The Secrets of Red Wine - Determination of Polyphenols by Comprehensive 2D LC with PDA and MS-MS (IT-TOF) Detection

March 1, 2011

The Application Notebook

The Application Notebook, The Application Notebook-03-01-2011, Volume 0, Issue 0

Shimadzu Application Note

Paola Donato,1 Francesco Cacciola,2 Paola Dugo1,2 and Luigi Mondello,1,2

1Università Campus Bio-Medico, Roma, Italy,

2Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Messina, Italy.


Produced and consumed world-wide, red wine is probably the best known "French paradox" contributor, being an excellent source of polyphenols and flavonoids, which confer important health benefits to the heart and blood vessels. In this application, we developed a comprehensive 2D LC technique for the separation of a mixture of 31 bioactive molecules commonly found in wine, whose complexity clearly overwhelms the peak capacity afforded by any single separation approach.


Comprehensive 2D LC-PDA analyses were performed on a Shimadzu prominence UFLC-XR system, coupled to an ion trap time-of-flight (IT-TOF) mass spectrometer with electrospray ionization interface (ESI).

Chromatographic separation was achieved in the first dimension by the following conditions:

Column: Ascentis Phenyl 250 × 1.0 mm, 5 µm d.p. (SigmaAldrich/Supelco)

Gradient: A: water (pH 3);

B: acetonitrile (pH 3)

0 min: 10% B;

60 min: 70% B;

110 min: 70% B;

111 min: 100% B;

120 min: 100% B;

121 min 10% B.

Flow-rate: 9.7 µL/min.

Fast repetitive gradient was performed in the second dimension by the following conditions:

Column: Ascentis Express 30 × 4.6 mm, 2.7 µm d.p. (Sigma-Aldrich/Supelco)

Gradient: A: water (pH 3);

B: acetonitrile (pH 3);

0 min: 0% B;

0.90 min: 20% B;

1.20 min: 30% B;

1.50 min: 100% B;

1.69 min: 100% B;

1.70 min: 0% B.

Flow-rate: 4 mL/min.

Oven temperature: 45 °C.

Modulation time of the switching valve was 120 sec. ESI was operated both in positive and negative mode, with CDL and block temperature of 200 °C, 1.5 mL/min nebulizer gas (nitrogen) flow, 1.60 kV detector voltage. Data acquisition by LCMSsolution software, ver. 3.50.346; 2D plot elaboration by Chromsquare Ver. 1.4 software (Chromaleont, Messina, Italy).

Figure 1: Software window with the 2D plot (top) and the corresponding raw chromatogram (bottom) extracted at 280 nm for the chromatographic separation of 31 bioactive polyphenols.


All of the 31 compounds tested have been separated perfectly by the comprehensive system, as shown in Figure 1. Tandem MS detection by IT-TOF is a powerful tool for confident structure elucidation, thanks to the high mass accuracy and high mass resolution independent of MS mode, as shown in Figure 2.

Figure 2: Peak identification of Procyanidin B by Comprehensive 2D LC–MS–MS (IT-TOF). Accuracy: 0.9 ppm. The peak is indicated by arrow in the plot of Figure 1.

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