Australian Centre for Research on Separation Science (ACROSS) and Pfizer Analytical Research Centre (PARC), School of Chemistry, Faculty of Science, Engineering and Technology, University of Tasmania, Private Bag 75, Hobart, Tas. 7001, Australia.
Australian Centre for Research on Separation Science (ACROSS) and Pfizer Analytical Research Centre (PARC), School of Chemistry, Faculty of Science, Engineering and Technology, University of Tasmania, Private Bag 75, Hobart, Tas. 7001, Australia.
J Chromatogr A. 2014 Aug 22;1356:180-7. doi: 10.1016/j.chroma.2014.06.055. Epub 2014 Jun 25.
The solvent dependency of the detection response is a major limitation of corona-charged aerosol detection (C-CAD). The present study empirically investigates the utility of temperature and flow-rate gradients to overcome solvent gradient limitations of C-CAD. In preliminary flow-injection investigations, it is demonstrated that the response of C-CAD remains relatively unaltered with variations in flow-rate when used with water-rich eluents. Based on these findings two separation approaches were developed and their utility for C-CAD response normalisation was demonstrated using a mixture of eight analytes. In the first approach the use of a solvent gradient is replaced with a temperature gradient performed under isocratic mobile phase conditions. Detection response is further enhanced by mixing a secondary stream of pure acetonitrile with the column effluent, yielding a 3-fold increase in detection response. In the second approach, flow-rate programming is used to improve speed of isocratic-temperature gradient separation. The use of simultaneous variation in flow-rate and column temperature reduced the separation time by 30%, with relatively uniform analyte response. Lastly, an inverse-gradient solvent compensation approach was used to evaluate the response homogeneity and the applicability of the above approaches for quantitative analysis. Good peak area reproducibility (RSD%<15%) and linearity (R(2)>0.994, on a log-scale) over the sample mass range of 0.1-10 μg was achieved. The response deviation across the mixture of eight compounds at seven concentration levels was 6-13% compared to 21-39% when a conventional solvent gradient was applied and this response deviation was comparable to that obtained in the inverse gradient solvent compensation approach. Finally, applicability of these approaches for typical pharmaceutical impurity profiling was demonstrated at a concentration of 5 μg/mL (0.1% of the principal compound).
电晕荷电气溶胶检测（C-CAD）的一个主要局限性是检测响应对溶剂的依赖性。本研究通过实验研究了利用温度和流速梯度来克服 C-CAD 中溶剂梯度限制的实用性。在初步的流动注射研究中，当使用富含水的洗脱液时，证明 C-CAD 的响应在流速变化时相对保持不变。基于这些发现，开发了两种分离方法，并使用八种分析物的混合物证明了它们对 C-CAD 响应归一化的用途。在第一种方法中，用在等度流动相条件下进行的温度梯度代替溶剂梯度。通过将纯乙腈的二次流与柱流出物混合，进一步增强了检测响应，检测响应提高了 3 倍。在第二种方法中，使用流速程序来提高等速-温度梯度分离的速度。同时改变流速和柱温可将分离时间缩短 30%，同时保持相对均匀的分析物响应。最后，使用逆梯度溶剂补偿方法来评估响应的均匀性以及上述方法在定量分析中的适用性。在 0.1-10μg 的样品质量范围内，获得了良好的峰面积重现性（RSD%<15%）和线性（在对数标度上 R（2）>0.994）。在七种浓度水平的八种化合物混合物中，响应偏差为 6-13%，而当应用常规溶剂梯度时，响应偏差为 21-39%，这与在逆梯度溶剂补偿方法中获得的响应偏差相当。最后，在 5μg/mL（主化合物的 0.1%）的浓度下，证明了这些方法在典型药物杂质分析中的适用性。
