Remmel R P, Kawle S P, Weller D, Fletcher C V
Departments of Medicinal Chemistry and Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA.
Clin Chem. 2000 Jan;46(1):73-81.
HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed.
Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 microL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 degrees C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma.
The extraction recovery was 87-92%. Within-run accuracy for quality-control samples was 6-8%, with CVs of 2-8%. Limits of quantification were 40-50 microg/L for indinavir, nelfinavir, and ritonavir, and 20 microg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r(2) >0.94) between methods. Larger variations at concentrations >4000 microg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined.
A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.
HIV蛋白酶抑制剂被推荐作为联合抗逆转录病毒治疗的一部分。双重蛋白酶抑制剂疗法也正在临床中使用。因此，开发了一种用于同时测定茚地那韦、奈非那韦、利托那韦和沙奎那韦的方法。
添加内标物（A - 86093）后，在碱性pH条件下用甲基叔丁基醚从血浆（250微升）中提取茚地那韦、奈非那韦、利托那韦和沙奎那韦。化合物在Keystone BetaBasic C4柱（250×3毫米内径）上于40℃分离，流动相为乙腈 - 50毫摩尔/升甲酸铵缓冲液，pH 4.1（体积比52:48），流速为0.5毫升/分钟。茚地那韦、奈非那韦、利托那韦和内标物（A - 86093）在218纳米处检测，沙奎那韦在235纳米处检测。通过对校准品进行五次重复分析以及对在人血浆中制备的三种不同浓度的质量控制样品进行分析来验证该方法。
提取回收率为87 - 92%。质量控制样品的批内准确度为6 - 8%，变异系数为2 - 8%。茚地那韦、奈非那韦和利托那韦的定量限为40 - 50微克/升，沙奎那韦的定量限为20微克/升。对患者样品进行了沙奎那韦和奈非那韦的液相色谱 - 质谱法交叉验证。回归分析显示两种方法之间具有良好的相关性（r²>0.94）。奈非那韦在浓度>4000微克/升时观察到较大变化。未观察到对艾滋病患者常用药物的干扰。测定了两名接受双重蛋白酶治疗患者的药代动力学曲线。
开发了一种可靠且耐用的同时测定四种HIV蛋白酶抑制剂的高效液相色谱法。该测定方法对于参与HIV蛋白酶抑制剂治疗药物监测的临床实验室很方便。该测定法具有足够的灵敏度，可对服用一种以上HIV蛋白酶抑制剂以及其他抗逆转录病毒药物的患者进行药代动力学研究。
