建立了超高效液相色谱-串联质谱快速测定蜂王幼虫中烟酰胺单核苷酸(nicotinamide mononucleotide,NMN)的方法。样品经乙醇和水提取后,采用Waters X Select HSS T3(2.1 mm×100 mm, 2.5 μm)色谱柱进行分离,串联质谱多反应监测模式定量分析。该方法中NMN在50~1 000 ng/mL线性关系良好(R2=0.999 7),检出限和定量限分别为11.2 μg/kg和37.4 μg/kg。在添加水平为20、40、80 μg/g时,样品的回收率为109.9%~111.2%,相对标准偏差为0.87%~3.31%。应用方法测定来自不同蜂场的蜂王幼虫样品中NMN的含量,11个新鲜幼虫样品均检出NMN,其含量为37.0~65.8 μg/g,1个蜂王幼虫冻干粉中NMN的含量为78.0 μg/g。稳定性实验结果显示,蜂王幼虫中NMN极易降解,在常温和4 ℃条件贮存7 d的降解率分别为96.5%和88.5%,低温或加工成冻干粉可有效抑制NMN的降解。该方法适用于蜂王幼虫中NMN的检测,实际样品分析结果显示,NMN可作为蜂王幼虫及其产品品质评价的潜在指标。
A simple and fast method for the determination of nicotinamide mononucleotide (NMN) in queen bee larva by ultra-high performance liquid chromatographic tandem mass spectrometry (UHPLC-MS/MS) was developed in this study. The NMN was separated on Waters X Select HSS T3 column (2.1 mm×100 mm,2.5 μm) and quantified on multiple reaction monitor (MRM). The method was validated using spiked samples with NMN standard of 20, 40 and 80 μg/g. Recoveries were 109.9%-111.2% with relative standard deviation (RSD) 0.87%-3.31%. The calibration curve was fitted with the correlation coefficient R2 = 0.999 7 in the range of 50-1 000 ng/mL. The limits of detection (LOD) and the limits of quantitation (LOQ) were 11.2 and 37.4 μg/kg, respectively. The proposed UHPLC-MS/MS method was applied to the analysis of queen bee larva samples. The results showed that the NMN in 11 fresh queen bee larva samples was from 37.0 to 65.8 μg/g, especially in 1 freeze-dried powder sample which was 78.0 μg/g. The NMN in queen bee larva was easily broken down, and the degradation rates at room temperature and 4 ℃ for 7 d were 96.5% and 88.5%, respectively. Low temperature storage or lyophilization could prevent the degradation of NMN in queen bee larva. As a functional and nutritional component, NMN can be used as a potential indicator for the quality evaluation of queen bee larva and their products.
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