一种用于母乳中地匹韦林定量分析的超灵敏液相色谱-串联质谱（LC-MS/MS）检测方法的验证与应用。
Validation and implementation of an ultrasensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assay for dapivirine quantitation in breast milk.
作者信息
Manohar Madhuri, Marzinke Mark A
机构信息
Department of Medicine, Johns Hopkins University School of Medicine, 4940 Eastern Avenue, Mason F. Lord Center Tower, Baltimore, MD 21224, USA.
Department of Medicine, Johns Hopkins University School of Medicine, 4940 Eastern Avenue, Mason F. Lord Center Tower, Baltimore, MD 21224, USA; Departments of Medicine and Pathology, Johns Hopkins University School of Medicine, 1800 Orleans Street, Sheikh Zayed Tower, B1020F, Baltimore, MD 21287, USA.
出版信息
Clin Biochem. 2020 Aug;82:66-72. doi: 10.1016/j.clinbiochem.2019.12.005. Epub 2019 Dec 13.
BACKGROUND
The non-nucleoside reverse transcriptase inhibitor dapivirine has been evaluated as a topical microbicidal agent to prevent HIV-1 acquisition. Several clinical trials have evaluated the pharmacokinetics of dapivirine when administered as a 25-mg intravaginal ring. Recent studies have focused on the distribution of dapivirine into breast milk. Drug distribution during lactation and breastfeeding can have implications in terms of infant drug exposure. Thus, sensitive bioanalytical tools are required to characterize the pharmacokinetics of dapivirine in breast milk.
METHODS
Whole breast milk was spiked with dapivirine and internal standard. Lipid content was disrupted via pre-treatment with n-hexane, and supernatants were subjected to solid phase extraction. Extracted materials were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. Separation occurred using a Waters BEH C8, 50 × 2.1 mm UPLC column with a 1.7 µm particle size and dapivirine was detected on an API 5000 mass analyzer. Methods were validated in accordance with FDA Bioanalytical Method Validation recommendations.
RESULTS
The analytical method was optimized for dapivirine extraction from breast milk. The analytical measuring range of the assay was 10-1000 pg/mL. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.6% and %DEVs ≤ ±12.7%. Stability and matrix effects studies were also conducted and deemed acceptable. The method was applied to a previously reported phase 1 clinical trial and demonstrated appropriate performance in the quantitation of dapivirine in breast milk samples from lactating women.
CONCLUSIONS
An ultrasensitive LC-MS/MS assay has been developed and validated for the quantitation of dapivirine in breast milk. The described method meets validation acceptance criteria and has been applied to a phase 1 clinical trial.
背景
非核苷类逆转录酶抑制剂达匹韦林已被评估作为一种局部杀菌剂用于预防HIV-1感染。多项临床试验评估了25毫克阴道环剂型达匹韦林的药代动力学。近期研究聚焦于达匹韦林在母乳中的分布情况。哺乳期和母乳喂养期间的药物分布可能对婴儿的药物暴露产生影响。因此，需要灵敏的生物分析工具来表征达匹韦林在母乳中的药代动力学。
方法
将达匹韦林和内标加入全脂母乳中。通过正己烷预处理破坏脂质成分，然后对上清液进行固相萃取。萃取后的物质进行液相色谱-串联质谱（LC-MS/MS）分析。使用粒径为1.7 µm的Waters BEH C8 50×2.1 mm超高效液相色谱柱进行分离，并在API 5000质量分析仪上检测达匹韦林。方法依据FDA生物分析方法验证建议进行验证。
结果
该分析方法针对从母乳中提取达匹韦林进行了优化。该检测方法的分析测量范围为10 - 1000 pg/mL。通过标准品的加权线性回归生成校准曲线。批内和批间精密度及准确度研究表明变异系数（%CVs）≤14.6%，偏差（%DEVs）≤±12.7%。还进行了稳定性和基质效应研究，结果均为可接受。该方法应用于先前报道的一项1期临床试验，在定量哺乳期妇女母乳样本中的达匹韦林时表现出良好性能。
结论
已开发并验证了一种超灵敏的LC-MS/MS检测方法用于定量母乳中的达匹韦林。所描述的方法符合验证验收标准，并已应用于一项1期临床试验。
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