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Received: 4 February 2019 Revised: 31 March 2019 Accepted: 17 April 2019
DOI: 10.1002/sscp.201900018
RESEARCH ARTICLE
Quantification of eight water soluble vitamins in Sutherlandia
frutescens species from Botswana using a validated reversed phase
HPLC method
Moshood A. Abibu1,2 David T. Takuwa1Kwenga Sichilongo1
1Department of Chemistry, Faculty of
Science, University of Botswana, Private Bag
UB 00704 Gaborone, Botswana
2Department of Science Laboratory
Technology, Federal Polytechnic Ede,
Osun-State, Nigeria
Correspondence
Professor KwengaSichilongo, University
of Botswana, Faculty of Science, Depart-
ment of Chemistry, PB UB 00704, Gaborone,
Botswana.
Email: kwenga.sichlongo@mopipi.ub.bw
A simple and rapid-reversed phase high-performance liquid chromatography method
for the separation and quantification of water-soluble vitamins; B1,B
2,B
3,B
4,B
6,B
9,
B12 and C from Sutherlandia frutescens (a traditionally used medicinal plant found in
Botswana), is presented. Vitamins were extracted using ultrasonic assisted extraction
while clean-up was done by solid-phase extraction with silica-based C-18 cartridges.
Eight vitamins were separated and identified using an Agilent 1260 Infinity HPLC
system fitted with a diode array detector. Two different types of silica-based bonded
C18 columns of equal length and internal diameter were compared for their separation
efficiencies using a methanol/formic acid mobile phase system at flow rates of 0.5 and
1.0 mL/min on a gradient elution programme. The separation of eight water-soluble
vitamins was achieved in 12 min at 266 nm. Analytical performance charactristics
were evaluated for percent recovery, precision as percent relative standard deviation,
limits of detection, limits of quantification and linearity for each vitamin. The method
developed herein was applied to determine some water soluble vitamins in a Suther-
landia frutescens plant from Botswana.
KEYWORDS
Botswana, solid-phase extraction, Sutherlandia frutescens, vitamins
1INTRODUCTION
Sutherlandia frutescens (S. frutescens), also known as “cancer
bush” is an indigenous Southern African herbal plant which
biologically belongs to the family of Fabaceae (legume, pea
and bean). It is the third largest family of flowering plants [1–
3]. In Botswana, this plant is locally known as “Lerumo
La Madi” and occurs naturally throughout the dry parts of
Southern Africa. From literature [4–6], it has been reported
that S. frutescens is traditionally used for the treatment of poor
appetite, indigestion, stomach complaints, dysentery, colds,
influenza, kidney conditions, fever, internal cancers, uterine
troubles, liver conditions, backache, rheumatoid arthritis, uri-
nary tract infections, stress and anxiety, dropsy, heart failure
and liver problems [7]. It has also traditionally been used as a
blood purifier and as an all-purpose tonic. It has been demon-
strated scientifically that S. frutescens regulates insulin levels
and glucose uptake in peripheral tissues and lowers glucose
uptake by the intestines [8,9]. It also inhibits resistance to
insulin and has been observed to significantly reverse lipid
accumulation in cell lines due to presence of fructose and
insulin. Other in vitro and in vivo experiments have also
shown its antidiabetic activities [10–12]. S. frutescens has
also been shown to possess anti-inflammatory properties via
promotion of tissue remodeling and immune regulation [13].
In Botswana, despite the availability of modern and
affordable medical facilities, the curative potential of plants
has long been recognized. Currently, plants contribute a sig-
nificant source of therapy to Batswana’s traditional medicine
practice and the consumption of herbal medicine is very
common among people living in South-West, North-East
and South-Eastern parts of the country [3,14,15]. There
Sep Sci plus 2019;1–10. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1www.sscp-journal.com
2ABIBU ET AL.
are different types of medicinal plants which have curative
potential and are sold as side-dish medicines in the market by
Batswana traditional healers. These have been employed in
the treatment of various ailments. It has been claimed that the
use of herbal tea leaves of the traditional medicinal plants,
provide health benefits due to the minerals, vitamins and
fibre contents. It has also been reported that, plants that are
rich in anti-oxidant vitamins play a protective role in health
and against incidental mortality from diabetis, cancers, heart
disease, fever, flu, hypertension and stroke [14].
Prevention and cure of many free radical diseases using
anti-oxidant compounds especially compounds or molecules
that bear an unpaired electron like vitamins have been docu-
mented [16]. Coincidentally, S. frutescens has been used else-
where traditionally for centuries for the treatment of various
ailments such as diabetis, cancers, heart diseases, fever, flu
and hypertension [5,6]. The presence of water-soluble vita-
mins in S. frutescens could probably add credit to its wide use
as a food supplement and medicine and this could also under-
score the medicinal value of water-soluble vitamins that could
be present in this plant which have not been investigated and
reported in literature.
Vitamins are a low molecular weight group of organic
compounds required in small quantities to perform various
chemical and physiological functions for normal growth, and
maintenance of human and animal bodies. They are derived
from plant and animal sources. There are 13 vitamins that
are classified according to their solubility into water-soluble
vitamins (B-group vitamins and vitamin C) and fat-soluble
vitamins (A, D, E, and K) [17]. Several of the vitamin
B-group including vitamin C, mainly serve as biological
catalysts for certain metabolic reactions in the body and are
only required in very small amounts. It has been reported that
plants rich in anti-oxidant vitamins, play a protective role in
health and against incident mortality from diabetic, cancers,
heart disease, fever, flu, hypertension and stroke [15]. Due to
these considerations, there has been a resurgence of research
on accurate and efficient analytical methods for the determi-
nation of vitamins in complex herbal preparations [18,19].
Several analytical methods are available, but most of them
are time-consuming or not accurate for quantification [20].
Some of these techniques include thin-layer chromatography
(TLC) [21], capillary electrophoresis (CE) [22], spectrome-
try [23], spectrofluorometry [24], enzymatic [25], microbi-
ological assay [26], gas chromatography (GC) [27,28] and
high-performance liquid chromatography (HPLC) [29–34]
TLC has been widely used for the separation and quan-
titation of plant metabolites [35]. The inexpensive and
disposable nature of TLC plates allows it to be used to
identify impurities that may cause damage to HPLC columns
and detectors. Therefore, TLC still plays a distinct role in the
analysis of mixtures of metabolites from crude plant extracts.
Recently however, TLC has been demonstrated to have a lot of
limitations and cannot be applied in the quantitative analysis
of vitamins [35].
Microbiological assays have been used for the routine
determination of vitamins [36,37]. However, these assays are
time consuming as they require tedious sample preparation
and multiple determinations to achieve the required precision
for estimation of the mean concentration value.
Spectrometry and CE techniques are not widely used in
vitamin determination owing to their low sensitivities [38].
GC is not widely used for the determination of vitamins due
to the limited volatility of many of the vitamins [27] as well as
their sensitivity to hot GC injectors and certain types of sta-
tionary phases. This raises the need for sample pre-treatment
with derivatization agents prior to analysis, thus, making the
analysis time consuming and the vitamins prone to sample
degradation due to the high temperatures that are involved in
the separation processes.
HPLC is one of the preferred techniques for separation
of various vitamins. It is favoured because it offers advan-
tages such as convenience, specificity, selectivity and pre-
serves analytes in their original state during and after sepa-
ration [39]. In contrast to methods discussed above, HPLC is
also relatively more accurate in the determination of vitamins
with ultraviolet-visible (UV/Vis) using diode array, fluori-
metric, electrochemical or mass spectrometric detections. The
aim of this study therefore, was to develop a simple, rapid and
sensitive HPLC-DAD method to allow simultaneous determi-
nation of 8 water-soluble vitamins in medicinal plants from
Botswana. The vitamins targeted in this study are shown in
Figure 1. It is worth noting that despite vitamin B4(adenine)
not being classified as a B vitamin, it was included in this
study due to the unavailability of vitamin B5.
2MATERIALS AND METHODS
2.1 Materials and reagents
Chemicals and reagents were purchased from Sigma-Aldrich,
(Poole, Dorset). Methanol, vitamin B1,B
2,B
3,B
4,B
6,B
9,
B12 standards were purchased from Rochelle Chemicals
(Johannesburg, South Africa) while vitamin C standard was
from Merck (Carolina, USA). Formic acid (99%), potassium
hydroxide pellets and potassium hydrogen carbonate salt
were from uniLAB (Mandaluyong, Philippines). All water-
soluble vitamin standards were of analytical grade. Methanol
and all other reagents/solvents were of HPLC grade. Double
distilled water used for HPLC analysis was prepared by
an Elix® Millipore deionizer (Saint-Quentin-en-Yvelines
Cedex, France). Sep-Pak C18 solid phase extraction (SPE)
cartridges (1 g) were purchased from the Waters Corporation
(Dublin, Ireland)
ABIBU ET AL.3
FIGURE 1 Chemical structures of the target vitamins in this study
Stock standard solutions of water-soluble i.e. vitamins B1
(thiamine), B2(riboflavin), B3(niacin), B4,B
6(pyridoxine),
B9(folic acid), B12 (cobalamin) and C (ascorbic acid) were
prepared individually by accurately weighing 25 mg of each
and quantitatively transferring to 25 mL volumetric flasks and
dissolving with double distilled water to make stock solutions
of 1.0 mg/mL i.e. 1000 ppm of each. Since vitamin C was
limited, it was freshly prepared at a higher concentration of
4000 mg/mL at the time of use. A 0.45 μm Nylon membrane
fil-ter type, HNWP, Millipore (Dublin, Ireland) were used as
required to remove undissolved particles from all solutions
before injection on the HPLC system. The solubility of vita-
min B2(riboflavin) and vitamin B9(folic acid) were poor in
water and as such 0.5 mg/mL (500 ppm) of vitamin B2was
prepared using 5 mM of KOH and 20 mM KHCO3for vita-
min B9. Working standard solutions were prepared from these
solutions and diluted with water prior to analysis to obtained
appropriate calibration standards. All the stock and working
standard solutions were prepared in dark brown bottles and
stored at +4.0 ◦C when not in use except vitamin C (ascorbic
acid) which was prepared at the time of use.
2.2 Instrumentation
The Agilent Technologies 1260 Infinity Series liquid chro-
matography system, (Mainz, Germany) was used throughout
4ABIBU ET AL.
and consisted of a G 1312C binary pump, a degasser, an
Agilent Technologies S 6020 manual injector fitted with
a20μL loop, a variable wavelength UV and fluorescence
detectors and a column thermostat. The instrument control,
data acquisition and analysis were performed using Chemsta-
tion software (Agilent Technologies). During all experiments
the column oven temperature was maintained at 25◦Cusing
G 1316A column thermostat. The G 1315D UV detector
wavelengths were set at 254, 266, 270 and 280 nm, depending
on the vitamins of interest. The pH of the aqueous mobile
phase was measured using the Hanna HI 2211 pH meter
(Bedfordshire, UK). Analytical columns Synergi C18, 150 ×
4.6, 4 μm particle size was from Phenomenex (California,
USA) while Ascentis express C18, 150 mm ×4.6 μm×3μm
particle size was from Supelco, Sigma-Aldrich (Taufkirchen,
Germany). A glass vacuum-filtration apparatus obtained
from Alltech Associates, was employed in the filtration of
the mobile phases using 0.45 μm membrane filters obtained
from Millipore (Bedford, MA, USA) and degassed before
use. To achieve better extraction each sample was vortex
using a vortex instrument from Scientific Industries, Inc.
(Bohema, New York, USA). Extraction was performed with
an ultrasonic bath SCIENTECH ultrasonic cleaner model:
704 (Johannesburg, South Africa).
2.3 Standard cocktail
A standard cocktail was prepared to give concentrations of
50 μg/mL for vitamins B1,B
3,B
4,B
6, 100 μg/mL for vitamin
C, 30 μg/mL of vitamin B12 and 15 μg/mL vitamin B2and B9.
625 μL of vitamins B1,B
3,B
4,B
6stock standard solution,
1.250 μL of vitamin C stock standard solution, 1.500 μLof
vitamin B12 stock standard solution, 1.500 μL of vitamins B2
and B9stock standard solutions were accurately measured and
mixed. The mixture was diluted with double distilled water to
a final volume of 50 mL. More dilute standards were prepared
as required from these stock solutions.
2.4 Samples
Samples of S. frutescens i.e. Lerumo La Madi dried herbs
were obtained from a traditional herbal medicine seller at the
Mall Station area of Central Gaborone, the capital city of
Botswana.
2.4.1 Sample preparation
Samples were protected from direct exposure to light and kept
in ice to minimized vitamin degradation during the extraction
process. About 2.5 g of each sample was accurately weighed
into a 50 mL centrifuge tube, and 1 mL of 0.00108 M
phenol solution was added as an internal standard. This was
followed by 20 mL of 10 mM ammonium acetate solution
as an extractant. Each of the samples was then thoroughly
vortexed for 5 min and subjected to extraction using a cold
ice ultrasonic bath. The extraction process was done for a
total time of 15 min. 10 mL of chloroform was then added to
the extract to precipitate proteins and fat. This was followed
by vortex shaking for 1 minute and centrifuging for 10 min
at 5000 rpm to remove suspended material. The supernatant
was filtered through a Whatman No. 1 filter paper to remove
large suspended particles. Smaller particles were removed by
re-filtering the supernatant using a 0.45 μm Teflon filter. The
filtrate was subjected to silica based solid phase extraction
(SPE) to trap the vitamins as a final sample clean-up before
injection into the HPLC system.
2.4.2 Sample clean-up
The SPE cartridge was conditioned by flushing with 10 mL
methanol and 10 mL hydrochloric acid (pH 2.7) to activate
the stationary phase. 5 mL of the sample extract was then
loaded. Elution was carried out using 5 mL of the mobile
phase (0.01% formic acid: methanol, 50:50) at a flow rate of
1.5 mL/min. The eluate was collected in an amber coloured
bottle and a fraction of it injected into the HPLC system. Chro-
matographic separation using optimized conditions was then
effected.
2.5 Preliminary experiments to afford
method development
The most suitable absorption wavelength for the standard
solutions at a concentration of 10 mg L−1for vitamins B1,
B3,B
4,B
6,B
12,Cand5mgL
−1for B2and B9were
determined individually using a scanning ultra-violet/visible
(UV/Vis) spectrophotometer. Based on the UV/Vis spectra
of each vitamin at 254, 266, 270 and 280 nm, the one that
had the highest intensity for each analyte was chosen for use
throughout. After selection of the wavelength, two different
columns were evaluated in order to select the best. This was
done because the behaviour of vitamins on different stationary
phases has been reported to change depending on the chem-
istry and physicochemical properties of a particular station-
ary phase [40,41]. The reversed phase C18 deactivated col-
umn has been shown to give excellent vitamin serapations
within reasonable times [42,43]. Subsequently, two C18 silica
based stationary phases were tested i.e. the Phenomenex Syn-
ergi, C18, (4.6 ×150 mm, 4.0 μm particles) and the Supelco
Ascentis, C18, (4.6 ×150 mm, 3.0 μm particles). The physic-
ochemical properties of the two columns that were tested are
given in Table 1. To evaluate separation performance of the
two columns, the number of theoretical plates (N), retention
factors (k´) and asymmetry factors were compared ultimately
selecting the best combination of those parameters.
After selecting the column, selection of a suitable acid
modifier became necessary. 0.01% formic acid and 0.01% tri-
fluoroacetic acid were examined as additives/modifiers for the
ABIBU ET AL.5
TABLE 1 Physico-chemical properties of the two that were compared for the separation of the water-soluble vitamins
Column Column size Particle size (𝛍m) Surface area (m2/g) Carbon load (%)
PhenomenexSynergi C18 4.6 mm ×15 cm 4.0 475 19
SupelcoAscentis C18 4.6 mms ×15 cm 3.0 450 25
separation of the vitamins. Results for these experiments are
given in Section 3.3.
This was followed by selection of the best mobile phase.
Methanol, acetonitrile and tetrahydrofuran (THF) mixed with
either formic acid or trifluoroacetic acids were investigated.
The presence of an acidic modifier in the mobile phase was
essential to obtain complete resolution of the vitamins, better
chromatographic efficiency and reduced peak tailing. The use
of 0.01% formic acid in water as a mobile phase, yielded bet-
ter separation and peak shapes compared to the use of 0.01%
trifluoroacetic acid in water.
2.6 Method validation
An analytical method is considered fully validated when the
associated parameters, such as precision, accuracy, linear-
ity, limit of detection and quantitation, recovery, specificity
and robustness fall within acceptable analytical levels. The
method developed in this study was validated in terms of lin-
earity using coefficients of determination (R2) values, limits
of detection (LODs) and quantification (LOQs), percent rel-
ative standard deviations (% RSDs) which defines the pre-
cision and percent mean recoveries which defines the accu-
racy. Validation of the method was performed using the Inter-
national Conference on Harmonization (ICH) guidelines and
literature [44] with the working range of standard mixtures
at five or six levels of concentration. For all chromatograms,
calibration graphs were constructed based on peak heights
versus concentration of each analyte. Peak heights were used
instead of peak areas because the standard deviations of peak
heights were better than those for peak areas. The limits of
detection (LODs) and quantification (LOQs) were determined
using calibration curves according to the method described by
the ICH and literature [45]. Equations (1) and (2) were used
for this purpose.
LOD=3s
b(1)
LOQ=6s
b(2)
Where b is the mean of the slope of calibration curves and sis
the standard deviation of the response. Optimised RP-HPLC
parameters were employed for this purpose. The average vita-
min content from each extract was calculated using linear
regression equation. Respective intra- and inter-day preci-
sions for area of responses were considered. The precision
was calculated on the basis of the relative standard deviation
(RSD%) using Equation (3).
%RSD = Standard deviation
Mean × 100 (3)
2.6.1 Recovery studies by matrix matching
Recovery experiments were carried out using matrix match-
ing. Two sets of extractions were carried out simultaneously
following optimization of the extraction procedure. In one
of them, the sample was spiked with a standard cocktail of
known concentrations of the target compounds followed by
extraction. This was the pre-extraction matrix (PrEM) spiked
sample. In the other, the same sample was extracted and then
the extract was spiked with the same cocktail of vitamins
afterwards. This was the post-extraction matrix (PoEM) spike
sample. The concentrations of the vitamins read from the cal-
ibration graph of the pre-extraction spike samples and the
post-extraction spike samples were compared to afford per-
cent recoveries. This procedure is a variant of the standard
addition method [46] in which the matrix is measured with
and without the analytes of interest and takes into account (i)
matrix effects i.e. that could give rise to loss or gain of the
analytical signal and (ii) recovery effects i.e. loss or gain of
analyte. The recovery of each vitamin was calculated by using
Equation (4).
%Recovery =Concentrations in the PrEMs
Concentrations in the PoEMs × 100 (4)
3RESULTS AND DISCUSSION
3.1 Preliminary experiments
In terms of wavelength selection, 266 nm gave excellent
peak shapes and intensities for all the vitamins and was
thus used throughout the experiments. Table 2 shows the
optimized gradient for the separation of the vitamins using
0.01% formic acid as solvent A and methanol as solvent B.
Most water-soluble vitamins are organic amine compounds
containing two to five nitrogens in their heterocyclic ring that
are extremely polar. From the two chromatograms obtained
using silica-based Phenomenex Synergi, C18, (4.6 ×150 mm,
4.0 μm particles) and Supelco Ascentis, C18, (4.6 ×150 mm,
3.0 μm particles) columns, performance data was tabulated
in Table 3 in order to compare their characteristics. Results in
6ABIBU ET AL.
TABLE 2 Optimum gradient elution program used for the
separation of the eight vitamins
Time (min)
Solvent A%
(Water +0.01%
formic acid)
Solvent B%
(Methanol)
Flow rate
(mL/min)
090 100.5
790 10 0.5
850 501.0
12 50 50 1.0
Table 3 indicated that the Phenomenex Synergi C18 column
was the most suitable for the separation of the vitamins
in this study. From all perspectives, performance charac-
teristics i.e.retention factor (k´), theoretical plates number
(N), asymmetry factor and resolution (Rs), the Phenomenex
Synergi (PS) column was superior to the Supelco Ascentis
(SA) column under the method conditions specified. The fact
that vitamin B6 coeluted with vitamin C underscored the
choice of the PS column as the most suitable to use in the
experiments.
3.2 Selection of a suitable acid modifier
0.01% formic acid as a mobile phase additive, gave better sep-
arations and peak shapes, compared to 0.01% trifluoroacetic
acid (TFA). Figure 2A and 2B show chromatograms for the
simultaneous separation of five vitamins using two different
modifiers. A mixture of 0.01% formic acid: methanol at a
90:10 v/v ratio for vitamins B1,B
3,B
4,B
6and C while
50:50 v/v ratio for B2and B9was the most suitable compo-
sition that achieved the best separations for all the analytes
of interest. The use of 0.01% TFA resulted in weak retention
using optimum conditions and this resulted in co-elution of
vitamins B3 and B4. Thus 0.01% formic acid which achieved
baseline separation for the five vitamins was selected as the
modifier.
3.3 Preliminary separation of the vitamins
The chromatogram of a cocktail of the eight vitamin standards
using the optimized elution gradient is shown in Figure 3. All
the vitamins were eluted within 12 minutes in the order: vita-
min B1,B
6,B
4,C,B
3,B
12,B
9and B2.
3.4 Method validation
Table 4 shows the regression equations, the R2values, the
LODs, LOQs and % RSDs obtained from the validation
experiments. The coefficients of determination (R2) ranged
between 0.9886 – 0.9991 with linear ranges spanning from 0
–3.5μg/mL for all the vitamins. The LODs of vitamins B1,
B2,B
3,B
4,B
6, and B9ranged between 0.03 – 0.23 μg/mL
while the LOQs were between 0.18 – 0.67 μg/mL. The % RSD
ranged between 1 and 15%. Values of% RSDs that were higher
than 5% were considered poor and attributed to signal varia-
tions caused by manual injections on the HPLC system. The
mean percentage recoveries ranged between 92 and 103%.
The run time for the six vitamins was 12 min. From the values
obtained, the method was fast with low solvent consumption
and yielded excellent accuracies and was reproducible.
3.5 Application to a real sample
Table 5 shows the concentrations of individual vitamins
in S. frutescens from Gaborone, Botswana. A representa-
tive chromatogram of a real sample in 12 min is given in
Figures 4. As seen in Table 5, six (6) vitamins were identified
and quantified in S. frutescens with the exception of vitamins
B12 and C which were not detected. Vitamin C was not
detected speculatively due to (i) seasonal variation and (ii)
the procedure that the medicinal plant seller used to dry the
fresh plant could have exposed the plant to light and heat
thus causing environmental factors to degrade it. Table 4
shows that, S. frutescens has the highest concentration of
vitamin B2at 54.3 mg/100 g. Perhaps further phytochemical
TABLE 3 Performance characteristics of the PhenomenexSynergi C18 and the SupelcoAscentis C18 columns on selected vitamins
Vitamin Retention factor (k´) Asymmetry factor Theoretical plates (N) Resolution (Rs)
PS SA PS SA PS SA PS SA
B1 2.29 2.52 0.55 0.55 2638 1127 1.26 -
B2 16.02 16.25 0.55 0.82 122317 46637 1.45 2.53
B3 8.26 7.59 0.53 1.28 4028 2457 1.94 1.58
B4 4.55 4.88 0.61 0.49 1903 2316 1.64 0.95
B6 3.38 4.48 (Vit C) 1.20 0.58 (Vit C) 2379 4017 (Vit C) 3.55 0.88 (Vit C)
B9 15.67 15.40 1.58 0.57 54574 34264 2.53 1.20
B12 16.10 15.06 0.95 1.18 151356 86961 18.31 14.70
C5.29 -0.63 12328 2.01
a) PS =PhenomenexSynergi C18 column
b) SA =SupelcoAscentis C18 column
c) VitaminB6andCcoelutedonSA
ABIBU ET AL.7
FIGURE 2 (A) Chromatogram of a cocktail of five vitamin standards using methanol-water containing 0.01% formic acid. 1 =B1; 2 =B6;
3=B4; 4 =C; 5 =B3. (B) Chromatogram of a cocktail of five vitamin standards using methanol-water containing 0.01% trifluoroacetic acid.
1=B1; 2 =C; 3 =B3 and B4; 4 =B6
FIGURE 3 Chromatogram of a mixture of eight water-soluble vitamin standards at 266 nm. 1 =B1; 2 =B6; 3 =B4; 4 =C; 5 =B3, 6 =B12;
7=B9; 8 =B2
8ABIBU ET AL.
TABLE 4 Method performance characteristics for the determination of water-soluble vitamins (n =6)
Vitamin Regression equation R2LOD (𝛍g/mL) LOQ (𝛍g/mL) Recovery (%) % RSD
B1 y=43.105x+8.1 0.9987 0.1 0.2 92 1
B2 y=19.444x+100.85 0.9886 0.3 0.7 103 10
B3 y=17.2x – 2.6111 0.9975 0.2 0.3 97 15
B4 y=143.71x+9.4756 0.9991 0.1 0.2 94 10
B6 y=24.819x+1.8556 0.9964 0.2 0.4 101 10
B9 y=20.336x+56.041 0.9956 0.2 0.5 96 9
B12 y=9.3695x+3.013 0.9975 0.2 0.3 95 5
Cy=47.615x+17.021 0.9940 0.3 0.6 95 4
TABLE 5 Mean concentrations of water-soluble vitamins in mg/100 g dried weight (DW) in Sutherlandia frutescens in a real sample collected
from Botswana and recommended dietary allowances
Vitamin Concentration (mg/100 g DW) s (n=3) Recommended dietary allowance (mg)
B1 6.6 1.6 1.0–1.5
B2 54.3 2.2 1.3–1.7
B3 5.3 0.9 15–19
B4 2.3 4.4 Not reported
B6 2.6 1.5 1.6–2.0
B9 28.9 1.6 180–200 (𝛍g)
DW =Dry weight
s=standard deviation
Recommended dietary allowances from reference [33]
FIGURE 4 Chromatogram of Sutherlandia frutescens i.e. Lerumo La Madi extract using optimum chromatographic conditions. 1 =B1;
2=B6; 3 =B4; 4 =B3; 5 =B9; 6 =B2
investigation of S. frutescens could shed more light on claims
that it is a blood purifier and all-purpose tonic [4–6]. The
large excessive amounts in contrast to the recommended
dietary allowance by the National Academy of Sciences Food
and Nutrition Board (1989) for vitamin B1,B
2and B9attest
to the fact that the plant could be useful for their extraction
for supplementation purposes in foods that may be deficient
of these vitamins. Here again, the authors recommend
further investigation and professional advice from nuitrition
experts.
4CONCLUDING REMARKS
A fast and simple method for the simultaneous determi-
nation of water soluble vitamins in S. frutescens has been
ABIBU ET AL.9
demonstrated and applied successfully on a real sample.
HPLC methods for analysing one or two water-soluble vita-
mins are common, but methods for the simultaneous anal-
ysis of all the water-soluble vitamins are rare. According
to the Food and Nutrition Board of the National Academy
of Sciences-National Research Council recommended dietary
allowances [47], two of the results obtained in this study fell
within the recommended daily dietary allowance while three
were excessively higher. From literature, this is the first report
of the content of vitamins in the indigenous medicinal plant
S. frutescens from Botswana.
ACKNOWLEDGEMENTS
Mr. AbibuM. A. would like to thank the Federal Polytechnic
Ede, Nigeria for financial support and the study leave for his
Ph.D study and University of Botswana for material support.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Kwenga Sichilongo
https://orcid.org/0000-0003-2965-3067
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How to cite this article: Abibu MA, Takuwa
DT, Sichilongo K. Quantification of eight water
soluble vitamins in Sutherlandia frutescens species
from Botswana using a validated reversed phase
HPLC method. Sep Sci plus 2019;1–10. https://doi.org/
10.1002/sscp.201900018
