High-Performance Liquid Chromatography/Mass Spectrometric Analysis of Middle-Chain Fatty Acids by 2-Nitrophenylhydrazide Derivatization

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 chromatography/mass spectrometric (LC/MS) detection. In the present study, LC/MS detection with electro spray ionization (ESI) was successfully applied to the analysis of middle-chain fatty acids (MFA): pentanoic, hexanoic, heptanoic, octanoic, nonanoic and decanoic acid, as their 2-nitrophenylhydrazide derivatives (2-NPHs). At the point of derivatization, microwave irradiation was successfully used to prepare 2-NPH derivatives. Of a range of analytical conditions that were examined, the optimum results were obtained by using reversed-phase liquid chromatography and a mixture of acetonitrile/ammonium formate. The effects of the chromatographic and ionization parameters, ionization mode and cone voltage on the sensitivity of 2-NPHs of MFA were examined. The negative-ion spectra of 2-NPHs of MFA showed molecular-related ions, [M - H](-), whose ions were the most abundant. Linear plots of the peak area versus the concentration were obtained for MS detection over the range 0.05 similar to 10 mu M for most 2-NPHs of MFA. The limits of detection of 5.5 similar to 10.5 nM (signal-to-noise ratio 3) were attained in a selected-ion monitoring (SIM) mode. These results showed that 2-NPHs of MFA contribute to the enhancement of LC/MS detection, and the 2-NPH derivatization using microwaves has a potential to simplify the conventional analytical method for MFA.