Liquid chromatography/mass spectrometric analysis of 2-nitrophenylhydrazide derivatives of low-molecular organic acids

Abstract

In chromatographic analysis, a suitable derivatization of the functional groups has sometimes been used to enhance the sensitivity and selectivity for detection. Transforming low molecular organic acids to the corresponding 2-nitrophenylhydrazide derivatives has been more extensively used as a suitable derivatization method for liquid chromatographic UV-VIS detection. In the present study, liquid chromatography/mass spectrometric (LC-MS) detection with electro spray ionization was successfully applied to the analysis of low-molecular organic acids, glycolic, acetic, lactic, 2-hydroxy-n-butyric, propionic and n-butyric acid as their 2-nitrophenylhydrazide derivatives. Of a range of analytical conditions that were tested, the optimum results were obtained by using reversed-phase liquid chromatography and a mixture of methanol/water or methanol/ammonium formate. The positive- and negative-ion spectra of 2-nitrophenylhydrazides of the organic acids showed molecular-related ions, [M + H]+ and [M -H]- whose ions were the most abundant, respectively. The effects of the chromatographic and ionization parameters, mobile phase, concentration of ammonium formate, pH and cone voltage on the sensitivity and linearity were examined. Linear plots of the peak area versus the concentration were obtained for MS detection over the range of 0.05-10 μM for propionic acid and 0.1-10 μM for lactic acid. Full-scan mass spectra were readily obtained from 34 nM of n-butyric acid derivative, while a limit of detection of 1.4 nM (signal-to-noise ratio 3) was attained in the selected-ion monitoring mode. These results showed that 2- nitrophenylhydrazide of low molecular organic acids contributes to the enhancement of the LC-MS detection compared with their UV-VIS detection.