Separation and identification of organic acid-coenzyme A thioesters using liquid chromatography/electrospray ionization-mass spectrometry

A method has been developed for the direct determination of coenzyme A (CoA) and organic acid-CoA thioesters in mixtures using directly combined liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). Mixtures of CoA and organic acid-CoA thioesters were analyzed by LC/ESI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of the CoA-thioesters were established based on LC retention times and simultaneously recorded mass spectra. Monitoring of the CoA specific fragment ion at m/z 428 throughout the chromatogram provides a unique fingerprint for CoA content in the samples that corroborates the identification of organic acid-CoA thioesters in the mixtures. Furthermore, fragment ions arising from the ester linkage portion of the molecule allow unambiguous identification of the CoA esters in the samples. A second LC elution system was developed that allows the simultaneous separation and identification of 2-hydroxypropionyl-CoA (lactyl-CoA) and 3-hydroxypropionyl CoA (3HP-CoA), which have the same mass and identical MS fragmentation behavior. The utility of LC/ESI-MS employing this elution system is demonstrated by the determination of 3HP-CoA and lactyl-CoA (converted to CoA-thioesters from their corresponding free acids using CoA-transferase) in fermentation broths from Escherichia coli strains engineered for the production of 3-hydroxypropionic acid (3HP). External calibration employing a purified 3HP-CoA standard allowed indirect quantification of 3HP content in the broth with a precision of 1% (RSD). The feasibility of extending the method described above to perform LC/selected reaction monitoring-tandem mass spectrometry for direct determination of organic acid-CoA thioesters in cells was also demonstrated.     

Determination of tea catechins

An overview of analytical methods for the measurement of biologically important tea catechins is presented. Liquid chromatography and capillary electrophoresis are the most cited techniques for catechin separation, identification and quantitation. Liquid chromatography with ultraviolet detection is frequently used; however, mass spectrometry, electrochemical, fluorescence and chemiluminescence detection are also utilized in cases where more sensitive or selective detection is needed. Two modes of capillary electrophoresis, capillary zone electrophoresis and micellar electrokinetic capillary chromatography, have been employed for the determination of catechins. Both modes of capillary electrophoresis are based on ultraviolet detection. Additional analytical techniques, such as gas chromatography, thin-layer chromatography, paper chromatography, spectrophotometry, biosensing, chemiluminescence and nuclear magnetic resonance spectroscopy have also been utilized for the determination of catechins and are reviewed herein.