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A multi-center evaluation (3 sites) of the LC/MS/MS MassTrak tacrolimus Immunosuppressants Kit (Kit) was undertaken.Ten aspects of the analytical performance of the Kit were investigated based on FDA and CLSI guidelines.The linear analytical range of the procedure was between 0.68 and 31.7ng/mL. Within-run and total imprecision were6% and8% (n=240), respectively. Recoveries of tacrolimus added to clinical samples that contained between 5 and 10ng/mL of tacrolimus before addition were 99, 102 and 105% at 5.0, 10 and 20ng/mL, respectively. Comparison of in-house and Kit procedures in samples from liver (n=50-58) or kidney (n=50 or 51) transplant recipients yielded method mean biases between -2.0 and +10.7% at 5 and 15ng/mL.This evaluation indicates that the Kit is suitable for the monitoring of tacrolimus in kidney and liver transplant recipients.

Therapeutic drug monitoring of critical dose immunosuppressant drugs is established clinical practice and there are similar good reasons to monitor antiretrovirals. The aim of this article is to review the recent literature (last five years), with particular reference to the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS offers many potential advantages. The superior selectivity of LC-MS/MS over immunoassays for immunosuppressant drugs has been widely reported. Simultaneous measurement of a number of drugs can be performed. It is currently routine practice for the four major immunosuppressants (cyclosporin, tacrolimus, sirolimus and everolimus) to be simultaneously measured in whole blood. While up to 17 antiretroviral drugs have been simultaneously measured in plasma. The exquisite sensitivity of LC-MS/MS also provides the opportunity to measure these drugs in alternative matrices, such as dried blood spots, saliva, peripheral blood mononuclear cells and tissue. However, the clinical utility of measuring these classes of drugs in alternative matrices is still to be determined.

Objectives: This study compared the performance of a fluorescent polarization immunoassay (FPIA) against HPLC-tandem mass spectrometry (HPLC-MS) for the measurement of everolimus in renal transplant recipients. Design and methods: A total of 333 pre-dose samples from 45 renal transplant patients were analyzed by FPIA and HPLC-MS. Results: The inter-batch inaccuracy and precision of the FPIA for control samples were ≤6% and ≤13.0%, respectively ( n = 17). The comparison of patient results yielded the Deming regression equation FPIA = 1.19 × HPLC-MS + 0.51. The mean bias was 24.4% (95% CI: −3.0 to 54.2%, range: −30.1% to 79.4%). Conclusions: The FPIA had acceptable analytical performance during the study but compared to HPLC-MS overestimated everolimus in patient samples. This overestimation is probably due to calibration differences between the methods and cross-reactivity of the FPIA antibody with everolimus metabolites. The clinical importance of the observed overestimation by FPIA requires further investigation.

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

High-performance liquid chromatography coupled by an electrospray ion source to a tandem mass spectrometer (HPLC–ESI–MS/MS) is the current analytical method of choice for quantitation of analytes in biological matrices. With HPLC–ESI–MS/MS having the characteristics of high selectivity, sensitivity, and throughput, this technology is being increasingly used in the clinical laboratory. An important issue to be addressed in method development, validation, and routine use of HPLC–ESI–MS/MS is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. These effects are unseen in the chromatogram but have deleterious impact on methods accuracy and sensitivity. The two common ways to assess matrix effects are either by the postextraction addition method or the postcolumn infusion method. To remove or minimize matrix effects, modification to the sample extraction methodology and improved chromatographic separation must be performed. These two parameters are linked together and form the basis of developing a successful and robust quantitative HPLC–ESI–MS/MS method. Due to the heterogenous nature of the population being studied, the variability of a method must be assessed in samples taken from a variety of subjects. In this paper, the major aspects of matrix effects are discussed with an approach to address matrix effects during method validation proposed.

Tacrolimus is an immunosuppressant drug with a narrow therapeutic window and thus requires therapeutic drug monitoring. This study evaluates the suitability of the second-generation microparticle enzyme immunoassay (MEIA II) against a specific method, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), for the measurement of tacrolimus in both heart- and lung-transplant groups. A secondary objective was to investigate the effect of tacrolimus concentration on MEIA II measurement.The HPLC-MS assay was conducted as per our reported method and MEIA II performed according to manufacturer's instructions. Quality-control samples at 5, 11, and 22 microg/L were run in each batch to ensure assay integrity in both methods. Multiple trough samples from 18 heart patients (n = 126) and 17 lung patients (n = 203) were analyzed.The inter-batch imprecision and analytical recovery over the quality-control range by HPLC-MS (n = 12) was6% and 98.2% to 104%, respectively, and by MEIA II (n = 16)15% and 92.0% to 99.1%, respectively. The mean overestimation by MEIA II between the two methods for heart- and lung-transplant patient samples was found to be 9.9% (range: -37.4-45.4%) and 13.2% (range: -29.2-64.3%), respectively. Stratification of these data based on the tacrolimus concentration determined by MEIA II, yielded no statistically significant differences in bias between concentration subgroups within the clinically relevant range (p0.4). However, a statistically significant difference was detected between the highest concentration subgroup (20.0 microg/L) and lower concentration subgroups in both transplant populations (p0.05).This study suggests that where HPLC-MS is not available, MEIA II may be suitable for the therapeutic drug monitoring of tacrolimus in heart- and lung-transplant recipients. However, the clinical importance of the observed mean bias, considering the wide range in overestimation in heart- and lung-transplant patient samples, is yet to be determined.