An automated workflow approach for the analysis of flavin adenine dinucleotide by HPLC with internal standard

Van Long Nguyen; Andrew Ah-Loo; Michael Fitzpatrick

doi:10.1016/j.ab.2020.113616

An automated workflow approach for the analysis of flavin adenine dinucleotide by HPLC with internal standard

Anal Biochem. 2020 Apr 1:594:113616. doi: 10.1016/j.ab.2020.113616. Epub 2020 Feb 7.

Authors

Van Long Nguyen¹, Andrew Ah-Loo², Michael Fitzpatrick²

Affiliations

¹ NSW Health Pathology, Department of Chemical Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia; School of Medicine, University of Sydney, Camperdown, NSW, 2006, Australia. Electronic address: vngu3382@uni.sydney.edu.au.
² NSW Health Pathology, Department of Chemical Pathology, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia.

PMID: 32035844
DOI: 10.1016/j.ab.2020.113616

Abstract

Flavin adenine dinucleotide (FAD) is an active coenzyme of vitamin B2 involved in oxidation and reduction reactions. In this study we have developed a fully automated method for the analysis of FAD by fluorescence detection. FAD is extracted from whole blood samples by trichloroacetic acid, with diethyl ribityl isoalloxazine used as an internal standard. Linearity for FAD was above 0.99 (r²) up to a concentration range of 1000 nmol/L. Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 8% (coefficient of variation) with recovery of FAD exceeding 90%. Accuracy of the protocol was compared against a previous cycle from an external quality assurance program (RCPAQAP) (n = 12) with satisfactory agreement. Overall, this method has increased laboratory workflow and reduced manual labour required in performing FAD analysis.

Keywords: FAD; Flavin adenine dinucleotide; HPLC-FLD; Vitamin B2.

MeSH terms

Chromatography, High Pressure Liquid / methods*
Flavin-Adenine Dinucleotide / blood*
Fluorescence
Humans
Workflow

Substances

Flavin-Adenine Dinucleotide