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目的:建立发酵液中咪唑立宾的鉴别及含量测定方法。方法:采用液质联用(HPLC-MS)技术对咪唑立宾进行定性鉴别,并以高效液相色谱法测定其含量。色谱柱:SinoChrom ODS-BP(250mm×4.6mm,5μm),以乙腈-0.1%甲酸(2∶98)为流动相,流速:0.8mL·min-1,检测波长:278nm,柱温:25℃;采用ESI-MS总离子流跟踪分析HPLC的洗脱液,并用低能量的CID-MS-MS(碰撞诱导裂解方式)进一步确定目标离子峰为咪唑立宾分子离子峰。结果:鉴定了发酵液存在咪唑立宾,含量在0.048～1.924mg·mL-1范围内线性关系良好(r=0.9991,n=6),平均回收率为100.3%(n=9)。结论:本方法简便、准确,重复性好,为筛选发酵合成咪唑立宾的高产菌株提供了可靠依据。
Objective: To establish the identification and determination of midazolam in fermentation broth. Methods: Imidazibine was qualitatively identified by liquid chromatography-mass spectrometry (HPLC-MS) and its content was determined by high performance liquid chromatography. Column: SinoChrom ODS-BP (250 mm × 4.6 mm, 5 μm) with acetonitrile-0.1% formic acid (2:98) as mobile phase at a flow rate of 0.8 mL · min -1 and a detection wavelength of 278 nm. The ESI-MS total ion current was used to trace and analyze HPLC eluate, and the target ion peak was determined to be the midazolam molecular ion peak with low energy CID-MS-MS (collision-induced lysis method). Results: The results showed that there was a good linear relationship (r = 0.9991, n = 6) in the range of 0.048-1.924 mg · mL-1 with an average recovery of 100.3% (n = 9). Conclusion: The method is simple, accurate and reproducible, which provides a reliable basis for the screening of high-yielding strains for the synthesis of mizoribine.