核苷酸化合物由含氮鹼基、核糖和磷酸基團所組成，以腺嘌呤(Adenine, Ade)所組成的三磷酸腺苷(Adenosine triphosphate, ATP)、二磷酸腺苷(Adenosine diphosphate, ADP)、單磷酸腺苷(Adenosine monophosphate, AMP)，在生理機能中扮演重要的角色。蜂王乳(Royal Jelly)為蜂巢中工蜂上顎腺(Mandibular gland)以及咽下腺(Hypopharyngeal gland)的分泌物，富含多種純天然成分，更有研究指出蜂王乳可提供人體所需之營養。本實驗以此為出發點，開發一種可靠且準確的方法，檢測蜂王乳中的核苷酸含量，並探討二日齡和三日齡蜂王乳的含量差異。由於蜂王乳為複雜的基質樣品，為了準確定量蜂王乳中的Ade、AMP、ADP和ATP含量，本實驗開發快速且有效的固相萃取法(Solid-Phase Extraction, SPE)以及分散式固相萃取法(Dispersive Solid-Phase Extraction, dSPE)兩種前處理方法，並優化參數以達到最佳的萃取效率。本實驗的分析方法，使用cyanopropryl (-CN)管柱搭配四元流動幫浦，藉由改變層析環境之pH值，使分析物在12分鐘內達到良好的分離，再結合串聯式質譜進行檢測。本實驗所開發的分析方法有良好的精確度(RSD% < 10.5%)與準確度(回收率81.3 - 118.4%)，線性範圍在2.5 - 1000 ng/mL之間，相關係數皆在r = 0.9995以上，偵測極限(LOD)和定量極限(LOQ)最低分別可達1.0 ng / mL和2.5 ng / mL。應用分散式固相萃取法搭配液相層析串聯式質譜法，成功定量蜂王乳中的核苷酸含量(6.2 - 2126.0 mg/kg)，二日齡與三日齡蜂王乳的核苷酸含量並無顯著差異。

Nucleotides are composed of nitrogenous base, ribose and phosphate groups. Adenine (Ade), adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all play important roles in physiological metabolism. Royal jelly is the worker bee’s secretions, it is rich in a variety of pure natural ingredients, several studies have shown that royal jelly can supply abundant nutrition to the human. In this study, a rapid and effective LC/MS method coupled with pre-processing methods were developed and validated for the accurate quantification of Ade, AMP, ADP and ATP contents in royal jelly. To achieve the best extraction efficiency, two pretreatment methods, solid-phase extraction (SPE) and dispersive solid-phase extraction (dSPE), were developed and investigated. The silica-based cyanopropyl (CN) liquid chromatography was employed using pH programming with quaternary mobile phase system. The total LC/MS run time was within 12 min with a constant flow rate of 0.25 mL/min. The linear range were 2.5-1000 ng/mL with correlation coefficient r = 0.9995. The limit of detection (LOD) and limit of quantitation (LOQ) of 1.0 ng/mL and 2.5 ng/mL were achieved, respectively. Precision (RSD% <10.5%) and accuracy (recovery 81.3-118.4%) were both satisfactory in two pre-processing methods. Eventually, nucleotides were successfully quantified from the 2-day and 3-day royal jelly samples, with concentrations within 6.2 to 2126.0 mg/kg.

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