Determination of cytidine 5'-monophospho-N-acetylneuraminic acid pool size in cell culture scale using high-performance anion-exchange chromatography with pulsed amperometric detection.
A simultaneous determination of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) and its metabolic products, cytidine, CMP and Neu5Ac by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Carbo-Pac PA1 column is described. Preparation of the samples involved a single purification step of the crude cell extract on DEAE-Sepharose. The method is adequate to quantify the amount of CMP-Neu5Ac produced by one culture dish; equivalent to 6.10(6) cells. In addition, a method for desalting and recovery of the separated material was developed to determine the cellular concentration of CMP-Neu5Ac in Madin Darby canine kidney (MDCK) cells. The addition of 5 mM N-acetylmannosamine to the culture medium gave rise to a 6.4-fold elevation of this value.