title: A hydrophilic-interaction chromatography tandem mass spectrometry method for quantitation of serum s-adenosylmethionine in patients infected with human immunodeficiency virus.
abstract: BACKGROUND
  s-Adenosylmethionine (SAM)1 has been suggested as a diagnostic test and surrogate marker for Pneumocystis jirovecii pneumonia (PCP) in HIV-positive patients. In this study, we report a robust hydrophilic-interaction liquid chromatography tandem mass spectrometry (LC-MS/MS) assay that can be used to quantitate serum SAM in clinical laboratories.
  
  
  METHODS
  Proteins in serum samples were precipitated using trichloroacetic acid. The supernatant was separated after centrifugation. D3d3-SAM was added as the internal standard. SAM and d3-SAM were extracted using a mixed-mode cation exchange column. Extracts were dried under nitrogen and reconstituted in H2O and acetonitrile (1:9, vol:vol). HPLC-tandem mass spectrometry analysis was performed with a silica column and multiple reaction monitoring for SAM and d3-SAM.
  
  
  RESULTS
  The limit of quantitation (LOQ) for SAM was 10 ng/mL. The assay was linear between 10 and 500 ng/mL. Intra-assay coefficient of variation (CV) was 8% and inter-assay CV was 17% at the LOQ. Turnaround time for each specimen was approximately 1 h. Using this method, we found that serum SAM concentration was correlated with fasting status, especially methionine intake. We also measured acute and convalescent serum SAM levels of 8 HIV-positive patients with PCP and non-PCP pneumonia. SAM concentrations in convalescent samples were significantly increased compared to acute levels only in patients with PCP.
  
  
  CONCLUSIONS
  The LC-MS/MS method had sufficient analytical sensitivity for detecting low levels of SAM found in HIV-infected patients and can be used for quantitative measurements in a clinical laboratory. This method facilitates research and possible clinical application of SAM as a marker for PCP.
authors.affiliation: Department of Lab Medicine, University of California, San Francisco, CA, United States. pwang@tmhs.org
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Laurence
authors.initials: L
authors.lastname: Huang
authors.forename: J Lucian
authors.initials: JL
authors.lastname: Davis
authors.forename: Alexandra
authors.initials: A
authors.lastname: Swartzman
authors.forename: Brenna
authors.initials: B
authors.lastname: Roth
authors.forename: Judy
authors.initials: J
authors.lastname: Stone
authors.forename: Alan H B
authors.initials: AH
authors.lastname: Wu
mesh_terms: D015415:Biomarkers / Q000097:blood; D005215:Fasting; D015658:HIV Infections / Q000097:blood*; D006801:Humans; D012436:S-Adenosylmethionine / Q000097:blood* / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014867:Water / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D014867:Water; D012436:S-Adenosylmethionine