Quantitation of endogenous analytes in biofluid without a true blank matrix

A method is presented that describes a reliable and practical procedure for quantitation of an analyte present at relatively high background levels in blank (untreated) biological matrixes. Using a 'surrogate analyte' approach, an endogenous analyte was quantitated in a variety of biological matrixes containing both very low (2000 ng/mL) background levels of the desired analyte. This quantitative 'surrogate analyte' approach was applied during the development of an HPLC/MS method for [alpha]-ketoisocaproic acid (KIC), which was identified as a potential biomarker for branched chain amino acid transferase inhibitor activity. Using deuterium-labeled KIC ([d.sub.3]) as a surrogate analyte, not an internal standard, to generate the calibration curve, the concentration of KIC in biofluid could be back-calculated based on the regression equation and response factor of KIC to KIC-[d.sub.3]. In particular, this approach made it possible to prepare standards in control biofluid such as plasma, which greatly facilitated the process of method development. For the validated method, a linear range of 10-5000 ng/mL for KIC-[d.sub.3] was observed. Intraday and interday experimental accuracy, calculated as percent error, were in the range of [less than or equal to] 10% for KIC-[d.sub.3]. This method is simple, rapid, and reliable for the quantitation of KIC in plasma, brain homogenate, cerebrospinal fluid, and other biological samples from discovery and pharmacological studies.