建立超高效能液相層析串聯式質譜偵測clopidogrel活性代謝物及其臨床運用性

Establishment of UPLC-MS/MS Method for Detection of Clopidogrel Active Metabolite and Its Clinical Application

Clopidogrel為抗血小板藥物，具有抑制血小板凝集及降低心血管疾病病患的缺血事件發生率。本研究開發及驗證UPLC-MS/MS定量血漿中clopidogrel活性代謝物(CAM)濃度之方法。於採血後在試管內加入穩定劑MPB(2-bromo-3’-methoxyacetophenone)，將CAM進行衍生化而轉變為穩定的CAMD(MPB-Derivatized Clopidogrel Active Metabolite)。經離心取得血漿樣品後，加入以同位素標定的clopidogrel做為內部標準品，以acetonitrile進行蛋白質沉澱，離心取得上清液。注入UPLC-MS/MS後，8分鐘內可定量CAMD濃度。此方法可校正濃度為0~200ng/mL，校正曲線之相關係數超過0.99；準確率為100±2.50%；而在Inter-和intra-assay的精準度皆±15％內，較低定量極限(LLOQ,Lower Limit of Qualification)為0.19ng/mL，沒有carryover干擾發生。運用此方法檢測21位服用600mg clopidogrel之檢體進行檢測，CAMD濃度於服藥後1小時達到最高，平均濃度為37.12ng/mL。我們建立了有效定量血漿中CAMD濃度之方法，並可運用在藥物動力學之研究中。

Clopidogrel, an antiplatelet agent, inhibits platelet aggregation and reduces ischemic events in people with cardiovascular diseases. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS) method was developed and validated to quantitate clopidogrel active metabolites (CAM) in plasma. CAM was derivatized and stabilized with MPB (2-bromo-3'-methoxyacetophenone) to form CAMD after blood sample collection. Plasma samples were obtained and deuterated analogs of clopidogrel were added as an internal standard. Proteinprecipitation was performed with acetonitrile, and then samples were centrifuged to pellet the proteins. The supernatant was injected into a UPLC-MS/MS. This UPLC-MS/MS method can quantify CAMD in less than 8 minutes. The calibration curves were linear with a correlation coefficient of over 0.99 in the ranged of 0 to 200 ng/mL. The accuracy was within 100±2.50%, and thecoefficient variation for inter- and intra-assay precision was within 15% at two different concentrations. The lower limit of qualification (LLOQ) was 0.19 ng/mL. There was no carry over observed in this assay. A total plasma samples were collected from 21 patients after administration of 600 mg clopidogrel. The highest concentration of CAMD was around 37.12 ng/mL at1 hour after receiving the loading dose. All in all, we have established an useful method to quantitate CAMD in plasma and could be used in pharmacokinetic study.