Facile derivatization of ultratrace carboxylic acids in saliva for quantification by HPLC-MS/MS

It remains an issue to directly quantify trace biologically important carboxyl compounds in body fluids. Herein we propose an innovative method to determine α-lipoic acid, 2-(β-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, prostaglandin E₂, cholic acid, and chenodeoxycholic acid in saliva. The method consists of two successive steps: fast and direct labeling of the target analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis. The method exhibited a wide linear range from 2.5 to 2500 pg/mL, with linear coefficients greater than 0.9963 and limits of detection and quantification as low as 0.10 and 0.33 pg/mL, respectively. The method precision was evaluated, with relative standard deviations ranging from 2.12% to 10.63% for intraday assays and from 2.98% to 12.88% for interday assays. The recoveries were measured by our spiking saliva samples with standards at three different levels, and ranged from 72.5% to 98.0%. Real applicability was validated by direct quantification of trace target analytes in human saliva, with simple pretreatment, use of a small sample volume, and a short analysis time. Graphical abstract Sequential steps to extract, label, and determine the ultratrace carboxylic acids in saliva. CDCA chenodeoxycholic acid, γ-CEHC 2-(β-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, α-LA α-lipoic acid, PGE₂ prostaglandin E₂, UHPLC-MS/MS ultrahigh-performance liquid chromatography-tandem mass spectrometry.