Notari Stefania, Bocedi Alessio, Ippolito Giuseppe, Narciso Pasquale, Pucillo Leopoldo Paolo, Tossini Gianna, Donnorso Raffaele Perrone, Gasparrini Francesco, Ascenzi Paolo
Istituto Nazionale per le Malattie Infettive-I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, I-00149 Rome, Italy.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):258-66. doi: 10.1016/j.jchromb.2005.12.016. Epub 2006 Jan 6.
Therapeutic drug monitoring (TDM) is pivotal to improve the management of HIV infection. Here, a HPLC-UV method has been developed to quantify simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine, zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the plasma sample was 600 microL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a water bath under nitrogen stream. The extracted samples were reconstituted with 100 microL methanol. Twenty microliters of these samples were injected into a HPLC-UV system, the analytes were eluted on an analytical C(18) Symmetry column (250 mm x 4.6mm I.D.) with a particle size of 5 microm. The mobile phase (0.01 M KH(2)PO(4) and acetonitrile) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The calibration curves were linear up to 10 microg/mL. The absolute recovery ranged between 88 and 120%. The in vitro stability of anti-HIV drugs (0.005-10 microg/mL) in plasma has been studied at 24.0 degrees C. On these bases, a two to four analyte method has been tailored to the individual needs of the HIV-infected patient. The HPLC-UV method here reported has been validated and is currently applied to monitor PIs, NRTIs, and NNRTIs in plasma of HIV-infected patients. It allows to monitor the largest number of anti-HIV drugs simultaneously, appearing useful in a routine laboratory, and represents an essential step to elucidate the utility of a formal therapeutic drug monitoring for the optimal follow-up of HIV-infected patients.
治疗药物监测（TDM）对于改善HIV感染的管理至关重要。在此，已开发出一种高效液相色谱-紫外检测法（HPLC-UV），用于同时定量测定人血浆中的七种HIV蛋白酶抑制剂（安普那韦、阿扎那韦、茚地那韦、洛匹那韦、奈非那韦、利托那韦和沙奎那韦；PIs）、七种核苷类逆转录酶抑制剂（阿巴卡韦、去羟肌苷、恩曲他滨、拉米夫定、司他夫定、扎西他滨和齐多夫定；NRTIs）以及两种非核苷类逆转录酶抑制剂（依非韦伦和奈韦拉平；NNRTIs）。血浆样本体积为600微升。该方法包括使用1毫升Oasis HLB柱（二乙烯苯和N-乙烯基吡咯烷酮）进行自动固相萃取，并在氮气流下于水浴中蒸发。萃取后的样本用100微升甲醇复溶。取20微升这些样本注入HPLC-UV系统，分析物在粒径为5微米的C(18) Symmetry分析柱（250毫米×4.6毫米内径）上洗脱。流动相（0.01 M磷酸二氢钾和乙腈）以1.0毫升/分钟的流速进行线性梯度洗脱。单次分析的总运行时间为35分钟，通过在240和260纳米处的紫外检测来检测抗HIV药物。校准曲线在高达10微克/毫升的范围内呈线性。绝对回收率在88%至120%之间。已研究了抗HIV药物（0.005 - 10微克/毫升）在24.0℃血浆中的体外稳定性。在此基础上，已根据HIV感染患者的个体需求定制了一种两至四种分析物的方法。本文报道的HPLC-UV方法已得到验证，目前用于监测HIV感染患者血浆中的PIs、NRTIs和NNRTIs。它能够同时监测数量最多的抗HIV药物，在常规实验室中似乎很有用，并且是阐明正式治疗药物监测对HIV感染患者进行最佳随访的效用的重要一步。
