A hydrophilic-interaction chromatography tandem mass spectrometry method for quantitation of serum s-adenosylmethionine in patients infected with human immunodeficiency virus

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 10ng/mL. The assay was linear between 10 and 500ng/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 1h. 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.