高效液相色谱法同时鉴别3种维生素K3类饲料添加剂

Simultaneous identification of three vitamin K_3 feed additive by HPLC

农业部《饲料添加剂品种目录(2013)》收录了3种维生素K_3类饲料添加剂,即亚硫酸氢钠甲萘醌(MSB)、亚硫酸氢烟酰胺甲萘醌(MNB)和二甲基嘧啶醇亚硫酸甲萘醌(MPB),目前发布的标准中分别采用甲萘醌的沉淀反应、二氧化硫的气味鉴别及烟酰胺的薄层色谱法来鉴别,方法繁锁,专属性不强。文章建立了用高效液相色谱法鉴别3种维生素K_3类化合物,采用Waters Atlantis~?HILIC Sili-ca色谱柱(4.6 mm×250 mm,5μm);以乙腈/水(80.20)为流动相,230 nm为检测波长,流速1 ml/min,结果显示MSB为1个主峰、MNB和MPB均出现2个主峰,即亚硫酸氢甲萘醌、烟酰胺和二甲基嘧啶醇,3个色谱峰的分离度R≥1.9,MSB、MPB、MNB在该色谱条件下得到较好的分离。MPB中亚硫酸氢甲萘醌和二甲基嘧啶醇峰的相对保留时间均为0.19,MNB中亚硫酸氢甲萘醌和烟酰胺的相对保留时间为0.30~0.32。该色谱条件下,用保留时间或相对保留时间即能有效鉴别3种维生素K_3类化合物,方法简便、专属性强。

The catalogue of feed additive variet- ies released by the Ministry of Agriculture includ- ed three vitamin K3 derivative feed additives, which are menadione sodium bisulfite （MSB）, menadione nicotinamide bisulfite （MNB） and men- adione dimethylpyrimidinol bisulfite （MPB）. Thecurrent standard uses menadione precipitation reaction, identification of sulfur dioxide smell and nico- tinamide thin-layer chromatography （TLC） respectively for the measurements of those three com- pounds. The method is tedious and not selective. Here, we report a method to identify three vitamin K3 derivatives using high performance liquid chromatography （HPLC） with a Waters Atlantis HILIC sili- ca column （4.6 mmX250 mm, 5μm）. The acetonitrile/water （80 ： 20） solution was used as the mobile phase, the detection wavelength was 230 nm and the flow rate was 1 ml/min. The results showed that the spectrum of MSB had a main peak while the spectra of MNB and MPB had two main peaks. The resolution（R） of three chromatographic peaks of menadione bisulfite, nicotinamide, and dimethylpyrim- idinol was higher than 1.9, indicating that MSB, MPB, and MNB were well separated under the chro- matographic conditions. The relative retention time of the menadione bisulfite and dimethylpyrimidinol peaks in MPB was 0.19 and the relative retention time of niobium and nicotinamide was 0.30-0.32. Under the given chromatographic conditions, three tion time or relative retention time, and the method vitamin K3 derivatives can be identified by reten- is simple and selective.