4.7 7.

Article

Multipronged Approach to Profiling

Metabolites in Beta vulgaris L.
Dried Pulp Extracts Using
Chromatography, NMR and Other
Spectroscopy Methods

Joshua Fiadorwu, Kiran Subedi, Daniel Todd and Mufeed M. Basti

https://doi.org/10.3390/foods12183510
 foods
Article
Multipronged Approach to Profiling Metabolites
in Beta vulgaris L. Dried Pulp Extracts Using Chromatography,
NMR and Other Spectroscopy Methods
Joshua Fiadorwu 1 , Kiran Subedi 2 , Daniel Todd 3 and Mufeed M. Basti 1, *

1 Department of Applied Science and Technology, College of Science and Technology, North Carolina
Agricultural and Technical State University, Greensboro, NC 27411, USA; jefiadorwu@aggies.ncat.edu
2 Analytical Services Laboratory, College of Agriculture and Environmental Sciences (CAES),
North Carolina Agricultural and Technical State University, Greensboro, NC 27411, USA; ksubedi@ncat.edu
3 Triad Mass Spectrometry Facility, University of North Carolina at Greensboro, Greensboro, NC 27412, USA;
todd.da@pg.com
* Correspondence: basti@ncat.edu; Tel.: +1-(336)-285-2229

Abstract: Beetroot (Beta vulgaris L.) is known for being a rich source of phytochemicals, minerals
and vitamins. This study aims to show how the combination of extraction/chromatography/mass
spectrometry and NMR offers an efficient way to profile metabolites in the extracts of beetroot. Such
combination may lead to the identification of more nutritional or medicinal compounds in natural
products, and it is essential for our ongoing investigation to study the selective adsorption/desorption
of these metabolites’ on/off nanoparticles. The aqueous and organic extracts underwent analyses
using UV-vis spectroscopy; GC-MS; LC-MS; 1 H, 13 C, 31 P, TOCSY, HSQC, and selective TOCSY NMR
experiments. Polar Extract: The two forms of betalain pigment were identified by UV-vis and LC MS.
Fourteen amino acids, sucrose, and other compounds, among which is riboflavin, were identified
by LC-MS. Two-dimensional TOCSY showed the spin coupling correlations corresponding to some
of these compounds. The HSQC spectrum showed 1 H/13 C spin correlation in sucrose, confirming
its high abundance in beetroot. Organic Extract: GC-MS data enabled the identification of several
Citation: Fiadorwu, J.; Subedi, K.; compounds including six fatty acid methyl esters (FAME) with higher than, on average, 90% similarity
Todd, D.; Basti, M.M. Multipronged score. Selective TOCSY NMR data showed the spin coupling pattern corresponding to oleic, linoleic,
Approach to Profiling Metabolites in and linolenic fatty acids. 31 P NMR spectra indicate that phospholipids exist in both the organic and
Beta vulgaris L. Dried Pulp Extracts
aqueous phase.
Using Chromatography, NMR and
Other Spectroscopy Methods. Foods
Keywords: beetroot (Beta vulgaris L.); Total Correlation Spectroscopy (TOCSY); Gas Chromatography-
2023, 12, 3510. https://doi.org/
Mass Spectrometry (GC-MS); selective TOCSY; homonuclear single quantum correlation (HSQC);
10.3390/foods12183510
fatty acid methyl esters (FAME)
Academic Editor: Diego
García-Gómez

Received: 31 July 2023

Revised: 29 August 2023 1. Introduction
Accepted: 13 September 2023 Red beet or beetroot (Beta vulgaris L.) is a root vegetable in the Chenopodiaceae
Published: 21 September 2023 family [1,2] which is now cultivated throughout the world. The main components in beets
are water (87.57%), carbohydrates (9.56%), protein (1.61%) and lipids (0.17%) [3]. It is
also a great source of micronutrients, including minerals such as copper, iron, manganese,
sodium, calcium, magnesium, potassium, phosphorus, and selenium [4]; and vitamins such
Copyright: © 2023 by the authors.
as ascorbic acid ©, choline (vitamin B4), riboflavin, and niacin (vitamin B3) [5,6] as well
Licensee MDPI, Basel, Switzerland.
as dietary nitrates [7]. Additionally, beets contain phytochemicals, such as polyphenols,
This article is an open access article
flavonoids, and betalains. The polyphenolic compounds in fruits and vegetables are
distributed under the terms and
responsible for their antioxidant effects [8]. The highest amount of phenolic content is found
conditions of the Creative Commons
Attribution (CC BY) license (https://
in the beets’ peel, followed by the crown, and then the flesh [9]. Beets contain a number of
creativecommons.org/licenses/by/
flavonoids, among which are quercetin (0.13 mg/100 g) and luetolin (0.37 mg/100 g) [10].
4.0/).
Flavonoids are powerful antioxidants with anti-inflammatory and immune system benefits.

Foods 2023, 12, 3510. https://doi.org/10.3390/foods12183510 https://www.mdpi.com/journal/foods

Foods 2023, 12, 3510 2 of 12

Diets rich in flavonoid-containing foods are sometimes associated with the prevention of
cancer, neurodegenerative disease and cardiovascular disease [11].
Beets also contain p-coumaric acid, feruloylamaranthin, and ferulic acid [9], where the
antioxidant properties of p-coumaric acid helps in lowering oxidative stress and inflamma-
tion [12]. Feruloylamaranthin helps in lowering inflammation and cholesterol levels and
facilitates weight loss [13]. Ferulic acid has significant antioxidant and anti-inflammatory
properties [14]. Beets also contain polysaccharides, including galacturonan, glucose polysac-
charides (28–39% as cellulose [15] and high-methylated pectin ~70%). Cellulose provides
structural support to the cell wall in plants, and when it is consumed, it serves as energy
storage and aids in gastrointestinal function [16]. High-methylated pectin helps to improve
blood sugar, reduce fat levels, and facilitate weight loss and the digestion of food [17]. Thus,
beets are a good source of many nutritional and medicinal compounds.
Betalains, the major phytochemicals found in beets, are water-soluble nitrogen-containing
pigments that impart the red–purple natural color to the beet. They are classified into
two structure-based groups: the red violet betacyanins and the yellow betaxanthins [18].
Betalain exhibits antioxidant, anti-inflammatory and antiviral properties [19,20]. Betalain, in
its form as betacyanin, contains a cyclic amine group and a partly glucosized phenolic group
that is responsible for its strong antioxidant effects [21]. The disadvantage of betalains is
that they have a relatively low bioavailability, which limits their physiological potential [22].
The application of ultraviolet-visible (UV-Vis), infrared (IR) and nuclear magnetic
resonance (NMR) spectroscopic methods in combination with chromatography are among
the key analytical techniques to profile metabolites in natural products [23]. One of the pur-
poses for such analysis is to identify active medicinal compounds among these metabolites.
NMR spectroscopy with its numerous approaches and nondestructive properties has been
shown to be an essential tool in plants’ biochemical and medicinal research.
In this study, several techniques, including GC-MS, LC-MS, and other spectroscopy
methods such as UV-Vis and NMR, were utilized to demonstrate how the collaborative
use of these tools offers an efficient approach to profile metabolites in both the organic
and aqueous extracts of dried beetroot. The multi-analytical-tool approach indicated the
advantages that each tool provides to yield a more comprehensive profiling of metabolites
in the extracts of a natural product. When such an approach is utilized with the organic
phase extract of beetroots, it reveals the presence of some bioactive compounds, such as
dehydroabietic acid [24], which has pronounced antiviral, antitumor [25], wound healing,
and antibacterial activities [26]. The use of 1D NMR selective TOCSY, which requires
less instrument time and is more sensitive than 2D TOCSY, enabled the quick profiling of
metabolites that are not that prevalent, such as the unsaturated fatty acids in the organic
phase extract whose percentage, as shown in the results section, is significantly less than the
aqueous phase. The 31 P NMR data (Figure S6) indicated that orthophosphate monoesters
and orthophosphate diesters exist in both aqueous and organic phases. Thus, the adopted
approach in this report can facilitate the discovery of more phytochemical and medicinal
metabolites. The approach is also essential prior to the investigation of the use of nanopar-
ticles to selectively adsorb certain metabolites from the aqueous and organic phase extracts,
which is ongoing in our lab.

2. Materials and Methods

2.1. Chemicals and Reagents
Fresh beetroots were obtained from a supermarket in Greensboro, NC, USA. Methanol,
HPLC grade chloroform and hexane were purchased from Fisher-Scientific, Waltham,
MA, USA. Deuterated chloroform (CDCl3 ) with 1% v/v 3-(Trimethylsilyl)propionic-2,
-3-(Trimethylsilyl)prop-98 atom % D (TSP) was obtained from Acros Organics, Morris
Plains, NJ, USA. Optima grade water and Acetonitrile for LCMS were obtained from
Fisher-Scientific, Waltham, MA, USA. Sodium phosphate dibasic (Na2 HPO4 , 99.0%) was
obtained from Alfa Aesar, Tokyo, Japan; sodium phosphate monobasic (NaH2 PO4 , 99.0%),
and sodium azide 99% extra pure were obtained from Acros Organics, Morris Plains, NJ,
Foods 2023, 12, 3510 3 of 12

USA. L-lysine (C6 H14 N2 O2 , 98.5%), L-leucine (C6 H13 NO2 , 98.5%), L-histidine (C6 H9 N3 O2 ,
98.5%), L-phenylalanine (C9 H11 NO2 , 98.5%) and sucrose (C12 H22 O11 ) were purchased from
Fisher BioReagents, Pittsburgh, PA, USA.

2.2. Sample Preparation

Beets were peeled and diced into small pieces on a watch glass and weighed. The
diced beets were dried in a convective air oven (ThermoScientific Heratherm OGS 180,
Waltham, MA, USA) for 24 h at 53 ◦ C [27]. Drying continued at 53 ◦ C for an additional 2 h
until a constant mass was reached.

2.3. Extraction
Dried samples were ground using a coffee grinder to obtain a fine powder. About
0.05 g of the powdered beets were placed in a microcentrifuge tube equipped with glass
beads; 0.5 mL aqueous methanol (66%/34% v/v) and 0.5 mL chloroform were added. The
sample was then placed in a BioSpec Mini BeadBeater16 at 3400 rpm for 10 min and then
centrifuged at 14.8 × 103 rpm for 10 min at 20 ◦ C using Legend Micro 2LR centrifuge,
Fisher-Scientific, Waltham, MA, USA.
Separation: After the centrifugation, three separate layers resulted from the extraction:
polar (top), non-polar (bottom) and a solid layer in between. The top phase contained polar
compounds dissolved in aqueous methanol. The bottom phase contained the non-polar
compounds dissolved in chloroform. The top polar and bottom non-polar phases were
micro-pipetted into separate microcentrifuge tubes and placed along the microcentrifuge
tube containing the middle layer into a Savant SpeedVac SPD1030 Integrated Vacuum
Concentrator, Fisher-Scientific, Waltham, MA, USA, at room temperature and a pressure of
8 torr for 4–6 h. The amounts of the three dried phases were then determined.

2.4. Instrumental Analysis

UV-Vis: A small portion of the polar extract was dissolved in 4 mL of sodium phos-
phate buffer. The UV-VIS absorption spectrum was recorded in the range 250–750 nm on
a Shimadzu UV-2501 PC Spectrophotometer in quartz cuvettes in the absorption mode,
where sodium phosphate buffer was the reference.
GC-MS: The methyl esterification of the non-polar phase was carried out using the
standard method [28]. Gas Chromatography-Mass Spectrometry (GC-MS) data was col-
lected using an Agilent 7890 B GC system featuring a 7693 A autosampler provided by
Agilent. Mass detection was accomplished utilizing the 5977 GC/MSD instrument.. For the
GC system, an Agilent GC HP-5 capillary column (30.0 m length, 0.25 mm i.d., 0.10 µm film
thickness) was used. The temperature program was set up starting at 100 ◦ C for 3 min and
programmed to increase to 200 ◦ C for 1 min, and ramped up to 250 ◦ C at 10 ◦ C/min, and
remained at 250 ◦ C for 10 min for a total program time of 15 min. Both the injector and de-
tector temperatures were 250 ◦ C and Helium gas was used as the carrier gas. The injection
volume was 2 µL. Ionization was conducted by electron impact (EI) and Ionization energy;
an IE of 70 eV was used for the mass spectroscopy detector with a source temperature at
230 ◦ C and transfer line temperature of 250 ◦ C. The scan range of the fragments was set to
40–500 amu. The fragmentation pattern in the experimental mass spectra were compared
with the NIST20.L Mass Spectral Library. Data were acquired using GC-MS acquisition
software (mass hunter qualitative analysis 10.0).
LC-MS: Liquid chromatography separation of the metabolites in the polar phase
was performed on a Thermo Fisher Q Exactive Plus Mass Spectrometer, Fisher-Scientific,
Waltham, MA, USA coupled to a Waters Acquity Ultra-Performance Liquid Chromatogra-
phy system using a Waters Acquity HSS (100 mm × 2.1 mm) column, Waters Corporation,
Milford, MA, USA. A 3 µL sample injection was eluted at a flow rate of 0.4 mL/min from
the column employing a binary solvent system comprising 0.1% formic acid in water
(designated as mobile phase A) and 0.1% formic acid in acetonitrile (designated as mobile
phase B). The gradient program was as follows: 0–1 min 100% A, 1–11 min 100% A–0%
Foods 2023, 12, 3510 4 of 12

A, 11–13.1 min 0% A–100% A, and 13.1–15 min 100% A. The LC eluent was directed into,
without splitting, a Thermo Fisher Q Exactive Plus mass spectrometer, Fisher-Scientific,
Waltham, MA, USA, fitted with a Heated Electrospray ion (HESI) source, and the MS was
operated using the following parameters: source, heated electrospray ionization (HESI);
polarity, Pos/Neg switching; capillary voltage, 2500 V; capillary temperature, 262.5 ◦ C;
sheath gas 50 L/min; auxiliary gas and spare gas set at 12.5 and 2.5 units, respectively, and
the heater temperature was set at 425 ◦ C. The LC-MS data were acquired over a scan range
of 75–1125 amu.
NMR: Deuterated chloroform (CDCl3 ) with 1% TSP as internal reference (0 ppm)
was used to dissolve the non-polar phase. The polar phase was dissolved in a sodium
phosphate buffer (pH 7.4) that contained TSP and 0.5% sodium azide in 90% water/10%
D2 O. The NMR spectra were acquired on a Bruker Ascend 400 MHz spectrometer at 25 ◦ C.
Standard 1D NOESY pulse sequence (with HDO presat pulse for the polar phase) was
used to acquire the 1 H spectrum. One-dimensional selective TOCSY data were collected
using the homonuclear Hartman–Hahn (HOHAHA) transfer pulse sequence, where the
MLEV17 sequence was used for mixing and the selective excitation was obtained using a
shaped pulse and Z-filter [29] with varying mixing times (0.03, 0.08, 0.12 s); the number
of scans was set to 256. The data were processed with LB of 0.1–1.0 Hz. Two-dimensional
NMR correlation spectroscopy (COSY) spectra were acquired using standard non-phase
sensitive sequence (2D homo-nuclear shift correlation [30]). Data were collected with
2KX256 data points matrix, then zero-filled to 2KX1K data points matrix. Total COSY
(TOCSY) 2D spectra were acquired using phase sensitive homonuclear Hartman–Hahn
(HOHAHA) transfer using MLEV17 sequence for mixing [31] with 2KX256 data points,
and zero filled to 2KX1K data point matrix.1 H- 13 C single quantum correlation (SQC) data
were acquired using the phase sensitive, 2D H-1/X correlation via double inept transfer
using the sensitivity improvement pulse sequence [32]. Data were acquired in 2KX256 data
points and zero-filled to 2KX1K data points.

3. Results and Discussion

3.1. Extraction
Based on four trials, the extraction data indicate that the average percentages of the
aqueous and organic phases are 33.50 and 3.05, respectively. The average percentage of the
middle solid layer that contains compounds that are not soluble in water/methanol or in
chloroform is 63.45.

3.2. Aqueous (polar) Phase

The UV-Vis spectrum of the aqueous phase in Figure 1 shows the two signals corre-
sponding to the two forms of the betalain pigment: the red–purple betacyanins at 538 nm
and the yellow betaxanthins at 484 nm [18]. The relative intensity of the two signals in
Figure 1 is consistent with the higher composition of the red–purple betacyanins relative to
that of the yellow betaxanthins [18].
Figure S1 in the supplementary material shows the MS fragmentation spectrum of the
compounds in the aqueous extract of beetroot powder in positive polarity mode. The major
difference in ionizability of the identified compounds in the polar phase (Table 1) rendered
the intensity of the signals in the LC-MS spectrum to be widely varied (Figure S1). Most
identified compounds by LC-MS data were found by searching for their corresponding
ions in the positive mode.
Foods 2023, 12, 3510 5 of 12

3.0

2.5

Absorption Intensity (a.u.)

2.0

1.5

1.0

0.5

0.0
300 400 500 600 700
Wavelength (nm)
Figure 1. UV-vis spectrum of aqueous beetroot extract.

Table 1. Identified compounds in the aqueous extract using LC-MS in positive polarity mode.

Molecular Measured Measured Standard Rt (min) of Confirmed by

Compound
Formula [M + H]+ (m/z) Rt (min) [M + H]+ (m/z) Standard NMR
Lysine C6 H14 N2 O2 147.1131 0.58 147.1126 NA No
Histidine C6 H9 N3 O2 156.0771 0.61 156.0765 0.61 No
Arginine C6 H14 N4 O2 175.1193 0.64 N/A NA No
Threonine C4 H9 NO3 120.0659 0.68 N/A NA No
Glutamic acid C5 H9 NO4 148.0606 0.68 N/A NA No
Valine C5 H11 NO2 118.0867 0.71 N/A NA Yes
Proline C5 H9 NO2 116.0710 0.77 N/A NA No
Sucrose C12 H22 O11 343.1239 0.98 343.1229 0.98 Yes
Glucose C6 H12 O6 181.0710 0.77 N/A NA Yes
Methionine C5 H11 NO2 S 150.0587 1.25 N/A NA No
Leucine C6 H13 NO2 132.1022 2.53 132.1018 2.49 Yes
Isoleucine C6 H13 NO2 132.1023 2.68 N/A NA Yes
Tyrosine C9 H11 NO3 182.0816 2.68 N/A NA No
Betacyanin C24 H26 N2 O13 551.1520 2.96 N/A NA Yes
Phenylalanine C9 H11 NO2 166.0867 3.02 166.0860 3.02 No
Tryptophan C11 H12 N2 O2 205.0975 3.48 N/A NA No
Riboflavin C17 H20 N4 O6 377.1442 3.85 N/A NA No
Betaxanthin C18 H18 N2 O6 359.1247 4.11 N/A NA Yes
Theanine C7 H14 N2 O3 175.1078 13.82 N/A NA No

Table 1 lists the identified compounds in the aqueous extract from the LC-MS results
(Figure S1) and the standards when applicable. Fourteen amino acids were identified. The
NMR signals corresponding to some of these amino acids were also identified, as shown
below (Figure 2).
Foods 2023, 12, 3510 6 of 12

Figure 2. 400 MHz 1 H-NMR spectrum of aqueous extract in phosphate/D2 O buffer with 0.5% 3-
(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP). Inset (A) shows 10× magnification of the
upfield region; (B) shows 20× magnification of the downfield region.

Figure 2 shows the 1 H NMR spectrum of the aqueous extract that indicates the sig-
nificant variance in the composition of the different compounds, where the intensity of
the signals corresponding to the aromatic compounds is much less than the intensity of
the signals corresponding to other compounds such as sugars and amino acids. Insert
A in Figure 3 shows an expansion of the up-field region of the 1D NMR spectrum be-
tween 0.5 and 3.2 ppm, and insert B shows the region of the spectrum where aromatic
compounds resonate.

Figure 3. A section of the TOCSY spectrum of aqueous extract shows the spin coupling between C1 H
of sucrose and of glucose and the corresponding protons.
Foods 2023, 12, 3510 7 of 12

Figure 3 is the section of the 2D TOCSY spectrum of the aqueous extract, which exhibits
spin coupling between C1 H of sucrose and the other protons in it. The spectrum also shows
the cross peaks corresponding to coupling of C1 H proton in the α- and β- forms of D-
glucopyranose to other protons in them. Table S1 in the supplementary material lists α the β
chemical shift of the identified protons in the two sugars and the corresponding literature
chemical shift values [33]. It is interesting to note that a part of the betacyanins dye is
a D-glucopyranose-like six-member ring [18], which means that some of the observed
couplings in Figure 4 could belong to the betacyanins pigment. The relative intensity
of C1 H signals of α- and β-forms of D-glucoyranose in Figure 3 is consistent with the
literature, indicating that the αβ formβis more abundant than the α form [34]. Figure 4 is
the section of 2D HSQC spectrum thatβshows the 1 H/13 C correlation corresponding
α to
13
sucrose; the chemical shift values of the sucrose C signals are listed in Table S1 along
with the corresponding literature values. Figure 5 shows the section of COSY spectrum
that exhibits the spin coupling between the two methyl groups of valine and C2 H proton.
Figures S2 and S3 in the supplementary material show the sections of COSY spectrum
that exhibit spin coupling corresponding to the isoleucine. Figure S4 in the supplementary
material shows combined sections from the COSY spectrum of the aqueous phase, which
display the coupling corresponding to leucine. Table S2 lists the chemical shift of the
identified protons in leucine, isoleucine and valine along with the literature chemical shift.
It Is interesting to note the similarity between the chemical shift of 1 H and 13 C signals of
the all unambiguously identified compounds and the literature data (Tables S1 and S2 ),
where the literature reported chemical shift values are from the spectra of pure compounds,
indicating that there is no significant matrix effect on the chemical shift. Many of the
bioactive compounds in beetroots, such as flavonoids and p-coumaric acid, are aromatic
compounds. The 1D NMR spectrum in Figure 2 and insert B show that the intensity of the
signals corresponding to the aromatic compounds is much lower than that of the sugar
signals, which are in the range of 3 to 5.5 ppm. This indicates that the composition of the
aromatic compounds is significantly lower than that of sugars, which made detecting the
spin systems corresponding to them, including the pigment’s signals, more difficult, even
while using the 1D selective TOCSY technique, which is more sensitive than 2D NMR
techniques. Insert A in Figure 2 shows the significant overlap of signals in the ppm range of
0.90 to 1.05 ppm, where the methyl groups usually resonate. Figures 5 and S2–S4 show how
2D NMR experiments can be utilized efficiently to identify some of the molecules whose
NMR signals show significant overlap. The figures also indicate how NMR techniques can
efficiently complement LC-MS data.

Figure 4. The section of the 400 MHz 2D-HSQC spectrum of aqueous extract shows the correlation
between 1 H and 13 C of sucrose.
Foods 2023, 12, 3510 8 of 12

Figure 5. A section of the COSY spectrum showing the spin coupling corresponding to Valine.

3.3. Organic Phase

Figure 6 shows the GC-MS chromatogram of the organic extract of dried beetroot
after chemically converting the fatty acids to methyl esters; the retention time of the
eluted compounds ranges between 6.50–25.50 min. For the identification of fatty acids
methyl esters (FAME), and other compounds in the organic phase, retention times and the
MS ionization spectra of the experimental data were compared with the corresponding
spectra from the NIST20 library [28]. For example, Figure S5 shows the MS experimental
and NIST20 library spectra of 9,12-Octadecadienoic acid methyl ester (RT 10.359 min).
Figures S6–S9 in the supplementary material show the matching MS spectra for Linolenic
acid, Oleic acid, Stearic acid, and Palmitic acid, respectively. The similarity between the
fragments in the two MS spectra was reported as matching/similarity score. Table 2
lists the identified compounds in the organic phase along with their molecular formula
and their corresponding retention times, base peak signal-to-noise ratio, base peak area,
and the similarity scores being 88% and above. Figure 7a shows the 1 H NMR spectrum
of the organic phase, while traces 7b, c and d are the selective TOCSY traces that were
used to identify the spin coupling in linolenic, linoleic and oleic acids [35], respectively,
where certain signals were selectively excited in each trace. The broad peak at 5.36 ppm
corresponds to the olefinic protons (protons 9, 10, 12, 13, 15, and 16) in linolenic acid,
(protons 9, 10, 12, and 13) in linoleic acids and protons 9 and 10 of oleic acid. The multiplet
at 2.77 ppm corresponds to the bis-allylic CH2 protons 11 and 14 in linolenic acid, and
protons 11 in linoleic acid. The multiplet at 2.31 ppm (designated as peak 2) corresponds
to the CH2 group adjacent to the carboxylic group in the three fatty acids. The multiplet
at 1.60 ppm (designated as peak 3) corresponds to the CH2 group next to the CH2 group
labeled as two in the three fatty acids. The multiplet at 2.06 ppm corresponds to the allylic
CH2 in the three fatty acids: protons 8 and 17 in linolenic acid, 8 and 14 in linoleic acid
and 8 and 11 in oleic acid. The intense peak at 1.26 corresponds to all other CH2 groups
in the three and the other saturated fatty acids. The peak at 0.88 ppm corresponds to the
terminal methyl in all fatty acids. When the peak at 1.26 ppm (the overlaping CH2 groups)
was selectively excited (trace 7b), the predicted spin coupling correlations in the three fatty
acids were observed according to the above-mentioned assignments. Similarly, when the
peaks at 2.06 ppm (allilic CH2 (s)) and 0.88 ppm (terminal methyl) were selectively excited
(traces 7c and d, respectively), the predicted spin coupling correlations in the three fatty
acids, according to the above-mentioned assignments, were observed. Table S3 shows the
Foods 2023, 12, 3510 9 of 12

chemical shift of the assigned protons in fattty acids, where the reported chemical shift
values in the literature are identical [35]. The selective TOCSY experiment proves to be
useful in assigning signals belonging to unsaturated fatty acids. Thus, in a mixture of
chemical compounds similar to the aqueous or organic phase of the extracts of a natural
product, selective TOCSY is a good tool to identify metabolites when these metabolites have
unique chemical properties such as unsaturated carbons or aromatic ring. Still, one has to be
aware of the signal overlaps that prohibit the identification of all the peaks in the selective
TOCSY traces, like the peak at 2.90 ppm in trace 7b. Figure S10 in the supplementary
material shows the proton-decoupled 31 P spectra of the aqueous phase extract in D2 O (A)
and the organic phase extract in CDCl3 (B). The chemical shift of 31 P peaks in both media
falls between 0 and 6.5 ppm. The peaks were then assigned to orthophosphate monoester
and diester [36]. Thus, both mono and diester exist in the two phases. The difference in the
chemical shifts of the phosphorus signals in the two phases has to do with R groups being
different and the solvent systems used (D2 O and CDCl3 ).

Figure 6. GC-MS chromatogram of esterified organic extract of dried beets.

Table 2. MS identification of compounds from esterified organic extract of dried beets. The methyl
esters of the fatty acids are presented in a bold label.

RT Base Peak Base Peak Match/Similarity

Label Compound Name Formula
(min) S/N Ratio Area Score (%)
I 8.41 9.73 × 102 6.15 × 106 Hexadecanoic acid, methyl ester C17 H34 O2 93.9
II 9.33 3.88 × 101 2.69 × 105 3-Methylbenzoic acid, 2,5-dichlorophenyl ester C14 H10 Cl2 O2 88.7
III 9.93 1.0 × 103 2.69 × 106 Methyl stearate C19 H38 O2 96.5
IV 10.03 2.35 × 102 7.04 × 105 9-Octadecenoic acid, methyl ester, (E)- C19 H36 O2 90.7
V 10.36 3.42 × 102 3.70 × 106 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C19 H34 O2 91.9
VI 10.85 3.08 × 102 6.45 × 105 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- C19 H32 O2 96.4
VII 11.99 3.58 × 103 2.42 × 107 Dibutyl phthalate C16 H22 O4 91.4
VIII 12.59 6.44 × 101 1.04 × 106 Pentacosane C25 H52 91.7
IX 13.74 1.66 × 102 1.57 × 106 n-Hexadecanoic acid C16 H32 O2 92.7
X 14.77 4.59 × 101 1.07 × 106 Octacosane C28 H58 90.1
XI 16.95 8.14 × 101 1.31 × 106 Octadecanoic acid C18 H36 O2 92.6
XII 19.46 2.31 × 102 4.92 × 105 3,5-di-tert-Butyl-4-hydroxyphenylpropionic acid C17 H26 O3 90.0
XIII 21.21 1.50 × 102 1.02 × 106 Oxybis(propane-1,2-diyl) dibenzoate C20 H22 O5 90.7
XIV 23.14 2.38 × 102 1.20 × 106 Diethylene glycol dibenzoate C18 H18 O5 97.2
XV 23.94 1.87 × 102 3.80 × 105 Dehydroabietic acid C20 H28 O2 88.1
Foods 2023, 12, 3510 10 of 12

Figure 7. Selective TOCSY data showing the spin coupling correlation in linolenic, linoleic and oleic
acids (mixing time 0.080 s). (a) Shows the 1 H-NMR spectrum of the organic phase, while traces
(b–d) are the selective TOCSY traces that were used to identify the spin coupling in linolenic, linoleic
and oleic acids, respectively.

4. Conclusions
The current report is an evidence of the efficacy of combining chromatography and
spectroscopy data in profiling metabolites and bioactive compounds in beetroot specifically,
and, more generally, in natural product research. The report shows how NMR in its
multifaceted tools, such as 1D of different nuclei and the variety of homo-nuclear and
hetero-nuclear 2D experiments, can efficiently complement LC/MS and GC/MS. For
example, the metabolites detected by LC/MS data (Table 1) include glucose, while 2D
TOCSY data (Figure 3) showed the spin coupling corresponding to the two forms of glucose
α and β. The projection along second dimension in Figure 3 shows the relative abundance
of these two glucose forms. The methyl groups of amino acids resonate up-field from most
signals. Albeit the significant signal overlap, Figures 5 and S2–S4 demonstrate how 2D
NMR can be utilized to assign overlapping signals. The chemical shift of the 1 H, 13 C and 31 P
signals that correspond to the unambiguously identified metabolites in both aqueous and
organic phases (Tables S1–S3) match very well with the chemical shift of the corresponding
metabolites in the literature, where these chemical shifts were obtained from the spectra of
pure metabolites. This indicates that the matrix effect on the chemical shift is negligible,
which means that the literature chemical shift values can be effectively used in identifying
metabolites in the NMR spectra of natural products. The low abundance of the aromatic
metabolites in the aqueous phase (Figure 2), such as polyphenols and the two forms of the
pigments, prohibited the unambiguous assignment of these compounds, even by using 1D
selective TOCSY experiments. UV-vis (Figure 1) and LC/MS (Figure S1 and Table 1) were
the effective tools to identify such metabolites, indicating the collaborative nature of the
tools used in this report. The 31 P NMR spectrum of the aqueous and organic phases indicate
that ortho monoester and diester phosphates are in both phases. The difference in polarity
of the two phases suggests that the R groups in the mono and dieters of the organic phase
are longer, which makes these esters more hydrophobic. Finally, the profiling of metabolites
and the analytical tools used in this report prove to be valuable in the ongoing investigation
in our lab to study the use of nanoparticles to selectively separate these metabolites as they
are selectively adsorbed on the nanoparticles.
Foods 2023, 12, 3510 11 of 12

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/foods12183510/s1, Figure S1 shows the LC/MS of the aqueous phase.
Figures S2–S4 show the COSY spectra used to display the spin couplings corresponding to the
identified amino acids. Figures S5–S9 show the matching between experimental and library MS
spectra corresponding to fatty acids in the organic phase. Figure S10 shows solution proton decoupled
31 P NMR spectra of the polar extract (A) in D O and the non-polar extract (B) in CDCl on a 400 MHz
2 3
spectrometer, ns = 10,000, 90◦ pulse, 12.38 µs, 2.0 s relaxation delay pulse delay, 25 ◦ C temperature,
and 20 Hz line-broadening. Tables S1–S3 list the NMR chemical shift of identified metabolites and
the corresponding literature values.
Author Contributions: J.F. is a graduate student in M.M.B.’s lab. He prepared all samples, acquired
UV-Vis and NMR spectra. Also, under the supervision of M.M.B., J.F. carried out the data analysis, and
prepared the manuscript. K.S. supervised the acquisition and analysis of GC-MS data. D.T. supervised
the acquisition and analysis of LC-MS data. M.M.B. was part of the project conceptualization and
supervised the NMR data acquisition and analysis, and supervised the manuscript’s preparation. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding. The dean of the College of Science and
Technology and the director of the Applied Science and Technology program offered financial
support to Mr. Fiadorwu.
Data Availability Statement: Between the data in the figures in the manuscript and the supplemen-
tary material most data are presented. Any other data that the science community shows interest in
can be shared upon request from the corresponding author.
Acknowledgments: We thank the Dean of the College of Science and Technology, Abdellah Ahmi-
douch, for his support, chemicals and materials, as well as the research assistant stipend for Fiadorwu.
We also acknowledge the current and former director of the Applied Science and Technology PhD
Program, Jenora D. Waterman and Keith Schimmel, for their support towards chemicals and ma-
terials and the research assistant stipend. We also acknowledge the guidance of Salam Ibrahim,
research professor of Food Sciences, Department of Family and Consumer Sciences at NC A&T State
University, in manuscript preparation. The authors extend their formal gratitude to the Analytical
Services Laboratory (ASL) at the College of Agriculture and Environmental Sciences (CAES) for their
invaluable contributions to the chromatographic and mass spectrometric analyses.
Conflicts of Interest: The authors declare no conflict of interest.

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