Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA

Abstract

Background: Therapeutic drug monitoring of immunosuppressive drugs in organ-transplanted patients is crucial to prevent intoxication or transplant rejection due to inadequate dosage. The commonly used immunoassays have been gradually undergoing replacement by mass spectrometry, since this physical method offers both a higher sensitivity and specificity. However, a switch should be carefully considered because it is a challenging procedure and needs to be thoroughly validated. From an economic perspective it is reasonable to include mycophenolic acid into the assay, because this saves the necessity for an additional measurement. However, to date very few validation protocols for the measurement of immunosuppressants, including mycophenolic acid, are available. In order to adequately compensate for matrix effects, the use of stable isotope labeled internal standards is advisable. Here, the authors describe a single method suitable for the quantification of cyclosporine A, tacrolimus, sirolimus, everolimus and mycophenolic acid, based on deuterated internal standards.

Methods: Plasma proteins were precipitated with zinc-sulfate, followed by an online solid phase extraction in the flow-through direction. Chromatographic separation was performed by a c18-phenyl-hexyl column. For subsequent mass spectrometric analysis stable-isotope-labeled internal standards were used. Results were available after 3.5 minutes.

Results: Low quantification limits (accuracy: 104 - 118%) and linearity resulted in 2 -1250 ng/ml for cyclosporine A; 0.5 - 42.2 ng/ml for tacrolimus; 0.6 - 49.2 ng/ml for sirolimus; 0.5 - 40.8 ng/ml for everolimus and 0.01 - 7.5 μg/ml for mycophenolic acid. Intra-assay precision revealed a coefficient of variation (CV) of 0.9 - 14.7%, with an accuracy of 89 - 138%. The CV of inter-assay precision was 2.5 - 12.5%, with an accuracy of 90 - 113%. Recovery ranged from 76.6 to 84%. Matrix effects were well compensated by deuterated internal standards.

Conclusions: The authors present a fast, economical and robust method for routine therapeutic drug monitoring comprising five immunosuppressants including mycophenolic acid.

Figure 1

Column Switching. Pos A: Conditioning of the analytical column and loading of SPE column with aqueous buffer containing 10% methanol. Pos B: Elution from the analytical column (97% methanolic solvent) and rinsing step of precolumn with pure methanol.

Figure 2

Microscopic images of whole blood (50 μl) incubated with water (125 μl) for various times. A: immediately after adding water B: 2 min incubation with water C: 2 min incubation with water plus 20 sec of ultrasonic treatment.

Figure 3

Exemplary chromatograms of original routine patient samples containing 103 ng/ml CSA, 9.6 ng/ml TAC, 6.1 ng/ml SIR, 5.3 ng/ml EVE and 4.9 μg/ml MPA. Mass transitions are stated.

Figure 4

Monitoring ion suppression (post column infusion method). The TIC of all immuno-suppressants when injected in methanolic solution, 1:3, 1:11 and 1:21 diluted sample-matrix respectively are shown. A: valve switch to methanolic buffer (97% methanol). B: valve switch to aqueous buffer (10% methanol; reconditioning of SPE column). C: strong loss of ions during straight forward elution of aqueous buffer from SPE column. D: second loss of signal during assumed elution of sample matrix (2.15 - 2.45 min).

Figure 5

Chromatographic separation of MPAG m/z 513.6/207.2 (dotted line) and MPA m/z 338.2/207.1 (solid line). The arrow indicates the in-source fragmentation of MPAG to MPA.