Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry

Abstract

A method for the determination of 15N enrichment and concentration of allantoin and uric acid simultaneously in urine using gas chromatography/mass spectrometry (GC/MS) is described. The urine samples contained [1,3-15N2] uric acid and its oxidation product allantoin. The uric acid and allantoin were isolated using an AG1-X8 (Cl-form) anion-exchange column and heated with a mixture containing 1:1 dimethylformamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). The tert-butyldimethylsilyl (TBDMS) derivatives of allantoin and uric acid formed were injected into a gas chromatograph interfaced with a mass spectrometer operated under electron impact ionization conditions. Isotope ratio measurements were made from the abundance of the M-57 ions at m/z 398, 399 and 400 for allantoin and at m/z 567 and 569 for uric acid. 15N2 allantoin (99 at.%) was produced from [1,3-15N2] uric acid by treatment with uricase and used as a standard. Quantitation of allantoin and uric acid was based on isotopic dilution by spiking the urine sample with known quantities of 99 at.% [15N] uric acid and allantoin internal standards. The observed isotope ratio measurements from the prepared standards matched the theoretical values. Coefficients of variation in measurements of isotope ratio and concentration were 0.2 and 0.5%, respectively. The method was applied in a study to measure the urinary recovery of [1,3-15N2] uric acid continuously infused for 8-10 h into the blood of four sheep each on two occasions. Within 24 h, 65.9 +/- 9.1% of the tracer was excreted in the urine unchanged. Little was converted into allantoin (approximately 7% of the dose). The total recovery (5 days) of the infused tracer averaged 69.5 +/- 7.6% as uric acid and 76.8 +/- 9.3% as the sum of uric acid and allantoin. Uricase activities in plasma, liver and kidney of sheep were also measured using [1,3-15N2] uric acid as a substrate. Uricase activity was estimated to be 0.6 mU g-1 wet tissue in the liver and there appeared to be none in plasma and kidney. The low uricase activities in sheep tissues appeared to explain the limited conversion of the intravenously administered [15N] uric acid to allantoin but did not explain the large quantities of allantoin excreted in urine (8.96 +/- 0.86 and 1.36 +/- 0.25 mmol d-1 for allantoin and uric acid, respectively). The GC/MS method for the determination of 15N enrichment and concentration of allantoin and uric acid in urine is accurate and precise and provides a useful tool for studies on uric acid and allantoin metabolism.