title: Measurement of posaconazole, itraconazole, and hydroxyitraconazole in plasma/serum by high-performance liquid chromatography with fluorescence detection.
abstract: BACKGROUND
  Itraconazole and posaconazole are used in the prevention and treatment of invasive fungal infections. However, the oral bioavailability of both compounds varies widely, and dose-serum concentration relationships are poorly defined for these analytes. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of therapeutic drug monitoring.
  
  
  METHODS
  Calibrators (n = 7) and internal quality control solutions (n = 3) were prepared in pooled human serum. Sample (100 μL), internal standard solution (25 μL), Tris solution (2 mol/L; pH 10.6), and extraction solvent (methyl tert-butyl ether, 600 μL) were vortex mixed and centrifuged. The solvent layer was removed and evaporated to dryness and the residue reconstituted in water:methanol (1 + 3, 50 μL). A portion (5 μL) of the reconstituted extract was analyzed using a 3-μm Gemini C6 phenyl column with fluorescence detection (excitation 260 nm, emission 350 nm). The method was used to measure itraconazole and hydroxyitraconazole, or posaconazole, in serum samples taken 1-2 hours before the next dose, from patients forming part of a study into management and diagnostic strategies for invasive aspergillosis.
  
  
  RESULTS
  Response was linear over the calibration ranges. Accuracy and imprecision were 92-111.4% and 3.2-13.4% (relative standard deviation), respectively. No interferences were noted. There was a good agreement with nominal values of each analyte in an external quality assessment scheme. In patients prescribed either 400 mg/d of itraconazole (n = 46) or 600-800 mg/d of posaconazole (n = 28) only 24% and 7% of samples, respectively, had serum itraconazole or posaconazole concentrations above the target threshold suggested in published guidelines.
  
  
  CONCLUSIONS
  A simple, sensitive high-performance liquid chromatographic method has been developed for the analysis of itraconazole, hydroxyitraconazole, and posaconazole in serum/plasma. Few of the samples measured from patients participating in the clinical study attained concentrations of the drug/metabolite in serum that have been recommended for effective antifungal therapy.
authors: Toxicology Unit, Department of Clinical Biochemistry, King's College Hospital NHS Foundation Trust, London, United Kingdom.
authors: Sara L
authors: SL
authors: Buckner
authors: M Mansour
authors: MM
authors: Ceesay
authors: Antonio
authors: A
authors: Pagliuca
authors: Phillip E
authors: PE
authors: Morgan
authors: Robert J
authors: RJ
authors: Flanagan
mesh_terms: D000328:Adult; D000368:Aged; D000935:Antifungal Agents / Q000008:administration & dosage / Q000097:blood*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D015331:Cohort Studies; D004305:Dose-Response Relationship, Drug; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000097:blood / Q000150:complications / Q000188:drug therapy; D017964:Itraconazole / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000097:blood*; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D009503:Neutropenia / Q000150:complications; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D014230:Triazoles / Q000008:administration & dosage / Q000097:blood*; D055815:Young Adult
chemical_list: D000935:Antifungal Agents; D014230:Triazoles; C084794:hydroxyitraconazole; D017964:Itraconazole; C101425:posaconazole