固相萃取-超高效液相色谱-串联质谱法同时检测食品中的烟酰胺单核苷酸α、β异构体和烟酰胺腺嘌呤二核苷酸

Simultaneous determination of nicotinamide mononucleotide α, β isomers and nicotinamide adenine dinucleotide in foods by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

中文关键词:  α-烟酰胺单核苷酸  β-烟酰胺单核苷酸  烟酰胺腺嘌呤二核苷酸  超高效液相色谱-串联质谱  异构体拆分  固相萃取

英文关键词:α-nicotinamide mononucleotide  β-nicotinamide mononucleotide  nicotinamide adenine dinucleotide  ultra performance liquid chromatography-tandem mass spectrometry  separation of isomers  solid phase extraction

中文摘要:

      目的 建立固相萃取-超高效液相色谱-串联质谱法同时测定食品中烟酰胺单核苷酸(nicotinamide mononucleotide, NMN) α、β异构体和烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD+)含量的方法。方法 样品经5%甲醇水溶液超声提取, HLB固相萃取柱和混合型阴离子交换固相萃取柱分别净化, 采用Waters ACQUITY UPLC?HSS T色谱柱进行分离, 流动相为5 mmol/L乙酸铵含0.1% (V/V)甲酸水-甲醇, 梯度洗脱; 流速0.2 mL/min; 柱温30℃; 多反应监测(multiple reaction monitoring, MRM)模式定量分析, 定量离子对NMN为m/z 335.0/123.0、NAD+为m/z 662.0/540.0。结果 在最优条件下, α-NMN和β-NMN两种异构体的分离度为5.56。该方法中α-NMN、β-NMN和NAD+均在10~1000 ng/mL范围内线性良好, 相关系数分别为0.9999, 0.9998和0.9995。α-NMN、β-NMN和NAD+的检出限分别为4.0、2.0和1.0 ng/mL, 定量限分别为10.0、5.0和3.0 ng/mL, 回收率为93.8%~103.8%, 相对标准偏差为2.1%~6.5%。结论 该方法灵敏度高、选择性好、结果准确可靠, 可同时快速检测各种基质食品中NMN α、β异构体和NAD+的含量。

英文摘要:

      Objective To establish a method for simultaneous determination of nicotinamide mononucleotide (NMN) α, β isomers and nicotinamide adenine dinucleotide (NAD+) in foods by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry. Methods The samples were extracted by 5% methanol aqueous solution, and purified by HLB solid-phase extraction column and mixed mode anion exchange solid phase extraction column. The Waters ACQUITY UPLC?HSS T3 chromatographic column was used for the chromatographic separation of target analytes at 30℃. The gradient elution method was used and the flow rate was 0.2 mL/min. The mobile phase composed of 5 mmol/L ammonium acetate aqueous solution containing 0.1% (V/V) formic acid and methanol. Quantitative determination was performed at the multi reaction monitoring mode of mass spectrometer. The quantitative ion pairs of NMN and NAD+ were m/z 335.0/123.0 and m/z 662.0/540.0, respectively. Results Under the above conditions, the separation degree of α-NMN and β-NMN isomers was 5.56. The method showed a good linear relationship between peak area and concentration over the range from 10 ng/mL to 1000 ng/mL and the correlation coefficients of α-NMN, β-NMN and NAD+ were 0.9999, 0.9998 and 0.9995, respectively. The limits of detection of α-NMN, β-NMN and NAD+ were 4.0, 2.0 and 1.0 ng/mL and the limits of quantitation were 10.0, 5.0 and 3.0 ng/mL. The recoveries of α-NMN, β-NMN and NAD+ ranged from 93.8%?103.8%, and the relative standard deviations of precision were 2.1%?6.5%. Conclusion This method has the advantages of high sensitivity, good selectivity, accurate and reliable results, which is suitable for the simultaneous quantitative analysis of NMN α, β isomers and NAD+ in various matrix foods.