Delahunty T, Lee B, Conte J E
Department of Clinical Pharmacology, University of California San Francisco, 94110, USA.
J Chromatogr B Biomed Sci Appl. 1998 Feb 13;705(2):323-9. doi: 10.1016/s0378-4347(97)00510-0.
The need to monitor the effectiveness of antimicrobial drugs in treating opportunistic infections such as tuberculosis in HIV-infected patients requires the development of sensitive assays. A suitable HPLC method was developed to measure the concentration of isoniazid (INH) in plasma 1 h after a standard 300 mg dose and to detect the low levels typically found in alveolar cells obtained by bronchoalveolar lavage of subjects maintained on a standard regimen of the drug. Following extraction with a chloroform-butanol mixture, the INH was back-extracted into dilute acid which was subsequently analyzed by HPLC using a CN reversed-phase column and an acetonitrile-isopropanol based mobile phase. Another HPLC method was developed using direct injection and a polymer based column to measure minute concentrations of INH in the cell-free lavage. In both systems, detection of the drug was accomplished with a sealed coulometric detector (+0.6 V) capable of giving a consistent daily response without adjustment. When saline, cellular extracts and plasma from untreated subjects were spiked with various amounts of INH and analyzed, the lowest level of quantitation was 10, 25 and 100 ng/ml, respectively. Calibration curves showed good linearity when spiked concentrations were compared to peak areas (r=0.991, 0.993 and 0.998, respectively). Alveolar cell extracts and cell-free bronchoalveolar fluid from HIV-positive patients maintained on a standard INH regimen had detectable levels of INH 4 h after a 300 mg oral dose. The plasma INH at 1 h had a range of 0.3-7.1 microg/ml (n = 50). Precision studies with plasma spiked at 0.1, 0.5, 1.0 and 5.0 microg/ml revealed within-run coefficients of variation (C.V.s) of 8.9, 7.2, 4.2 and 4.9%, respectively and analytical recoveries of 97, 108, 108 and 98%, respectively. The day-to-day C.V.s for the plasma method were 7.6, 4.9 and 3.8% at concentrations of 0.5, 1.0 and 3.0 microg/ml, respectively. The results suggest that this rugged HPLC technique can quantitate INH in 1 h plasma with good precision and can be used to estimate the very low INH concentrations found in alveolar cells and cell-free lavage recovered from patients undergoing anti-tuberculosis therapy.
监测抗微生物药物治疗艾滋病毒感染患者中结核病等机会性感染的有效性，需要开发灵敏的检测方法。已开发出一种合适的高效液相色谱法，用于测定标准300毫克剂量给药1小时后血浆中异烟肼（INH）的浓度，并检测在接受该药物标准治疗方案的受试者经支气管肺泡灌洗获得的肺泡细胞中通常发现的低水平异烟肼。用氯仿 - 丁醇混合物萃取后，将异烟肼反萃取到稀酸中，随后使用CN反相柱和基于乙腈 - 异丙醇的流动相通过高效液相色谱法进行分析。还开发了另一种高效液相色谱法，采用直接进样和基于聚合物的柱子来测定无细胞灌洗液中微量的异烟肼。在这两种系统中，药物检测均通过密封库仑检测器（+0.6 V）完成，该检测器能够在无需调整的情况下给出一致的每日响应。当向未治疗受试者的生理盐水、细胞提取物和血浆中加入不同量的异烟肼并进行分析时，最低定量水平分别为10、25和100纳克/毫升。当将加标浓度与峰面积进行比较时，校准曲线显示出良好的线性（r分别为0.991、0.993和0.998）。在口服300毫克剂量4小时后，接受标准异烟肼治疗方案的艾滋病毒阳性患者的肺泡细胞提取物和无细胞支气管肺泡灌洗液中可检测到异烟肼水平。1小时时血浆中异烟肼的范围为0.3 - 7.1微克/毫升（n = 50）。对加标浓度为0.1、0.5、1.0和5.0微克/毫升的血浆进行精密度研究，结果显示批内变异系数（C.V.s）分别为8.9%、7.2%、4.2%和4.9%，分析回收率分别为97%、108%、108%和98%。血浆方法的日间C.V.s在浓度为0.5、1.0和3.0微克/毫升时分别为7.6%、4.9%和3.8%。结果表明，这种耐用的高效液相色谱技术能够精确地定量1小时血浆中的异烟肼，可用于估计接受抗结核治疗患者的肺泡细胞和无细胞灌洗液中极低的异烟肼浓度。
