An LC-MS/MS Assay for Detection and Quantification of the Dipeptide Gly-Gln in Rat Brain

The enzymatic cleavage products of β-endorphin (β-endorphin 1-27 and Gly-Gln) reduce voluntary alcohol consumption in alcohol preferring P rats. Gly-Gln also inhibits the reward benefiting effects of morphine and nicotine. It would be useful for the investigation of this effect to have an analytical method suitable for Gly-Gln detection and quantitation. Given the now widespread availability of LC-MS/MS instruments, the development of an LC-MS/MS-based approach seemed a viable option. An LC-MS/MS method for Gly-Gln quantitation was developed based on derivatization with Marfey’s reagent. The Marfey’s adduct of Gly-Gln (Mar-Gly-Gln) was chromatographically resolved and readily detected and quantitated by LC-MS/MS. Precursor/product positive ions of 456.2/366.2, 456.2/237.2, 456.2/147.0 were used for detection and quantitation. This method shows good linearity from 1 to 500 pmole of Mar-Gly-Gln (R 2 >0.99). The assay also demonstrated good accuracy and precision, with an average percent standard deviation for Gly-Gln over the range of the assay of