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.

Andries A ，De Rechter S ，Janssens P ，Mekahli D ，Van Schepdael A ... -  《-》

Improved HPLC method for the simultaneous determination of allantoin, uric acid and creatinine in cattle urine.

George SK ，Dipu MT ，Mehra UR ，Singh P ，Verma AK ，Ramgaokar JS ... -  《JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES》

Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection.

Czauderna M ，Kowalczyk J 《-》

Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.

The development of a simple HILIC-LC-MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine, and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1 × 150 mm, 3.5 μm) was achieved under gradient elution with two mobile phases: 0.1% formic acid-ACN (5:95) and 0.1% formic acid-ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency and the U.S. Food and Drug Administration guidelines. The lower LOQ was 50 nM, 5 nM, 20 nM, and 2 nM for allantoin, inosine, hypoxanthine, and adenosine, respectively. The recovery was repeatable and stable. The intraday precision ranged from 1.6% to 6.5%, while the interday precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix-matched calibration curve each day to overcome this issue. Since the quality control samples' stability did not always comply with the guidelines, the samples need to be analyzed soon after collection.

Andries A ，Feyaerts A ，Mekahli D ，Van Schepdael A ... -  《-》

Quantitative determination of an extremely polar compound allantoin in human urine by LC-MS/MS based on the separation on a polymeric amino column.

Berthemy A ，Newton J ，Wu D ，Buhrman D ... -  《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》