Ultra Fast Liquid Chromatography-Tandem Mass Spectrometry Routine Method for Simultaneous Determination of Cyclosporin A, Tacrolimus, Sirolimus, and Everolimus in Whole Blood Using Deuterated Internal Standards for Cyclosporin A and Everolimus

Abstract

Specific chromatographic methods for the measurement of cyclosporin A, tacrolimus, sirolimus, and everolimus blood levels in patients with organ transplants are time consuming when large numbers of samples must be processed. The authors developed a robust and fast (1 minute) online solid-phase extraction liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of cyclosporin A, tacrolimus, sirolimus, and everolimus. After protein precipitation of the whole blood with zinc sulphate and methanol, the supernatant was loaded on a wide pore reversed-phase column and cleansed of potential interferences with high flow for 20 seconds. After column switching, the analytes were transferred within 20 seconds in the back-flush mode to a short phenyl-hexyl column. The valve was then returned to its initial position and the chromatographic separation performed within 20 seconds. In the meantime, the loading column was prepared for the next injection. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes. Multiple-reaction mode transitions for each immunosuppressant and the internal standards were used for quantification. The working range of the method was 10-1500 microg/L for cyclosporin A, 1.0-44 microg/L for tacrolimus, 1.0-48 microg/L for sirolimus, and 1.2-48 microg/L for everolimus. Within and between-run assay coefficients of variation ranged from 1.8% to 13.0%. The described liquid chromatography/tandem mass spectrometry method shows best performance using the internal standards cyclosporin A-d4 for cyclosporin A, everolimus-d4 for everolimus and ascomycin for tacrolimus and sirolimus. In conclusion, the authors present a very fast, robust, and economical analytical method for therapeutic monitoring of multiple immunosuppressants in daily clinical practice.

Therapeutic drug monitoring of immunosuppressants by liquid chromatography–mass spectrometry

“…One is the use of ultra-performance liquid chromatography (UPLC) [46][47][48]. UPLC increases throughput by shortening the LC run time, while retaining resolution due to small diameter separation media and high pressure.…”

“…A short LC-MS/MS method (chromatography time of 1 min) was accomplished by online solid phase extraction before chromatography was performed on a Luna phenyl-hexyl column (50 × 2.1 mm, 5 μm) [48]. The between-run CV was 1.8%-13.0% for cyclosporine A, tacrolimus, sirolimus, and everolimus.…”

“…Lastly, a fourth group found that differences in hematocrit (20% to 45%), spotted blood volume (25,35,50,75, and 100 μL), and punch location did not affect immunosuppressant quantitation [39]. [46,47] Online solid phase extraction (analytical run time of 1 min) [48] No LC separation (analytical run time of 13 s) [49] Liquid handler [50] Commercialization/ Standardization 7-Lab tacrolimus unification [52] 3-Lab cyclosporine A, tacrolimus, and sirolimus unification [53] Much of these DBS validation studies involve the deposition of venous blood from a collection tube onto the paper. Two recent studies evaluated capillary blood from a finger stick.…”

Antineoplastic drugs and their analysis: a state of the art review

“…589 The sensitivity and selectivity provided by MS helped to ease sample preparation in clinical chemistry laboratories. Column-switching systems coupling the extraction support to the chromatographic column before MS detection were also used for the analysis of sirolimus [598][599][600][601][602][603][604][605][606][607][608][609][610][611] and everolimus 598,599,602,603,[605][606][607]609,[612][613][614] in whole blood. LLE 615 or SPE 616 on 96-well plates constitutes another approach for sample preparation automatisation.…”

Development of Reference Measurement Procedures with LC-MS in Clinical Chemistry

“…The reduction of anti-rejection therapy decreases opportunistic infections and development of malignant tumor (22) The available analytical methods for therapeutic drug monitoring can divided into two groups such as immunological (33)(34)(35) and chromatographic (HPLC) procedures. (34,(36)(37)(38)(39)(40)(41)(42)(43)(44)(45).…”