Eluted folates

Fig. 11.8.5). The eluting folates were detected by monitoring their UV absorbance at 284 nm. Reversed phase chromatographic systems have been used to detect folates in serum (Birmingham and Green, 1983) and in multivitamin preparations (Holcombe and Fusari, 1981). 

The insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

The final product is analyzed by reverse phase HPLC (Biocad Sprint, USA). The HPLC machine was equipped with a Vydac C4 column. Analysis of DSPE-PEG g -folate is performed with isocratic elution using a solvent mixture of 92 8 v/v methanol and 10 mM sodium phosphate (pH 7) at a flow rate of 1 ml/min (18). 

After the buffer has eluted from the column, load up to 2 mL of the crude reaction mixture containing the protein-folate conjugate onto the column. 

Even though addition of stable isotope labeled internal standards is useful for quantitation, in SIM mode both analytes and internal standards co-elute. Further, enhanced specificity and sensitivity were achieved by using a tandem mass spectrometer (MS/MS), operated in selected reaction monitoring (SRM) mode to monitor isolated folate-specific ion fragmentation [15,26,33]. 

Folate and five related compounds (tetrahydro-, 5-methyltetrahydro-, 5-fomtyl-tetrahydro-, 10-formyl-, and 10-formyldihydrofolate) were extracted fitom foods and analyzed on a Cjg column (A = 280 nm). A complex 20-min 95/5 - 90/10 water (33 mM phosphate bulfer at pH 2.1)/acetonitrile gradient was used [1118]. Elution was complete in 18min. Tetrahydropfolate was incompletely resolved from,coextracted materials. Calibration standards ran from 8 to lOOng/mL. 

Following HPLC separation of different folate forms, a number of detection systems can be used including UV, fluorometric, or electrochemical activity (65). Due to its sensitivity and selectivity, fluorescence detection is most commonly used, particularly for reduced folate forms (86). Quantitation is usually based on external standard methods, although an internal method using a stable isotope label has been recently reported (see below) (87). More cumbersome identification and quantifications are needed in methods in which folates are eluted in clusters (69). It is important that the actual concentrations of folate calibration solutions are determined using known extinction factors (30) because most commercially available folate calibrants are of variable purity (7). 

With a mobile phase of 0.2 M ammonium phosphate buffer (pH 5.1) in a reversed-phase system, folate, pyridoxine, nicotinamide, and thiamine could be separated, in this order, within 20 min, followed by vitamin Bn and riboflavin (17). The latter two compounds eluted after a step gradient to 30% aqueous methanol. This procedure has the advantage of completely separating at least nine coenzyme forms of water-soluble vitamins, including TPP. A similar mobile phase, composed of methanol-water (50 50) was used in connection with a LiCh-rosorb RP-18 column to separate thiamine, pyridoxine, vitamin and riboflavin in 3 min, as shown in Figure 4 (23). The detection limit at 254 nm is 5 ng (15 pmol) for thiamine and 10 to 20 ng for the other three vitamins, with a coefficient of variation of <4%. 

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