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 mononucleotideα,βisomers and nicotinamide adenine dinucleotide in foods by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

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NMNα、β异构体和NAD+的含量。

Objective To establish a method for simultaneous determination of nicotinamide mononucleotide(NMN)α,βisomers 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 ofα-NMN andβ-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 ofα-NMN,β-NMN and NAD+were 0.9999,0.9998 and 0.9995,respectively.The limits of detection ofα-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 ofα-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 NMNα,βisomers and NAD+in various matrix foods.