Therapeutic drug monitoring (TDM) is gaining importance for improving the success

Therapeutic drug monitoring (TDM) is gaining importance for improving the success of antiretroviral treatment in human immunodeficiency virus-infected patients. 12 h after administration of 90 mg of ENF and 23% and 58% of patients are expected to have concentrations below 1 0 ng/ml and 2 200 ng/ml respectively. Both values have been proposed as cutoffs for virological efficacy. The median maximum concentration of drug in serum (for 6 min. An aliquot of 200 μl of the supernatant was transferred into a clean low-binding microreaction vial and evaporated to dryness in a vacuum centrifuge (Bachofer Reutlingen Germany). The dry extract was then reconstituted in reconstitution solution (mobile phase A-ACN [70:30 vol/vol]) vortexed gently for 30 min and centrifuged at 13 0 × for 6 min. HPLC conditions. A 50-μl volume of the reconstituted sample was injected onto a Eurogel 100 polymery reversed phase column (8 μm 100 by 2 mm) with an integrated guard column (Knauer Berlin Germany). Mobile phase A was H2O containing acetic acid (0.002%) and trifluoroacetic acid (0.0002%). Mobile phase B was acetonitrile containing acetic acid (0.08%). The high-pressure liquid chromatography (HPLC) system consisted of the following components: a mobile-phase delivery pump (Rheos 2000; Flux Instruments Basel Switzerland) a mobile-phase degasser (Degasys DG 1210; Uniflows Tokyo Japan) and an autosampler (Micro 215 liquid handler; Gilson S.A. Villiers-le-Bel France). HPLC separation was achieved with mobile-phase-gradient elution (flow 0.25 ml/min) using the following sequence: 0 min 65 A; ?1.0 min 65 A; ?1.5 min 20 A; ?3.0 min 20 A; ?3.5 min 10 PHA 291639 A; and ?6.0 min 10 A. The total run time was 15 min. After every injection the HPLC components with direct contact with the sample (injection needle injection port and loop) were intensely rinsed with ACN-H2O (60:40 vol/vol). The total effluent entered the interface of the mass spectrometer. MS-MS conditions. An API 3000 (Applied Biosystems Ontario Canada) tandem mass spectrometer (MS-MS) equipped with an electrospray ionization ion source and run PHA 291639 with Analyst software (version 1.2 service pack 1) was used for detection. ENF and the internal STMN1 standard were monitored in the positive ion mode with the following transitions of precursor to product ions: 1 123.7 to 1 1 343 (ENF) and 1 126.4 to 1 1 346.6 (deuterium-labeled ENF). This apparent mass gain after fragmentation represents the transition of quadruply charged parent ions into triply charged fragment ions. The ion source temperature was set to 400°C. The mass spectrometric parameters for ENF and the internal standard (IS) were optimized manually and are shown in Table ?Table11. TABLE 1. MS-MS parameters Calibration and QC. Standards and quality control (QC) samples were prepared in blank pool serum from inpatients treated in our clinic for reasons other than HIV infection. With each batch containing a maximum of 36 unknown samples an eight-point standard calibration curve run in duplicate was analyzed with samples containing ENF in concentrations ranging from 110 ng/ml to 14 24 ng/ml. A weighted (1/being the analyte concentration) linear regression was used to PHA 291639 generate the regression formula. QC samples analyzed during the validation process were prepared from a different stock solution (ENF in stock solution buffer) at concentrations of 110 ng/ml 297 ng/ml 2 971 ng/ml and 14 857 ng/ml. Assay performance during analytical runs was controlled for by analyzing samples PHA 291639 at concentrations of 297 ng/ml 2 971 ng/ml and 14 24 ng/ml. Population PK analysis. PK model building for describing the typical concentration-time profile and characterizing the variability in the population was performed using the nonlinear mixed-effects modeling approach implemented in NONMEM version V 1.1. First-order conditional estimation with interaction was used as the estimation method. In a stepwise approach first a structural model was developed by investigating different numbers of compartments as well as mono- and bidirectional transport processes. Afterwards interindividual and residual variability were characterized. The model was parameterized in terms of clearance (CL) and distribution volumes with PREDPP subroutines ADVAN 4 and TRANS 4. The steady-state routine SS 4 was used to account for the mere steady-state concentrations in the data set. Interindividual variability in e.g. PK parameter clearance was modeled using an exponential error PHA 291639 term according to the equation where CLrepresents.