Buprenorphine and buprenorphine/naloxone combination are maintenance treatments used worldwide. However, since their marketing, despite ceiling respiratory effects, poisonings and fatalities have been attributed to buprenorphine misuse and overdose. Therefore, to better understand the mechanisms of buprenorphine-related toxicity in vivo, experimental investigations have been conducted, mainly in the rat. We developed a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method with electrospray ionization for the simultaneous quantification of buprenorphine, naloxone and their metabolites (norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide and naloxone glucuronide) in rat whole blood. Compounds were extracted from whole blood by protein precipitation and chromatographically separated using gradient elution of aqueous ammonium formate and methanol in a Raptor Biphenyl core-shell column (100 mm x 3,0 mm x 2,7 μm). Following electrospray ionization, quantification was carried out in the multiple reaction monitoring (MRM) mode by the tandem mass spectrometer API 3200 system. The LC-MS/MS method was validated according to the currently accepted criteria for bioanalytical method validation. The method required small sample volumes (50 μL) and was sensitive with limits of quantification of 6.9, 6.2, 3.6, 3.3, 1.3 and 57.7 ng/mL for buprenorphine, norbuprenorphine, buprenorphine glucuronide, norbuprenorphine glucuronide, naloxone and naloxone glucuronide respectively. The upper limit of quantification was 4000 ng/ml for all the studied compounds. Trueness (88-115 %), repeatability and intermediate precision (both <15%) were in accordance with the international recommendations. The procedure was successfully used to quantify these compounds in the whole blood sample from one rat 24 h after the intravenous administration of buprenorphine/naloxone (30.0/7.5 mg/kg).

中文翻译：

---

通过LC-MS / MS测定大鼠全血中的丁丙诺啡，纳洛酮以及I和II期代谢物。

丁丙诺啡和丁丙诺啡/纳洛酮组合是世界范围内使用的维持疗法。然而，自从上市以来，尽管有上限呼吸作用，但丁丙诺啡的滥用和服用过量已导致中毒和死亡。因此，为了更好地了解体内丁丙诺啡相关毒性的机制，已经进行了主要在大鼠中的实验研究。我们开发了一种采用电喷雾电离的液相色谱-串联质谱（LC-MS / MS）方法，用于大鼠中丁丙诺啡，纳洛酮及其代谢产物（诺丁丙诺啡，丁丙诺啡葡糖醛酸，诺丁丙诺啡葡糖醛酸和纳洛酮葡糖醛酸）的同时定量。通过蛋白质沉淀从全血中提取化合物，并在Raptor联苯核壳色谱柱（100 mm x 3.0 mm x 2,7μm）中使用甲酸铵水溶液和甲醇的梯度洗脱进行色谱分离。电喷雾电离后，通过串联质谱仪API 3200系统在多反应监测（MRM）模式下进行定量。LC-MS / MS方法已根据当前公认的生物分析方法验证标准进行了验证。该方法需要少量样品（50μL），并且对丁丙诺啡，去甲丁丙诺啡，丁丙诺啡葡糖苷酸，去甲丁丙诺啡葡糖醛酸，纳洛酮和纳洛酮葡萄糖醛酸的定量限分别为6.9、6.2、3.6、3.3、1.3和57.7 ng / mL。所有研究化合物的定量上限为4000 ng / ml。真实度（88-115％），可重复性和中间精度（均<15％）符合国际建议。在静脉注射丁丙诺啡/纳洛酮（30.0 / 7.5 mg / kg）后24小时，该程序已成功用于定量一只大鼠全血样品中的这些化合物。