Phlorotannins are an important class of polyphenolic compounds only found in brown algae. The chemical analysis of these bioactive polyphenols is rather difficult due to the great chemical variability and complexity of the natural composition of these components in algae, forming large phloroglucinol polymers. In the present work, a new approach based on the use of comprehensive 2D LC (LC × LC) is shown to analyze this complex family of compounds. The developed LC × LC methodology is based on the coupling of a hydrophilic-interaction LC (HILIC)-based separation in the first dimension and an RP-based separation in the second dimension. The employment of this online coupling together with DAD and MS/MS allowed the separation and identification of more than 50 compounds in a Cystoseira abies-marina brown alga extract. Phlorotannins containing from 5 to 17 phloroglucinol units were identified in this sample by HILIC × RP-DAD-MS/MS. Besides, using the 2,4-dimethoxybenzaldehyde assay, it was possible to determine that the total amount of phlorotannins present in the extract was 40.2 mg phloroglucinol equivalents per gram of extract. To our knowledge, this work is the first demonstration of the usefulness of HILIC × RP-DAD-MS/MS for the determination of phlorotannins.
In this work, the development and optimization of a new methodology to analyze grape seed procyanidins based on the application of two-dimensional comprehensive LC is presented. This two-dimensional method involves the use of a microbore column containing a diol stationary phase in the first dimension coupled to either a C18 partially porous short column or a C18 monolithic column in the second dimension. The orthogonal hydrophilic interaction × reversed phase liquid chromatography (HILIC×RP-LC) system is interfaced through a ten-port two-position switching valve. The optimized HILIC×RP-LC separation followed by diode array and tandem mass spectrometry detection (HILIC×RP-LC-DAD-MS/MS) made possible the direct analysis of a complex grape seed extract and allowed the tentative identification of 43 flavan-3-ols, including monomers and procyanidin oligomers till a polymerization degree of 7 units with different galloylation degrees. To the best of our knowledge, this is the first time that this powerful analytical technique is employed to characterize complex procyanidin samples. This work successfully demonstrates the great capabilities of the HILIC×RP-LC-DAD-MS/MS coupling for the direct analysis of very complex natural samples like grape seeds.
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