title: LC-ESI-MS/MS analysis of testosterone at sub-picogram levels using a novel derivatization reagent.
abstract: Testosterone analysis by LC-MS/MS is becoming the analytical method of choice over immunoassays due to its specificity and accuracy. However, neutral steroid hormones possess poor ionization efficiency in MS/MS, resulting in insufficient sensitivity for analyzing samples with trace concentrations of the hormones. The method presented here utilizes a derivatization step involving a novel, permanently charged, quaternary aminooxy (QAO) reagent or MS-tag that reacts to the ketone functionality of testosterone and significantly enhances its ESI-MS/MS sensitivity. This derivatization method enabled quantitation of total testosterone in human serum (200 μL) with a lower limit of quantitation (LLOQ) of 1 pg/mL (3.47 pmol/L), total testosterone in dried blood spots (8-10 μL) with a LLOQ of 40 pg/mL, and free testosterone in serum ultrafiltrate (400 μL) with a LLOQ of 0.5 pg/mL. The linearity of each of the high sensitivity applications was maintained over a broad dynamic range of 1-5000 pg/mL for the serum samples and 40-10,000 pg/mL for the dried blood spots (DBS) with R(2) >0.998. The %CV at the LLOQ was <15 for all applications. The QAO derivatization and sample preparation workflows are quick, simple, and robust. Comparison of the derivatization method with an LC-ESI-MS/MS nonderivatization method yielded high correlation and agreement. The derivatization reagent is universal and reacts with other compounds containing ketone or aldehyde functionality.
authors.affiliation: AB SCIEX, Chemistry and Consumables R&D, 500 Old Connecticut Path, Framingham, Massachusetts 01701, United States. michal.weinstock@absciex.com
authors.forename: Michal
authors.initials: M
authors.lastname: Star-Weinstock
authors.forename: Brian L
authors.initials: BL
authors.lastname: Williamson
authors.forename: Subhakar
authors.initials: S
authors.lastname: Dey
authors.forename: Sasi
authors.initials: S
authors.lastname: Pillai
authors.forename: Subhasish
authors.initials: S
authors.lastname: Purkayastha
mesh_terms: D000293:Adolescent; D000328:Adult; D000588:Amines / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D002648:Child; D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D055815:Young Adult
chemical_list: D000588:Amines; D007202:Indicators and Reagents; D013739:Testosterone