title: Determination of sirolimus blood concentration using high-performance liquid chromatography with ultraviolet detection.
abstract: BACKGROUND
  Different HPLC methods have been developed and used to determined sirolimus blood concentrations. These methods show different performance characteristics, mostly related to peak interference, recovery, assay sensitivity, and turnaround times.
  
  
  OBJECTIVE
  We adapted, improved, and validated an HPLC method with UV detection for measurement of sirolimus in whole blood clinical samples.
  
  
  METHODS
  The standards, quality controls, or patient samples (0.25 or 0.5 mL) and internal standard (desmethoxysirolimus) were extracted with 1-chlorobutane. After evaporation, the extract was reconstituted in a 70% acetonitrile/water mixture and analyzed onto a reverse-phase C18 column at 50 degrees C under a flow rate of 1.0 mL/min in the HPLC system. Ultraviolet detection was performed at 278 nm, with sensitivity setting of 0.010 AUFS. Identification of peaks of interest was by retention time; quantification of sirolimus was based on a peak area ratio.
  
  
  RESULTS
  Analytic recovery ranging from 96 to 120% (CV = 3.7 to 16.8%; bias = -4.2 to 16.7%) was observed throughout the assay's linear range (2.5-150.0 ng/mL). The lower limit of quantification for both sample volumes (0.25 or 0.5 mL) was 2.5 ng/mL (CV = 12 and 15%, bias = -1.2 and 4%, respectively). The intra- and interassay imprecision ranged from 6.2 to 14.4% and from 9.1 to 18.6%, with bias ranging from 1.3 to 12.9% and -1.8% to 7.1, for quality control levels of 3, 10, and 20 ng/mL. Whole blood and extracted samples are stable at room temperature and at 4 and -20 degrees C for 1 week and 3 days, respectively. Chromatograms showed good separation free of interfering peaks. A set of 45 samples can be extracted in 2 h, allowing results within 24 h.
  
  
  CONCLUSIONS
  This HPLC-UV method shows good and reproducible performance, satisfying all requirements of an assay designated to be applied in therapeutic drug monitoring strategies after organ transplantation.
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring; D004355:Drug Stability; D006801:Humans; D012680:Sensitivity and Specificity; D020123:Sirolimus / Q000097:blood*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D020123:Sirolimus