Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography tandem mass spectrometry
Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography tandem mass spectrometry
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DOI：
10.1002/bmc.463
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发表时间：
2005-05-01
影响因子：
1.8
通讯作者：
Ogden, MW
中科院分区：
文献类型：
--
作者：
Heavner, DL;Richardson, JD;Ogden, MW
建立了一种hplc - lc -MS/MS测定人体尿液中尼古丁和5种主要代谢物：可替宁、反式-3′-羟基可替宁、尼古丁- n -葡萄糖醛酸盐、可替宁- n -葡萄糖醛酸盐和反式-3′-羟基coti - 9 - o -葡萄糖醛酸盐的方法。取500 μ L尿样，用含有nicotineinethyl-d、cotinine-methyl-d和trail -3′-hydroxycotinine-methyl-d(3)内标的磷酸盐缓冲液（1.5 mL）调整ph。对于未结合的代谢物，将等分液（800 μ L）应用于30 mg Oasis (TM) HLB-SPE柱，用2% nh4 (4)OH/H(2)O （3.0 m，L）和H(2)O （3.0 mL）冲洗，用甲醇（500 μ L）洗脱。洗脱液在二醇柱（50 x 2.1 mm）上采用LC-NIS/MS等浓度（100%甲醇）分析。对于总代谢物，在剩余的缓冲液中加入β -葡萄糖醛酸酶/缓冲液（100 μ L），在37℃（20 h）下孵育。提取800 μ L经酶处理的缓冲液，用同样的方法进行分析。结合代谢物通过减法间接测定。该方法的定量范围（ng/mL）为尼古丁14 ~ 10320，可替宁15 ~ 9800，反式-3′-羟基可替宁32 ~ 19220。该验证方法用于观察吸烟者现场尿样的日变化，吸烟者24小时尿样的消除半衰期以及现场和24小时尿样中的代谢物分布概况。版权所有(c) 2005 John Wiley & Sons, Ltd。
An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycoti nine-O-glucuronicle. A 500 mu L urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotineinethyl-d, cotinine-methyl-d, and traiis-3'-hydroxycotinine-methyl-d(3) internal standards. For the unconjugated metabolites, an aliquot (800 mu L) of the buffered solution was applied to a 30 mg Oasis (TM) HLB-SPE column, rinsed with 2% NH(4)OH/H(2)O (3.0 m,L) and H(2)O (3.0 mL) and eluted with methanol (500 mu L). The eluate was analyzed isocratically (100% methanol) by LC-NIS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 mu L) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 mu L) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples. Copyright (c) 2005 John Wiley & Sons, Ltd.