LC-ESI-QTOF-MS/MS Characterization of Seaweed Phenolics and Their Antioxidant Potential

Seaweed is an important food widely consumed in Asian countries. Seaweed has a diverse array of bioactive compounds, including dietary fiber, carbohydrate, protein, fatty acid, minerals and polyphenols, which contribute to the health benefits and commercial value of seaweed. Nevertheless, detailed information on polyphenol content in seaweeds is still limited. Therefore, the present work aimed to investigate the phenolic compounds present in eight seaweeds [Chlorophyta (green), Ulva sp., Caulerpa sp. and Codium sp.; Rhodophyta (red), Dasya sp., Grateloupia sp. and Centroceras sp.; Ochrophyta (brown), Ecklonia sp., Sargassum sp.], using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS). The total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) were determined. The antioxidant potential of seaweed was assessed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, a 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Brown seaweed species showed the highest total polyphenol content, which correlated with the highest antioxidant potential. The LC-ESI-QTOF-MS/MS tentatively identified a total of 54 phenolic compounds present in the eight seaweeds. The largest number of phenolic compounds were present in Centroceras sp. followed by Ecklonia sp. and Caulerpa sp. Using high-performance liquid chromatography-photodiode array (HPLC-PDA) quantification, the most abundant phenolic compound was p-hydroxybenzoic acid, present in Ulva sp. at 846.083 ± 0.02 μg/g fresh weight. The results obtained indicate the importance of seaweed as a promising source of polyphenols with antioxidant properties, consistent with the health potential of seaweed in food, pharmaceutical and nutraceutical applications.


Introduction
Seaweed has been utilized as a food for humans for centuries, and the current global market is valued at more than USD 6 billion per annum with an annual volume of approximately 12 million tonnes in 2018 [1,2]. Seaweeds (macroalgae) are classified into three major groups including Chlorophyta (green algae), Rhodophyta (red algae) and Ochrophyta (brown algae) based on their color. It is estimated that 1800 different green macroalgae, 6200 red macroalgae, and 1800 brown macroalgae are found in previous studies [22,23] and supported by our current study. The highest total flavonoid content was found in red seaweed Grateloupia sp. (54.4 ± 0.74 µg QE/g f.w. ) (p < 0.05) as compared to brown and green seaweeds. However, compared to previous studies [24], the total flavonoid content of red seaweed we found was relatively low compared with that of brown and green seaweed. The inconsistency might be explained by Chan, et al. [25], who reported that the total flavonoid content of seaweeds is impacted by sunlight, climate, region and extraction solvent. The data are shown as mean ± standard error (n = 3); the superscript letters (a-f), indicate the means within a column with significant difference (p < 0.05) using a one-way analysis of variance (ANOVA) and Tukey's test. Data of seaweed is reported on a fresh weight basis. *: total polyphenol content of brown seaweeds was significantly higher than green and red seaweeds; total flavonoid content of red seaweeds was significantly higher than green and brown seaweeds (p < 0.05). The phenolic content, as measured by total phenolic content (TPC), total flavonoid content (TFC), total tannin contents (TTC). GAE stands for gallic acid equivalents, QE stands for quercetin equivalents and CE stands for catechin equivalents.
Regarding seaweed groups, brown seaweeds presented statistically higher TPC and TTC values than green and red seaweeds (p < 0.05). This is in agreement with previous research which reported that brown seaweed had a higher total phenolic content than red and green seaweeds [26]. In addition, a study conducted by Cox, Abu-Ghannam and Gupta [24] also indicated that the total tannin content of brown seaweeds was significantly higher than that of green and red seaweed, which is explained by the presence of the unique polyphenolic components of phlorotannin in brown seaweed [27].

Antioxidant Activities (ABTS, DPPH and FRAP)
The antioxidant activities were determined using ABTS, DPPH and FRAP assays (Table 2.). The brown seaweed Ecklonia sp. had a significantly higher level of antioxidant potential than other seaweeds (958 ± 0.4 µg AAE/g f.w. for ABTS, 510 ± 3.4 µg AAE/g f.w. for DPPH and 170 ± 2.0 µg AAE/g f.w. for FRAP, p < 0.05). The result was consistent with a previous study where phlorotannins were successfully isolated from Ecklonia sp. and exhibited strong DPPH radical scavenging activity [28]. In the present work, although Ulva sp., Caulerpa sp. and Codium sp. exhibited ABTS radical scavenging activities, no DPPH radical scavenging activities were detected. This might be due to limitations of the DPPH assay [29]. Firstly, unlike water-soluble ABTS + , hydrophobic DPPH must be performed in organic solvent, which interferes with the hydrogen atom transfer reaction by disturbing the release of hydrogen atoms. Secondly, DPPH reacts rapidly, mainly through single electron transfer, with ascorbic acid and simple phenols with no ring adducts, but slowly with complex phenolic compounds with side chains and ring adducts. Therefore, the application of organic solvent and the complex structure of phenolic compounds in seaweed might lead to underestimation of DPPH scavenging activities. The data are shown as mean ± standard error (n = 3); the superscript letters (a-d), indicate the means within a column with significant difference (p < 0.05) using a one-way analysis of variance (ANOVA) and Tukey's test. Data of seaweed is reported on a fresh weight basis. *: Antioxidant capacities of brown seaweeds are significantly higher than that of green and red seaweeds (p < 0.05). DPPH stands for 2,2-diphenyl-1-picrylhydrazyl, ABTS stands for 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and FRAP stands for ferric reducing antioxidant power assay. AAE stands for ascorbic acid equivalents.
Within the seaweed groups, brown seaweed species presented significantly higher antioxidant properties for all assays than green and red seaweed species (p < 0.05). This result was in accordance with a previous study, which also found brown seaweed had higher ABTS radical scavenging activity than red or green seaweeds [30].
The relationship between TPC and antioxidant potential of all three type of (green, red and brown) seaweeds was confirmed by performing a regression model between the values of TPC and each antioxidant assay. Results showed a significant positive correlation between TPC and antioxidant activity (r 2 = 0.926 for ABTS, r 2 = 0.714 for DPPH and r 2 = 0.899 for FRAP, p < 0.05). A positive correlation between total phenolic content and antioxidant assay results was also supported by previous studies, suggesting that phenolics are the major contributor to the excellent antioxidant properties of seaweeds [21,30].

LC-ESI-QTOF-MS/MS Characterization of The Phenolic Compounds
LC-MS has been widely used for the characterization of the phenolic profiles of different plant and marine samples [31]. A qualitative analysis of the phenolic compounds from different seaweed extracts were achieved by LC-ESI-QTOF-MS/MS analysis in negative and positive ionization modes (Table S1, Figures S1 and S2-Supplementary Materials). Phenolic compounds present in eight different seaweeds were tentatively identified from their m/z value and MS spectra in both negative and positive ionization modes ([M − H] − /[M + H] + ) using Agilent LC-MS Qualitative Software and Personal Compound Database and Library (PCDL). Compounds with mass error < ± 5 ppm and PCDL library score more than 80 were selected for further MS/MS identification and m/z characterization purposes.
In the present work, LC-MS/MS enabled the tentative identification of 54 phenolic compounds, including 22 phenolic acids, 17 flavonoids, 11 other polyphenols and 4 lignans (Table 3).

Phenolic Acids
Phenolic acids have been reported as the most abundant phenolic compounds in red, green and brown algae [21]. In the present work, four sub-classes of phenolic acid were detected, including hydroxybenzoic acids, hydroxycinnamic acids, hydroxyphenylpentanoic acids and hydroxyphenylacetic acids.

Hydroxybenzoic Acids Derivatives
Six hydroxybenzoic acid derivatives were detected in six out of eight seaweeds. The typical neutral losses of CO 2 (44 Da) and hexosyl moiety (162 Da) were observed in phenolic acids [32]. Compound 2 with [M − H] − m/z at 169.0138 was only detected from red seaweed Centroceras sp., and characterized as gallic acid based on the product ion at 125 m/z, corresponding to the loss of CO 2 (44 Da) from precursor ion [32]. Gallic acid was also previously reported as abundant in the brown seaweed Himanthalia elongate [33]. p-Hydroxybenzoic acid (Compound 5 with [M − H] − ion at m/z 137.0240) present in Ulva sp., Caulerpa sp. and Centroceras sp. was identified and confirmed by MS 2 experiments (Figure 1). In the MS 2 spectrum of m/z 137.0240, the product ion at m/z 93 was due to the loss of a CO 2 (44 Da) from the parent ion [32]. This is consistent with p-hydroxybenzoic acid also being found in seaweeds from the Danish coastal area [34]. Hydroxybenzoic Acids Derivatives Six hydroxybenzoic acid derivatives were detected in six out of eight seaweeds. The typical neutral losses of CO2 (44 Da) and hexosyl moiety (162 Da) were observed in phenolic acids [32]. Compound 2 with [M -H] − m/z at 169.0138 was only detected from red seaweed Centroceras sp., and characterized as gallic acid based on the product ion at 125 m/z, corresponding to the loss of CO2 (44 Da) from precursor ion [32]. Gallic acid was also previously reported as abundant in the brown seaweed Himanthalia elongate [33]. p-Hydroxybenzoic acid (Compound 5 with [M − H] − ion at m/z 137.0240) present in Ulva sp., Caulerpa sp. and Centroceras sp. was identified and confirmed by MS 2 experiments ( Figure 1). In the MS 2 spectrum of m/z 137.0240, the product ion at m/z 93 was due to the loss of a CO2 (44 Da) from the parent ion [32]. This is consistent with p-hydroxybenzoic acid also being found in seaweeds from the Danish coastal area [34].

Hydroxycinnamic Acids and Other Phenolic Acid Derivatives
Thirteen hydroxycinnamic acids derivatives, two hydroxyphenylpentanoic acids and one hydroxyphenylacetic acid were tentatively identified in our study.
Compound (7) [38]. To the best of our knowledge, caffeoyl tartaric acid and caffeoyl glucose were previously reported primarily in fruit samples such as grape, however, it was the first time that they were reported in seaweeds [39].  [40]. Chlorogenic acid was also present in the green seaweed Capsosiphon fulvescens from Korea, according to previous research [41].
Sinapic acid (Compound 16) were detected in both positive (ESI + ) and negative (ESI − ) modes in Ulva sp. Caulerpa sp. and Grateloupia sp. with an observed [M − H] − m/z at 223.0621. In the MS 2 spectrum of sinapic acid, the product ions at m/z 205, 179 and 163 were due to the loss of H 2 O (18 Da), CO 2 (44 Da) and two CH 2 O units (60 Da) from the parent ion, respectively, which was comparable with the fragmentation rules of sinapinic acid [42].
Coumaric acid (compound 18 with [M − H] − m/z at 163.0406), yielding a main product ion at m/z 119, which corresponded to loss of CO 2 (44 Da), was found in Caulerpa sp. [43]. The presence of coumaric acid in marine seaweeds was also previously reported [34].
Three other phenolic acid derivatives were also detected, including two hydroxyphenylpentanoic acid derivatives and one hydroxyphenylacetic acid derivative. To our best knowledge, this is the first time these other phenolic acid derivatives have been reported in seaweeds. Phenolic acids are the predominant polyphenol compounds found in different seaweeds, which were characterized by using LC-MS in previous studies, and displayed remarkable antioxidant potential [44,45].

Flavonoids
Flavonoid is the main class of phenolic compounds responsible for the antioxidant and free radical scavenging properties observed in seaweed [24]. In the present study, a total of 17 flavonoids were tentatively identified, which were further divided into anthocyanins (03), flavanols (03), flavonols (03), flavone (01) and isoflavonoids (07).

Anthocyanins, Flavanols and Flavonols Derivatives
Anthocyanins are naturally occurring pigments that belong to the subclass of flavonoids, which were previously reported in brown Irish seaweeds [46]. In our study, three anthocyanin derivatives were detected only in the red seaweeds Grateloupia sp. and Centroceras sp., in positive ionization mode. This is the first time all of these anthocyanins derivatives have been reported in seaweeds.
Three flavanols (Compound 26, 27 and 28) were detected in all seaweeds except Centroceras sp. and  [50]. To the best of our knowledge, this is the first time that isoflavonoids derivatives were identified and characterized in seaweeds. Flavonoids in different seaweeds with high antioxidant potential have already been reported, which are promising as functional food ingredients or dietary supplements for daily intake [51].

Hydroxybenzaldehydes, hydroxycoumarins and hydroxyphenylpropenes Derivatives
Three other polyphenols derivatives were detected, including compound (49) with [M − H] − at m/z 125.0242, which was proposed as phloroglucinol appearing in brown seaweed Ecklonia sp. and Sargassum sp. The identity was confirmed by the MS 2 spectrum, which produced a major fragment ion at m/z 97, resulting from the loss of CO (28 Da) from the precursor ion [9]. The presence of phloroglucinol in Irish brown seaweed Himanthalia elongate was previously reported by Rajauria, Foley and Abu-Ghannam [9] according to the precursor and product ions, and further confirmed by the UV spectrum and retention time using phloroglucinol standard.

Lignans
Lignans were minor components present in the seaweeds. In the present study, a total of four lignans were shown to be present in seven out of eight seaweeds. . Lignans are abundant in seaweeds, however, the lignans in the present study have not previously been reported in seaweeds [62]. Previously, it was reported that lignans are abundant in seaweeds with various health-promoting properties, including antioxidant, anti-inflammatory and antitumor activities [62,63]. In addition, some epidemiological studies have proposed the therapeutic potential of lignans in chronic diseases, such as cardiovascular disease, type 2 diabetes and cancers [64,65].

Lignans Derivatives
The screening and characterization of polyphenolic compounds showed that some of the polyphenols presented in these seaweeds have strong antioxidant potential. Hydroxycinnamic acid derivatives, hydroxybenzoic acids and their derivatives, protocatechuic acid, anthocyanins, flavonoids and their derivatives, hydroxybenzaldehydes, hydroxytyrosol, phloroglucinol and quercetin derivatives are regarded as potential compounds showing considerable free radical scavenging capacity [66][67][68][69][70][71]. The presence of these antioxidant compounds indicates that seaweeds can be good sources of polyphenols and could be utilized in food, feed, and pharmaceutical industries.

HPLC Quantitative Analysis
The quantitative analysis of targeted phenolic compounds was performed based on peak area computation using the calibration of corresponding standards and the result are presented as µg/g fresh weight of seaweeds (Table 4.). In total, seven polyphenols were targeted to quantify by HPLC-PDA, including six phenolic acids (gallic acid, caftaric acid, chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid) and one flavonoid (catechin). The most abundant targeted phenolic compound was p-hydroxybenzoic acid (Compound 5), which was present in Ulva sp. with the concentration of 846.0 ± 0.02 µg/g f.w. The p-hydroxybenzoic acid content of eight green and red seaweeds in South Africa was previously reported as ranging from 0.51 ± 0.01 to 13.53 ± 0.03 µg/g dry weight (d.w.) [72], which was significantly lower than that of Ulva sp. in the present study. Gallic acid (Compound 1), chlorogenic acid (Compound 2) and caftaric acid (Compound 4) were detected in Centroceras sp. with the concentration of 138.9 ± 0.02 µg/g f.w. , 122.7 ± 0.01 µg/g f.w. and 19.7 ± 0.01 µg/g f.w. , respectively. Coumaric acid (Compound 6) was quantified in Ulva sp. with concentrations of 505.4 ± 0.03 µg/g f.w . Caffeic acid (Compound 3) and catechin (Compound 7) were present in Caulerpa sp. with a concentration of 612.9 ± 0.02 µg/g f.w. and 29.5 ± 0.03 µg/g f.w. , respectively. Concentrations of gallic acid, chlorogenic acid and caffeic acid in brown seaweed Himanthalia elongate were also previously reported, being measured as 96.3 ± 3.12 µg/g d.w. , 38.8 ± 1.94 µg/g d.w. and 44.4 ± 2.72 µg/g d.w. , respectively [33]. About 10 marine-derived pharmaceutical drugs were approved by the Food and Drug Administration (FDA), and 30 candidates were in different stages of clinical trials for application in a number of disease areas [73]. The presence of these abundant polyphenols provide evidence for seaweeds as a good source of antioxidants for application in food and pharmaceutical industries, while further toxicity, pharmacological and clinical studies are needed.

Sample Preparation and Extraction of Polyphenols
Eight seaweeds which were identified as Chlorophyta (green; Ulva sp., Caulerpa sp. and Codium sp.), Rhodophyta (Red; Dasya sp., Grateloupia sp. and Centroceras sp.) and Ochrophyta (Brown; Ecklonia sp. and Sargassum sp.) were freshly collected from Brighton Beach in March 2019, VIC, Australia. Seaweeds were morphologically identified to the genus level. Classifications for Rhodophyta and Chlorophyta were verified using cytochrome c oxidase subunit I (COI-5P) and Elongation factor Tu 1-Escherichia coli (strain K12) tufA sequence data, respectively, following the protocol of Saunders and Kucera [74].
Extracts were prepared by modifying the previous studies [75,76], 2 g of each seaweed was grounded and mixed with 10 mL of 80% ethanol followed by homogenization using an Ultra-Turrax ® T25 homogenizer (Rawang, Selangor, Malaysia) at 10,000 rpm for 20 s. Then, incubation was carried out in a shaking incubator (ZWYR-240, Labwit, Ashwood, VIC, Australia) at 120 rpm at 4 • C for 16 h. Then, all the samples were centrifuged (Hettich Rotina 380R, Tuttlingen, Germany) at 10,000 rpm for 10 min. The supernatant was collected and stored at −20 • C for further analysis. For HPLC and LC-MS analysis, the extracts were filtered through a 0.45 µm syringe filter (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Estimation of Polyphenols and Antioxidant Assays
For polyphenol estimation, TPC, TFC and TTC were measured, while for antioxidant potential, three different antioxidant assays, including DPPH, FRAP, and ABTS, were performed using the method of Feng, et al. [77]. The data were obtained by the Multiskan ® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA).

Total Phenolic Content (TPC)
The total phenolic content of seaweed was determined using the Folin-Ciocalteu's method [13] with some modifications. Twenty-five microliters of standards and samples (supernatant), 25 µL of 25% (v/v) folin reagent solution and 200 µL water were added to the wells in a 96-well plate (Corning Inc., Corning, NY, USA) and incubated at 25 • C for 5 min. Then, 25 µL of 10% (w/w) sodium carbonate was added and further incubated for 1 h at 25 • C. The absorbance was measured at 765 nm against a blank using a Multiskan ® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). The calibration curve was plotted using a gallic acid standard ranging from 0 to 200 µg/mL in ethanolic solution and the results were presented as microgram equivalents of gallic acid equivalents (GAE) per gram ± standard error (SE) on the basis of fresh weight (f.w.) (y = 0.0059x + 0.0593, R 2 = 0.9996).

Total Flavonoid Content (TFC)
The total flavonoid content was measured by aluminum chloride colorimetry according to Chan, Matanjun, Yasir and Tan [25], with some modifications. Methanolic quercetin standards and samples (80 µL) were added to the 96-well plate. Then, 80 µL of 2% (w/v) aluminum chloride (diluted with analytical grade ethanol) and 120 µL 50 g/L sodium acetate was added the wells in the plate followed by the incubation at 25 • C for 2.5 h in the dark. The calibration curve was plotted using quercetin standards ranging from 0 to 50 µg/mL and the results are presented as microgram equivalents of quercetin equivalents (QE)/g f.w. ± SE (y = 0.0195x + 0.0646, R 2 = 0.999).

Total Tannins Content (TTC)
Total tannin content was measured by modifying the method of Rebaya, et al. [78]. Sample/standard (25 µL of supernatant or standard), 150 µL 4% (w/v) methanolic vanillin solution and 25 µL 32% (v/v) sulfuric acid (diluted with methanol) were mixed in a 96-well plate and incubated at room temperature for 15 min. The absorbance was measured at 500 nm wavelength against a blank using the microplate reader. The calibration curve was plotted by catechin methanolic solution ranging from 0 to 1000 µg/mL and the results are presented as microgram equivalents of catechin (CE)/g f.w. ± SE (y = 0.0005x + 0.0578, R 2 = 0.9854).
3.3.4. 2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay DPPH radical scavenging activities of different extracts were determined based on Chan et al. [25] with some modifications. Quantities of 40 µL samples/standards and 260 µL of 0.1 mM methanolic DPPH were added to a 96-well plate. The reaction mixture was incubated for 30 min in the dark at room temperature, and the absorbance was measured under 517 nm wavelength against a blank. The standard curve was plotted by ascorbic acid aqueous solution ranging from 0 to 50 µg/mL and the results are expressed as the microgram equivalents of ascorbic acid (AAE)/g f.w. ± SE (y = −0.0089x + 0.5988, R 2 = 0.9708).

Ferric Reducing Antioxidant Power (FRAP) Assay
The ferric reducing capabilities of the samples were measured using the FRAP method described by Matanjun, et al. [79], with slight modifications. The FRAP reagent was freshly prepared by mixing 300 mM acetate buffer, 10 mM TPTZ solution and 20 mM ferric chloride in the ratio of 10:1:1 (v/v). 20 µL samples/standards were added into the 96-well plate and mixed with 280 µL FRAP reagent. The mixture was incubated at 37 • C in the plate reader for 10 min before absorbance was measured at 593 nm. A standard curve was generated using ascorbic acid aqueous solution ranging from 0 to 50 µg/mL and the results are expressed as the microgram AAE/g f.w. ± SE (y = 0.009x + 0.403, R 2 = 0.9819).
3.3.6. 2,2 -Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) assay The antioxidant activities of seaweeds were also measured by an ABTS assay according to Matanjun, Mohamed, Mustapha, Muhammad and Ming [79], with some modifications. ABTS + was prepared by mixing 5 mL of 7 mM ABTS solution and 88 µL of 140 mM potassium persulfate solution, and the mixture was placed in the dark for 16 h to allow free radical generation. The stock solution was further diluted with 45 mL analytical-grade ethanol while the absorbance of the dye was fixed at approximately 0.7 at 734 nm. Quantities of 10 µL of sample/standards and 290 µL prepared dye solution were added into a 96-well plate followed by incubation at room temperature for 6 min and the absorbance was measured at 734 nm wavelength. The standard curve was plotted using ascorbic acid aqueous solution ranging from 0 to 200µ/mL and the results are expressed as the microgram AAE/g f.w. ± SE (y = -0.0042x + 0.6923, R 2 = 0.9962).

LC-ESI-QTOF-MS/MS Characterization of Phenolic Compounds
LC-ESI-QTOF-MS/MS analysis was performed with an Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 6520 Accurate-Mass Q-TOF LC-MS (Agilent Technologies, Santa Clara, CA, USA) via an electrospray ionization source (ESI). The separation was achieved by a Synergi Hydro-RP 80 Å, LC Column (250 mm × 4.6 mm, 4 µm) (Phenomenex, Lane Cove, NSW, Australia) at room temperature and the sample temperature was set at 10 • C. LC-MS/MS analysis were performed by modifying the method of Chao et al [66]. The mobile phase consisted of water/acetic acid (98:2, v/v; eluent A) and acetonitrile/acetic acid/ water (50:0.5:49.5, v/v/v; eluent B). The gradient profile was described as follows: 10 (87-90 min). A volume of 6 µL was injected for each standard or sample and the flow rate was set at 0.8 mL/min. Nitrogen gas nebulization was set at 45 psi with a flow rate of 5L/min at 300 • C and the sheath gas was set at 11 L/min at 250 • C. The capillary and nozzle voltage were set at 3.5 kV and 500 V, respectively. A complete mass scan ranging from m/z 50 to 1300 was used, MS/MS analyses were carried out in automatic mode with collision energy (10, 15 and 30 eV) for fragmentation. Peak identification was performed in both positive and negative modes while the instrument control, data acquisition and processing were performed using MassHunter workstation software (Qualitative Analysis, version B.03.01) (Agilent Technologies, Santa Clara, CA, USA).

HPLC-PDA Quantitative Analysis of Individual Phenolic Compounds
The quantitative measurement of individual phenolic compounds present in seaweed samples was performed with an Agilent 1200 HPLC equipped with a photodiode array (PDA) detector by adopting the protocol of Peng et al. [68]. The same column and conditions were used as described above in LC-ESI-QTOF-MS/MS, except for a sample injection volume of 20 µL. The compositions of extracts were detected under λ 280 nm, 320 nm, and 370 nm by PDA detector simultaneously with 1.25 scan/s (peak width = 0.2 min) spectral acquisition rate. The targeted phenolic compounds were quantified based on linear regression of external standards peak area against concentration. Data acquisition and analysis were performed by MassHunter workstation software-version B.03.01 (Agilent Technologies, Santa Clara, CA, USA).

Statistical Analysis
All analyses were performed in triplicates and the results are presented as mean ± standard error (n = 3). Data were analyzed using Tukey's one-way analysis of variance (ANOVA) by Minitab ® 19 for windows (Minitab, NSW, Australia). A significant difference was considered at the level of p ≤ 0.05 using Tukey's HSD test.

Conclusions
Brown seaweed species showed significantly higher polyphenolic content and potential antioxidant capacity than green and red seaweeds. The antioxidant properties varied across different species. Application of LC-ESI-QTOF-MS/MS enabled the isolation and identification of 54 phenolic compounds present in seaweeds. Quantitative analysis of targeted compounds was achieved by calibration of standards using HPLC-PDA. Seven targeted compounds were quantified in seaweeds, with p-hydroxybenzoic acid being the most abundant. This is the first report that applied different antioxidant assays to estimate the antioxidant potential and applied LC-MS technique to isolate and characterize the polyphenols in some abundant Australian seaweed species. The presence of the various polyphenols with antioxidant potential was identified. Further toxicity, pharmacological and clinical studies should be explored before the application of these Australian seaweeds as ingredients in food, nutraceuticals and pharmaceutical products.