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An On-Column Enzyme Mediated Fluorescence-Amplification Method for Plasma Total Cholesterol Measurement by Capillary Electrophoresis with LIF Detection

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Abstract

Total cholesterol monitoring in human plasma is one of the most important routine analysis performed in the clinical laboratory. Here we report a new capillary electrophoresis method for determination of plasma total cholesterol based on on-column enzymatic assay and laser-induced fluorescence detection. In this method, the capillary is used not only as a separation medium but also as the reaction chamber which minimizes the amount of samples and the consumption of the reagents. The whole procedure can be automated which minimizes the possible errors and saves experimental time. The analytical characteristics of the proposed method, such as linearity and repeatability of the peak area and the migration time, were evaluated. The RSD of the migration time and peak area were 1.0 and 3.5 % (intraday), and 2.2 and 2.9 % (interday), respectively. The obtained LOQ values were at the nanomolar concentration level, therefore the developed method is sufficient for determination and quantification of plasma total cholesterol.

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Abbreviations

CE:

Capillary electophoresis

LIF:

Laser-induced fluorescence

APF:

2-[6-(4’-Amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid

ChOx:

Cholesterol oxidase

HRP:

Horseradish peroxidase

LOD:

Limit of detection

H2O2 :

Hydrogen peroxide

ChEt:

Cholesterol esterase

DMF:

Dimethylformamide

EtOAc:

Ethyl acetate

hROS:

Highly reactive oxygen species

ROS:

Reactive oxygen species

RNS:

Reactive nitrogen species

EMMA:

Electrophoretically mediated microanalysis

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Acknowledgments

The work was supported by Science and Technology Project of Taizhou (No. 131KY07), Scientific Research Fund of Zhejiang Provincial Education Department (No. Y201329469) and the Research Foundation for Post-doctoral Scientists of Taizhou (No. 2013BSH01).

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Authors and Affiliations

  1. Department of Chemistry, Taizhou University, Taizhou, 318000, China

    Guobin Zhou

  2. Department of Biological and Chemical Engineering, Taizhou Vocational and Technical College, Taizhou, 318000, China

    Yueqing Guan

Authors
  1. Guobin Zhou
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  2. Yueqing Guan
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Correspondence to Yueqing Guan.

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All authors have no conflicts of interests.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Zhou, G., Guan, Y. An On-Column Enzyme Mediated Fluorescence-Amplification Method for Plasma Total Cholesterol Measurement by Capillary Electrophoresis with LIF Detection. Chromatographia 79, 319–325 (2016). https://doi.org/10.1007/s10337-016-3023-2

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  • DOI: https://doi.org/10.1007/s10337-016-3023-2

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