Ultra fast liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of cyclosporin A, tacrolimus, sirolimus, and everolimus in whole blood using deuterated internal standards for cyclosporin A and everolimus.

Specific chromatographic methods for the measurement of cyclosporin A, tacrolimus, sirolimus, and everolimus blood levels in patients with organ transplants are time consuming when large numbers of samples must be processed. The authors developed a robust and fast (1 minute) online solid-phase extraction liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of cyclosporin A, tacrolimus, sirolimus, and everolimus. After protein precipitation of the whole blood with zinc sulphate and methanol, the supernatant was loaded on a wide pore reversed-phase column and cleansed of potential interferences with high flow for 20 seconds. After column switching, the analytes were transferred within 20 seconds in the back-flush mode to a short phenyl-hexyl column. The valve was then returned to its initial position and the chromatographic separation performed within 20 seconds. In the meantime, the loading column was prepared for the next injection. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes. Multiple-reaction mode transitions for each immunosuppressant and the internal standards were used for quantification. The working range of the method was 10-1500 microg/L for cyclosporin A, 1.0-44 microg/L for tacrolimus, 1.0-48 microg/L for sirolimus, and 1.2-48 microg/L for everolimus. Within and between-run assay coefficients of variation ranged from 1.8% to 13.0%. The described liquid chromatography/tandem mass spectrometry method shows best performance using the internal standards cyclosporin A-d4 for cyclosporin A, everolimus-d4 for everolimus and ascomycin for tacrolimus and sirolimus. In conclusion, the authors present a very fast, robust, and economical analytical method for therapeutic monitoring of multiple immunosuppressants in daily clinical practice.

Drugs and brain death diagnostics: determination of drugs capable of inducing EEG zero line.

BACKGROUND: Several drugs may affect the diagnosis of brain death by depressing the electroencephalographic signal. Serum levels of these drugs must be below their respective therapeutic ranges. METHODS: A high performance liquid chromatography-based fast and simple method was developed for determination of thiopentone, pentobarbitone, phenobarbitone, methohexital and propofol in serum and validated according to international recommendations. RESULTS: Separation of extracted analytes was performed on a reversed phase column [Agilent Zorbax SB C18, 5 microm, 4.6 x 250 mm; mobile phase 50% 50 mM NaH(2)PO(4) pH 4.6 mixed with 35% (v/v) acetonitrile and 15% (v/v) methanol]. Calibration curves were linear throughout the selected ranges (microg/mL, thiopentone 0.25-50, pentobarbitone 0.25-25, phenobarbitone 2.5-50, methohexital 0.125-2.50, propofol 0.25-5.0). The standard deviations for the regression line, recovery, imprecision and accuracy results were all highly satisfactory. The lower limits of quantification for propofol, thiopentone and pentobarbitone were set at 0.25 microg/mL, for phenobarbitone 2.5 microg/mL, and for methohexital 0.125 microg/mL, which are below the lowest pharmacologically relevant serum concentrations. Intra- and inter-day coefficients of variation were less than 10% throughout as determined with six replicates. CONCLUSIONS: The method presented is suitable for drug monitoring to help enhance the reliability of the diagnosis of brain death.