Observation and quantification of self-associated adenosine extracted from royal jelly products purchased in USA by HPLC

The concentration of adenosine in pure royal jelly creams and dietary supplements purchased in the United States was determined by reversed phase high performance liquid chromatography (HPLC). Preliminary studies revealed the existence of several forms of adenosine via self-association and base-pairing in solutions. Therefore, proper optimisation of pH and compositions of extraction solvents and mobile phases was critical for successful separation and quantification. In this work, adenosine in samples was extracted by sonication in a mixture consisting of 5% ethanol and 95% deionized water and separated using a Zorbax Eclipse XDB-C18 column (150 × 4.6 mm) and a mobile phase of 93% deionized water and 7% acetonitrile at 25 °C. The flow rate of a mobile phase was set to 1.0 ml/min and the UV detection was performed at 260 nm. The average recovery rate of adenosine was 92.8-99.2% with the relative standard deviation (RSD) of 0.1-1.3% over concentrations ranging from 5 to 160 μg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were found to be ∼0.01 and ∼0.05 μg/ml, respectively. Quantification was carried out using calibration curves constructed by both internal standard (ISTD) and external standard (ESTD) methods. Our results show that the concentration of adenosine lies between ∼27 and 50 μg/g for pure royal jelly creams and between ∼2 and ∼173 μg/g for royal jelly supplements.