Measurement of hypoxanthine and xanthine in late-gestation human amniotic fluid by reversed-phase high-performance...

A robust analytical method was developed for measurement of hypoxanthine and xanthine in late-gestation human amniotic fluid by reversed-phase high-performance liquid chromatography using diodearray detection. Purity of analyte peaks was confirmed via on-line analysis of peak spectra utilizing the purity parameter treatment of spectral data. Amniotic fluid obtained by amniocentesis was deproteinized by centrifugal ultrafiltration and chromatographed on an octadecylsilica column using isocratic elution with 1% (v/v) acetonitrile in 0.05 M ammonium dihydrogenphosphate pH 6.0; hypoxanthine and xanthine were resolved, but the hypoxanthine peak was not pure. Chromatography on a column of polar endcapped octadecylsilica, using similar mobile phase conditions, yielded spectrally pure peaks of hypoxanthine xanthine. Hypoxanthine and xanthine levels in amniotic fluid from fourteen patients, gestational age 34-39 weeks, ranged from 0.56 to 2.74 microM and 1.62 to 5.52 microM, respectively.