摘要:
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。
Abstract:
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
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