Development and validation of HPLC-MS2 methodology for the accurate determination of C4\u2013C8 B-type flavanols and procyanidins

Ugo Bussy,Alan Crozier,Catherine Kwik-Uribe,Yusuf Olanrewaju,Javier Ottaviani

doi:10.1038/s41598-021-93993-0

Ugo Bussy, Alan Crozier + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-93993-0

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Abstract

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits. Research on their safety and efficacy is critically dependent upon on the ability to reliably characeterize the research materials that are utilized, and with growing consumer availability of CF-based products, reliable methods for the detection of potential adulteration are of increasing importance. This research focused on the development of a high performance liquid chromatography-tandem mass spectrometry method (HPLC-MS2) using primary standards and 13C-labelled procyanidins as internal standards. The ability of MS2 detection to discriminate A- and B-type procyanidins was demonstrated. Method performances were validated for degrees of polymerization up to four in seven model food matrices. Accuracy ranged from 90.9 to 125.4% and precision was < 10% at lower concentrations. Finally, the method was applied to cocoa-based samples and compared to the AOAC 2020.05 analytical protocol, supporting the use of NIST 8403 as reference material for HPLC-MS2 analysis.

Highlights

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits
When higher selectivity is sought and CF measurement is required to be specific to B-type procyanidins, HPLC-MS2 is the method of choice
HPLC-MS2 represents a more selective quantification tool for cocoa flavanols and procyanidins than the HPLCFLD method currently used for routine testing

Summary

Introduction

Cocoa flavanols and procyanidins (CFs), natural dietary bioactives, have been studied extensively over the past two decades for their potential health benefits. The use of HPLC–MS for the direct and accurate quantification of flavanols and procyanidins has not been reported and is dependent upon the the availability of analytical standards for method calibration and quantitative analysis. CF oligomeric fractions including DP2–7 isolated from the seeds of Theobroma cacao L. were used in combination with commerciallyavailable procyanidin dimer standards, NIST cocoa extract reference material, and 13C-labeled procyandins (Fig. 2) to (1) develop HPLC-MS2 conditions for cocoa procyanidins, (2) study M S2 selectivity toward A-type and B-type procyanidins, (3) identify and compensate for matrix effects, and (4) validate and apply the method to commercial samples.

Results

Conclusion