HPLC法测定复合维生素B片中的微量叶酸、维生素B₁₂和生物素

目的：应用高效液相色谱（HPLC）技术，建立同时测定复合维生素B片中微量叶酸、维生素B₁₂和生物素含量的方法。方法：采用Alltima C₁₈色谱柱（250 mm×4.6 mm，5 μm），以50 mmol·L^-1磷酸二氢铵溶液（磷酸调至pH 3.0）-乙腈为流动相，梯度洗脱，流速1.0 mL·min^-1，检测波长200 nm，柱温30℃。结果：在本方法中，3个成分的线性关系良好，线性相关系数（r）为1.000；精密度良好，RSD小于1.9%；回收率良好，叶酸、维生素B₁₂和生物素的3个加样浓度的平均回收率（n=9）分别为100.6%、99.7%和99.7%；稳定性良好，3种成分在24 h内保持稳定，RSD小于1.0%。3批复合维生素B片中叶酸、维生素B₁₂和生物素的含量测定结果分别为480.9~493.2、47.31~48.38、50.80~52.63 μg·片^-1。结论：该方可作为复合维生素B片中微量叶酸、维生素B₁₂和生物素同时测定的方法。

Objective: To develop a high-performance liquid chromatography(HPLC)method for simultaneous determination of trace folic acid,vitamin B₁₂ and biotin in complex vitamin B tablets.Methods: An Alltima C₁₈ column(250 mm×4.6 mm,5 μm)was used for the separation,with gradient elution of 50 mmol·L^-1 ammonium dihydrogen phosphate(adjusted pH to 3.0 with phosphoric acid)and acetonitrile as the mobile phase at the flow rate of 1.0 mL·min^-1.The detection wavelength was set at 200 nm and the column temperature was 30℃.Results: The method demonstrated good linearity for folic acid,vitamin B₁₂ and biotin with all correlation coefficients(r)of 1.000.The precision was satisfactory with all RSDs less than 1.9%;the average spike recoveries(n=9)at three concentration levels for folic acid,vitamin B₁₂ and biotin were 100.6%,99.7% and 99.7%,respectively.The stabilities were satisfactory and the three compounds remained stable in 24 hours with all RSDs less than 1.0%.The contents of folic acid,vitamin B₁₂ and biotin in three batches of complex vitamin B tablets were 480.9-493.2,47.31-48.38,50.80-52.63 μg per tablet,respectively.Conclusion: The established method can be utilized for the simultaneous determination of trace folic acid,vitamin B₁₂ and biotin in complex vitamin B tablets.