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 (<10 ng/mL) and high (>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(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(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(3) was observed. Intraday and interday experimental accuracy, calculated as percent error, were in the range of < or =10% for KIC-d(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.
Bioanalytical Research, Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA.
Wenlin
W
Li
Lucinda H
LH
Cohen