On-line determination of carboxylic acids, aldehydes and ketones by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionisation mass spectrometry after derivatization with 2-nitrophenylhydrazine

J Chromatogr A. 2004 Mar 26;1031(1-2):35-50. doi: 10.1016/j.chroma.2003.10.100.


2-Nitrophenylhydrazine (2-NPH) is widely used for the derivatization of carboxylic acids, aldehydes and ketones, in industrial and biological samples. These compounds react with 2-NPH to form derivatives, which are separated by high-performance liquid chromatography (HPLC) and detected with diode array detection (DAD). The UV spectra give information about the functionality of the compounds: carboxylic acid or ketone/aldehyde. Most of the eluting compounds in "known" samples are well characterised by the retention time (comparison with those of standards) of the 2-NPH derivative and their UV spectrum. The identification of different unknown 2-NPH derivatives of carboxylic acids, ketones and/or aldehydes, in industrial or biological samples, based on retention time and/or UV spectrum is not sufficient. These unknown 2-NPH compounds can be identified with on-line atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) based on the molecular mass or/and the fragmentation of the derivative. A novel and specific on-line HPLC-DAD-APCI(-)-MS method is described for the determination of carboxylic acids, ketones and aldehydes, after on-line pre-column derivatization with 2-NHP. The fragmentation of different 2-NPH derivatives were investigated and the possibilities of APCI(-)-MS detection were demonstrated by the on-line identification of an unknown derivative, which turned out to be a side product between 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-NPH in the presence of high concentrations of a cyclic amide in the sample solution.

MeSH terms

  • Aldehydes / analysis*
  • Aldehydes / isolation & purification
  • Carboxylic Acids / analysis*
  • Carboxylic Acids / isolation & purification
  • Chromatography, High Pressure Liquid
  • Data Display
  • Hydrazines / chemistry*
  • Indicators and Reagents
  • Ketones / analysis*
  • Ketones / isolation & purification
  • Mass Spectrometry
  • Online Systems
  • Phenylhydrazines
  • Solvents
  • Spectrophotometry, Ultraviolet


  • Aldehydes
  • Carboxylic Acids
  • Hydrazines
  • Indicators and Reagents
  • Ketones
  • Phenylhydrazines
  • Solvents
  • 2-nitrophenylhydrazine