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 μL) spiked with FT, 5-FU, and 5-chlorouracil (internal standard) were diluted using 40 μL of 2 M ammonium acetate and 250 μL of acetonitrile with 2% formic acid; 20 μL of plasma spiked with the two drugs and internal standard was diluted with 80 μL of 2 M ammonium acetate and 500 μL of acetonitrile with 2% formic acid. After centrifugation, the clear supernatant extract (15 μL) was directly injected into the HILIC/MS/MS instrument, and each drug was separated on a Unison UK-Amino column (50 mm × 3 mm i.d., 3 μm 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 μg/mL for the tear and plasma samples with detection limits at 0.02-0.04 μg/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.

中文翻译：

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使用亲水相互作用液相色谱/串联质谱分析人眼和血浆中的替加福和5-氟尿嘧啶的高通量方法。

原理我们使用亲水相互作用色谱（HILIC）/串联质谱（MS / MS）开发了一种新的高通量方法，用于分析眼泪和血浆样品中的替加福（FT）和5-氟尿嘧啶（5-FU）。方法用40μL的2 M醋酸铵和250μL的乙腈（含2％甲酸）稀释掺有FT，5-FU和5-氯尿嘧啶（内标）的泪液样品（10μL）；用两种药物和内标加标的20μL血浆分别用80μL2 M乙酸铵和500μL乙腈和2％甲酸稀释。离心后，将澄清的上清液提取物（15μL）直接注入HILIC / MS / MS仪器，每种药物在Unison UK-Amino柱（50 mm×3 mm内径，粒径为3μm），线性梯度洗脱系统由10 mM乙酸铵（pH 6.8）和乙腈组成，流速为0.7 mL / min。我们通过负离子大气压化学电离的多反应监测（MRM）进行了定量分析。结果在2分钟内，每个MRM通道上的药物均出现了明显的峰。对于泪液和血浆样品，回归方程在0.04-4.0μg/ mL范围内显示出良好的线性，检出限为0.02-0.04μg/ mL。眼泪和血浆样品的目标分析物（FT和5-FU）的回收率分别在94-128％和94-104％范围内。两种药物的日内和日间变异系数均低于10.8％。两种样品的定量准确度均为97-115％。结论我们建立了高通量，可重现的，HILIC / MS / MS分析和氨丙基键合混合模式分离柱分析人眼泪和血浆样品中FT和5-FU的方法和实用程序 该方法可以应用于临床分析中使用的高通量程序。