Uracil in plasma: comparison of two in-house-developed LC-MS/MS methods

To prevent intoxication with 5-fluorouracil (5-FU) during chemotherapy, the initial measurement of plasma uracil levels has recently been recommended by the European Medicines Agency. Here we present and compare two new chromatographic methods for analysis of uracil in plasma. Two independent laboratories analyzed the same set of samples using in-house-developed analytical methods. The aim of this comparison is to demonstrate that our methods are harmonized, providing comparable concentrations and consistent clinical interpretations. The approaches presented employ mass spectroscopic detection in negative ion mode but differ in sample preparation and chromatographic techniques. Method 1: Protein precipitation using acetonitrile, followed by injection of a small sample volume for hydrophilic interaction liquid chromatography (HILIC). Method 2: Protein precipitation with acetonitrile combined with filtration through a 96-well filter plate, followed by evaporation and reconstitution in the chromatographic mobile phase for C18 reverse-phase chromatography. Both methods are demonstrated robust and accurate quantification of uracil. Results obtained from patient samples (n=161) show a strong correlation between the two methods, with a relative mean deviation of 2 %. The same clinical interpretation is obtained for the majority of samples (150/161). The remaining samples (11/161) were close to the lower clinical decision limit (16 μg/L). For the analysis of uracil, both presented methods, which are based on simplified sample preparation procedures, provide accurate results without significantly impacting the clinical interpretation. However, the results indicate a potential gray area of analytical imprecision from 14 to 20 μg/L, encompassing the lower clinical decision limit (16 μg/L).