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目的:建立一种反相高效液相色谱法测定核黄素的含量及其有关物质。方法:采用Agilent Zarbox C18(250 mm×4.6mm,5μm)色谱柱,以甲醇(A)-0.1%醋酸(B)为流动相梯度洗脱(0～15 min,30%A;15～25 min,30%A→60%A;25～35min,60%A),流速1.0 mL.min-1,检测波长267 nm,柱温为室温,进样量20μL,外标法定量。结果:在该色谱条件下,核黄素及其有关物质能够得到很好的分离,分离度大于1.5,理论塔板数按核黄素计大于5000;核黄素浓度在1～30μg.mL-1范围内与峰面积呈良好的线性关系(r=0.9999);定量限和检测限分别为1.08 ng(S/N=10)和0.36 ng(S/N=3)。结论:该高效液相色谱法准确、灵敏、可靠,专属性好,可用于核黄素的含量及其有关物质的测定。
Objective: To establish a reversed-phase high performance liquid chromatography for the determination of riboflavin content and related substances. METHODS: The mobile phase was eluted with a mobile phase of methanol (A) - 0.1% acetic acid (B) with an Agilent Zarbox C18 column (250 mm × 4.6 mm, 5 μm) , 30% A → 60% A; 25-35min, 60% A) at a flow rate of 1.0 mL.min-1. The detection wavelength was 267 nm. The column temperature was at room temperature and the injection volume was 20 μL. Results: Under the chromatographic conditions, riboflavin and its related substances can be well separated, the resolution is more than 1.5, the number of theoretical plates is more than 5000 according to riboflavin, the concentration of riboflavin is between 1 ~ 30μg.mL- 1 (r = 0.9999). The limits of quantitation and detection limit were 1.08 ng (S / N = 10) and 0.36 ng (S / N = 3), respectively. Conclusion: The HPLC method is accurate, sensitive, reliable and specific. It can be used to determine the content of riboflavin and its related substances.