Ceccato A, Vanderbist F, Pabst J Y, Streel B
Galephar MF, Marche-en-Famenne, Belgium.
J Chromatogr B Biomed Sci Appl. 2000 Oct 1;748(1):65-76. doi: 10.1016/s0378-4347(00)00318-2.
Pharmacokinetic studies require sensitive analytical methods to allow the determination of low concentrations of drugs and metabolites. When drugs present an asymmetric center, the enantiomeric determination of the compounds of interest should be performed. The method developed is based on on-line LC-MS-MS using atmospheric pressure chemical ionization as an interface determination of enantiomers of tramadol (T) and its active metabolite O-desmethyltramadol (ODT) in human plasma. This determination is preceded by an off-line solid-phase extraction (SPE) on disposable extraction cartridges (DECs), performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and water. The washing step was performed with water and the analytes were finally eluted by dispensing methanol. The collected eluate was then evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The enantiomeric separation of tramadol and ODT was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine (97:3:0.1, v/v). The LC system was then coupled to a tandem mass spectrometry system with an APCI interface in the positive ion mode. The chromatographed analytes were detected in the selected reaction monitoring mode. The MS-MS ion transitions monitored were 264-->58 for tramadol, 250-->58 for ODT, and 278-->58 for ethyltramadol, used as internal standard. The method was validated. The recoveries were around 90% for both T and ODT. The method was found to be linear for each enantiomer of both compounds (r2>0.999). The mean RSD values for repeatability and intermediate precision were 3.5 and 6.4% for T enantiomers and 5.0 and 5.6% for ODT enantiomers, respectively. Moreover, the method was found to be selective towards other metabolites, N-desmethyltramadol and N,O-desmethyltramadol (NODT). The method developed was successfully used to investigate plasma concentration of enantiomers of T and ODT in a pharmacokinetic study.
药代动力学研究需要灵敏的分析方法，以便测定低浓度的药物和代谢物。当药物存在不对称中心时，应对感兴趣的化合物进行对映体测定。所开发的方法基于在线液相色谱-串联质谱法，使用大气压化学电离作为接口，测定人血浆中曲马多（T）及其活性代谢物O-去甲基曲马多（ODT）的对映体。在进行该测定之前，先在一次性萃取柱（DEC）上进行离线固相萃取（SPE），该操作通过配备机械臂的样品处理器（ASPEC系统）自动完成。填充有乙基硅胶（50 mg）的DEC先用甲醇和水进行预处理。用水进行洗涤步骤，最后通过加入甲醇洗脱分析物。收集的洗脱液然后蒸发至干，再溶解于液相色谱流动相中并注入液相色谱系统。在以直链淀粉三（3,5-二甲基苯基氨基甲酸酯）作为手性选择剂的Chiralpak AD柱上实现了曲马多和ODT的对映体分离。流动相为异己烷-乙醇-二乙胺（97:3:0.1，v/v）。然后将液相色谱系统与具有大气压化学电离接口的串联质谱系统相连，采用正离子模式。在选择反应监测模式下检测色谱分离后的分析物。监测的质谱-质谱离子跃迁对于曲马多为264→58，对于ODT为250→58，对于用作内标的乙基曲马多为278→58。该方法经过了验证。T和ODT的回收率均约为90%。发现该方法对两种化合物的每种对映体均呈线性（r2>0.999）。T对映体重复性和中间精密度的平均相对标准偏差值分别为3.5%和6.4%，ODT对映体分别为5.0%和5.6%。此外，发现该方法对其他代谢物N-去甲基曲马多和N,O-去甲基曲马多（NODT）具有选择性。所开发的方法成功用于药代动力学研究中T和ODT对映体血浆浓度的研究。
