A User-Friendly Sample Preparation Alternative for Manual and Automated LC-MS/MS Quantification of Testosterone

We describe an LC-MS/MS method for serum testosterone using a novel extraction media, AC Extraction Plate™ (AC Plate,Tecan Schweiz). The AC Plate principle is essentially that of a liquid-liquid extraction (LLE) but employs a stationary nonpolar phase coated on the wells of 96-well plates instead of a nonmiscible organic solvent for partitioning testosterone out of serum, leaving interfering substances behind. This low complexity sample preparation protocol has been validated for and used in production in our laboratory with both manual and automated liquid handling. The primary advantage of this method is the highly reproducible nature of an extraction method that does not require LC-MS/MS expertise or specialized extraction equipment. We modified the existing vendor application and validated the method for matrix effect, recovery, precision, trueness [accuracy relative to certified reference material (CRM)], specificity, reportable range, sample stability, various sample containers, and correlation with other methods.Method performance is excellent, with a reportable range of 4-750 ng/dL, between-day quality control coefficient of variation (CV) over 12 months of <8%, mean accuracy of <4.0% bias against CRM, no interference from hemolysis, icterus, lipemia, serum separator tube gel, or common steroids/metabolites, and mean bias of 1.3% versus 4 other LC-MS/MS testosterone methods. An investigation of calibration stability and robustness supports sparse (3 versus 6 calibrators) and/or historical calibration for routine use.