Page 62 - Application Handbook - Liquid Chromatography

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500000000 1,5 Comprehensive Two-dimensional Liquid
250000000 Gallic acid 1,0 339,1
0,5 Technical Chromatography for Determination of
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Report Polyphenols in Red Wines
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400000000 2:577.00( − ) LC×LC-PDA-MS/MS for polyphenol analysis in red wine
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300000000 Procyanidin B1 1,0
200000000 Procyanidin B2
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0,5 212,1 443,2 Paola Dugo 1, 2 , Francesca Rigano , Francesco Cacciola , Paola Donato , Luigi Mondello 1, 2
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Abstract:
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750000000 2:289.00( − ) 2,5 289,1 A comprehensive two-dimensional liquid chromatography method was developed and applied to the determination of polyphenols in red wines.
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500000000 To fulŠl such a task, a micro cyano and a partially porous octadecylsilane columns were employed in the Šrst and the second dimension, respec-
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Catechin 1,0 tively in combination with photodiode array and mass spectrometry (LC×LC-PDA-MSMS) detection. To increase the peak capacity values by using
250000000 Epicatechin RP modes in both dimensions, a comparison of a conventional full-in-fraction and shifted second dimension gradient was carried out. The
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Inten. (×100,000) Keywords: comprehensive LC, polyphenols, red wines, mass spectrometry
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Quercetin 212,1
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2. Experimental
1. Introduction 2. Experimental
1.
oduction
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Phenolic compounds are secondary metabolites synthesized by plants 2-1. Reagents and Materials
Fig. 6 Ion extracted chromatograms (on the left) along with relative mass spectra (on the right) of the main polyphenolic compounds identi ed during normal development and in response to stress conditions. They em-
in the red wine sample investigated LC-MS grade solvents for LC×LC analyses: water (H 2 O), acetonitrile
brace a considerable range of substances possessing an aromatic ring bear-
(ACN); Acetic acid 99–100%, (glacial). All the solvents and chemicals
ing one or more hydroxyl moieties. Produced and consumed world-wide,
wine is an excellent natural source of various polyphenol families that go were purchased from Sigma-Aldrich (Milan, Italy).
5. Conclusions
5. Conclusions from phenolic acids (benzoic- or cinnamic-like derivatives) to different Chromatographic separations were carried out using columns provid-
A comprehensive two-dimensional liquid chromatography system, classes of flavonoids (flavones, flavan-3-ols, flavonols and anthocyanins). ed by Supelco (Bellefonte, PA, USA): Ascentis Cyano (250 mmL. × 1
based on the use of a micro cyano column and a partially porous (C 18 ) Sometimes, the polyphenol content in real world-samples can be so com- mmI.D., 5 µm d.p.), and Ascentis Express C 18 (30 mmL. × 4.6 mmI.D.,
column in the Šrst and second dimension, respectively, in combination plex that they cannot be resolved in a one-dimensional HPLC analysis. In 2.7 µm d.p.).
with photodiode array and mass spectrometry detection, is presented. order to overcome this problem, comprehensive two-dimensional liquid The red wine was purchased in a local market. The sample was œl-
Two second dimension gradient approaches, namely full in fraction chromatography (LC×LC) employing two columns with different selectiv- tered through a 0.45 µm Acrodisc nylon membrane (Pall Life Scienc-
and shifted were investigated and compared in terms of peak capacity. ity in the two dimensions could be a viable tool. In addition, in order to es, Ann Arbor, MI, USA) before injection.
improve the peak distribution, different gradient elution strategies could
The shifted gradient method used increased the effective peak-distri-
be investigated to enhance the orthogonality degree, by means of specif-
bution area in the LC×LC analysis of a red wine sample.
ic elution gradient approaches to be used in the second dimension.
Therefore, the use of a shifted gradient in an LC×LC system brings This technical report describes a novel LC×LC-PDA-MSMS (Fig. 1) instru-
about a signiŠcant improvement in separation power and is a great ment, capable of extremely high-resolution power, as well as targeted
advantage in the analysis of such complex samples.
and untargeted analysis, that was successfully applied to the character-
ization of the polyphenol content a red wine sample (Fig. 2).
First Edition: December, 2015

For Research Use Only. Not for use in diagnostic procedures.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. Fig. 2 RP-LC×RP-LC Plot of a red wine sample
The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject Fig. 1 LC×LC-PDA-MSMS instrumentation
to change without notice.
www.shimadzu.com/an/ © Shimadzu Corporation, 2015 1 University of Messina, Italy
2 Chromaleont S.r.l.