title: Sensitive simultaneous quantitation of testosterone and estradiol in serum by LC-MS/MS without derivatization and comparison with the CDC HoSt program.
abstract: BACKGROUND
  Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators.
  
  
  METHODS
  Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C18column and mass spectrometry was performed in both positive and negative ion modes.
  
  
  RESULTS
  The lower limits of quantitation (LLOQs) of E2and Te were 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E2was 5-600pg/mL and 1-1,170ng/dL for Te. Assay accuracy was determined both by comparison with a LC-MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N=10) and between-day (N=20) CVs at concentrations spanning the AMR were less than 7% for both analytes.
  
  
  CONCLUSIONS
  We have developed an accurate and highly sensitive assay to measure E2and Te levels in serum by HPLC-MS/MS without chemical derivatization and using commercially available calibrators.
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Schofield
authors.affiliation: Department of Laboratory Medicine, Montefiore Medical Center, Bronx, NY, United States.
authors.forename: Damodara R
authors.initials: DR
authors.lastname: Mendu
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors.forename: Lakshmi V
authors.initials: LV
authors.lastname: Ramanathan
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States.
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Pessin
authors.affiliation: Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States. Electronic address: carlowd@mskcc.org.
authors.forename: Dean C
authors.initials: DC
authors.lastname: Carlow
mesh_terms: D000085:Acetates / Q000737:chemistry; D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004958:Estradiol / Q000097:blood* / Q000302:isolation & purification; D006586:Hexanes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000097:blood* / Q000302:isolation & purification
chemical_list: D000085:Acetates; D006586:Hexanes; D013739:Testosterone; D004958:Estradiol; C007650:ethyl acetate
keywords: CDC HoSt; Estradiol; Liquid chromatography; Mass spectrometry; Method correlation; NIST SRM 971; Testosterone