Multivariate DoE Optimization of Asymmetric Flow Field Flow Fractionation Coupled to Quantitative LC-MS/MS for Analysis of Lipoprotein Subclasses

Chromatography

Zsuzsanna Kuklenyik²  Michael S. Gardner²  Bryan A. Parks²  David M. Schieltz²  Jon C. Rees²  Lisa G. McWilliams²  Yulanda M. Williamson²  James L. Pirkle²  John R. Barr¹ 

[1] id="af1-chromatography-02-00096">Division of Laboratory Sciences, Centers for Disease Control and Prevention, National Center for Environmental Health, Atlanta, GA 30341, U

关键词: asymmetric flow field flow fractionation;    mass spectrometry;    lipoproteins;    design of experiment;    quality by design;   

DOI  :  10.3390/chromatography2010096

来源: mdpi

【 摘 要 】

In this report we demonstrate a practical multivariate design of experiment (DoE) approach for asymmetric flow field-flow fractionation (AF4) method optimization using separation of lipoprotein subclasses as an example. First, with the aid of commercially available software, we built a full factorial screening design where the theoretical outcomes were calculated by applying established formulas that govern AF4 channel performance for a 5–35 nm particle size range of interest for lipid particles. Second, using the desirable ranges of instrumental parameters established from theoretical optimization, we performed fractional factorial DoE for AF4 separation of pure albumin and ferritin with UV detection to narrow the range of instrumental parameters and allow optimum size resolution while minimizing losses from membrane immobilization. Third, the optimal range of conditions were tested using response surface DoE for sub-fractionation of high and low density lipoproteins (HDL and LDL) in human serum, where the recovery of the analytes were monitored by fraction collection and isotope-dilution LC-MS/MS analysis of each individual fraction for cholesterol and apolipoproteins (ApoA-1 and ApoB-100). Our results show that DoE is an effective tool in combining AF4 theoretical knowledge and experimental data in finding the most optimal set of AF4 instrumental parameters for quantitative coupling with LC-MS/MS measurements.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.