Therapeutic Drug Monitoring Of Immunosuppressants Research Articles

B-303 Comparison of Tacrolimus Concentrations Obtained By the Newly Approved Alinity i Tacrolimus Assay With a Reference Liquid-Chromatography Combined With Tandem Mass spectrometry Assay

Abstract Background Tacrolimus belongs to the calcineurin-inhibitor class of medications and used for preventing and maintenance of organ rejection in transplant recipients alone or in combination with other medications. Due to narrow therapeutic window and significant nephrotoxicity, this drug is routinely monitored with a suggested therapeutic range of 5–15 ng/mL. Recently, Abbott Laboratories (Abbott Park, IL) received FDA approval for using tacrolimus immunoassay on the Alinity i platform. We compared tacrolimus values in 101 transplant patients using Alinity i analyzer and a reference method based on liquid chromatography combined with mass tandem spectrometry (LC-MS/MS). Methods Tacrolimus immunoassay on the Alinity i analyzer is a chemiluminescent microparticle immunoassay (CMIA) requiring pretreatment of whole blood specimen prior to analysis. The analytical measurement range of the assay is 2–30 ng/mL. The left-over blood specimens were sent to Eurofins-Viracor reference laboratory in Lenexa, KS for analysis using LC-MS/MS method. After liquid-liquid extraction, specimens were analyzed using ascomycin as the internal standard for the presence of tacrolimus (assay is capable of analyzing other immunosuppressants simultaneously). UPLC C-18 column (waters) was used for separation using two mobile phases (A: 2.0 mM ammonium acetate in 0.1% formic caid in water and B: 2.0 mM ammonium acetate in 0.1% formic acid in methanol). Mass spectrometer was operated in positive ionization electrospray mode (tacrolimus m/z 821.6 > 768.6, ascomycin m/z 809.756.6. Results The total precision of the low control was 3.6% (mean: 4.48 ng/mL; CV 0.16 ng/mL, n = 20) while the total precision of the high control was 5.1% (mean: 25.4 ng/mL, SD: 1.29 ng/mL, n = 20). The linearity of the assay (2.0–30.0 ng/mL) was verified by running various calibrators as unknown. Comparing tacrolimus values in specimens from 101 transplant patients (49 kidney, 19 bone marrow transplant, 16 liver and 15 heart transplant) using the reference method (LC-MS/MS) as the x-axis and the corresponding values obtained by the tacrolimus immunoassay using Alinity I analyzer, we observed the following regression equation: y = 0.97 x + 1.005 (n = 101, r = 0.95). For kidney transplant recipients, the regression equation was y = 0.96 + 1.20 (n = 49, r = 0.94). Discussion Excellent correlation between tacrolimus values obtained by the newly FDA approved tacrolimus immunoassay with the reference LC-MS/MS based assay indicates further improvement in the performance of immunoassays for therapeutic drug monitoring of immunosuppressants. This may be due to reduced cross-reactivity of metabolites as indicated in the package insert. Conclusions The newly approved tacrolimus immunoassay for the application on the Alinity I analyzer can be used for routine therapeutic drug monitoring of tacrolimus in clinical laboratories.

A Drug Safety Concept (I) to Avoid Polypharmacy Risks in Transplantation by Individual Pharmacotherapy Management in Therapeutic Drug Monitoring of Immunosuppressants.

For several, also vital medications, such as immunosuppressants in solid organ and hematopoietic stem cell transplantation, therapeutic drug monitoring (TDM) remains the only strategy for fine-tuning the dosage to the individual patient. Especially in severe clinical complications, the intraindividual condition of the patient changes abruptly, and in addition, drug-drug interactions (DDIs) can significantly impact exposure, due to concomitant medication alterations. Therefore, a single TDM value can hardly be the sole basis for optimal timely dose adjustment. Moreover, every intraindividually varying situation that affects the drug exposure needs synoptic consideration for the earliest adjustment. To place the TDM value in the context of the patient's most detailed current condition and concomitant medications, the Individual Pharmacotherapy Management (IPM) was implemented in the posttransplant TDM of calcineurin inhibitors assessed by the in-house laboratory. The first strategic pillar are the defined patient scores from the electronic patient record. In this synopsis, the Summaries of Product Characteristics (SmPCs) of each drug from the updated medication list are reconciled for contraindication, dosing, adverse drug reactions (ADRs), and DDIs, accounting for defined medication scores as a second pillar. In parallel, IPM documents the resulting review of each TDM value chronologically in a separate electronic Excel file throughout each patient's transplant course. This longitudinal overview provides a further source of information at a glance. Thus, the applied two-arm concept of TDM and IPM ensures an individually tailored immunosuppression in the severely susceptible early phase of transplantation through digital interdisciplinary networking, with instructive and educative recommendations to the attending physicians in real-time. This concept of contextualizing a TDM value to the precise patient's condition and comedication was established at Halle University Hospital to ensure patient, graft, and drug safety.

Liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants and creatinine from a single dried blood spot using the Capitainer® qDBS device

In recent years, a lot of attention has been given to a more patient-centric therapeutic drug monitoring (TDM) of immunosuppressant drugs (tacrolimus, sirolimus, everolimus and cyclosporin A) by the use of microsampling techniques. By adopting Dried Blood Spots (DBS) after a finger prick, instead of conventional venous blood draws, follow-up can (partially) be established from patients’ homes. Despite the many advantages of DBS, one of the major disadvantages associated with this technique is the well described hematocrit (hct) effect. In order to overcome the hct area bias, different strategies have been proposed, amongst which the use of dried blood sampling techniques based on the volumetric collection of blood.The aim of this study was to evaluate the use of the Capitainer® qDBS (quantitative Dried Blood Spot) device for the combined TDM of four immunosuppressants and creatinine from a single qDBS. The set-up of an adequate sample preparation allowing both immunosuppressants and creatinine quantification was one of the key challenges in the method development due to device-specific interferences. Liquid chromatography tandem-mass spectrometry methods for the quantification of tacrolimus, sirolimus, everolimus, cyclosporin A and creatinine from qDBS (10 μL) were developed and validated based on international guidelines, also taking into account DBS-specific parameters. The methods proved to be accurate and reproducible, with absolute biases below 10% and within-run CVs (%) below 8% over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus, 20–1500 ng/mL for cyclosporin A, and 15–700 μmol/L for creatinine. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained, showing no hematocrit-dependence (compared to a hct of 0.37), except for cyclosporin A at higher hct values. Application on venous blood left-over patient samples showed good agreement between the results of Capitainer® qDBS and whole blood with 98% (47/48), 93% (41/44), 89% (41/46), 88% (38/43) and 89% (116/131) of the samples lying within 20% of the whole blood result for tacrolimus, sirolimus, everolimus, cyclosporin A and plasma/serum for creatinine, respectively. For creatinine a blood/plasma ratio of 0.85 was found and used to convert qDBS results to plasma/serum results. As a next step, capillary finger prick samples will need to demonstrate the clinical applicability of the method in a real life setting.

Application of a Fully Automated Dried Blood Spot Method for Therapeutic Drug Monitoring of Immunosuppressants: Another Step Toward Implementation of Dried Blood Spot Analysis.

The follow-up of patients under lifelong immunosuppressant therapy is pivotal to prevent allograft rejection after transplant. Part of the difficulties associated with routine monitoring of immunosuppressant concentrations can be alleviated by home sampling using dried blood spots (DBSs). To evaluate the applicability of a DBS method for the determination of immunosuppressants in venous blood samples, making use of an automated extraction platform. Paired venous DBSs and whole blood samples were analyzed for tacrolimus (n = 162), sirolimus (n = 47), everolimus (n = 45), and cyclosporin A (n = 61) with liquid chromatography coupled to tandem mass spectrometry, using fully automated extraction for DBSs. Agreement between the automated DBS and whole blood method was assessed by using Bland-Altman comparison. Both an analytical and a clinical acceptance limit were predefined at more than 67% of all paired samples within 20% of the mean of both samples and more than 80% of all paired samples within 20% of the whole blood concentration, respectively. An impact of the hematocrit (hct) on DBS quantitation was observed for all analytes, which could be alleviated for all analytes by using a hct conversion formula based on a tacrolimus data subset: [DBScorrected] = [DBSmeasured]/(1.6305 - 1.559*hct). After correction, both analytical and clinical acceptance criteria were met for all analytes. Automated DBS analysis shows great potential for routine therapeutic drug monitoring of immunosuppressants, avoiding any manual sample handling.

Volumetric microsampling for simultaneous remote immunosuppressant and kidney function monitoring in outpatient kidney transplant recipients.

Immunosuppressant and kidney function monitoring are crucial for kidney transplant recipient follow-up. Microsamples enable remote sampling and minimise patient burden as compared to conventional venous sampling at the clinic. We developed a liquid chromatography-tandem mass spectrometry assay to quantify tacrolimus, mycophenolic acid (MPA), creatinine and iohexol in dried blood spot (DBS), and volumetric absorptive microsample (VAMS) samples. The assay was successfully validated analytically for all analytes. Clinical validation was conducted by direct comparison of paired DBS, VAMS and venous reference samples from 25 kidney transplant recipients. Patients received iohexol 5-15 minutes before immunosuppressant intake and were sampled 0, 1, 2 and 3hours thereafter, enabling tacrolimus and MPA area under the concentration-time curve (AUC) and creatinine-based and iohexol-based glomerular filtration rate (GFR) estimation. Method agreement was evaluated using Passing-Bablok regression, Bland-Altman analysis and the percentages of values within 15-30% of the reference (P15 -P30 ) with a P20 acceptance threshold of 80%. For DBS samples, method agreement was excellent for tacrolimus trough concentrations (n =25, P15 = 92.0%) and AUCs (n =25; P20 = 95.8%) and adequate for creatinine-based GFR trend monitoring (n =25; P20 = 80%). DBS-based MPA AUC assessment showed suboptimal agreement (n =16; P20 = 68.8%), but was considered acceptable given its P30 of 100%. The assay performed inadequately for DBS-based iohexol GFR determination (n =24; P20 = 75%). The VAMS technique generally showed inferior performance, but can be considered for certain situations. The assay was successfully validated for tacrolimus, MPA and creatinine quantification in DBS samples, enabling simultaneous remote kidney function trend monitoring and immunosuppressant therapeutic drug monitoring in kidney transplant recipients.

Development and evaluation of a new test kit for determination of immunosuppressants in blood by UHPLC-MS/MS

In this study, we have developed and validated a new test kit that applied pure solvents instead of blood samples for determination of immunosuppressant drugs in blood by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). We have used commercially available quality control samples to verify this new test kit, and the results showed that the performance of our new kit was comparable with commercially available kits by immunoassays. The new test kit is pure solvent-based, which saves the cost and can be directly loaded without pre-treatment, thus shortening the detection time. The agreement between pure solvent-based kit and blood-based kit was also evaluated by Bland-Altman plot and two-tailed Student’s T-test. For 97.08% of the 137 Tacrolimus clinical samples, the concentration difference between these two kits was laid within 20% of the mean concentration. The concentrations of 12 cyclosporine samples and 15 sirolimus samples obtained by both kits showed no significant difference evaluated by the two-tailed Student’s T-test. These results further demonstrated the good agreement between these two types of test kits. Meanwhile, our pure solvent-based test kit could be stored stably for 14 weeks at -30 °C. Therefore, the newly developed pure solvent-based test kit can be used for detecting the whole blood immunosuppressant concentration and therapeutic drug monitoring of immunosuppressants.

Fully Automated Dried Blood Spot Extraction coupled to Liquid Chromatography-tandem Mass Spectrometry for Therapeutic Drug Monitoring of Immunosuppressants

Patients receiving immunosuppressant therapy, require intensive follow-up via therapeutic drug monitoring (TDM). This puts quite a burden on the patient involving frequent hospital visits and venipunctures and could (partially) be resolved by the use of dried blood microsamples (e.g. dried blood spots, DBS). One of the drawbacks of the use of DBS is the requirement for a dedicated, manual sample preparation. Fully automated DBS extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, could provide a solution for that. The aim of this study was to evaluate the use of the DBS-MS 500, online coupled to an LC-MS/MS system, for the TDM of immunosuppressants using DBS. Two methods for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A, in both DBS and whole blood, were developed and validated based on international guidelines. For the DBS method also DBS-specific parameters were taken into account. Both methods proved to be accurate and reproducible with biases below 11% (20% for the LLOQ) and CVs (%) below 14% (with a single exception) (20% for the LLOQ) over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus and 20 to 1500 ng/mL for cyclosporin A. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained. However, a hematocrit-dependent relative recovery was observed for DBS, with lower hematocrit values yielding higher relative recoveries (and vice versa). Relative to the reference hematocrit of 0.37, this difference exceeded 15% at hematocrit extremes (0.18 and 0.60). Application on venous left-over patient samples showed reasonable agreement between the results of both methodologies (8,6,9 and 9/10 mean DBS results within 20% of the mean whole blood result for tacrolimus, sirolimus, everolimus and cyclosporin A, respectively), although also here an impact of the hematocrit could be discerned. As a next step, larger patient sets are needed to allow a better insight on how (correction for) the hct effect affects the quantification of immunosuppressants via fully automated DBS analysis.

Feasibility of Using High-Contrast Grating as a Point-of-Care Sensor for Therapeutic Drug Monitoring of Immunosuppressants.

Point-of-care (POC) testing has demonstrated great transformative potential in personalized medicine. In particular, patients undergoing transplantation require POC testing to ensure appropriate serum immunosuppressant levels so as to maintain adequate graft function and survival. However, no suitable POC device for monitoring immunosuppressant levels is currently available. Exploiting the latest advances in metamaterials can lead to a breakthrough in POC testing. A high-contrast grating (HCG) biosensor is a low-cost, compact, simple-to-fabricate, and easy-to-operate structure. It is highly sensitive and robust in surface-based biomarker detection, which is favorable for the efficiency of a POC device. In this study, the feasibility of using an HCG as a POC sensor for therapeutic drug monitoring of immunosuppressants was evaluated. The detection efficiency of the most commonly prescribed immunosuppressive medication cyclosporine A by using this sensor was demonstrated to be comparable to those of conventional commercial kits, suggesting that the sensor has the potential to be used as a rapid detection and feedback platform for increasing drug compliance and improving new organ transplant survival.

Immunosuppressant therapeutic drug monitoring and trough level stabilisation after paediatric liver or kidney transplantation.

Immunosuppressive therapy must be guided by therapeutic drug monitoring (TDM) in paediatric liver (LT) and kidney transplantation (KT) patients to prevent under- and overdosing, which have clinical consequences. The purpose of our study was to analyse TDM results in our institutions and evaluate factors associated with blood level stabilisation after LT and KT. Blood levels of immunosuppressants were measured by immunoassay analysis. We compared blood level stabilisation between LT and KT, and evaluated associated factors in a retrospective study in two Swiss university hospitals. Forty-six patients (27 LT [median age 1.0 y], 19 KT [15.1 y]) were included. During the first month after transplantation, 32.8% (LT) and 41.2% (KT) of tacrolimus, and 22.1% (KT) of ciclosporin trough levels (measured before the next dose) were within target. In KT, trough levels stabilised earlier for tacrolimus than for ciclosporin (p = 0.02). Intensive care and hospital discharge occurred earlier in KT patients (p &lt;0.001). Living-donor LT was associated with an earlier intensive care discharge compared with deceased donor (5.5 vs 11 days, p = 0.02). Primary metabolic disease and graft/recipient weight-ratio &ge;0.03 was associated with earlier tacrolimus level stabilisation (14 vs 18 days, p = 0.01 and 15 vs 22 days, p = 0.05, respectively). In KT, recipient age (&ge;15.1 years) and weight (&ge;39.4 kg) were associated with an earlier trough level stabilisation (both 13 days vs not reached, p &lt;0.001), and age with earlier hospital discharge (10 vs 14 days, p = 0.02). Immunosuppressant trough levels were often outside the target range in the first month after LT and KT. Organ-specific factors were associated with trough stabilisation.

Preanalytical considerations in therapeutic drug monitoring of immunosuppressants with dried blood spots.

Background Dried blood spots (DBSs) could allow patients to prepare their own samples at home and send them to the laboratory for therapeutic drug monitoring (TDM) of immunosuppressants. The purpose of this review is to provide an overview of the current knowledge about the impact of DBS-related preanalytical factors on TDM of tacrolimus, sirolimus and everolimus. Content Blood spot volume, blood spot inhomogeneity, stability of analytes in DBS and hematocrit (Hct) effects are considered important DBS-related preanalytical factors. In addition, the influence of drying time has recently been identified as a noteworthy preanalytical factor. Tacrolimus is not significantly influenced by these factors. Sirolimus and everolimus are more prone to heat degradation and exhibited variations in recovery which were dependent on Hct and drying time. Summary and outlook DBS-related preanalytical factors can have a significant impact on TDM for immunosuppressants. Tacrolimus is not significantly influenced by the studied preanalytical factors and is a viable candidate for DBS sampling. For sirolimus and everolimus more validation of preanalytical factors is needed. In particular, drying conditions need to be examined further, as current protocols may mask Hct-dependent effects on recovery. Further validation is also necessary for home-based self-sampling of immunosuppressants as the sampling quality is variable.

Target Enzyme Activity and Phosphorylation of Pathway Molecules As Specific Biomarkers in Transplantation.

Therapeutic drug monitoring of immunosuppressants in the clinic is based on the measurement of blood concentration (pharmacokinetics). However, pharmacokinetic monitoring of immunosuppressants does not allow prediction of individual differences in pharmacological effects on immune cells. Pharmacodynamic (PD) monitoring via direct determination of target enzyme activity and phosphorylation of pathway molecules may enhance therapeutic drug monitoring and allow prediction of individual responses to immunosuppressants. This review discusses the clinical relevance of monitoring the activity of inosine-5'-monophosphate dehydrogenase (IMPDH), the target enzyme of mycophenolic acid, and of the phosphorylation of mechanistic target of rapamycin (mTOR) molecules. Significant progress regarding a robust and practicable assay for the determination of IMPDH activity as a specific PD parameter of mycophenolic acid activity has been achieved. The development of a rapid and reliable IMPDH assay system suitable for use in clinical practice was an important step that allowed thorough pharmacokinetics-PD investigations in large numbers of mycophenolic acid-treated patients. A reproducible and validated IMPDH assay was used in a few clinical trials by different research groups and is based on the chromatographic determination of newly generated xanthine monophosphate in mononuclear cell lysates. This assay requires only small volumes of blood and can be reliably used in multicenter trials; however, more clinical data from larger cohorts are needed to determine its clinical utility. Regarding monitoring of mTOR inhibitors (mTORis), the results of the first study that provided data on measurement of mTOR pathway molecules [p70 ribosomal protein S6 kinase (p70S6 kinase) and phosphorylated ribosomal protein S6] suggest that they are suitable targets for individualized PD monitoring of sirolimus and everolimus after organ transplantation. At present, only the phospho-flow phosphorylated ribosomal protein S6 assay has been validated in vitro and evaluated for confounding factors in vivo. The reported specific biomarkers for IMPDH activity and phosphorylation of mTOR molecules must be validated in clinical settings and multicenter studies to prove their clinical validity.

Clinical applications of MS-based protein quantification.

Mass spectrometry-based assays are increasingly important in clinical laboratory medicine and nowadays are already commonly used in several areas of routine diagnostics. These include therapeutic drug monitoring, toxicology, endocrinology, pediatrics, and microbiology. Accordingly, some of the most common analyses are therapeutic drug monitoring of immunosuppressants, vitamin D, steroids, newborn screening, and bacterial identification. However, MS-based quantification of peptides and proteins for routine diagnostic use is rather rare up to now despite excellent analytical specificity and good sensitivity. Here, we want to give an overview over current fit-for-purpose assays for MS-based protein quantification. Advantages as well as challenges of this approach will be discussed with focus on feasibility for routine diagnostic use.

Simultaneous Measurement of Cyclosporine A, Everolimus, Sirolimus and Tacrolimus Concentrations in Human Blood by UPLC–MS/MS

Liquid chromatography coupled with tandem mass spectrometry for therapeutic drug monitoring of immunosuppressants has been widely adopted in clinical chemistry laboratories. However, UPLC is replacing classical LC techniques, providing higher resolution and speed. We developed and validated an UPLC–MS/MS method for the simultaneous measurement of cyclosporine A, everolimus, sirolimus and tacrolimus concentrations in human blood. Following extraction with a zinc sulfate solution and acetonitrile, the chromatographic separation was achieved using an Acquity® UPLC® BEH™ (2.1 × 30 mm id, 1.7 µm) reverse-phase C18 column, with a water/methanol linear gradient containing 2 mM ammonium acetate with 0.1 % formic acid at a 0.5 mL min−1 flow rate. All immunosuppressants were detected by ESI mass spectrometry in positive ion multiple reaction monitoring mode using mass-to-charge transitions of 1219.8 → 1202.6/1184.4, 975.5 → 908.3/891.6, 931.5 → 864.3/883.3, 821.4 → 768.2/719.9 for cyclosporine A, everolimus, sirolimus and tacrolimus, respectively. Coefficients of variation and relative bias were less than 5.8 and 9.7 % for cyclosporine A, 8.7 and 6.4 % for everolimus, 8.5 and 7.2 % for sirolimus and 6.7 and 4.7 % for tacrolimus. Limits of quantification were 15.4 µg L−1 for cyclosporine A, 1.42 µg L−1 for everolimus, 1.58 µg L−1 for sirolimus and 0.65 µg L−1 for tacrolimus. Mean recoveries were greater than 77.6 % for all immunosuppressants. Evaluation of the matrix effect showed ion suppression for all the immunosuppressants, except for cyclosporine A, which suffered ion enhancement. No carry-over was observed. The validated method appears to be well adapted for therapeutic drug monitoring of multiple immunosuppressants in daily clinical practice.

TDMの落とし穴

Therapeutic drug monitoring (TDM) is an essential tool for the optimal use of drugs. Although rare, the drug concentration in blood determined by TDM can be higher or lower than that predicted from the dosage amount and patient information. In such cases, it is necessary to consider the occurrence of false positives and false negatives in the measurement process. Dose adjustment based on the measured blood drug concentration poses potential risks of underdosing and overdosing in patients suspected of false positive and false negative data, respectively. Therefore, pharmacists involved in TDM are required to become familiar with false positives and false negatives in TDM. In particular, the possibility of false positives in TDM of immunosuppressants by the affinity column-mediated immunoassay (ACMIA) method should be considered, while TDM of vancomycin or phenytoin requires attention to false negatives during measurement by the particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method. Moreover, interference from IgM in the measurement process may be of concern in false-negative results; therefore, the measured blood drug concentration needs to be verified when the blood IgM level is high. This mini-review outlines false positives and false negatives that should be considered in TDM and presents a discussion of the relevant literature.

Immunosuppressant therapeutic drug monitoring by LC-MS/MS: Workflow optimization through automated processing of whole blood samples

ObjectivesAlthough, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. MethodsSix-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. ResultsCalibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. ConclusionsOur findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.

Two rapid ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) methods with common sample pretreatment for therapeutic drug monitoring of immunosuppressants compared to immunoassay

Therapeutic drug monitoring of immunosuppressive agents is a critical and essential part of patient therapy after organ transplantation. We have developed high-throughput, robust, and rapid liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) methods with common pretreatment procedures for simultaneous quantification of four immunosuppressive agents (everolimus, sirolimus, tacrolimus, and cyclosporin A) in whole blood and one immunosuppressant (mycophenolic acid) in plasma. The new approach used in this work is based on improved sample preparation procedures allowing the analysis of five immunosuppressive drugs. Whole blood was prepared by transferring 100μL of blood into a 1.5-mL silanized conical test tube. Zinc sulfate solution (150μL), containing deuterated internal standards, was added to perform hemolysis. The samples were vortexing for 10s, followed by the addition of 250μL acetonitrile, containing internal standard for cyclosporin A, to precipitate proteins. The mixture was vortexed for 1min and centrifuged for 2min at 14,000rpm. The whole supernatant was transferred to a vial. To prepare blood plasma, the hemolysis step involving the addition of zinc sulfate was omitted and, instead of acetonitrile, methanol was used as the solvent for the internal standard (mycophenolic acid-d3). The volumes of chemicals used in this procedure were the same as those used in the procedure for immunosuppressants in whole blood. The basic validation parameters for the analytical methods were limits of detection (0.5ng/mL for everolimus, sirolimus and tacrolimus, 25ng/mL for cyclosporin A and 100ng/mL for mycophenolic acid), precision (<15%), recovery (>84%), repeatability and reproducibility. Possible mutual ion suppression was eliminated in the presence of internal standards. The method developed for the quantitation of immunosuppressants in whole blood was used to analyze 276 patient samples containing tacrolimus and 55 samples containing cyclosporin A. The results from LC/MS/MS were compared to those obtained from immunoassays of the same samples. Immunoassays significantly overestimated the concentrations of immunosuppressants.

High-throughput LC-MS/MS method for monitoring sirolimus and everolimus in the routine clinical laboratory

Immunosuppressant therapeutic drug monitoring (TDM) is an important requirement in post-transplant patient care. In recent years, high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a valid alternative to antibody-based immunoassays in TDM due to its high specificity and sensitivity. Furthermore, this technology allows for the simultaneous measurement of several immunosuppressive drugs. The aim of the present study was to establish a straightforward, robust, and high-throughput LC-MS/MS method for the simultaneous determination of sirolimus and everolimus in whole blood in order to replace immunoassays in our routine practice. Five-level blood calibrators were employed for assay development, while three materials at different concentrations were used for internal quality control. The proposed method uses protein precipitation for sample preparation. Analyses were performed using a triple quadrupole LC-MS/MS, with a C18 held at 60°C. Using an appropriate gradient elution profile and SPE on-line, elution times for all compounds analysed were 2.6 min with a total run-time of 3.5 min. Calibration curves were linear throughout the selected ranges. The intra- and inter-assay CVs (<7%), the limit of quantification (0.2 μg/L) and accuracy were highly satisfactory. On testing the results using the international proficiency testing scheme (UK-NEQAS), the performance of the proposed method was found to be highly reliable. The findings made by us indicate that the proposed method is of value, since it is speedy, straightforward, accurate, and applicable to different LC-MS/MS instruments for the routine TDM of organ transplant recipients.

Evaluation of the MassTrak Immunosuppressant XE Kit for the determination of everolimus and cyclosporin A in human whole blood employing isotopically labeled internal standards

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for therapeutic drug monitoring of immunosuppressants given to transplant recipients. This study evaluated the performance of the newly introduced MassTrak Immunosuppressant XE Kit (Waters Corporation; "the Kit") in the determination of everolimus and cyclosporin A (CsA) using LC-MS/MS. The linearity, precision, detection limit, carryover and matrix effect of the Kit and comparison of the in-house method and Kit procedure were evaluated according to Clinical and Laboratory Standards Institute guidelines. The Kit afforded good linearity in the measurement of everolimus from 2 to 26 ng/mL (R² = 0.996) and of CsA from 42.6 to 1407 ng/mL (R² = 0.995). The between- and within-run assay coefficients of variation were 2.3% - 8.0% for everolimus and 1.1% - 3.1% for CsA. The lower limit of quantitation of everolimus and CsA were 1.1-4.1 ng/mL, respectively. A comparison of CsA values obtained using inhouse reagents indicated good correlation with Kit results (R = 0.9909). The average matrix effect ranged from 15% - 39% for everolimus and 3% -10% for CsA, and ion enhancement was mostly observed for the matrix effect. The effect on analyte/internal standard ratios ranged from 1%-8% for everolimus and 1%-16% for CsA. The Kit employing isotopically labeled internal standards provides reliable measurements of immunosuppressant levels over a broad range of concentrations.

LC–MS/MS for Immunosuppressant Therapeutic Drug Monitoring

Due to their narrow therapeutic indices and highly variable pharmacokinetics, therapeutic drug monitoring is necessary to individualize immunosuppressant dosage following organ transplantation. Until recently, monitoring was performed primarily using immunoassays, however, there is an increasing shift to HPLC coupled with MS/MS, due to its greater sensitivity and specificity. Online sample clean-up with either a single analytical column or with 2D chromatography significantly reduces manual handling and is essential to minimize matrix effects and maximize specificity and, coupled with rapid chromatography, allows the simultaneous analysis of the major immunosuppressants, with rapid sample throughput. Thus, LC-MS/MS is an attractive and versatile technique that facilitates rapid development of analytical methods, including new immunosuppressants as they become approved for clinical use.

Immunosuppressant drug monitoring – a routine undertaking?1

Abstract The quantitative assessment of immunosuppressant drug levels is still one of the most challenging therapeutic drug monitoring procedures in clinical routine. During the past years, several technical developments matured to usable methods. In addition to immunoassays, liquid chromatography-tandem mass spectrometry has become a key method in immunosuppressant therapeutic drug monitoring. This overview should aid in understanding the advantages and disadvantages of the various assays and methods. UK-NEQAS proficiency testing results are used for an inter-assay comparison approach.