Liao Wan-Rou, Huang Jen-Pang, Chen Sung-Fang
Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan.
MSonline Scientific Co., Ltd., Taipei, Taiwan.
J Food Drug Anal. 2020 Sep 15;28(3):365-374. doi: 10.38212/2224-6614.1007.
Nucleotides are composed of nitrogen bases, ribose units and phosphate groups. Adenine (Ade), adenosine monophosphate (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 cyanopropyl (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 2.5-1000 ng/mL with a correlation coefficient r = 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 81.3-118.4%) were satisfactory for both two pre-processing methods. Nucleotides were successfully quantified from 2-day and 3-day royal jelly samples, with concentrations within 6.2-2126.0 mg/kg.
核苷酸由氮碱基、核糖单元和磷酸基团组成。腺嘌呤（Ade）、一磷酸腺苷（AMP）、二磷酸腺苷（ADP）和三磷酸腺苷（ATP）在生理代谢中均发挥着重要作用。蜂王浆是工蜂分泌的一种物质，含有多种天然成分，多项研究表明蜂王浆可作为人类的营养来源。在本研究中，开发并验证了一种结合预处理方法的快速有效的液相色谱/质谱法，用于准确定量蜂王浆中的Ade、AMP、ADP和ATP。为实现最佳提取效率，开发并研究了两种预处理方法，即固相萃取（SPE）和分散固相萃取（dSPE）。采用基于硅胶的氰丙基（CN）液相色谱，使用pH梯度洗脱和四元流动相系统进行分析。液相色谱/质谱总运行时间小于12分钟，恒定流速为0.25 mL/min。线性范围为2.5 - 1000 ng/mL，相关系数r = 0.9995。Ade、AMP、ADP和ATP的检测限分别为1、1、2.5和5 ng/mL；定量限分别为2.5、2.5、5和10 ng/mL。两种预处理方法的精密度（RSD% <10.5%）和准确度（回收率81.3 - 118.4%）均令人满意。成功定量了2日龄和3日龄蜂王浆样品中的核苷酸，浓度在6.2 - 2126.0 mg/kg范围内。
