Therapeutic Drug Monitoring of Immunosuppressants with MassTox® Series A

Therapeutic drug monitoring of immunosuppressants is indispensable for therapy following organ transplantation. Cyclosporin A, everolimus, sirolimus and tacrolimus are mainly prescribed for avoiding organ rejection. Laboratories working with MassTox® TDM Series A can perform therapeutic drug monitoring of more than 200 drugs with one column and an identical sample preparation. So far, it excluded the analysis of immunosuppressants. Here, we describe that MassTox® Series A can also be used for the analysis of cyclosporin A, everolimus, sirolimus and tacrolimus

  Download Article as pdf

Sample Preparation

The sample preparation was performed in line with the MassTox® Immunosuppressants protocol (Chromsystems). In brief: 50 µl ­of sample (EDTA-whole blood), reconstituted 6PLUS1® calibrator (order no 28039/XL) or MassCheck® control (order no 0081-0085, 0089) was pipetted into a 1.5 ml reaction vial. 25 µl of the reconstituted Internal Standard Mix (order no. 93946/RUO) and 100 µl Extraction Buffer (order no 93005/RUO) were added and mixed briefly. 250 µl of Precipitation Reagent (order no. 93003/RUO) was added, vortexed for 1 min and incubated for 2 min at ambient temperature, then centrifuged for 5 min at 15000 g. The supernatant was diluted with Dilution Buffer 1 (92007/RUO; ratio 1:1).

LC-MS/MS

10 – 50 µl were injected at a temperature for the autosampler of 8°C – 15°C. Substances were separated on MasterColumn® A (order. no 92110) with a column temperature of 70°C. Flow rate, position of selection valve and binary gradient can be found in table 1. Detection was performed with electrospray ionisation in positive ion mode with a Sciex 4500 mass spectrometer. Used multiple reaction monitoring (MRM) transitions are found in table 2.

Table 1: Binary gradient profile

A full calibration of the analysis system for each series of measurements has been performed with 6PLUS1® Multilevel ­Calibrator Set (order no. 28039/XL). Calibration curves were constructed by calculating the analyte to internal standard (ISTD) peak area ratio on the y axis against calibrator concentrations on the x axis. Then a calibration curve was plotted for all analytes using linear regression and 1/x weighting.

The analysis of the four immunosuppressants was performed with an analysis time of 3.5 min (see chromatogram in fig. 1). Coefficients of variation were below 10% [Inter-assay (CV = 2.1 % - 8.6 %) and intra-assay (CV = 1.9 % - 8.3 %)] for all clinically relevant concentration ranges (see table 3). The lower limit and upper limits of quantification were determined and cover published therapeutic ranges for all four analytes (table 4).

Table 2: MRMs and corresponding ISTD

Table 3: Intra- and inter-assay data

Fig. 1: Chromatogram for the four immunosuppressants and its internal standards.

Table 4: Upper and lower limit of quantification

Ordering Information (To order the products or for a quote, please contact us here)

RUO: For research use only and not for diagnostic purposes