采用LC-DAD-ESI-MS/MS法分离鉴定新品红标准试剂的组分

Characterization of new fuchsin reference reagent by LC-DAD-ESI-MS /MS

目的：对新品红标准试剂的组分进行分离、鉴定,以了解掌握该标准试剂的组分情况,为该批标准试剂的标化提供依据。方法：采用液相色谱-二极管阵列检测器-电喷雾离子化串联质谱法（LC-DAD-ESI-MS/MS）进行分离鉴定。色谱柱为Phenomenx Kinetex C18柱（100 mm!2.1 mm,2.6$m）,柱温35℃;流动相A为乙腈,流动相B为0.05 mol·L-1醋酸铵溶液,梯度洗脱（0～30 min,流动相A的比例从5%线性改变至45%）,流速为0.2 mL·min^-1;二极管阵列检测器的扫描范围为200～700 nm（提取波长：550 nm）,质谱的扫描范围为m/z 110～400,裂解电压为200 V。结果：新品红标准试剂（Sigma公司产品）含3种组分,百分比含量分别为71.5%、25.7%和2.8%。根据其色谱保留行为、UV提取图谱、质谱裂解信息,并结合相关的文献报道,将其分别归属为品红Ⅲ（magentaⅢ或new fuchsin（NF）,主组分）、品红Ⅱ（magentaⅡ）和品红Ⅰ（magentaⅠ）。薄层色谱结果验证了其组分情况。结论：采用串联色谱技术,利用目标化合物的色谱保留行为、紫外吸收光谱和质谱裂解规律等综合信息可为新品红标准试剂的定性鉴别提供技术依据。

Objective： To isolate and identify the components in new fuchsin reference reagent,so as to provide valuable information for its standardization. Methods： Reversed- phase liquid chromatography coupled with photo-diode array（ DAD） detection and electrospray ionization tandem mass spectrometry（ ESI- MS /MS） was adopted.The separation was performed on a Phenomenx Kinetex C18column（ 100 mm !2. 1 mm,2. 6 $m） at 35 ℃ in gradient elution mode（ 0 ～ 30 min,5%,45% A）,with the mixture of acetonitrile（ A） and 0. 05 mol·L- 1ammonium acetate solution（ B） as the mobile phase at a flow rate of 0. 2 mL·min- 1. Ultraviolet detections were performed from 200 nm to 700 nm（ extraction： 550 nm）. The MS spectra were scanned in the range of m /z 110- 400,with the Frag set at 200 V. Results： Three components were identified in the new fuchsin reference reagent（ Sigma）,and their normalized peak area percentages were 71. 5%,25. 7%,and 2. 8%,as determined by LC- UV. Based on their chromatographic behavior,UV spectra,fragmentation pathway and related literature,the components were deduced as magenta Ⅲ /new fuchsin（ NF）（ major component）,magenta Ⅱ and magenta Ⅰ,respectively. The components ratio was verified by thin- layer chromatography. Conclusion： The comprehensive information of the chromatographic retention behavior,UV spectra,ESI- MS /MS data and related literature provides an effective and reliable approach for qualitative identification of the new fuchsin reference reagent.