With the exception of one patient (ID11), all participants Navitoclax in vivo reported adequate adherence (>95%) between days 1 and 14 of treatment, and the 0-h blood sample for day 14 was drawn 24 h from the estimated time of the last dose for each patient. The majority of patients in this study had no watches or clocks in their homes, and hence could only give estimated and not actual times for dosing from days 2 to 13. Twenty-four-hour sampling was carried out on days 1 and 14 before observed medication and then
at 1, 2, 3, 4, 6, 8, 16 and 24 h after treatment. At each of these time-points, 4 mL of whole blood was drawn using an antecubital cannula and immediately centrifuged at 3000 rpm (1560 g) for 10 min; plasma was stored
at -70 °C until it was transported to Sweden for high-performance liquid chromatography (HPLC) analysis. HPLC analysis was carried out at the Department of Laboratory Medicine, Karolinska University Hospital Huddinge (Karolinska Institute, Stockholm, Sweden), where reverse-phase HPLC with UV detection was used to determine the plasma efavirenz concentration. For HPLC, an Agilent Series 1100 (Agilent Technologies, Santa Clara, CA, USA), consisting of column compartment G1316A, degasser G132A, Quat pump G1311A, auto-sampler G1329A ALS, and diode array detector G1315B was used. The column used was Ace3C18, 3 µm, 50 × 30 mm (Advanced Z-VAD-FMK solubility dmso Chromatography Technologies, Aberdeen, UK) and the mobile phase consisted of 30% acetonitrile, 30% methanol, 4 mmol/L potassium hydroxide and 10 mmol/L acetic acid (pH 4.3). Plasma proteins were precipitated with acetonitrile before centrifuging, after which 6 µL of the supernatant was injected and eluted at 0.80 Exoribonuclease mL/min for 3.5 min. The reference material was 99.9% efavirenz supplied by the WHO Collaborating Center for
Chemical Reference Substances through Apoteket AB (Stockholm, Sweden), and the retention time was 2.42 min as detected at UV-VIS 1, 210 nm, UV-VIS 2, 220 nm. This method was linear, and the within-day coefficient of variation was 3.2, 3.3 and 5.1% at concentrations of 0.63 (n=17), 2.53 (n=17) and 6.31 mg/L (n=16), respectively, with a between-day coefficient of variation of 4.1% (n=50) and a limit of quantification of 0.11 mg/L. The Karolinska University hospital laboratory is accredited by the Swedish Board for Accreditation and Conformity Assessment (SEWDAC), accreditation number 6695, and the laboratory participates in proficiency testing programmes under the same quality control board. HPLC of the samples yielded 924 data points for efavirenz plasma concentrations that were utilized to study the pharmacokinetics of the drug using noncompartmental analysis (NCA).