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 UV-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 microM (sample size 50 microl) for 1-methylhistidine, 3-methylhistidine, histidine, creatinine, anserine, carnosine, and homocarnosine, and from 0.5 to 2000 microM 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.
Yokoyama
Y
Y
Laboratory of Analytical Chemistry, Faculty of Engineering, Yokohama National University, Hodogaya, Japan. yokyuk@ynu.ac.jp
Horikoshi
S
S
Takahashi
T
T
Sato
H
H
D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008661:Metabolism, Inborn Errors / Q000175:diagnosis / Q000652:urine; D013056:Spectrophotometry, Ultraviolet; D013451:Sulfonic Acids / Q000737:chemistry*; D016482:Urinalysis
D002411:Cation Exchange Resins; D013451:Sulfonic Acids