Enhanced LC-MS Detection of γ-Aminobutyric Acid via Trimethylation Enhancement Using Diazomethane

Neurotransmitters are critical for the proper function, signal transmission, and physiological balance of the brain, with γ-aminobutyric acid (GABA) being the main inhibitory neurotransmitter in the central nervous system. GABA is present at relatively low concentrations compared to other neurotransmitters, therefore requiring sensitive analytical methods for accurate identification and quantitation. Described herein is a rapid and facile liquid chromatography mass spectrometry (LC-MS)-based chemical derivatization method to enhance the detection of GABA, demonstrated in both saline culture media and Carassius auratus (goldfish) retina samples. We have expanded the use of trimethylation enhancement using diazomethane (TrEnDi) to permethylate GABA ([GABA^Tr]⁺) at 98-100% yields across all matrix types. Quantitative methylation of the carboxylic acid and amino moieties nullifies any zwitterionic character and fixes a permanent positive charge on [GABA^Tr]⁺, leading to MS sensitivity enhancement. In biological triplicates of goldfish retina samples, [GABA^Tr]⁺ boasted 6.3-27.9-fold increases in MS sensitivity compared to its unmodified counterpart, enabling quantitation with concentrations ranging between 78.6 and 806.5 nM. Calibration curve linearity for [GABA^Tr]⁺ and unmodified GABA was R² = 0.9996 and R² = 0.9923, respectively. Limits of detection and quantitation (LOD/LOQ) for [GABA^Tr]⁺ were 0.053 nM (1.1 fmol)/0.18 nM (3.6 fmol), compared to 2.5 nM (50 fmol)/8.3 nM (167 fmol) for unmodified GABA. This work demonstrates that TrEnDi has the ability to rapidly enhance LC-MS detection of GABA in a relatively facile manner, reducing the probability of reporting false negatives in the analysis of complex biological samples.