Low-capacity cation-exchange chromatography of ultraviolet-absorbing urinary basic metabolites using a reversed-phase column coated with hexadecylsulfonate

A low-capacity cation-exchange HPLC method for the determination of W-absorbing organic cations such as amino acids, histidine dipeptides, and creatinine was developed. A commercially available reversed-phase column was dynamically coated with hexadecylsulfonate, and was successfully used for the cation-exchange separation with ethylenediammonium eluting ion at pH 2.5. The coated column was enough stable for the specific use with a completely aqueous mobile phase at low and constant pH; and the day-to-day reproducibility for retention time was 0.9-1.7% of RSD (relative standard deviation). The linear relation between concentrations and detector responses (area) by using a photodiode-array UV detection at 210 nm ranged from 0.2 to 1000 mu M (sample size 50 mu l) for l-methylhistidine, 3-methylhistidine, histidine, creatinine, anserine, carnosine, and homocarnosine, and from 0.5 to 2000 mu M for creatine, tyrosine, and phenylalanine, with less than 5% of RSD. The UV spectrum (190-300 nm) obtained during chromatography was very indicative for each analyte. Overall recoveries were 97-104%. The developed HPLC method in conjunction with preliminary fractionation technique could be applied to the analysis of urine of patient with metabolic disorder such as phenylketonuria. (C) 2000 Elsevier Science B.V. All rights reserved.