Gdańsk University of Technology, Faculty of Chemistry, Department of Analytical Chemistry, G. Narutowicz 11/12 Street, 80-233 Gdansk, Poland; Institute of Sport - National Research Institute, Trylogii 2/16 Street, 01-982 Warsaw, Poland.
Institute of Sport - National Research Institute, Department of Anti-Doping Research, Trylogii 2/16 Street, 01-982 Warsaw, Poland.
J Pharm Biomed Anal. 2018 Apr 15;152:279-288. doi: 10.1016/j.jpba.2018.02.004. Epub 2018 Feb 5.
A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL, 0.62 μg mL, 4.15 μg mL, 0.58 μg mL and 4.04 μg mL for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.
本研究建立并详细阐述了一种用于同时测定马尿液中布洛芬（IBU）及其四种可能代谢物（1-羟基布洛芬（1-OH IBU）、2-羟基布洛芬（2-OH IBU）、3-羟基布洛芬（3-OH IBU）和羧基布洛芬（CBX IBU）的新型分析方法，该方法采用气相色谱-电子电离-质谱联用技术（GC-EI-MS）。本研究采用弱阳离子固相萃取与强阳离子固相萃取相结合的方法，最大限度地减少基质效应，对样品前处理方法进行了改进。采用基质匹配的质控样品，对该方法的专属性、基质效应、线性、检测限（LOD）和定量限（LOQ）、精密度、准确度、交叉污染和稳定性进行了验证。此外，还评估了提取回收率。该方法对 IBU、1-OH IBU、2-OH IBU、3-OH IBU 和 CBX IBU 的检测限分别为 1.75μg·mL、0.62μg·mL、4.15μg·mL、0.58μg·mL 和 4.04μg·mL，定量限分别为 5.00μg·mL、0.62μg·mL、4.15μg·mL、0.58μg·mL 和 4.04μg·mL。该方法已成功应用于马的排泄研究，给 12 匹马（母马和公马）单次口服 IBU 后，在给药后 5 或 6 天内采集马尿液样本。首次报道了马尿液中 3 种 IBU 代谢物（2-OH IBU、3-OH IBU 和 CBX IBU）的检测和测定数据。结果表明，IBU 及其代谢物在给药后第 1 天即可被检测到，排泄迅速。在马尿液样本中，IBU 是最主要的化合物（有两个例外，即来自两只马的样本，其中 CBX IBU 的仪器响应最高）。结果表明，CBX IBU 和 2-OH IBU 这两种主要的 IBU 代谢物可能是马运动中 IBU 滥用的重要标志物，可用于赛马运动中的兴奋剂检测。
