SENSITIVE LIQUID-CHROMATOGRAPHIC TECHNIQUE TO MEASURE ISONIAZID IN ALVEOLAR CELLS, BRONCHOALVEOLAR LAVAGE AND PLASMA IN HIV-INFECTED PATIENTS

The need to monitor the effectiveness of antimicrobial drugs in treati ng opportunistic infections such as tuberculosis in HIV-infected patie nts requires the development of sensitive assays. A suitable HPLC meth od was developed to measure the concentration of isoniazid (INH) in pl asma 1 h after a standard 300 mg dose and to detect the low levels typ ically found in alveolar cells obtained by bronchoalveolar lavage of s ubjects maintained on a standard regimen of the drug, Following extrac tion with a chloroform-butanol mixture, the INH was back-extracted int o dilute acid which was subsequently analyzed by HPLC using a CN rever sed-phase column and an acetonitrile-isopropanol based mobile phase. A nother 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 d aily response without adjustment. When saline, cellular extracts and p lasma from untreated subjects were spiked with various amounts of INH and analyzed, the lowest level of quantitation was 10, 25 and 100 ng/m l, 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 fl uid from HIV-positive patients maintained on a standard INH regimen ha d detectable levels of INH 4 h after a 300 mg oral dose. The plasma IN H at 1 h had a range of 0.3-7.1 mu g/ml (n=50). Precision studies with plasma spiked at 0.1, 0.5, 1.0 and 5.0 mu g/ml revealed within-run co efficients of variation (C.V.s) of 8.9, 7.2, 4.2 and 4.9%, respectivel y 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 conc entrations of 0.5, 1.0 and 3.0 mu g/ml, respectively. The results sugg est that this rugged HPLC technique can quantitate INH in 1 h plasma w ith good precision and can be used to estimate the very low INH concen trations found in alveolar cells and cell-free lavage recovered from p atients undergoing anti-tuberculosis therapy. (C) 1998 Elsevier Scienc e B.V.