title: A validated method for quantification of efavirenz in dried blood spots using high-performance liquid chromatography-mass spectrometry.
abstract: 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.
authors.affiliation: *Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, United Kingdom; †Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria; and ‡National Center in HIV Epidemiology and Clinical Research (NCHECR), University of New South Wales, Australia.
authors.forename: Alieu B
authors.initials: AB
authors.lastname: Amara
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Else
authors.forename: John
authors.initials: J
authors.lastname: Tjia
authors.forename: Adeniyi
authors.initials: A
authors.lastname: Olagunju
authors.forename: Rebekah L
authors.initials: RL
authors.lastname: Puls
authors.forename: Saye
authors.initials: S
authors.lastname: Khoo
authors.forename: David J
authors.initials: DJ
authors.lastname: Back
mesh_terms: D000480:Alkynes; D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000097:blood*; D048588:Benzoxazines / Q000008:administration & dosage / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003521:Cyclopropanes; D004305:Dose-Response Relationship, Drug; D004311:Double-Blind Method; D015658:HIV Infections / Q000188:drug therapy; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; C098320:efavirenz