Koal Therese, Burhenne Heike, Römling Regina, Svoboda Michal, Resch Klaus, Kaever Volkhard
Medical School Hannover, Institute of Pharmacology, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Rapid Commun Mass Spectrom. 2005;19(21):2995-3001. doi: 10.1002/rcm.2158.
For the first time approved antiretroviral drugs, i.e. protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI), were quantified in dried blood spots (DBS) from HIV/AIDS patient whole blood samples as the basis for therapeutic drug monitoring (TDM) by a robust simultaneous liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. This study included seven PI (amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, atazanavir) and two NNRTI (nevirapine, efavirenz). LC/MS/MS coupling was realized using a Phenomenex Synergy Max RP LC column (150 x 2 mm, 4 micro) in combination with a tandem mass spectrometer (API 2000, Applied Biosystems/MDS Sciex Concord) operating in positive and negative multiple reaction monitoring (MRM) mode with reserpine as internal standard. DBS samples were punched out and extracted with 50:50 MeOH/0.2 M ZnSO4 (v/v) as extraction reagent. The method performance data for the drugs in DBS like limits of detection (LOD, 8-70 ng/mL), lower limits of quantification (LLOQ, 41-102 ng/mL), linearity (R2, 0.9981-0.9999), linear concentration ranges (41-10.000 ng/mL), accuracies (92-113%), recoveries (62-94%), and ion suppression were investigated and are comparable to data obtained from human plasma, which is the current standard matrix for TDM of PI and NNRTI. In this case, off-line plasma sample preparation was performed by means of simple protein precipitation with 80:20 methanol/0.2 M ZnSO4 (v/v) as precipitation reagent. Significant correlations between real patient plasma and DBS were obtained for samples containing lopinavir, atazanavir, ritonavir, saquinavir, and efavirenz. DBS preparation as sampling alternative is well suited and practicable for TDM minimizing the high infection risk of HIV/AIDS samples and may facilitate sample mailing.
首次采用稳健的同时液相色谱/串联质谱（LC/MS/MS）方法，对来自HIV/AIDS患者全血样本的干血斑（DBS）中的首批获批抗逆转录病毒药物，即蛋白酶抑制剂（PI）和非核苷类逆转录酶抑制剂（NNRTI）进行定量，作为治疗药物监测（TDM）的基础。本研究包括7种PI（安普那韦、奈非那韦、茚地那韦、洛匹那韦、沙奎那韦、利托那韦、阿扎那韦）和2种NNRTI（奈韦拉平、依非韦伦）。使用Phenomenex Synergy Max RP LC柱（150×2 mm，4μm）结合串联质谱仪（API 2000，Applied Biosystems/MDS Sciex Concord）实现LC/MS/MS联用，该串联质谱仪在正、负多反应监测（MRM）模式下运行，以利血平作为内标。将DBS样本冲压出来，并用50:50的甲醇/0.2 M硫酸锌（v/v）作为提取试剂进行提取。研究了DBS中药物的方法性能数据，如检测限（LOD，8 - 70 ng/mL）、定量下限（LLOQ，41 - 102 ng/mL）、线性（R2，0.9981 - 0.9999）、线性浓度范围（41 - 10,000 ng/mL）、准确度（92 - 113%）、回收率（62 - 94%）和离子抑制情况，这些数据与从人血浆中获得的数据相当，而人血浆是目前PI和NNRTI TDM的标准基质。在这种情况下，离线血浆样本制备通过用80:20的甲醇/0.2 M硫酸锌（v/v）作为沉淀试剂进行简单的蛋白质沉淀来进行。对于含有洛匹那韦、阿扎那韦、利托那韦、沙奎那韦和依非韦伦的样本，在实际患者血浆和DBS之间获得了显著相关性。作为采样替代方法的DBS制备非常适合且适用于TDM，可将HIV/AIDS样本的高感染风险降至最低，并可能便于样本邮寄。
