背景:文拉法辛(VEN)及其O-去甲基化代谢物,O-去甲基文拉法辛(ODV),是常用的5-羟色胺-去甲肾上腺素再摄取抑制剂,批准用于治疗抑郁症和焦虑症。两者都通过细胞色素P450酶代谢成非活性代谢物。虽然以前的研究集中在量化VEN和ODV,需要同时测量所有代谢物的生物分析方法来充分表征VEN和ODV的药理学。
方法:用VEN掺入K2EDTA血浆,ODV,N-去甲基文拉法辛(NDV),N,O-二去甲基文拉法辛(NODDV),N,N-二去甲基文拉法辛(NNDDV)。通过蛋白沉淀提取药物和代谢物,并使用液相色谱-串联质谱法(LC-MS/MS)进行定量。多重测定根据监管建议进行验证,并在服用文拉法辛的人的残余血浆样本中进行了评估。
结果:文拉法辛和所有四种代谢物的分析测量范围为5-800ng/mL。通过加权二次(NNDDV)或线性(VEN,ODV,NDV,NODDV)校准品回归。对于所有水平的所有分析物,测定间不精确性在1.9-9.3%之间。观察到轻微的基质效应,所有分析物的回收效率和工艺效率均>96%。所有其他测定验证评估均符合验收标准。从当前使用文拉法辛处方(37.5-450mg/天)的患者获得的残余血浆标本中测量的药物浓度产生了NDV,NDV,6/21、14/21和20/21样品中的NODDV代谢物浓度,分别。活性与非活性分析物的比率范围为0.74至14.5,中值为6.39。
结论:开发并验证了一种有效且准确的LC-MS/MS方法用于VEN的定量,ODV,和血浆中所有三种无活性代谢物。该试验符合所有验收标准,并可能用于这些药物的药代动力学的未来研究。
BACKGROUND: Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both are metabolized to inactive metabolites via cytochrome P450 enzymes. While previous studies have focused on quantifying VEN and ODV, bioanalytical methods for the simultaneous measurement of all metabolites are needed to fully characterize the pharmacology of VEN and ODV.
METHODS: K2EDTA plasma was spiked with VEN, ODV, N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV). Drugs and metabolites were extracted via protein precipitation and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The multiplexed assay was validated in accordance with regulatory recommendations, and evaluated in remnant plasma samples from persons prescribed venlafaxine.
RESULTS: The analytical measuring range for venlafaxine and all four metabolites was 5-800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was between 1.9-9.3% for all levels of all analytes. Minor matrix effects were observed, and both recovery efficiency and process efficiency were >96% for all analytes. All other assay validation assessments met acceptance criteria. Drug concentrations measured from remnant plasma specimens obtained from patients with current venlafaxine prescriptions (37.5-450 mg/day) yielded NDDV, NDV, and NODDV metabolite concentrations in 6/21, 14/21, and 20/21 samples, respectively. The ratio of active to inactive analytes ranged from 0.74 to 14.5, with a median of 6.39.
CONCLUSIONS: An efficient and accurate LC-MS/MS method was developed and validated for the quantification of VEN, ODV, and all three inactive metabolites in plasma. The assay met all acceptance criteria, and may be used in future studies of the pharmacokinetics of these drugs.