Simultaneous determination of allantoin and adenosine in human urine using liquid chromatography - UV detection

We report a HPLC-UV method for the quantitative determination of allantoin and adenosine in human urine, validated according to the acceptance criteria of both the USA Food and Drug Administration (FDA) guideline for bioanalytical method validation and the European Medicines Agency (EMA) validation guidelines. Both allantoins and adenosine are compounds of the purine catabolic pathway. Adenosine is situated at the top as a uric acid (UA) precursor, while allantoin is the best-known degradation product of UA. These two compounds are endogenously present in human urine. Chromatographic separation was achieved with a gradient elution at 0.6 mL/min using a Zorbax SB-Aq column coupled to a Zorbax SB-Aq guard column. Three different mobile phases were used: mobile phase A consisted of 10 mM KH₂PO₄ (pH 4.7) in milli-Q water, mobile phase B was 12.5 mM KH₂PO₄ (pH 4.7) - ACN (80:20) and mobile phase C consisted of ACN - H₂O (50:50). The linear response range in human urine was 14-800 μM for allantoin and 1.25-50 μM for adenosine. The recoveries of allantoin, adenosine and the internal standard were greater than 93.8%. The intra-day accuracy ranged between 99.5 and 104.9% for allantoin and between 96.6 and 107.3% for adenosine, while the inter-day accuracy ranged respectively from 91.2 to 103.0% and from 94.5 to 107.8%. The intra-day precision range was from 0.8 to 6.2% RSD for allantoin and from 0.6 to 15.0% for adenosine. The inter-day precision ranged from 2.1-17.5% for allantoin and from 2.9-17.9% for adenosine. This method was successfully applied to analyze both compounds in urine samples of healthy volunteers. In conclusion, an accurate, precise and stable HPLC-UV method was developed and validated to quantify the endogenously present compounds allantoin and adenosine in human urine samples.