Development and validation of a rapid and sensitive UPLC-MS/MS method for determination of uracil and dihydrouracil in human plasma

作者：Jacobs Bart A W; Rosing Hilde; de Vries Niels; Meulendijks Didier; Henricks Linda M; Schellens Jan H M; Beijnen Jos H

来源：Journal of Pharmaceutical and Biomedical Analysis, 2016, 126: 75-82.

DOI：10.1016/j.jpba.2016.04.039

• Uracil
• Dihydrouracil
• Dihydropyrimidine dehydrogenase
• UPLC-MS/MS
• Capecitabine
• 5-Fluorouracil

Quantification of the endogenous dihydropyrimidine dehydrogenase (DPD) substrate uracil (U) and the reaction product dihydrouracil (UH2) in plasma might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity as a result of DPD deficiency. In this paper, we describe the development and validation of a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantification of U and UH2 in human plasma.
Analytes were extracted by protein precipitation, chromatographically separated on an Acquity UPLC (R) HSS T3 column with gradient elution and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. U was quantified in the negative ion mode and UH2 in the positive ion mode. Stable isotopes for U and UH2 were used as internal standards.
Total chromatographic run time was 5 min. Validated concentration ranges for U and UH2 were from 1 to 100 ng/mL and 10 to 1000 ng/mL, respectively. Inter-assay bias and inter-assay precision for U were within +/- 2.8% and <= 12.4%. For UH2, inter-assay bias and inter-assay precision were within +/- 2.9% and <= 7.2%. Adequate stability of U and UH2 in dry extract, final extract, stock solution and plasma was demonstrated. Stability of U and UH2 in whole blood was only satisfactory when stored up to 4 hours at 2-8 degrees C, but not at ambient temperatures.
An accurate, precise and sensitive UPLC-MS/MS assay for quantification of U and UH2 in plasma was developed. This assay is now applied to support clinical studies with fluoropyrimidine drugs.

• 出版日期2016-7-15