Therapeutic Drug Monitoring of Immunosuppressants with MassTox® Series A

Introduction

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

Material and Methods

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

Quantification

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.

Results

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

Conclusions

We demonstrate in this application note that the immunosuppressants cyclosporin A, everolimus, sirolimus and tacrolimus in whole blood can be analysed by using a hybrid of the two Chromsystems kits: MassTox® Series A and the MassTox® ONEMINUTE Immunosuppressants assay. The sample preparation is performed in line with the immunosupressants assay and with a dilution using a reagent of Series A. The sample is then applied to the analytical system of MassTox® Series A without modification to the chromatographic setup.

While this approach is not CE-IVD compliant, customers can use this assay on a research-use basis, enabling the analysis of more than 200 drugs plus immunosuppressants in the laboratory while also eliminating the need for changes to the chromatographic setup.

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

MassTox® Series A

Order No.  Products
92001/RUO

Mobile Phase 1

92002/RUO

Mobile Phase 2

92009/RUO

Rinsing Solution

92110

MassTox® TDM Master Column Series A

92007/RUO

Dilution Buffer 1

RUO: For research use only and not for not for diagnostic purposes

MassTox® Immunosuppressants

Order No.  Products
29039/XL

6PLUS1® Multilevel Whole Blood Calibrator Set

0081-0085, 0089

MassCheck® Immunosuppressants Whole Blood Controls Level I-IV, Blank

93946/RUO

Internal Standard Set

93003/RUO

Precipitation Reagent

93005/RUO

Extraction Buffer

33006

Reaction vials, transparent

93915/RUO

Tuning Mix