Division of Analytical and Measuring Instruments, Shimadzu Corporation, 1 Kuwabaracho Nishinokyo Nakagyo-ku, Kyoto, 604-8511, Japan; Department of Systems Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, 812-8582, Japan.
Department of Systems Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, 812-8582, Japan; Division of Metabolomics/Mass Spectrometry Center, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, 812-8582, Japan.
Anal Chim Acta. 2023 Mar 15;1246:340863. doi: 10.1016/j.aca.2023.340863. Epub 2023 Jan 20.
Supercritical fluid chromatography (SFC) is often coupled with electrospray ionization mass spectrometry (ESI-MS) for analyte detection because of its detection capability to a wide range of chemical properties. However, MS sensitivity is highly dependent on the chromatographic conditions, so that it is important to understand the ionization mechanism to determine the optimal chromatographic conditions. The ionization mechanism in SFC/ESI-MS is different to that of liquid chromatography because of the use of CO as a mobile phase. Some studies have suggested that alkoxycarbonic acids are formed in the mixture of CO and the alcohol modifier, and these species contribute to ionization in CO-assisted SFC/ESI-MS. Therefore, in this study, we investigated CO-assisted ESI to test this hypothesis, and we confirmed that methoxylcarbonic acid is generated in CO/methanol mixtures and contributed to ion generation and detection because it acts as a proton donor in positive-ion mode. However, methoxylcarbonic acid interfered with ionization in negative-ion mode. Addition of ammonium acetate, which is often added to the modifier for negative ion detection in SFC/MS analysis, did not contribute to the recovery of MS sensitivity, although it tended to suppress the formation of metoxylcarbonic acid. This is likely due to ion suppression and neutralization of the negative sites of the analytes by anions or cations derived from ammonium acetate in the negative ion mode. Thus, additive-free methanol/CO was the most suitable mobile phase for obtaining high sensitivity in SFC/MS. To demonstrate the practicality of these findings, we tested our optimal mobile phase selection for pesticide analysis. In addition, we tested the addition of 0, 1, and 5 mM ammonium formate to the modifier and make-up solvent, and found that the addition of 1 mM ammonium formate gave the best results in pesticides analysis. In SFC/MS, salt is often added to improve separation or prevent desorption, but our findings suggest that the concentration of salt must be kept as low as possible to achieve highly sensitive MS detection. The results of this study reveal the best selection of the optimal conditions for the modifier and make-up solvent for CO-assisted SFC/MS analysis and will be useful for the method development in SFC/MS.
超临界流体色谱（SFC）通常与电喷雾电离质谱（ESI-MS）联用进行分析物检测，因为它能够检测广泛的化学性质。然而，MS 灵敏度高度依赖于色谱条件，因此了解电离机制以确定最佳色谱条件非常重要。由于 SFC/ESI-MS 使用 CO 作为流动相，因此其电离机制与液相色谱不同。一些研究表明，在 CO 与醇改性剂的混合物中形成烷氧基碳酸，这些物质有助于 CO 辅助 SFC/ESI-MS 中的电离。因此，在本研究中，我们研究了 CO 辅助 ESI 以验证该假设，并证实甲醇/CO 混合物中生成了甲氧基碳酸，并通过在正离子模式下作为质子供体促进离子生成和检测。然而，甲氧基碳酸在负离子模式下会干扰电离。尽管添加乙酸铵通常有助于 SFC/MS 分析中负离子检测的改性剂，但其并未有助于恢复 MS 灵敏度，尽管它倾向于抑制甲氧基碳酸的形成。这可能是由于在负离子模式下，来自乙酸铵的阴离子或阳离子会抑制分析物的负电荷部位的离子抑制和中和作用。因此，无添加剂甲醇/CO 是获得 SFC/MS 高灵敏度的最适流动相。为了证明这些发现的实用性，我们测试了用于农药分析的最佳流动相选择。此外，我们测试了在改性剂和补加溶剂中添加 0、1 和 5 mM 甲酸铵，并发现添加 1 mM 甲酸铵在农药分析中效果最佳。在 SFC/MS 中，通常添加盐以改善分离或防止解吸，但我们的研究结果表明，为了实现高度敏感的 MS 检测，盐的浓度必须尽可能低。本研究的结果揭示了 CO 辅助 SFC/MS 分析中改性剂和补加溶剂的最佳条件选择，将有助于 SFC/MS 方法的开发。
