title: Determination of efavirenz in human dried blood spots by reversed-phase high-performance liquid chromatography with UV detection.
abstract: BACKGROUND
  Previously published methods for determination of efavirenz (EFV) in human dried blood spots (DBS) use costly and complex liquid chromatography/mass spectrometry. We describe the validation and evaluation of a simple and inexpensive high-performance liquid chromatography method for EFV quantification in human DBS and dried plasma spots (DPS), using ultraviolet detection appropriate for resource-limited settings.
  
  
  METHODS
  One hundred microliters of heparinized whole blood or plasma were spotted onto blood collection cards, dried, punched, and eluted. Eluates are injected onto a C-18 reversed phase high-performance liquid chromatography column. EFV is separated isocratically using a potassium phosphate and acetonitrile mobile phase. Ultraviolet detection is at 245 nm. Quantitation is by use of external calibration standards. Following validation, the method was evaluated using whole blood and plasma from HIV-positive patients undergoing EFV therapy.
  
  
  RESULTS
  Mean recovery of drug from DBS is 91.5%. The method is linear over the validated concentration range of 0.3125-20.0 μg/mL. A good correlation (Spearman r = 0.96) between paired plasma and DBS EFV concentrations from the clinical samples was observed, and hematocrit level was not found to be a significant determinant of the EFV DBS level. The mean observed C DBS/C plasma ratio was 0.68. A good correlation (Spearman r = 0.96) between paired plasma and DPS EFV concentrations from the clinical samples was observed. The mean percent deviation of DPS samples from plasma samples is 1.68%.
  
  
  CONCLUSIONS
  Dried whole blood spot or dried plasma spot sampling is well suited for monitoring EFV therapy in resource-limited settings, particularly when high sensitivity is not essential.
authors.affiliation: Skagg's School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, CA 92093-0831, USA.
authors.forename: Justin T
authors.initials: JT
authors.lastname: Hoffman
authors.forename: Steven S
authors.initials: SS
authors.lastname: Rossi
authors.forename: Rowena
authors.initials: R
authors.lastname: Espina-Quinto
authors.forename: Scott
authors.initials: S
authors.lastname: Letendre
authors.forename: Edmund V
authors.initials: EV
authors.lastname: Capparelli
mesh_terms: D000480:Alkynes; D019380:Anti-HIV Agents / Q000097:blood*; D048588:Benzoxazines / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003521:Cyclopropanes; D059788:Dried Blood Spot Testing / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; C098320:efavirenz