Browning products HPLC separation

Casein heated in aqueous solution with furfural rapidly turns orange-brown. Melanoidins (> 10 kDa), isolated by ultracentrifugation, were enzymically hydrolysed and the product was separated by HPLC. Two peaks led to red compounds in the ratio 1 7, which were unequivocally identified by Hofmann191 as the lysine analogues of 24a and 24b (MM = 0.476 kDa), respectively. This is the first demonstration of the attachment of a specific type of coloured Maillard product to a protein... 

Thus, RP-HPLC-MS has been employed for the analysis of sulphonated dyes and intermediates. Dyes included in the investigation were Acid yellow 36, Acid blue 40, Acid violet 7, Direct yellow 28, Direct blue 106, Acid yellow 23, Direct green 28, Direct red 79, Direct blue 78 and some metal complex dyes such as Acid orange 142, Acid red 357, Acid Violet 90, Acid yellow 194 and Acid brown 355. RP-HPLC was realized in an ODS column (150 X 3 mm i.d. particle size 7 /.an). The composition of the mobile phase varied according to the chemical structure of the analytes to be separated. For the majority of cases the mobile phase consisted of methanol-5 mM aqueous ammonium acetate (10 90, v/v). Subsituted anthraquinones were separated in similar mobile phases containing 40 per cent methanol. The flow rate was 1 ml/min for UV and 0.6 ml/min for MS detection, respectively. The chemical structure of dye intermediates investigated in this study and their retention times are compiled in Table 3.28. It was found that the method is suitable for the separation of decomposition products and intermediates of dyes but the separation of the original dye molecules was not adequate in this RP-HPLC system [162],... 

Amadori compounds (N-substituted-l-amino-l-deoxy-2-ketoses) are potential precursors to the formation of many of these heterocyclic volatile products. The secondary nitrogen in most Amadori compounds is weakly basic and is therefore a likely site for rapid nitrosation reactions via normal reactions with nitrous acid, under mildly acidic conditions. However, purified Amadori compounds are usually obtained only after tedious isolation procedures are invoked to separate them from the complex mixtures of typical Maillard browning systems. Takeoka et al. ( 5) reported high performance liquid chromatographic (HPLC) procedures to separate Amadori compounds in highly purified form on a wide variety of columns, both of hydrophilic and hydrophobic nature. They were able to thus demonstrate that reaction products could be followed for kinetic measurements as well as to ensure purity of isolated products. 

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

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