A Validated Method for Quantification of Efavirenz in Dried Blood Spots Using High-Performance Liquid Chromatography-Mass Spectrometry
A Validated Method for Quantification of Efavirenz in Dried Blood Spots Using High-Performance Liquid Chromatography-Mass Spectrometry
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DOI：
10.1097/ftd.0000000000000127
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发表时间：
2015-04-01
影响因子：
2.5
通讯作者：
Back, David J.
中科院分区：
文献类型：
--
作者：
Amara, Alieu B.;Else, Laura J.;Back, David J.
背景：依法韦仑（EFV）是一线抗逆转录病毒治疗的首选成分之一。EFV的特点是血浆半衰期长（40-55小时），患者间变异性大，这提高了个体化治疗的可能性。血浆中EFV水平的分析需要专门的设施（冷藏/运输），在资源有限的环境中，这可能是有问题的;干血斑（DBS）-EFV测量因此为治疗药物监测提供了一种廉价、简便的替代方法。我们的目的是开发和验证一种液相色谱-质谱法，以量化EFV DBS收集的临床试验的一部分，在资源有限的setting.Methods：DBS标准，质量控制样品，和患者样品被切除，然后用乙酸乙酯/正己烷（50/50体积/体积）萃取后，加入内标己巴比妥，和1 mol/L K2 CO 3。将提取物蒸干，残留物在移动的相中复溶，并直接通过液相色谱-质谱法进行分析。在反相C-18柱上使用1 mmol/L乙酸铵水溶液和乙腈进行梯度洗脱。通过在负电离模式下的选择反应监测进行定量。DBS样品在几个时间点超过24小时，从HIV+患者在400或600毫克EFV与恩曲他滨/替诺福韦。结果：内标和EFV洗脱在2.68和3.54分钟，分别在5分钟的运行时间。基质效应极轻微（-5.4%）。在25-5000 ng/mL浓度范围内验证校准曲线。不精密度的批内和批间变异范围为6.7%-8.7%，准确度的批内和批间变异范围为100.3%-104.2%。平均回收率> 64%。DBS数据显示出与经验证的血浆EFV测定的强正相关（R = 0.9764，P < 0.001）。从DBS EFV浓度约42%，低于配对血浆值，血液/血浆的比例并没有改变给药interval.Conclusions：经验证的分析，现在常规应用于临床样本测量DBS EFV药代动力学分析。该方法具有耐用性、准确性和灵敏度。
Background: Efavirenz (EFV) is one of the preferred components of first-line antiretroviral treatment. EFV is characterized by a long plasma half-life (40-55 hours) with large interpatient variability, which raises the potential for individualization of therapy. Analyses of EFV levels in plasma require specialized facilities (cold storage/transport) which, in resource-limited settings, can be problematic; dried blood spots (DBS)-EFV measurements thus provide a cheap easy alternative for therapeutic drug monitoring. Our aim was to develop and validate a liquid chromatography-mass spectrometry method to quantify EFV in DBS collected as part of clinical trials in resource-limited settings.Methods: DBS for standards, quality control samples, and patient samples were excised and then extracted with ethyl acetate/n-hexane (50/50 vol/vol) after addition of internal standard hexobarbital, and 1 mol/L K2CO3. The extract was evaporated to dryness, the residue reconstituted in mobile phase and analyzed directly by liquid chromatography-mass spectrometry. Gradient elution was on a reverse-phase C-18 column using 1 mmol/L ammonium acetate in water and acetonitrile. Quantification was by selected reaction monitoring in negative ionization mode. DBS samples were obtained at several time points over 24 hours from HIV+ patients on either 400 or 600 mg EFV in combination with emtricitabine/tenofovir.Results: The internal standard and EFV eluted at 2.68 and 3.54 minutes, respectively in a 5-minute run time. Matrix effects were minimal (-5.4%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra-assay and interassay variations ranged between 6.7% and 8.7% for imprecision and 100.3% and 104.2% for accuracy. Mean recovery was >64%. The DBS data showed a strong positive correlation with a validated plasma EFV assay (R = 0.9764, P < 0.001). EFV concentrations from DBS were approximately 42% lower than the paired plasma values, and the ratio of blood/plasma did not change over the dosing interval.Conclusions: The validated assay is now routinely applied to clinical samples measuring DBS EFV for pharmacokinetic analysis. The methodology is robust, accurate, and sensitive.