高效液相色谱分析真菌液态发酵产物中的麦角硫因

利用高效液相色谱(HPLC)建立了定量检测发酵产物中麦角硫因的方法。该方法采用Zorbax SB-Aq色谱柱,检测条件为流动相A(纯水:甲醇体积比99:1,使用20%的乙酸和50%的氨水调节流动相的pH至5.0)和B(甲醇),进样量5 μL,柱温30 ℃,流速0.7 mL/min,检测波长为257 nm。建立的梯度洗脱的模式进一步提高了方法的稳定性、延长了色谱柱的使用时间。经高效液相色谱(HPLC)和高效液相色谱质谱联用仪(HPLC-MS)验证,该方法特异性良好,检出限和定量限分别为0.045和0.900 μg/L。在2.5~1000 mg/L的浓度范围内,线性关系良好,相关系数R2=0.9991。精密性、日内稳定性和日间稳定性的RSD分别为0.33%、1.89%和0.026%。平均加标回收率在92.17%和93.92%之间。该方法简便、迅速、稳定、准确,可以用于麦角硫因的研发以及应用的定量分析。

Determination of Ergothioneine Content in Liquid Fermentation Broth of Fungi by HPLC

An efficient and stable method was developed for quantitative analysis of ergothioneine (EGT) by high performance liquid chromatography (HPLC). The method was carried out on Zorbax SB-Aq Column with elution A (the volume ratio of urtrapure water vs methanol was 99:1, adjusted to pH5 by 20% of acetic acid and 50% of ammomnia and elution B (methanol) as mobile phase at 0.7 mL/min. An UV-VIS detector with a wavelength of 257 nm was employed. The injection volumn was 5 μL, with the column temperature being 30℃. The gradient elution program makes the method more stable and prolong utility time of the chromatographic column. Based on the results of HPLC and HPLC-MS, we detected the specificity of this method was good. The limit of detection (LOD) and limit of quantification (LOQ) were 0.045 and 0.900 μg/L. Good linearity (correlation coefficient R2=0.9991) could be achieved for EGT quantification at the range of 2.5 to 1000 mg/L.The RSD of precision, stability in 12 h and stability in 14 d were 0.33%, 1.89%, and 0.026% respectively. Meanwhile, the average recoveries of EGT were within 92.17%~93.92%. The method was simple, rapid, stable and accurate, which could be used in the research and application of EGT biosynthesis