Quantification of adenosine mono-, di-and triphosphate from royal jelly using liquid chromatography-tandem mass spectrometry

Nucleotides are composed of nitrogen bases, ribose units and phosphate groups. Adenine (Ade), adenosine mono-phosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all play important roles in physiological metabolism. Royal jelly, a secretion produced by worker bees, contains a variety of natural ingredients and several studies have shown that royal jelly can serve as a source of nutrition for humans. In this study, a rapid and effective LC/ MS method coupled with pre-processing methods was developed and validated for the accurate quantification of Ade, AMP, ADP and ATP in royal jelly. To achieve the best extraction efficiency, two pretreatment methods, namely, solid-phase extraction (SPE) and dispersive solid-phase extraction (dSPE), were developed and investigated. Silica-based cya-nopropyl (CN) liquid chromatography was employed using pH programming with a quaternary mobile phase system for the analyses. The total LC/MS run time was less than 12 min with a constant flow rate of 0.25 mL/min. The linear range were 2.5e1000 ng/mL with a correlation coefficient r ¼ 0.9995. The limit of detection (LOD) of Ade, AMP, ADP and ATP was 1, 1, 2.5 and 5 ng/mL; the limit of quantitation (LOQ) was 2.5, 2.5, 5 and 10 ng/mL, respectively. Precision (RSD% <10.5%) and accuracy (recovery 81.3e118.4%) were satisfactory for both two pre-processing methods. Nucleotides were successfully quantified from 2-day and 3-day royal jelly samples, with concentrations within 6.2e2126.0 mg/kg.