Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD.
Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD.
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DOI：
10.1016/j.jchromb.2022.123417
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发表时间：
2022-10-15
影响因子：
3
通讯作者：
Rawle, Robert J.
中科院分区：
文献类型：
--
作者：
Graceffa, Oliva;Kim, Eunice;Broweleit, Rachel;Rawle, Robert J.
关键词：
蒸发光散射检测器 (ELSD) 通常与高效液相色谱 (HPLC) 一起使用来分离和定量脂质，而脂质通常不易通过更传统的方法（例如紫外可见检测器）检测到。在许多分析脂质的 HPLC-ELSD 方法中，流动相中含有挥发性缓冲液，以控制 pH 值并促进脂质种类之间的分离。在这里，我们报告了缓冲液选择在脂质 HPLC-ELSD 分析中可能产生的意想不到的影响 - 缓冲液的特性和浓度可以显着影响所得的 ELSD 峰面积。为了分离这种影响，我们使用简单的等度甲醇流动相，辅以不同浓度的常用缓冲液进行 ELSD 分析，并量化七种不同脂质（POPC、DOPE、胆固醇、鞘磷脂、DOTAP、DOPS 和乳糖神经酰胺）对峰宽、峰形和峰面积的影响。我们发现，即使在峰宽和形状不变的情况下，不同脂质的 ELSD 峰面积也会根据流动相缓冲液成分发生显着变化。对于具有 UV 活性的分析物子集，我们还证明了 UV 定量的峰面积在不同缓冲条件下保持不变，表明这种效应是 ELSD 定量所特有的。我们推测这种 ELSD 缓冲效应可能是多种物理现象的结果，包括：气溶胶液滴尺寸的改变、流动相蒸发过程中分析物聚类的改变以及质量放大或离子对效应，所有这些都可能导致观察到的峰面积的差异。预计这种效应是分子特异性的，与我们的数据一致。我们预计这份报告将对研究人员设计和实施 HPLC-ELSD 方法（尤其是脂质）有用。
Evaporative light scattering detectors (ELSD) are commonly used with high-performance liquid chromatography (HPLC) to separate and quantify lipids, which are typically not easily detectable by more conventional methods such as UV-visible detectors. In many HPLC-ELSD methods to analyze lipids, a volatile buffer is included in the mobile phase to control the pH and facilitate separation between lipid species. Here, we report an unintended effect that buffer choice can have in HPLC-ELSD analysis of lipids – the identity and concentration of the buffer can substantially influence the resulting ELSD peak areas. To isolate this effect, we use a simple isocratic methanol mobile phase supplemented with different concentrations of commonly used buffers for ELSD analysis, and quantify the effect on peak width, peak shape, and peak area for seven different lipids (POPC, DOPE, cholesterol, sphingomyelin, DOTAP, DOPS, and lactose ceramide). We find that the ELSD peak areas for different lipids can change substantially depending on the mobile phase buffer composition, even in cases where the peak width and shape are unchanged. For a subset of analytes which are UV-active, we also demonstrate that the peak area quantified by UV remains unchanged under different buffer conditions, indicating that this effect is particular to ELSD quantification. We speculate that this ELSD-buffer effect may be the result of a variety of physical phenomenon, including: modification of aerosol droplet size, alteration of clustering of analytes during evaporation of the mobile phase, and mass-amplification or ion-pair effects, all of which could lead to differences in observed peak areas. Such effects would be expected to be molecule-specific, consistent with our data. We anticipate that this report will be useful for researchers designing and implementing HPLC-ELSD methods, especially of lipids.
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影响因子：
5.8
作者：
Varache, Mathieu;Ciancone, Mathieu;Couffin, Anne-Claude
通讯作者：
Couffin, Anne-Claude
DOI：
10.1016/j.jchromb.2018.05.028
发表时间：
2018-08-01
影响因子：
3
作者：
Kothalawala, Nuwan;Mudalige, Thilak K.;Linder, Sean W.
通讯作者：
Linder, Sean W.
DOI：
10.3390/foods10020428
发表时间：
2021-02-16
期刊：
Foods (Basel, Switzerland)
影响因子：
--
作者：
Melis S;Foubert I;Delcour JA
通讯作者：
Delcour JA
影响因子：
7.4
作者：
ALLEN, LB;KOROPCHAK, JA;SZOSTEK, B
通讯作者：
SZOSTEK, B
影响因子：
5.4
作者：
Roces CB;Kastner E;Stone P;Lowry D;Perrie Y
通讯作者：
Perrie Y