Abstract：
BACKGROUND: Efavirenz (EFV) is a drug used to treat HIV. Low plasma concentrations of EFV result in suboptimal viral suppression, whereas high concentrations can cause adverse neuropsychiatric side reactions. Some studies have identified a correlation between the plasma concentrations of EFV metabolites and neurotoxicity. To our knowledge, no studies have investigated the metabolism of EFV in young children and its effect on treatment outcomes. Therefore, the aim of this study was to develop and validate a method for quantifying EFV and its metabolites in human plasma derived from children.
METHODS: Sample preparation was performed using protein precipitation of 100 µL plasma. Thereafter, an aliquot of the supernatant was used to quantify EFV, 7-hydroxyefavirenz (7-OH-EFV), 8-hydroxyefavirenz (8-OH-EFV), and a newly discovered metabolite (\"EFAdeg\") associated with 8-OH-EFV. A second aliquot of the supernatant was hydrolyzed using β-glucuronidase/arylsulfatase and used with the first aliquot to quantify phase II metabolites. The analyses were performed using a Dionex Ultimate 3000RS LC-system coupled with a Q Exactive Orbitrap mass spectrometer.
RESULTS: The method has a measuring range of 100-50,000 ng/mL (EFV, 8-OH-EFV), 125-25,000 ng/mL (7-OH-EFV), and 200-10,000 ng/mL (\"EFAdeg\"). All criteria of the European Medicines Agency guidelines regarding precision, accuracy, and selectivity were met. Of note, carryover must be considered for 8-OH-EFV. Overall, the validated method was successfully applied to plasma samples obtained from children and confirmed the presence of the newly discovered metabolite, \"EFAdeg.\"
CONCLUSIONS: An LC-HRMS/MS method for the quantification of EFV and its phase I and II metabolites was developed and validated. This method is suitable for analyzing plasma samples from children. Furthermore, studies using this method identified an additional metabolite that may influence the concentration of 8-OH-EFV in patient samples.
METHODS: Sample preparation was performed using protein precipitation of 100 µL plasma. Thereafter, an aliquot of the supernatant was used to quantify EFV, 7-hydroxyefavirenz (7-OH-EFV), 8-hydroxyefavirenz (8-OH-EFV), and a newly discovered metabolite (\"EFAdeg\") associated with 8-OH-EFV. A second aliquot of the supernatant was hydrolyzed using β-glucuronidase/arylsulfatase and used with the first aliquot to quantify phase II metabolites. The analyses were performed using a Dionex Ultimate 3000RS LC-system coupled with a Q Exactive Orbitrap mass spectrometer.
RESULTS: The method has a measuring range of 100-50,000 ng/mL (EFV, 8-OH-EFV), 125-25,000 ng/mL (7-OH-EFV), and 200-10,000 ng/mL (\"EFAdeg\"). All criteria of the European Medicines Agency guidelines regarding precision, accuracy, and selectivity were met. Of note, carryover must be considered for 8-OH-EFV. Overall, the validated method was successfully applied to plasma samples obtained from children and confirmed the presence of the newly discovered metabolite, \"EFAdeg.\"
CONCLUSIONS: An LC-HRMS/MS method for the quantification of EFV and its phase I and II metabolites was developed and validated. This method is suitable for analyzing plasma samples from children. Furthermore, studies using this method identified an additional metabolite that may influence the concentration of 8-OH-EFV in patient samples.
摘要：
背景：依非韦仑（EFV）是一种用于治疗HIV的药物。低血浆浓度的EFV导致次优的病毒抑制，而高浓度会引起不良的神经精神副反应。一些研究已经确定了EFV代谢物的血浆浓度与神经毒性之间的相关性。据我们所知,尚无研究调查幼儿EFV的代谢及其对治疗结局的影响.因此,这项研究的目的是开发和验证一种定量儿童血浆中EFV及其代谢物的方法.
方法:使用100μL血浆的蛋白沉淀进行样品制备。此后,上清液的等分试样用于定量EFV，7-羟基法韦仑(7-OH-EFV),8-羟基法韦仑(8-OH-EFV),以及一种新发现的与8-OH-EFV相关的代谢物(“EFADeg”)。使用β-葡糖醛酸糖苷酶/芳基硫酸酯酶水解上清液的第二等分试样,并与第一等分试样一起用于定量II相代谢物。使用与QExactiveOrbitrap质谱仪耦合的DionexUltimate3000RSLC系统进行分析。
结果：该方法的测量范围为100-50,000ng/mL（EFV，8-OH-EFV)，125-25,000ng/mL（7-OH-EFV），和200-10,000ng/mL（“EFADeg”）。欧洲药品管理局指南关于精确度的所有标准，准确度,和选择性得到满足。值得注意的是，结转必须考虑8-OH-EFV。总的来说,经过验证的方法已成功应用于从儿童获得的血浆样本，并证实了新发现的代谢物的存在，\"Efadeg。\"
结论：开发并验证了用于定量EFV及其I和II相代谢物的LC-HRMS/MS方法。该方法适用于分析来自儿童的血浆样品。此外，使用该方法的研究确定了可能影响患者样品中8-OH-EFV浓度的其他代谢物.
方法:使用100μL血浆的蛋白沉淀进行样品制备。此后,上清液的等分试样用于定量EFV，7-羟基法韦仑(7-OH-EFV),8-羟基法韦仑(8-OH-EFV),以及一种新发现的与8-OH-EFV相关的代谢物(“EFADeg”)。使用β-葡糖醛酸糖苷酶/芳基硫酸酯酶水解上清液的第二等分试样,并与第一等分试样一起用于定量II相代谢物。使用与QExactiveOrbitrap质谱仪耦合的DionexUltimate3000RSLC系统进行分析。
结果：该方法的测量范围为100-50,000ng/mL（EFV，8-OH-EFV)，125-25,000ng/mL（7-OH-EFV），和200-10,000ng/mL（“EFADeg”）。欧洲药品管理局指南关于精确度的所有标准，准确度,和选择性得到满足。值得注意的是，结转必须考虑8-OH-EFV。总的来说,经过验证的方法已成功应用于从儿童获得的血浆样本，并证实了新发现的代谢物的存在，\"Efadeg。\"
结论：开发并验证了用于定量EFV及其I和II相代谢物的LC-HRMS/MS方法。该方法适用于分析来自儿童的血浆样品。此外，使用该方法的研究确定了可能影响患者样品中8-OH-EFV浓度的其他代谢物.
