High-performance liquid chromatographic separation and quantitation of nucleosides in urine and some other biological fluids.

Precise, quantitative high-performance liquid chromatography of samples equivalent to 25 l of urine can be done in less than 1 h with excellent resolution and recovery of pseudouridine, 1-methyladenosine, 1-methylinosine, N2-methylguanosine, adenosine, and N2,N2-dimethylguanosine. These six nucleosides are present in normal urine in concentrations ranging from 0.4 to 60 mg/liter. The use of an affinity chromatograph column with a bound boronic acid functionality has enabled us to retain nudeosides selectively as boronate complexes from biological samples as complex as urine. The nucleosides are readily eluted with dilute formic acid from the boronate affinity column, concentrated, and separated by reversed-phase liquid chromatography on “hz Bondapak/C18” columns. In addition, about 10 other nucleosides are resolved and present in normal urine, 0.2 to 4 mg/liter. Isocratic chromatographic quantitation gave a minimum detection limit of 5 pmol or less for each of the nine nucleosides examined; the relation between peak area and concentration was linear with average CV’s of less than 3% for six nucleosides over the range of 100 to 1000 pmol for each nucleoside injected. Analytical recoveries of nucleosides from standard mixtures exceeded 90% at concentrations comparable to those found in normal urine; those for eight nucleosides added to urine exceeded 85% at a concentration of 5 nmol (about 1 tg) added per milliliter. The method is being used to determine the concentrations, and their ratios (to creatinine), of nucleosides in urine from patients with different types of cancer. Data on urine samples from normal persons and persons with cancer of the colon are compared by gas chromatography and the present method. Application of the method to serum and amniotic fluid is demonstrated.