recovery was between 97.0% and 102%, respectively. The interbatch and intrabatch coefficients of variation of the method for both analytes, at the three quality control concentrations (0.5, 20, and 80 &mgr;g/L), were <16% and <10%, respectively. The analytical recovery, at the three control concentrations, ranged from 99.2% to 104% of the nominal concentration. The mean absolute recovery (±standard deviation) of tacrolimus, sirolimus, and internal standard was 82 ± 7%, 89 ± 12%, and 77 ± 8%, respectively (n = 12). In conclusion, the method presented can be used for simultaneous determination of tacrolimus and sirolimus and will aid in pharmacokinetic studies and therapeutic drug monitoring of these drugs. Furthermore, this method has economic benefits in the clinical setting where these drugs are coadministered.

The quantification of sirolimus by high-performance liquid chromatography-tandem mass spectrometry and microparticle enzyme immunoassay in renal transplant recipients

BACKGROUND Sirolimus, an immunosuppressive agent, is undergoing clinical trials in the prophylaxis of organ rejection. OBJECTIVES The aim of this study was to compare the performance of the semi-automated prototype (mode IA) microparticle enzyme immunoassay (MEIA) against a validated high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for measuring sirolimus concentrations. A secondary objective was to identify potential factors that may influence sirolimus measurement. METHODS The comparison was based on predose samples (n = 841) from 74 renal transplant patients receiving sirolimus therapy. Samples were collected up to 12 months after transplantation. RESULTS The mean (+/- SD) overestimation by MEIA was 42.5%+/-16.9%. Several variables were investigated to determine potential contributors to the observed overestimation. Stratification of the data based on the mean sirolimus concentrations determined by both assays yielded no statistically significant differences in bias between concentration subgroups within the clinically relevant range. Multiple linear regression analysis identified HPLC-MS sirolimus concentration (P = 0.03), hemoglobin concentration (P < 0.001), and time after transplantation (P < 0.001) as significant variables in the prediction of overestimation by MEIA. Analysis of the effect of time after transplantation on overestimation yielded a statistically significant difference up to 6 months after transplantation (35.6% to 46.4%) compared with 9 (23.9%) and 12 months (24.4%). A relationship between hemoglobin concentration and time after transplantation may explain the reduction in bias observed after 6 months. CONCLUSION The MEIA overestimates sirolimus concentrations in renal transplant patients compared with HPLC-MS. The clinical importance of this observed overestimation requires further investigation.

Liquid chromatography-tandem mass spectrometry method for the simultaneous quantitative determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood.

Simultaneous determination of the organophosphorus pesticides dimethoate, fenthion, diazinon and chlorpyrifos in human blood by HPLC-tandem mass spectrometry was developed and validated. The pesticides were extracted by a simple one-step protein precipitation procedure. Chromatography was performed on a Luna C(18) (30mmx2.0mm, 3microm) column, using a step-gradient at a flow rate of 0.4ml/min. The assay was linear from 0.5 to 100ng/ml (r(2)>0.992, n=24) for all pesticides. The inter- and intra-day accuracy and precision for the method was 96.6-106.1% and <10%, respectively. The lower limit of quantification was 0.5ng/ml. In conclusion, the method described displays analytical performance characteristics that are suitable for the quantification of these pesticides in cases of acute poisoning.

Quantification of free mycophenolic acid by high-performance liquid chromatography–atmospheric pressure chemical ionisation tandem mass spectrometry

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 microl) using ultrafiltration. The analytes were extracted from the ultrafiltrate (200 microl) using C18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 microg/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 microg/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was <8 and <9%, respectively. Further, analysis of pooled patient plasma produced an intra-day imprecision of 6.6%. The signal-to-noise ratio at the limit of quantification (2.5 microg/l) was approximately 5:1. The mean absolute recovery (n=6) of mycophenolic acid and the internal standard were 76.0+/-13.5% and 86.0+/-9.1%, respectively. The method reported provides an accurate and precise quantification of free mycophenolic acid over a wide analytical range and thus can be used for routine monitoring and pharmacokinetic studies.

Cloned enzyme donor immunoassay tacrolimus assay compared with high-performance liquid chromatography-tandem mass spectrometry and microparticle enzyme immunoassay in liver and renal transplant recipients.

The immunosuppressant drug tacrolimus has a narrow therapeutic index and is subject to a large variation in individual bioavailability and clearance. With its narrow therapeutic index, therapeutic drug monitoring is standard clinical practice in the management of transplant recipients. In this study, we report the evaluation of the cloned enzyme donor immunoassay (CEDIA) for the determination of whole-blood tacrolimus concentrations compared with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and microparticle enzyme immunoassay (MEIA) using samples obtained from liver (n = 100) and renal (n = 88) transplant recipients. Linear regression analysis showed a relationship of CEDIA = 1.24 HPLC-MS/MS −0.18 (r2 = 0.81). The mean bias (±SEM) for all patients when compared with HPLC-MS/MS was 22.2% (±2.1%). The precision of the CEDIA method for all samples showed a root mean square error of 3.1 μg/L. Liver transplant recipient samples showed a mean (±SEM) bias compared with HPLC-MS/MS of 12.5% (±1.6%). The precision of the CEDIA method for these samples showed a root mean square error of 1.5 μg/L. The data suggest that in the renal transplant group, the CEDIA and MEIA methods have a bias of 33.3% and 20.1%, respectively, compared with HPLC-MS/MS. The CEDIA tacrolimus immunoassay has been shown to be a rapid method for the determination of whole-blood tacrolimus concentrations and may be considered when HPLC-MS/MS is not available. When used in the clinical setting with other parameters, it would be a useful adjunct in the management of liver transplant recipients, but a significant bias in renal transplant patients needs to be further investigated.

A high-performance liquid chromatography-mass spectrometry method using a novel atmospheric pressure chemical ionization approach for the rapid simultaneous measurement of tacrolimus and cyclosporin in whole blood.

Concentration monitoring and dose individualization is required to optimize either tacrolimus or cyclosporin therapy. In this study, the validation of a simple, rapid high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous measurement of tacrolimus and cyclosporin in whole blood is reported. Blood samples (100 μL) were prepared by protein precipitation with zinc sulphate followed by acetonitrile (containing the internal standards ascomycin and cyclosporin D). The chromatographic run time was 1.5 minutes per sample. Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in negative ionization mode (tacrolimus: m/z 802.5 → 560.6, cyclosporin: m/z 1200.8 → 1088.4). The assay had an analytical range of 1.0 to 30 μg/L (r2 > 0.998, n = 6) for tacrolimus and 25 to 2000 μg/L (r2 > 0.999, n = 6) for cyclosporin. Tacrolimus inter- and intraday inaccuracy and precision [coefficient of variation (CV)] using quality control samples (2.5, 12.5, 25 μg/L) was less than ±10.0% and CV less than 5.0%, respectively (n = 5). Similarly, cyclosporin inter- and intraday inaccuracy and precision using quality control samples (70, 400, 1500 μg/L) was less than ±2.0% and CV less than 5.0%, respectively (n = 5). The lower limit of quantification for tacrolimus was 1.0 μg/L and cyclosporin 25 μg/L. The assay had an absolute mean recovery of 86.7% for tacrolimus and 89.0% for cyclosporin (n = 15). Intersubject variability, as a measure of potential matrix effects on results, was less than 6.0% CV for both analytes (n = 15). Extracted samples were stable for at least 20 hours. Results obtained from external proficiency testing samples measured by this method compared with the mean of all liquid chromatography-tandem mass spectrometry methods used by scheme participants revealed a strong correlation and good agreement for tacrolimus (r = 0.993, mean bias = −10.3%, n = 19) and cyclosporin (r = 0.996, mean bias = 3.0%, n = 20). In conclusion, this is the first reported high-throughput method that uses negative atmospheric pressure chemical ionization for the simultaneous measurement of tacrolimus and cyclosporin in whole blood.

High-performance liquid chromatography-tandem mass spectrometry as a reference for analysis of tacrolimus to assess two immunoassays in patients with liver and renal transplants

The accuracy and imprecision of three assays used for therapeutic monitoring of tacrolimus were tested using blood-containing weighed-in amounts of the drug, an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA I), and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) assay. Accuracy was acceptable for the HPLC-MS2 assay at all concentrations tested (< 10% deviation) and for the ELISA at 1.0 and 4.0 microg/l. Accuracy was not acceptable for the ELISA at 15.0 and 50.0 microg/l or for the MEIA I at all concentrations tested. Imprecision was acceptable for the HPLC-MS2 assay at all concentrations tested (coefficient of variation < 10%), for the ELISA at 15.0 and 50.0 microg/l, and for the MEIA I at 15.0 and 50.0 microg/l. Imprecision was not acceptable for the ELISA at 1.0 and 4.0 microg/l or for the MEIA I at 1.0 and 4.0 microg/l. This assessment with weighed-in amounts of tacrolimus verified the HPLC-MS2 assay as a reference method. The performance of the two immunoassays with HPLC-MS2 was then compared in the clinical setting using blood from patients with liver (n = 30) and renal (n = 37) transplants. In the liver transplant group (127 samples), the range of tacrolimus concentrations measured by HPLC-MS2, ELISA, and MEIA I was 1.9 to 31.8, 2.1 to 35.0, and less than 0.1 to 36.5 mg/l, respectively. In the renal transplant group (129 samples), the ranges were 1.7 to 26.1, 1.9 to 24.4, and 0.9 to 28.5 microg/l, respectively. Compared with the HPLC-MS2, the ELISA had minimal bias (0.1 to 0.2 microg/l) but unacceptable variability in values (SD > 13%). The MEIA I had unacceptable bias (1.7-1.8 microg/l) and variability (SD > 23%). These data indicated that neither the ELISA nor MEIA I is interchangeable with HPLC-MS2. Moreover, in view of the current trend to reduce the therapeutic dose of tacrolimus, quantitative results using the MEIA I would not be obtainable during therapeutic drug monitoring in some patients in whom effective therapeutic concentrations can be less than 5.0 microg/l.

A reliable high-performance liquid chromatography assay for high-throughput routine cyclosporin A monitoring in whole blood.

We report here a reliable high-performance liquid chromatography-ultraviolet assay for routine assay of cyclosporin A (CsA) in whole blood using solid-phase extraction. This assay is linear, between 20 and 2,000 μg/L, with correlation coefficients >0.998 for five consecutive standard curves. All coefficients of variation (CV) were <8% at CsA concentrations of 45, 480, and 1,800 μg/L, with the exception of the between-day CV at 45 μg/L, which was <15%. The relative accuracy of the method is >94% at 45, 480, and 1,800 μg/L. The mean recoveries for CsA and cyclosporin D (internal standard) were 38.2 ± 4.8% (n = 45) and 40.1 ± 6.7% (n = 45), respectively. This method has proven to be reliable and robust in a high-throughput therapeutic drug monitoring laboratory.