Wang Ling-Zhi, Goh Sok-Hwei, Wong Andrea Li-Ann, Thuya Win-Lwin, Lau Jie-Ying Amelia, Wan Seow-Ching, Lee Soo-Chin, Ho Paul C, Goh Boon-Cher
Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore 117599, Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University Health System, Singapore 117599, Singapore.
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.
PLoS One. 2015 Mar 20;10(3):e0118553. doi: 10.1371/journal.pone.0118553. eCollection 2015.
A novel, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the evaluation of exemestane pharmacokinetics and its metabolites, 17β-dihydroexemestane (active metabolite) and 17β-dihydroexemestane-17-O-β-D-glucuronide (inactive metabolite) in human plasma. Their respective D3 isotopes were used as internal standards. Chromatographic separation of analytes was achieved using Thermo Fisher BDS Hypersil C18 analytic HPLC column (100 × 2.1 mm, 5 μm). The mobile phase was delivered at a rate of 0.5 mL/min by gradient elution with 0.1% aqueous formic acid and acetonitrile. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionisation (ESI) and monitored by multiple reaction monitoring (MRM) in positive mode. Mass transitions 297 > 121 m/z, 300 > 121 m/z, 299 > 135 m/z, 302 > 135 m/z, 475 > 281 m/z, and 478 > 284 m/z were monitored for exemestane, exemestane-d3, 17β-dihydroexemestane, 17β-dihydroexemestane-d3, 17β-dihydroexemestane-17-O-β-D-glucuronide, and 17β-dihydroexemestane-17-O-β-D-glucuronide-d3 respectively. The assay demonstrated linear ranges of 0.4-40.0 ng/mL, for exemestane; and 0.2-15.0 ng/mL, for 17β-dihydroexemestane and 17β-dihydroexemestane-17-O-β-D-glucuronide, with coefficient of determination (r2) of > 0.998. The precision (coefficient of variation) were ≤10.7%, 7.7% and 9.5% and the accuracies ranged from 88.8 to 103.1% for exemestane, 98.5 to 106.1% for 17β-dihydroexemestane and 92.0 to 103.2% for 17β-dihydroexemestane-17-O-β-D-glucuronide. The method was successfully applied to a pharmacokinetics/dynamics study in breast cancer patients receiving exemestane 25 mg daily orally. For a representative patient, 20.7% of exemestane in plasma was converted into 17β-dihydroexemestane and 29.0% of 17β-dihydroexemestane was inactivated as 17β-dihydroexemestane-17-O-β-D-glucuronide 24 hours after ingestion of exemestane, suggesting that altered 17-dihydroexemestane glucuronidation may play an important role in determining effect of exemestane against breast cancer cells.
开发并验证了一种新颖、快速且灵敏的液相色谱 - 串联质谱（LC-MS/MS）方法，用于评估依西美坦在人血浆中的药代动力学及其代谢产物17β - 二氢依西美坦（活性代谢产物）和17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷（非活性代谢产物）。它们各自的D3同位素用作内标。使用Thermo Fisher BDS Hypersil C18分析型HPLC柱（100×2.1 mm，5μm）实现分析物的色谱分离。流动相通过0.1%甲酸水溶液和乙腈的梯度洗脱以0.5 mL/min的流速输送。柱流出物通过API 4000三重四极杆质谱仪采用电喷雾电离（ESI）进行检测，并在正模式下通过多反应监测（MRM）进行监测。分别监测依西美坦、依西美坦 - d3、17β - 二氢依西美坦、17β - 二氢依西美坦 - d3、17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷和17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷 - d3的质量跃迁297 > 121 m/z、300 > 121 m/z、299 > 135 m/z、302 > 135 m/z、475 > 281 m/z和478 > 284 m/z。该测定法显示依西美坦的线性范围为0.4 - 40.0 ng/mL，17β - 二氢依西美坦和17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷的线性范围为0.2 - 15.0 ng/mL，决定系数（r2）> 0.998。依西美坦的精密度（变异系数）≤10.7%，17β - 二氢依西美坦为7.7%，17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷为9.5%，准确度范围分别为依西美坦88.8%至103.1%，17β - 二氢依西美坦98.5%至106.1%，17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷92.0%至103.2%。该方法成功应用于接受每日口服25 mg依西美坦的乳腺癌患者的药代动力学/药效学研究。对于一名代表性患者，摄入依西美坦24小时后，血浆中20.7%的依西美坦转化为17β - 二氢依西美坦，29.0%的17β - 二氢依西美坦失活为17β - 二氢依西美坦 - 17 - O - β - D - 葡萄糖醛酸苷，这表明17 - 二氢依西美坦葡萄糖醛酸化的改变可能在决定依西美坦对乳腺癌细胞的作用中起重要作用。
