Development, validation, and application of a surrogate analyte method for determining N-acetyl-l-aspartyl-l-glutamic acid levels in rat brain, plasma, and cerebrospinal fluid

•We developed a surrogate analyte method to quantify NAAG in biological matrices.•We show strategy for the method development and validation with surrogate analytes.•We analyzed NAAG levels in naïve rats and in NAAG peptidase inhibitor-treated rats.

A bioanalytical strategy for the simple and accurate determination of endogenous substances in a variety of biological matrices using liquid chromatography-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compounds as a surrogate analyte and an internal standard to construct calibration curves with authentic matrices that can be applied to determine N-acetyl-l-aspartyl-l-glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extraction and with a short analysis time of 4 min. The validated lower limits of quantification were 1.00 nmol/g for brain and 0.0100 nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naïve rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the determination of NAAG in different matrices in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biological matrices.