LC-PDA-MS/MS analysis of polyphenols in red wine 

Polyphenols in red wine are responsible for the taste and color, and confer astringency and structure of the beverage. The knowledge about the composition of anthocyanins is important predicting the aging and color stability of wine. Moreover, polyphenols are considered as major compounds responsible for benefits of wine consumption because of their antioxidant properties and positive effects in the treatment of circulatory disorders.

The LC-PDA-MS/MS analytical technique is the best approach for the characterization of polyphenols, aglycones and attached sugar moieties available nowadays.

Instrumental: Orbitrap Discovery hybrid FT mass spectrometer coupled with Accela UPLC (Thermo Scientific). The mass spectrometer was operated in positive and negative ESI modes using scan and MSn (MS2 and MS3) options. Chromatographic separations were carried out using Gemini Hexyl-Phenyl column (3×250 mm, 3µ particle size, Phenomenex) using linear gradients of acetonitrile/water (with 0.1% AcOH). 

Negative ESI TIC scan (black), MSn (red), and UV (blue) chromatograms of concentrated red wine. While just about a dozen peaks can be observed in the chromatograms from first sight, there were identified about 40 polyphenols and detected many more unknown compounds.

An example (1) of identification of "known unknown" using the high-resolution MSn option.

Coutaric acid was detected upon negative ESI in the scan mode as [M-H]‾ ion. The parent ion gave rise to two daughter ions, namely 4-hydroxycinnamate and tartrate, in the first MS/MS event. The atomic composition calculated from the accurate masses of the parent and two daughter ions (A) supported the detection of coutaric acid. Moreover, the MSn option of the LTQ Orbitrap was used to generate granddaughter ions i.e. when the coumarate (B) and tartrate (C) anions were isolated and fragmented, which gave additional data about the structural composition and support that 4-hydroxycinnamoyltartaric acid indeed was detected.

An example (2) of identification of "known unknown" using the high-resolution MSn option.

Many beneficial effects are attributed to a stilbene polyphenol resveratrol. Though, the amount of resveratrol in wine is so minor that it makes to believe that its role is probably overestimated. Nonetheless, resveratrol is a biologically active compound (phytoalexin) produced by some plants in response to injury, where it acts against pathogens, including bacteria and fungi, and possesses high antioxidant potential.

Well, the matter of mass spectrometry is the reliable identification of compounds. Resveratrol is rarely present in plants in a free form rather it is often found as a glycoside, piceid. Deprotonated piceid loses sugar moiety at the first MS/MS event giving rise to the [M-H]‾ ion of resveratrol (m/z 227.0713) which can be isolated and subjected to further MS/MS using the MSn option of the LTQ Orbitrap.

It is important to note that plants produce derivatives of trans-resveratrol which slowly converts into cis- isomer upon light. The trans and cis isomers have identical MS/MS spectra and cannot be differentiated by mass spectrometry. However trans and cis isomers are eluted at different retention times and have different UV spectra.

Structures of compounds identified in red wine in negative ESI mode.

Structures of compounds identified in red wine in positive ESI mode.

References for the structures of polyphenols identified in red wine in this study

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