高效液相色谱法同时测定烟酰胺单核苷酸中5种有关物质

Simultaneous determination of 5 kinds of related substances in nicotinamide mononucleotide by high performance liquid chromatography

中文关键词:  烟酰胺单核苷酸  烟酰胺氧化物  三磷酸腺苷  二磷酸腺苷  一磷酸腺苷  烟酰胺  HPLC

英文关键词:nicotinamide mononucleotide  nicotinamide oxide  adenosine triphosphate  adenosine diphosphate  adenosine monophosphate  nicotinamide  high performance liquid chromatography

中文摘要:

      目的 构建同时检测烟酰胺单核苷酸(nicotinamide mononucleotide, NMN)中5种有关物质[烟酰胺氧化物、三磷酸腺苷(adenosine triphosphate, ATP)、二磷酸腺苷(adenosine diphosphate, ADP)、一磷酸腺苷(adenosine monophosphate, AMP)及烟酰胺]含量的高效液相色谱法。方法 采用C18色谱柱(250 mm×4.6 mm, 5 μm), 以50 mmol/L磷酸二氢钾缓冲盐溶液(用磷酸调节pH为4.5)-甲醇为流动相进行梯度洗脱, 流速为1 mL/min, 进样量为20 μL, 检测波长210 nm, 柱温20℃, 试样盘控温: 5℃, 并对方法的专属性、灵敏性、准确性、重复性、耐用性及适用性进行验证。结果 烟酰胺氧化物、ATP、ADP、AMP及烟酰胺分别在质量浓度0.4060~16.2402、0.4110~16.4403、0.4150~16.6010、0.4163~16.6507、0.4028~16.1206 μg/mL范围内, 质量浓度与峰面积呈现良好的线性关系, 线性系数r2值均大于0.998; 加标回收率范围分别为98.5%~102.6%、95.6%~104.1%、98.9%~104.6%、100.0%~102.8%、100.6%~101.3%; 3批样品中5种杂质含量检测结果分别为0.18%、0.20%、0.16%、0.14%、0.26%; 5种杂质的检出限范围为0.04028~0.04163 μg/mL, 定量限范围为0.4028~0.4163 μg/mL, 灵敏度高; 专属性、重复性和耐用性均良好。结论 该方法简便易行、适用性广, 适用于NMN中5种有关物质的检测, 为提升其质量标准奠定基础, 为食品安全提供保障。

英文摘要:

      Objective To construct a method for simultaneous detection of 5 kinds of related substances [nicotinamide oxide, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and nicotinamide] in nicotinamide mononucleotide (NMN) by high performance liquid chromatography. Method A C18 column (250 mm×4.6 mm, 5 μm) was used for gradient elution with 50 mmol/L potassium dihydrogen phosphate buffered salt solution (pH 4.5 adjusted by phosphoric acid) and methanol as mobile phase. The flow rate was 1 mL/min, the sample size was 20 μL, the detection wavelength was 210 nm, the column temperature was 20℃, and the sample disk temperature was 5℃. The specificity, sensitivity, accuracy, repeatability, durability and applicability of the method were verified. Results Nicotinamide oxide, ATP, ADP, AMP and nicotinamide showed a good linear relationship with peak area in the mass concentration range of 0.4060?16.2402, 0.4110?16.4403, 0.4150?16.6010, 0.4163?16.6507, 0.4028?16.1206 μg/mL. The linear coefficients r2>0.998. Standard addition recoveries were 98.5%?102.6%, 95.6%?104.1%, 98.9%?104.6%, 100.0%?102.8% and 100.6%?101.3%, respectively. The content of 5 kinds of impurities in 3 batches of samples were 0.18%, 0.20%, 0.16%, 0.14%, 0.26%, respectively. The limits of detection for the 5 kinds of impurities ranged from 0.04028 to 0.04163 μg/mL, and the limits of quantification ranged from 0.4028 to 0.4163 μg/mL, showing high sensitivity, good specificity, repeatability and durability. Conclusion The method is simple, convenient and widely applicable for the detection of 5 kinds of related substances in NMN, which lays a foundation for improving its quality standards and provides a guarantee for food safety.