UPLC-QTOF-MS fingerprinting combined with chemometrics to assess the solvent extraction efficiency, phytochemical variation, and antioxidant activities of Beta vulgaris L.

Abstract

Accurate assays of plant antioxidants and other phytochemicals require efficient extraction conditions and enable rigorous assessments of crop varieties and production systems. This study assessed the extraction of phytochemicals and antioxidants from conventionally or organically grown red and golden beets (Beta vulgaris L.), using twenty solvent (S1eS20) mixtures containing water, methanol, and ethanol alone or with acids (ascorbic, formic, acetic). Red beetroot extracted with methanol with or without acid had the highest betanin content (2791.0 mg/g and 8222.3 mg/g of fresh weight [FW], respectively) and golden beetroot extracted with methanol/ascorbic acid/water had the highest vulgaxanthin I (193.7 mg/g and 15.0 mg/g of FW, respectively). The radical-scavenging activity and total phenolics in beetroot extracts reflected the different extraction efficiency of each solvent. UHPLC-QTOF-MS was used to identify twenty-seven phytochemicals, including 23 betalains, 2 amino acids, and 2 phenolic acids. Chemometric approaches discriminated the beet varieties and different extracts within one variety based on the composition and abundance of the key phytochemicals. The red beetroot extracted with aqueous ethanol with or without acid (S5, S7, S8, S9), and golden beetroot extracted with methanol-containing solvents (S15 for conventionally and S20 for organically) had the highest levels of phytochemicals, suggesting that these conditions efficiently extract key phytochemicals.

Betalains and their applications in food: The current state of processing, stability and future opportunities in the industry

“…The roots of different varieties of the beet Beta vulgaris L. represent one of the most studied natural sources due to their high content of betalains, at between 30.9 and 445 mg of betacyanins/100 g and 16.3 to 242 mg of betaxanthins/100 g ( Silva et al, 2020 , Swamy et al, 2014 ). This natural source has shown a high ABTS scavenging activity of 229.83–300.76 mg of AAE/g in contents of 260–436.5 mg of betanin/100 g of fresh root ( Wang et al, 2020 ). In addition, 0.19 mmol ET/g (by DPPH assay), 0.15 mmol ET/g (by FRAP), and 4.88 mmol ET/g (by ABTS) were found at concentrations of 4.6 mg of betacyanins and 2.6 mg of betaxanthins/g ( Silva et al, 2020 ).…”

“…Other extraction methods have been studied in the search to improve the efficiency of conventional extraction when using enzymatic treatment with pectinases ( Naderi et al, 2010 ), ultrasound-assisted extraction ( Haq et al, 2020 , Silva et al, 2020 , Wang et al, 2020 ), β-cyclodextrin (CD)-enhanced ultrasound-assisted extraction ( Tutunchi et al, 2019 ), microwave-assisted extraction ( Ferreres et al, 2017 , Melgar et al, 2019 , Singh et al, 2017 ), and the application of pulsed electric fields (PEFs) ( Jiménez-Alvarado et al, 2015 , Nowacka et al, 2019 ) as well as the use of supercritical fluid extraction (SFE) as a safe alternative for the environment due to the lack or minimal use of solvents compared to conventional extraction ( Fathordoobady et al, 2016 , Nunes et al, 2015 ). Some of the alternative methods to conventional extraction as well as the primary process conditions are summarized in Table 3 , again indicating that the nature of the sample will determine the conditions, time and intensity of the process as well as the type of solvent and the appropriate R L/S .…”

“…Through tools such as liquid chromatography (LC), in the high-pressure liquid chromatography (HPLC) modality ( Ferreres et al, 2017 , García-Cruz et al, 2017 , Wang et al, 2020 ), ultra-performance liquid chromatography (UPLC) ( Melgar et al, 2019 ), or ultra-high-pressure liquid chromatography (UHPLC) ( Cejudo-Bastante et al, 2014 , Wang et al, 2020 ) in reverse-phase (RP), the separation of betalain mixtures has been very effective. Some of the solvents used as mobile phases to separate betalains by LC are acetonitrile–water ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Sawicki et al, 2016 , Sawicki et al, 2017 , Wang et al, 2020 ), methanol–water ( Cejudo-Bastante et al, 2014 , Slavov et al, 2013 ), and acetonitrile buffer of KH 2 PO 4 (pH 2.74) ( Sanchez-Gonzalez et al, 2013 ) in different proportions, which are applied by eluting in isocratic mode or modifying the gradient concentration of the phase. The acidification of the mobile phase with 0.012–1.0% formic acid ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Sawicki et al, 2016 , Sawicki et al, 2017 , Wang et al, 2020 ) or with trifluoroacetic acid (TFA) 0.05% ( Naderi et al, 2010 ) is common in this analysis to maintain the stability of the betalains structure during the process.…”

“…Some of the solvents used as mobile phases to separate betalains by LC are acetonitrile–water ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Sawicki et al, 2016 , Sawicki et al, 2017 , Wang et al, 2020 ), methanol–water ( Cejudo-Bastante et al, 2014 , Slavov et al, 2013 ), and acetonitrile buffer of KH 2 PO 4 (pH 2.74) ( Sanchez-Gonzalez et al, 2013 ) in different proportions, which are applied by eluting in isocratic mode or modifying the gradient concentration of the phase. The acidification of the mobile phase with 0.012–1.0% formic acid ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Sawicki et al, 2016 , Sawicki et al, 2017 , Wang et al, 2020 ) or with trifluoroacetic acid (TFA) 0.05% ( Naderi et al, 2010 ) is common in this analysis to maintain the stability of the betalains structure during the process. The coupling of LC to detectors, such as UV–Vis spectroscopy, diode array detection (DAD), and mass spectrometry (MS), has enabled the qualitative and quantitative analysis applied to the characterization of the betalain pigments profile from several natural sources, stability studies, and evaluations of the extraction efficiencies of specific betalains of particular interest.…”

“…In detection by UV–Vis spectroscopy, monitoring at a wavelength of 480 or 540 nm is recommended ( Melgar et al, 2019 , Naderi et al, 2010 , Slavov et al, 2013 ). For DAD, using a monitoring window at 477–484 and 535 nm is recommended ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Melgar et al, 2019 , Wang et al, 2020 ). In detection by MS by time-of-flight (TOF) applying electrospray ionization (ESI) ( Fathordoobady et al, 2016 , Ferreres et al, 2017 , García-Cruz et al, 2017 , Melgar et al, 2019 , Sawicki et al, 2016 , Wang et al, 2020 ) or by tandem mass spectrometry (MS/MS) ( Sawicki et al, 2017 ), operating the electrospray ionization source in positive mode is suggested.…”