Selective, sensitive and fluorometric determination of urinary cytosine with 4-trifluoromethylbenzamidoxime and N,N-dimethylformamide.

BACKGROUND

Cytosine in urine is one of the biomarkers for the diagnosis of metabolic immunodeficiency. It has been mentioned that a high level of cytosine is found in urine of children having immunodeficiency. In this study, we have developed a fluorescence (fluorescence) derivatization reaction of cytosine using 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent.

METHODS

In this reaction, cytosine was mixed with 4-TFMBAO, K3[Fe(CN)6], N,N-dimethylformamide (DMF) and KOH in an aqueous solution. The mixture was heated at 100°C for 20 min. The fluorescence intensity of the mixture was measured with a spectrofluorometer.

RESULTS

Under the optimized reaction conditions, a strong fluorescence was produced only from cytosine amongst 62 compounds including structurally related bio-substances. The selectivity and sensitivity of this method were compared with a conventional fluorescence one using 2-bromoacetophenone that reacts with cytosine, adenine and their related substances. The present method was sufficiently selective toward cytosine, and approximately 50 times more sensitive than the conventional one.

CONCLUSIONS

Our method permitted the quantitative determination of cytosine in human urines without any pretreatment for a primary screening test of inborn disorder in pyrimidine metabolism with immunodeficiency, and indicated the lower detection limit of 0.1 μmol/l cytosine which gave 3 times greater fluorescence intensity than that observed for the reagent blank.