title: A direct assay for the routine measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone by liquid chromatography tandem mass spectrometry.
abstract: BACKGROUND
  The measurement of androgens in many laboratories is often limited to testosterone. To more accurately determine the androgen status in both sexes, the measurement of other androgens such as dihydrotestosterone, the more potent metabolite of testosterone, and androstenendione and dehydroepiandrosterone, the most abundant circulating androgens in women would be informative. We report a combined liquid chromatography tandem mass spectrometry method for the measurement of these androgens.
  
  
  METHODS
  Internal standards in methanol (10 µL) were added to 100 µL serum followed by the addition of zinc sulphate (100 µL). After mixing, 100 µL of acetonitrile was added and was further mixed. The samples were centrifuged and the steroids extracted using an automated online solid phase extraction on a C18 cartridge by a Waters Acquity with online sample manager coupled to a TQS mass spectrometer.
  
  
  RESULTS
  Separation of the androgens was achieved by liquid chromatography. The run time was 6.5 min per sample. The lower limit of quantitation was 0.1 nmol/L for testosterone, androstenedione and dihydrotestosterone and 1 nmol/L for dehydroepiandrosterone. The coefficient of variation of the assay in serum for testosterone was <6%, androstenedione <8% and dihydrotestosterone and dehydroepiandrosterone <10%.
  
  
  CONCLUSIONS
  We have developed a rapid assay for the liquid chromatography tandem mass spectrometry measurement of testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone in a routine clinical laboratory. The assay requires a small volume of serum, and all analytes are measured simultaneously. The assay is rapid and simple to execute offering the potential for routine clinical application.
authors.affiliation: Manchester Academic Health Science Centre, Biochemistry Department, University Hospital of South Manchester, Manchester, UK laura.owen@uhsm.nhs.uk.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Owen
authors.affiliation: Andrology Research Unit, The University of Manchester, Manchester, UK.
authors.forename: Frederick C
authors.initials: FC
authors.lastname: Wu
authors.affiliation: Endocrine Laboratory, Department of Clinical Chemistry, VU University Medical Center Amsterdam, Amsterdam, The Netherlands.
authors.forename: Rahel M
authors.initials: RM
authors.lastname: Büttler
authors.affiliation: Manchester Academic Health Science Centre, Biochemistry Department, University Hospital of South Manchester, Manchester, UK.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Keevil
mesh_terms: D000735:Androstenedione / Q000097:blood* / Q000302:isolation & purification; D001774:Blood Chemical Analysis / Q000592:standards; D002138:Calibration; D002853:Chromatography, Liquid / Q000592:standards; D003687:Dehydroepiandrosterone / Q000097:blood* / Q000302:isolation & purification; D013196:Dihydrotestosterone / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D008297:Male; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D013739:Testosterone / Q000097:blood* / Q000302:isolation & purification
chemical_list: D013196:Dihydrotestosterone; D013739:Testosterone; D000735:Androstenedione; D003687:Dehydroepiandrosterone
keywords: Androgens; DHEA; DHT; LC-MS/MS; androstenedione; testosterone