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Background: The aim of this study was to determine the utility of dried blood spots (DBS) compared with conventional plasma collection methods for characterization of efavirenz pharmacokinetics, in the setting of a large-scale, global clinical trial (ENCORE1)., Methods: Six hundred thirty patients were recruited from 38 sites and had single matched whole blood DBS and plasma samples (mid-dose interval) taken at weeks 4 and 12 of treatment. In addition, a subgroup of patients underwent intensive DBS and plasma sampling (0-24 hours) to provide full-profile data for pharmacokinetic parameters. Efavirenz concentrations were determined by validated high-performance liquid chromatography-mass spectrometry methods. A DBS-predicted plasma concentration was derived and linear regression and Bland-Altman plots were used to compare DBS-predicted plasma concentrations with that of measured plasma concentrations., Results: Efavirenz DBS and plasma concentrations were significantly correlated (R = 0.904, P < 0.001; n = 1094), and DBS concentrations were, on average, 53% ± 9.5% lower than plasma. In the main study, the DBS-predicted plasma values significantly underestimated the true measured concentration of efavirenz in plasma; the mean difference (95% confidence interval) between efavirenz DBS-predicted concentrations and measured plasma concentrations was -0.451 mg/L (-0.504 to -0.398) at week 4 (n = 561). However, in the intensive study, the mean difference was only 0.086 mg/L (-0.006 to 0.178) at 12 hours after dose (n = 46) and was not statistically significant., Conclusions: Our data show a high correlation between measurements of efavirenz concentrations in plasma and in DBS. However, DBS concentrations significantly underestimated the true measured plasma concentrations in the sparse samples taken in this large multinational ENCORE1 trial.

Background: Efavirenz (EFV) is one of the preferred components of first-line antiretroviral treatment. EFV is characterized by a long plasma half-life (40-55 hours) with large interpatient variability, which raises the potential for individualization of therapy. Analyses of EFV levels in plasma require specialized facilities (cold storage/transport) which, in resource-limited settings, can be problematic; dried blood spots (DBS)-EFV measurements thus provide a cheap easy alternative for therapeutic drug monitoring. Our aim was to develop and validate a liquid chromatography-mass spectrometry method to quantify EFV in DBS collected as part of clinical trials in resource-limited settings., Methods: DBS for standards, quality control samples, and patient samples were excised and then extracted with ethyl acetate/n-hexane (50/50 vol/vol) after addition of internal standard hexobarbital, and 1 mol/L K2CO3. The extract was evaporated to dryness, the residue reconstituted in mobile phase and analyzed directly by liquid chromatography-mass spectrometry. Gradient elution was on a reverse-phase C18 column using 1 mmol/L ammonium acetate in water and acetonitrile. Quantification was by selected reaction monitoring in negative ionization mode. DBS samples were obtained at several time points over 24 hours from HIV+ patients on either 400 or 600 mg EFV in combination with emtricitabine/tenofovir., Results: The internal standard and EFV eluted at 2.68 and 3.54 minutes, respectively in a 5-minute run time. Matrix effects were minimal (-5.4%). Calibration curves were validated over a concentration range of 25-5000 ng/mL. Intra-assay and interassay variations ranged between 6.7% and 8.7% for imprecision and 100.3% and 104.2% for accuracy. Mean recovery was >64%. The DBS data showed a strong positive correlation with a validated plasma EFV assay (R = 0.9764, P < 0.001). EFV concentrations from DBS were approximately 42% lower than the paired plasma values, and the ratio of blood/plasma did not change over the dosing interval., Conclusions: The validated assay is now routinely applied to clinical samples measuring DBS EFV for pharmacokinetic analysis. The methodology is robust, accurate, and sensitive.