基于可靠HPLC定量方法的β-烟酰胺单核苷酸降解动力学研究

Degradation kinetics ofβ-nicotinamide mononucleotide based on reliable HPLC quantitative method

为探讨β-烟酰胺单核苷酸(β-nicotinamide mononucleotide,NMN)的稳定性特征,为NMN的生产制备和相关产品研发提供数据支撑,该研究建立简单基质下NMN的HPLC含量测定方法,探讨NMN在不同化学、物理学和生物学条件下的降解行为、降解产物和降解动力学特征。HPLC方法的色谱柱为Welch Xtimate AQ-C_(18)(4.6 mm×250 mm,5μm),检测波长266 nm,柱温30℃,流速1.0 mL·min^(-1),进样量5μL,流动相甲醇(A)-10 mmol·L^(-1)甲酸铵水溶液(B),梯度洗脱(0~6.7 min,0~4%A;6.7~13 min,4%~18%A;13~14.2 min,18%A;14.2~15 min,18%~0 A;15~22 min,0 A)。该方法下NMN与潜在杂质和降解产物分离度良好,线性范围广,分析时间短,耐用性好,准确度高,平均加样回收率为98.71%(RSD 1.2%),仪器精密度良好(RSD 0.26%),在考察范围内的线性良好(R^(2)≥0.9999),可以应用于简单基质下NMN的含量测定。NMN水溶液降解过程符合表观一级反应,降解速率主要受高温和pH影响,在低温、中性或弱酸弱碱环境较为稳定;强酸、强碱可加速其降解,降解速率受胃蛋白酶和胰蛋白酶影响小。在室温水溶液中符合动力学方程lg C_(t)=0.0057t+4.8172,t_(0.9)和t_(1/2)分别为95.58、860.26 h。结果提示:pH、温度是NMN在水溶液中稳定性的主要影响因素,低温、防潮和弱酸性环境更有利于NMN及其制品的贮存、应用。

To explore the stability characteristics ofβ-nicotinamide mononucleotide(NMN)and provide data support for NMN production,preparation,and related product development,this study established a simple HPLC content determination method for NMN in simple substrate and investigated the degradation behavior,degradation products,and degradation kinetics of NMN under various chemical,physical,and biological conditions.The HPLC method employed a Welch Xtimate AQ-C_(18) column(4.6 mm×250 mm,5μm),a detection wavelength of 266 nm,a column temperature of 30℃,a flow rate of 1.0 mL·min^(-1),an injection volume of 5μL,and a mobile phase consisting of methanol(A)and a 10 mmol·L^(-1) ammonium formate aqueous solution(B)with a gradient elution(0-6.7 min,0-4%A;6.7-13 min,4%-18%A;13-14.2 min,18%A;14.2-15 min,18%-0 A;15-22 min,0 A).This method provided good separation between NMN and potential impurities and degradation products,and had a wide linear range,short analysis time,good durability,high accuracy,an average sample recovery rate of 98.71%,and an RSD of 1.2%.The instrument precision had an RSD of 0.26%,and the linearity within the examined range was excellent(R^(2)≥0.9999).This method can be applied for NMN content determination in simple substrate.The degradation process of NMN in aqueous solution followed apparent first-order kinetics,with the degradation rate primarily influenced by high temperature and pH.NMN was more stable in lowtemperature,neutral,or weakly acidic/alkaline environments.Strong acids or strong alkalis could accelerate its degradation,and its degradation rate was less affected by pepsin and trypsin.In an aqueous solution at room temperature,it followed the kinetic equation lg C_(t)=0.0057t+4.8172,with t_(0.9) and t_(1/2) values of 95.58,860.26 h,respectively.The results suggest that pH and temperature are the main factors affecting the stability of NMN in aqueous solution,and low temperature,moisture protection,and a weakly acidic environment are more conducive to the storage and application of NMN and its products.