DETERMINATION OF CYTIDINE 5'-MONOPHOSPHO-N-ACETYLNEURAMINIC ACID POOLSIZE IN CELL-CULTURE SCALE USING HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY WITH PULSED AMPEROMETRIC DETECTION

A simultaneous determination of cytidine 5'-monophospho-N-acetylneuram inic acid (CMP-Neu5Ac) and its metabolic products, cytidine, CMP and N eu5Ac by high-performance anion-exchange chromatography with pulsed am perometric detection (HPAEC-PAD) using a Carbo-Pac PA1 column is descr ibed. Preparation of the samples involved a single purification step o f the crude cell extract on DEAE-Sepharose. The method is adequate to quantify the amount of CMP-Neu5Ac produced by one culture dish: equiva lent to 6 . 10(6) cells. In addition, a method for desalting and recov ery 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 r ise to a 6.4-fold elevation of this value.