Abstract
|
Procedure for Determination of Immunosuppressive Drugs in Whole Blood with Liquid Chromatography-Isotope Dilution Mass Spectrometry
by Petra Magdolna Molnár, László Dux, Hans Reinauer, Michael Kress, Theodorus Akerboom, Edit Szederkényi, Patricia Kaiser
|
|
|
Background: Cyclosporin A, sirolimus, tacrolimus, and everolimus are immunosuppressive drugs used for therapy after organ transplantation. There are several analytical procedures for monitoring the drug level in blood, e.g. immunological methods and high-performance liquid chromatography combined with mass spectrometry (MS).
From external quality assessment schemes, it became evident that the analytical results show high dispersion and further standardization is required.
Methods: Liquid/liquid extraction of the drugs from whole blood samples was performed using ammonium acetate buffer, pH 9.5, and tert-butylmethyl ether/ethyl acetate (1:1 v/v). Separation of the immunosuppressive drugs was achieved by HPLC using a phenyl-hexyl-RP column with a ternary gradient elution profile, consisting of water, methanol, and acetonitrile containing 0.1 % v/v formic acid and 0.1 mmol/L Cs+. Quantification of immunosup-pressive drugs was performed by isotope-dilution mass spectrometry using [2H]-Cyclosporin A [13C, 2H3]-Rapa-mycin, [13C,2H2]-Tacrolimus, and [13C2,2H4]-42-O-(2-Hydroxyethyl)rapamycin as internal standards.
Results: The recovery of the new procedure was determined by analysis of spiked blood samples. The recovery in spiked EDTA whole blood samples was 100.8 - 102.5 % for cyclosporin A, 101.6 - 103.0 % for sirolimus, 100.0 - 101.2 % for tacrolimus, and 99.5 - 102.4 % for everolimus. The imprecision of the new measurement procedure, expressed as the coefficient of variation (CV), was 1.17 - 2.60 % for cyclosporin A in the concentration range be-tween 8.1 and 979 µg/L, 0.92 - 1.72 % for sirolimus in the concentration range between 2.1 and 33.2 µg/L, 0.44 -1.06 % for tacrolimus in the concentration range between 2.0 and 30.8 µg/L and 0.82 - 4.34 % for everolimus in the concentration range between 2.1 and 31.4 µg/L.
Conclusions: An isotope dilution LC-MS/MS procedure for determination of four immunosuppressive drugs was developed to provide a basis for further development toward a reference measurement procedure.
DOI: Clin. Lab. 2011;57:983-992
|
