Derivatization for the simultaneous LC/MS quantification of multiple neurotransmitters in extracellular fluid from rat brain microdialysis.

Quantification of amino acid based neurotransmitters in extracellular fluids, such as those in the neuron synapse, presents a challenge to the analytical chemistry because of the absence of UV- or fluorescence-detectable functional groups and the low sensitivity in mass spectrometric detection. This report describes a novel use of the succinimide reagent, N-α-Boc-l-tryptophan hydroxysuccinimide ester (Boc-TRP), for the pre-column derivatization to simultaneously quantify multiple neurotransmitters in the rat brain microdialysis samples. The Boc-TRP derivatization was rapid and quantitative in phosphate the buffer (pH 7.4) at room temperature. The derivatized neurotransmitters were suitable for rapid LC/MS quantification with less than 3-min chromatographic separation. The Boc-group in the derivatized product generated unique fragmentation patterns in the triple quadrupole mass spectrometric analysis under Multiple Reaction Monitoring mode and significantly increased the specificity and sensitivity. The derivatization and rapid LC/MS quantification method developed in this study showed a linear dynamic range from single digit nM to 1000nM with coefficient greater than 0.990. At the LOQ, the accuracy ranged from 95 to 108% and the precision (CV%) was less than 20%. Since there was no concentration and reconstitution in the sample workup process, this derivatization approach simplified the neurotransmitter quantification of the brain microdialysis samples.