46. Testosterone Analysis Using Derivatization Chemistry and LC/MS/MS
Poster: Mon 6:30-7:30PM
Michal Weinstock
Applied Biosystems
Michal Weinstock, Subhakar Dey, Brian WIlliamson, Sasi Pillai, Subabish Purkayastha,

Applied Biosystems, 500 Old Connecticut Path Framingham MA, 01701
Immunoassays have been widely used for clinical applications of human Testosterone (Te) analysis. These assays are lacking the sensitivity and specificity that is needed for samples in which Te levels fall below 100pg/mL (10ng/DL). LC-MS/MS is becoming more widely used in steroid analysis due to its specificity and accuracy, but the ionization efficiency for many neutral steroids may not be sufficient to reliably quantify low pg/mL levels. Derivatization with ionizable moiety could significantly improve the ionization efficiency of neutral steroids.

Herein presented a novel derivatization chemistry for high sensitivity analysis of Testosterone in serum samples.

Materials and Methods:
Testosterone is enriched from 200uL of serum samples by protein precipitation with MeOH. The samples are dried and the derivatization reaction is performed in MeOH+10% Acetic acid for 30min in RT. LC-MS/MS separation and analysis is performed using a C-18 column (50x2.0mm), MeOH/H2O/Formic acid gradient and API 4000 MS/MS (Applied Biosystems/ MDS Analytical Technologies) ESI source, operating in MRM mode. Quantitation of the derivatized Te is enabled by using an isotopically enriched reagent as an Internal Standard (IS). A known amount of IS is spiked into the sample before analysis and the unknown concentration is extrapolated from the analyte/IS peak area ratio of a known standard curve.

Preliminary Data:
Product ion spectra of the derivatized Te produced one strong signature ion from the derivatization reagent. The resultant signal enhancement factor relatively to the underivatized Te, is >100. The LOD for the derivatized Te in solvent is 30fg on column and for Te derivatized in serum (double charcoal stripped) is 5pg/mL (~0.35pg on column). The reaction is complete within 30min in RT. Two positional isomers are observed for the drivatized Te which are baseline separated under the method’s chromatographic conditions. Reliable quantitation can be achieved by integrating both peaks or each peak separately. The method is linear (R2=0.994) over the range of 10-2500 pg/mL. The %CV within day at the concentration level of 25pg/mL is 6.8 and between day reproducibility %CV=13. The method is suitable for the quantitation of Testosterone in samples with low pg/mL concentrations.