Robust, High-Throughput LC-MS/MS Method for Therapeutic Drug Monitoring of Cyclosporine, Tacrolimus, Everolimus, and Sirolimus in Whole Blood

Abstract

The authors describe a fast, robust, and straightforward liquid chromatography and tandem mass spectrometry (LC-MS/MS) method with the use of a single LC-MS/MS system for cyclosporine A, tacrolimus, sirolimus, and everolimus in whole blood. The purpose of this method was to replace the immunoassay (IA) methods used in the laboratory of a hospital performing most organ transplantations (including heart, lung, liver, kidneys, bone marrow, and intestinal tract). Several LC-MS/MS methods have been described so far; however, most of them require complicated online extraction procedures. The described LC-MS/MS method uses a chromatographic gradient in combination with protein precipitation as sample preparation. The chromatographic method is capable of separating otherwise interfering peaks, with an analysis time of 2.6 minutes. Analyses were performed on a triple quadrupole LC-MS/MS system, with a C18 column held at 60 degrees C. Sample preparation required only 1 precipitation/dilution step. Sirolimus and everolimus are prepared and measured separately from tacrolimus and cyclosporine. During method development, it was found that the use of zinc sulfate provides process efficiency results of about 100% for tacrolimus and cyclosporine A, but only 81% and 87% for sirolimus and everolimus, respectively. With the developed sample preparation without zinc sulfate for sirolimus and everolimus, process efficiencies were 99% and 108%, respectively. The methods have been fully validated, and in a comparative study, patient samples were analyzed with IA and our developed LC-MS/MS methods. In the comparative studies, correlations (R2 values) of more than 0.85 were found between the IA and the new LC-MS/MS patient blood levels. There was a systematic deviation in blood levels measured by LC-MS/MS compared with IAs for cyclosporine A (17% lower than with immunoassay) and everolimus (30% lower than with IA). There seemed to be little or no systematic deviation for sirolimus and tacrolimus. The controls determined by the LC-MS/MS method over the past 10 months showed coefficient of variations of no more than 8.0% for each of the 4 immunosuppressants. In conclusion, the authors found the developed methods to be cost saving, more flexible, and more sensitive and that these methods have larger linear ranges than the previously used IA methods. The methods are already used for more than 20,000 patient samples in the daily routine, analyzing approximately 70 patient samples per day.

Comparison of blood sirolimus, tacrolimus and everolimus concentrations measured by LC-MS/MS, HPLC-UV and immunoassay methods

“…Several LC-MS/MS methods have been published for the quantitation of individual [16][17][18][19][20] or multiple immunosuppressants [21][22][23] in whole blood. Our laboratory initially introduced a previously published LC-MS/MS method for the analysis of sirolimus [20], which has subsequently been modified to also allow the additional simultaneous quantitation of tacrolimus and everolimus.…”

Pharmacogenetic testing and therapeutic drug monitoring are complementary tools for optimal individualization of drug therapy

“…Finally, since inadequate immunosuppression is linked to graft rejection and because high tacrolimus plasma levels have been associated to serious adverse effects, TDM may be helpful in the first stages of the treatment in determining an appropriate starting dosage, rapidly achieving adequate immunosuppression through controlling the evolution of tacrolimus levels and ultimately improving the outcome of renal transplantation (Table 2) [59].…”

Effects of bile duct stricture on the pharmacokinetics of the immunosuppressant tacrolimus in rats

“…Analysis of TAC levels in the blood and the level of demethyl- TAC and hydroxy-TAC in the hepatic microsomes was performed using LC-MS/MS [21]. The LC-MS/MS system (Applied Biosystems, Foster City, California) consisted of an LC-10AD micropump (Shimadzu Corporation, Kyoto, Japan) and an AS8020 automatic sample injector (Toso, Tokyo, Japan).…”