A liquid chromatography–tandem mass spectrometry assay for detection and quantitation of the dipeptide Gly-Gln in rat brain

Abstract

The enzymatic cleavage products of β-endorphin (β-endorphin1-27 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 (R2 >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 <5%. A combination of MRM fragment ratio normalization and chromatographic peak shifting were used to ensure that the LC-MS/MS peak for Mar-Gly-Gln was free from possible isobar interferences. This assay was then demonstrated for the determination of in vivo Gly-Gln levels in P and Sprague-Dawley rat cortex and nucleus accumbens samples.

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers

“…8). No matrix effects were apparent with human serum diluted samples (data not shown), consistent with our prior studies on LC-MS/MS quantification of other Marfey's reagent derivatized amino acid analytes [49-51]. …”

“…Alternative gradients were investigated – including isocratic conditions, and inclusion of methanol in the solvent system – which we have previously found useful for differential shifting of overlapping Marfey's derivative peaks [51]. The best resolution obtained was with the gradient described above.…”

A review of sample preparation methods for quantitation of small-molecule analytes in brain tissue by liquid chromatography tandem mass spectrometry (LC-MS/MS)

“…In order to utilize the properties of buffer systems as well as organic solvents, a mixture of more than one of these agents can be used as the homogenization media. 10,23,48,60,64,66,78 Instead of using pure organic solvents that have very strong precipitating abilities, a mixture of organic solvents (ACN or MeOH) and water (or aqueous solutions) was used as the homogenization media, so that the proteins in the brain tissue were not drastically precipitated and, therefore, the resulting homogenate was more stable. Moreover, the presence of water in the homogenization media can make it possible to add ionic or polar additives, inorganic salts or acids for example, which otherwise are not soluble in pure organic solvents.…”

“…It is capable of simple and fast treatment of biological samples, and can be considered as the most widely used sample preparation method. 9,10,13,15,16,[19][20][21][22]24,25,[29][30][31][34][35][36][37][38][39]41,42,46,48,49,[51][52][53]55,57,58,[60][61][62][63][66][67][68]72,74,[76][77][78][79][80]87,88,90,92,94 Proteins are large biological molecules that are composed of amino acids linked by peptide bonds. Under physiological conditions, a solub...…”

“…Protein precipitants with more than one component are also used in multiple published studies. 10,16,30,31,38,48,76,78 In a recent study about the quantitation of loxapine, amoxapine and their hydroxylated metabolites, combinations of MeOH and PCA at different relative ratios were tested in the sample preparation of brain tissue homogenate, suggesting that one volume of MeOH and 7 volumes of PCA yielded the best analyte recovery. 78 In another method published by Karinen et al in 2009, a mixture of ACN and MeOH was used for the protein precipitation of brain homogenate.…”