High-throughput method to analyze tegafur and 5-fluorouracil in human tears and plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Rationale We developed a new high-throughput method to analyze tegafur (FT) and 5-fluorouracil (5-FU) in tear and plasma samples using hydrophilic interaction liquid chromatography (HILIC)/tandem mass spectrometry (MS/MS). Methods The tear samples (10 mu L) spiked with FT, 5-FU, and 5-chlorouracil (internal standard) were diluted using 40 mu L of 2 M ammonium acetate and 250 mu L of acetonitrile with 2% formic acid; 20 mu L of plasma spiked with the two drugs and internal standard was diluted with 80 mu L of 2 M ammonium acetate and 500 mu L of acetonitrile with 2% formic acid. After centrifugation, the clear supernatant extract (15 mu L) was directly injected into the HILIC/MS/MS instrument, and each drug was separated on a Unison UK-Amino column (50 mm x 3 mm i.d., 3 mu m particle size) with a linear gradient elution system composed of 10 mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.7 mL/min. We performed quantification by multiple reaction monitoring (MRM) with negative-ion atmospheric-pressure chemical ionization. Results Distinct peaks were observed for the drugs on each MRM channel within 2 min. The regression equations showed good linearity within the range 0.04-4.0 mu g/mL for the tear and plasma samples with detection limits at 0.02-0.04 mu g/mL. Recoveries for target analytes (FT and 5-FU) for the tear and plasma samples were in the 94-128% and 94-104% ranges, respectively. The intra- and inter-day coefficients of variation for the two drugs were lower than 10.8%. The accuracies of quantitation were 97-115% for both samples. Conclusions We established a high-throughput, reproducible, and practical procedure for analyzing FT and 5-FU in human tear and plasma samples using HILIC/MS/MS analysis with an aminopropyl-bonded mixed-mode separation column. This method can be applied to the high-throughput routines used in clinical analyses.