Byrd G D, Caldwell W S, Bhatti B S, Ravard A, Crooks P A
Biological Chemistry Division, R.J. Reynolds Tobacco Co., Winston-Salem, NC 27102, USA.
Drug Metabol Drug Interact. 2000;16(4):281-97. doi: 10.1515/dmdi.2000.16.4.281.
[Methyl-d3]-N-1-beta-D-glucopyranosyl-(+/-)-nicotinium inner salt ((+/-)-[methyl-d3]nicotine N-1-glucuronide) was synthesized from (+/-)-[methyl-d3]nicotine via reaction with methyl-2,3,4-tri-O-acetyl-1-bromodeoxy-alpha-D-glucopyranouronate, followed by deprotection with 1 M aqueous NaOH and purification by preparative TLC. Nicotine N-glucuronide was identified and determined directly in smokers' urine. A solid phase extraction method was used to partially isolate the material from urine. Subsequent determination was by thermospray-LC/MS using the synthetic d3-labeled nicotine N-glucuronide as internal standard. The identified urinary component had the same retention time as a synthetic standard and gave the same mass spectrum. The thermospray mass spectrum was characterized from the protonated molecular ion (m/z 339) and the protonated aglycone ion (m/z 163). Quantitative results from this direct method were compared with those from an indirect method, which calculated the nicotine glucuronide in the biological sample from the amount of nicotine released following treatment of the sample with the deconjugating enzyme, beta-glucuronidase. On average, the concentration of nicotine N-glucuronide determined by the direct method was 34% greater than that determined by the indirect method. Concentrations of nicotine N-glucuronide in urine ranged from 2.2 to 7.6 nmol/ml with a limit of detection of 1.3 nmol/ml.
[甲基 - d3]-N - 1 - β - D - 吡喃葡萄糖基-(±)-烟碱内盐((±)-[甲基 - d3]烟碱N - 1 - 葡萄糖醛酸苷)由(±)-[甲基 - d3]烟碱与2,3,4 - 三 - O - 乙酰基 - 1 - 溴脱氧 - α - D - 吡喃葡萄糖醛酸甲酯反应合成，随后用1 M氢氧化钠水溶液脱保护，并通过制备型薄层色谱法纯化。烟碱N - 葡萄糖醛酸苷在吸烟者尿液中被直接鉴定和测定。采用固相萃取法从尿液中部分分离该物质。随后以合成的d3标记烟碱N - 葡萄糖醛酸苷作为内标，通过热喷雾液相色谱/质谱法进行测定。鉴定出的尿液成分与合成标准品具有相同的保留时间，并给出相同的质谱图。热喷雾质谱以质子化分子离子(m/z 339)和质子化苷元离子(m/z 163)为特征。将该直接法的定量结果与间接法的结果进行比较，间接法是根据用β - 葡萄糖醛酸苷酶处理样品后释放的烟碱量来计算生物样品中的烟碱葡萄糖醛酸苷。平均而言，直接法测定的烟碱N - 葡萄糖醛酸苷浓度比间接法测定的高34%。尿液中烟碱N - 葡萄糖醛酸苷的浓度范围为2.2至7.6 nmol/ml，检测限为1.3 nmol/ml。
