Abstract
Thiopurine drugs (azathioprine, 6-mercaptopurine) show wide interindividual variability and a narrow therapeutic range thus making therapeutic monitoring of their active metabolite 6-thioguanine nucleotides (6-TGN) desirable. We improved the currently available laborious and complex methodology of therapeutic drug monitoring of 6-TGN and the metabolite 6-methylmercaptopurine (6-MMP) in washed erythrocytes (ery) based on a whole-blood method. The analytes were hydrolyzed and extracted from 25-µL ethylenediaminetetraacetic acid-anticoagulated whole-blood spiked with isotope labeled 6-TG-C2N and 6-MMP-d3 internal standards. Chromatography was performed in 5.1 minutes on a C18 reverse phase column followed by detection via electrospray interface-coupled API 4000 mass spectrometer set up in the positive multiple reaction monitoring mode. The hemoglobin concentration was measured in 20 µL of the original sample (AHD575 method), and the results were standardized to 120 g/L of hemoglobin. Calibration curves were linear with r > 0.999 (6-TGN and 6-MMP up to 10,000 pmol/0.2 mL). The limit of quantification was 30 pmol/0.2 mL for 6-TGN and 6-MMP. Intraassay and interassay imprecision was <7.5% at 3 tested levels for 6-TGN and 6-MMP, respectively. Method comparisons were as follows: Ery 6-TGN: y = 1.3x - 11 and ery 6-MMP y = 1.1x - 124. The new method compares favorably with established ones, allowing for rapid single run determination of 6-TGN and 6-MMP from <50 µL of fresh or frozen whole blood. Linearity and limits of quantification cover the clinically relevant range. Variability during sample preparation and matrix effects are compensated by the use of isotope-labeled internal standards. The whole-blood method is hemoglobin standardized to avoid falsely low results in the case of anemia. The method correlates well with 6-TGN measured in washed erythrocytes, but it requires significantly less hands-on time. Preliminary therapeutic ranges for the most common indications of azathioprine and 6-MP are provided.
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