title: Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry.
abstract: BACKGROUND
  Challenges for steroid analysis by LC-MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface.
  
  
  METHODS
  250μL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC-DMS-MS/MS. The method was validated and compared with immunoassays and LC-MS/MS methods of reference laboratories.
  
  
  RESULTS
  Inter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r(2)) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively.
  
  
  CONCLUSIONS
  We developed and validated an LC-DMS-MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States. Electronic address: julie.ray@aruplab.com.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Ray
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States.
authors.forename: Mark M
authors.initials: MM
authors.lastname: Kushnir
authors.affiliation: Department of Chemistry, University of Florida, Gainesville, FL, United States.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Yost
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States; Department of Pathology, University of Utah, Salt Lake City, UT, United States.
authors.forename: Alan L
authors.initials: AL
authors.lastname: Rockwood
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States; Department of Pathology, University of Utah, Salt Lake City, UT, United States; Department of Medicine, University of Utah, Salt Lake City, UT, United States.
authors.forename: A
authors.initials: A
authors.lastname: Wayne Meikle
mesh_terms: D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D002853:Chromatography, Liquid; D003345:Corticosterone / Q000097:blood*; D003350:Cortodoxone / Q000097:blood*; D003900:Desoxycorticosterone / Q000097:blood*; D006801:Humans; D011374:Progesterone / Q000097:blood*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods / Q000592:standards*; D053719:Tandem Mass Spectrometry
chemical_list: D003900:Desoxycorticosterone; D011374:Progesterone; D019326:17-alpha-Hydroxyprogesterone; D003345:Corticosterone; D003350:Cortodoxone
keywords: Chemiluminiscent immunoassay (CIA); Congenital adrenal hyperplasia (CAH); Differential ion mobility spectrometry (DMS); Immunoassay (IA); Liquid chromatography–tandem mass spectrometry (LC–MS/MS); Steroids