Separation and characterization of phlorotannins from brown algae Cystoseira abies‐marina by comprehensive two‐dimensional liquid chromatography

Abstract

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.

Anti-proliferative activity and chemical characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry of phlorotannins from the brown macroalga Sargassum muticum collected on North-Atlantic coasts

“…For this reason, in order to elucidate the compounds present in the most active S. muticum extracts, a LC × LC approach was used. We have previously presented the ability of this analytical tool to separate and tentatively identify phlorotannins of other brown algae, C. abies-marina, reaching degrees of polymerization of 17 phloroglucinol units (PGU) [16]. By using a combined HILIC separation in the first dimension (D1) and a RP-based separation in the second dimension (D2), 52 different phlorotannins could be effectively separated in C. abies-marina extracts.…”

“…D1 separation was run on a Lichrospher diol-5 (150 × 1.0 mm, 5 m d.p., HiChrom, Reading, UK) column, following the separation conditions developed in our previous work [16]. Briefly, the mobile phases were (A) acetonitrile/acetic acid (98:2, v/v) and (B) methanol/water/acetic acid (95:3:2, v/v/v) eluted at 15 L min −1 according to the following gradient: 0 min, 0% B; 3 min, 0% B; 5 min, 7% B; 30 min, 15% B; 70 min, 15% B; 75 min, 25% B; 85 min, 25% B.…”

“…Results revealed that these techniques were useful for discriminating among species, giving a differentiated profile but only determining the class of phlorotannins in the sample, without elucidating the entire structure of any compound. In a recent work carried out in our laboratory [16], a new comprehensive two-dimensional liquid chromatography coupled to DAD and tandem mass spectrometry (LC × LC-DAD-MS/MS) methodology was developed based on the coupling of a hydrophilic-interaction chromatography (HILIC)-based separation in the first dimension and an RP-based separation in the second dimension that allowed the separation and identification of more than 50 compounds in a Cystoseira abies-marina brown algal extract. By using this approach, phlorotannins containing from 5 to 17 phloroglucinol units were identified in this sample [16].…”

“…In a recent work carried out in our laboratory [16], a new comprehensive two-dimensional liquid chromatography coupled to DAD and tandem mass spectrometry (LC × LC-DAD-MS/MS) methodology was developed based on the coupling of a hydrophilic-interaction chromatography (HILIC)-based separation in the first dimension and an RP-based separation in the second dimension that allowed the separation and identification of more than 50 compounds in a Cystoseira abies-marina brown algal extract. By using this approach, phlorotannins containing from 5 to 17 phloroglucinol units were identified in this sample [16]. The application of this methodology to S. muticum could therefore imply a definitive step forward for the characterization of its phlorotannin composition.…”

Theory and Practice of UHPLC and UHPLC–MS

“…In this regard, remarkable efforts have been made to extend the pressure capability of the pumping systems to 1300 bar. In addition, compared to old generation HPLC instruments, the contribution of the system to band broadening was dramatically reduced by using short and narrow connecting tubes, low injected volumes, small UV-cell volumes, and fast acquisition rates Jandera, 2012, 2016;Krol-Kogus et al, 2014;de Villiers, 2009, 2010;Beelders et al, 2015;Kalili et al, 2013;Montero et al, 2014;Willemse et al, 2014Scoparo et al, 2012).…”