title: Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay calibrated to NIST SRM 971.
abstract: BACKGROUND
  At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house.
  
  
  METHODS
  A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed.
  
  
  RESULTS
  Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded <15% CV at the limit of quantitation and <7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed.
  
  
  CONCLUSIONS
  An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds.
authors.affiliation: Department of Laboratory Medicine, University of California-San Francisco, San Francisco, CA 94107, United States. deborah.french@ucsf.edu
authors.forename: Deborah
authors.initials: D
authors.lastname: French
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001681:Biological Assay / Q000592:standards*; D002138:Calibration; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods / Q000592:standards*; D013739:Testosterone / Q000097:blood*
chemical_list: D013739:Testosterone