Online Cleanup of Accelerated Solvent Extractions for Determination of Adenosine 5′-Triphosphate (ATP), Adenosine 5′-Diphosphate (ADP), and Adenosine 5′-Monophosphate (AMP) in Royal Jelly Using High-Performance Liquid Chromatography

Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5-diphosphate (ADP), and adenosine 5-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in <25 min using a Waters XBridge Shield RP18 column with 0.05 mol L-1 NH4H2PO4 (pH 5.70) and acetonitrile as the mobile phase. Detection was performed at 257 nm. The method was sensitive (LOD <= 0.32 mg L-1), repeatable (RSD <= 4.5%), accurate (recovery rates of 87.6-94.2% with RSD <= 5.4), and precise (intraday RSD <= 8.5%; interday RSD <= 3.4%). The ASE extraction procedures developed here were compared with the classical adenosine phosphate extraction procedures (perchloric acid). The results indicate that the two techniques are similar in terms of recovery and reproducibility, but when other factors such as extraction time,. environmental protection, and worker's health are considered, ASE is preferable to the classical extraction method. With this ASE-HPLC method, a minisurvey of ATP, ADP, and AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels.