Online Coupling of Isotachophoresis – Contactless Conductivity Detection - Mass Spectrometry for the Qualitative and Quantitative Analysis of Precursors of the Shikimate Pathway

POSCH, TJORBEN N.; KLER, PABLO A.; HUHN, CAROLIN

Essen (NRW)

Congreso; ANAKON 2013; 2013

GDCH

Precise qualitative and quantitative determination of the small precursors of the shikimate pathway is of great importance to facilitate a deeper understanding of the metabolism of the model plant Arabidopsis thaliana, including e.g. phosphoenolpyruvate and erythrose-4-phosphate. These small molecules are not directly accessible by CE with UV-detection and conductivity detection (C4D) or mass spectrometry have to be used instead. However, a precise quantification of these fragile organic acids via mass spectrometry is bias affected and provides only high levels of detection. To overcome these limitations we chose capillary isotachophoresis for separation, well suitable for anion analysis in combination with conductivity detection for the quantitative analysis and with mass spectrometry for the qualitative, unambigious identification. For the first time we here present anion ITP-C4D-MS with excellent separation of the analytes with sharp boundaries in both detection systems together with its inherent ability for preconcentration and matrix removal. ITP here is combined with precise quantification in C4D or in mass spectrometry, the latter being independent of the signal intensity. In addition, accurate mass spectral data in a quenching free ion spray are obtained. For method optimization, different counter-ions in the sheath-liquid were tested and caproic- and ascorbic acid were employed as terminating electrolytes. With the method presented here, a precise identification of organic acids with an m/z-value of at least 60 is possible. For quantification, an algorithm for the data evaluation of the conductivity measurements was developed and provided precise readings of the inflection points after derivation of the ITP-steps calculating the step length via the distance between to neighboring peaks.