Food Chemistry 365 (2021) 130514

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Comparison of ultraviolet and refractive index detections in the HPLC

analysis of sugars
Iqbal Jalaludin a, Jeongkwon Kim a, b, *
a
Department of Chemistry, Chungnam National University, Daejeon 34134, Republic of Korea
b
Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Refractive index (RI) detection is the standard approach for quantitatively detecting sugars via high-performance
Honey liquid chromatography (HPLC), while ultraviolet (UV) absorbance detection is the most commonly used
HPLC detection method for general HPLC analysis. We compared the two detection approaches of UV and RI in the
Refractive index
HPLC analysis of small sugars to investigate whether UV detection could be an alternative method to RI
Sugar
Ultraviolet
detection. UV detection was performed using a photodiode array scanning from 190 to 400 nm. We obtained
comparable limit of detection (LOD) results for RI and UV detection in the HPLC analysis of monosaccharides,
while HPLC-RI provided better LOD results than HPLC-UV in disaccharide analysis. Both HPLC-RI and HPLC-UV
methods were applied to analyze a real honey sample, and similar results were obtained in terms of precision and
recovery. The study conclusively shows that the UV-based HPLC analysis of sugars offers a sufficient alternative
to RI-based HPLC analysis.

1. Introduction absence of UV chromophores in sugars, RI detection may be preferable

Ultraviolet (UV)-based detection is widely used in high-performance
liquid chromatography (HPLC), primarily because it is easy to operate
and relatively sensitive (Swartz, 2010). However, a refractive index (RI)
detector is typically used as a universal detector to detect compounds
such as carbohydrates that do not have UV chromophores (Zhang, Li, &
Yan, 2008). These detection methods share some similarities, but the
sensitivity and stability for a specific analyte vary when using different
detectors (Scott, 1986).
Carbohydrate analysis is a standard practice in many food industries
and is generally used for food labeling, quality assurance, and testing of
food additives (BeMiller, 2017). The use of chromatography improves
carbohydrate testing, and is considered a better alternative to colori­
metric tests (e.g., phenol-sulfur assay), which quantify total carbohy­
drates rather than individual sugar components (BeMiller, 2017). There
are several benefits of quantifying individual sugars instead of an overall
carbohydrate estimation, including the investigation of honey adulter­
ation (Se, Wahab, Syed Yaacob, & Ghoshal, 2019). Assessments for
honey adulteration are typically conducted by evaluating the percentage
of monosaccharides and disaccharides, with the former expected to be
within 70% and the latter about 5% (Wang et al., 2015). Due to the
over UV-based detection for sugar analyses. However, this limitation
does not completely restrict UV-based detection, as UV absorption of
sugars is observed below 200 nm (Uchiho et al., 2015). To our knowl­
edge, only a few studies have been performed using the UV-based
identification of unlabeled sugars, with sugars in fruits and juices
detected at 190 nm using HPLC-UV (Karkacier, Erbas, Uslu, & Aksu,
2003; Shaw & Wilson, 1983).
The most apparent superiority of UV-based detection in HPLC over
RI detection is its compatibility with gradient elution. Gradient elution
can either shorten the acquisition time or increase the retention time
difference between two adjacent peaks, ultimately improving the chro­
matogram resolution. Moreover, gradient elution minimizes the use of
solvents, which is undoubtedly beneficial to many industrial sectors.
Stability is not always an issue with UV detection, unlike in RI detection
where temperature and pressure must be precisely controlled to main­
tain a consistent baseline (Hirata, Kawaguchi, & Funada, 1996). In
general, the lack of chromophore-containing compounds, such as sugars,
does not benefit greatly from UV detection, because individual sugars do
not have unique UV absorptions. However, a detector such as a photo­
diode array (PDA) can measure absorbance over a broad wavelength
range while discerning sugars from other components whose UV

* Corresponding author at: Department of Chemistry, Chungnam National University, Daejeon 34134, Republic of Korea.
E-mail address: jkkim48105@cnu.ac.kr (J. Kim).

https://doi.org/10.1016/j.foodchem.2021.130514
Received 4 March 2021; Received in revised form 21 May 2021; Accepted 29 June 2021
Available online 1 July 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
I. Jalaludin and J. Kim
absorbance is beyond the intended wavelength range. Consequently, a
more reliable HPLC quantification can be performed for a complex
sample.
In this study, we compared the performance of UV and RI detectors in
carbohydrate analysis, where UV detection was performed using a PDA
detector. Two elution modes, i.e., isocratic and gradient, were compared
to assess the reliability of the approaches for their subsequent use on a
real honey sample.
2. Materials and methods
2.1. Materials and reagents
Fructose was purchased from Kanto Chemical Co. Inc. (Tokyo,
Japan). Glucose, sucrose, and maltose monohydrate were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (HPLC-grade)
was obtained from Merck (Darmstadt, Germany). A commercial honey
sample was obtained from a local market in South Korea. The honey
sample used in this study had previously passed the standard tests
required by the Korea Food and Drug Administration (KFDA) for quality
control purposes. Water was purified using the Human Power I+ Water
Purification System (Seoul, South Korea).
2.2. Standard and sample preparation
A stock solution (10 mL) containing 100 mg each of fructose,
glucose, sucrose, and maltose was prepared by homogeneously dis­
solving each sugar in a small amount of water with mild heating,
combining all sugar solutions into a volumetric flask, and finally adding
water to make up a 10-mL solution. Standard working solutions for
generating a calibration curve were prepared from the stock solution by
two-fold serial dilutions (10–0.039 mg/mL).
To prepare the honey sample solution, 100 μL of pure honey was
diluted with water to prepare a 10-mL solution (100-fold dilution). The
mass of the 100 μL of pure honey was measured using an analytical
balance (OHAUS Adventurer, Parsippany, NJ, USA) for the accurate
determination of the sugar content in honey. The honey was allowed to
immerse in the water for 1 h and the solution was then vortexed to
ensure complete dissolution. Both standards and samples were filtered
using a 0.2-µm pore size syringe filter (Whatman™ Puradisc, Buck­
inghamshire, UK) before HPLC analysis.
2.3. HPLC operating conditions
All HPLC analyses in this study were conducted using an Alliance
HPLC system (Waters, Milford, MA, USA), which consisted of an e2695
separation module, 2998 PDA detector, and 2414 RI detector. Both
detector units were used simultaneously in isocratic mode, but only the
UV detector was used in gradient mode. The HPLC system was equipped
with a Shodex Asahipak NH2P-50 4E column (4.6 mm internal diameter
× 250 mm; Showa Denko KK, Tokyo, Japan). The system was semi-
controlled with the Waters Empower 3 Chromatography software.
In isocratic mode, the mobile phase was acetonitrile:water (75:25, v/
v) (Tihomirova, Dalecka, & Mezule, 2016), which was retained for 15
min at a flow rate of 1.0 mL/min. In gradient mode, the mobile phase
consisted of acetonitrile (solvent A) and water (solvent B), from which
the gradient procedure started at 35% of solvent B for 4 min and
decreased linearly to 20% from 4 to 10 min (Supplementary Fig. S1).
Other gradient conditions were also attempted (Supplementary Fig. S2).
In both isocratic and gradient elution modes, the sample injection vol­
ume was 20 μL and the column temperature was 35 ◦ C. The wavelength
range of the UV detector was set from 190 to 400 nm for simultaneous
detection. The peak area was measured at 190 nm.
2
Food Chemistry 365 (2021) 130514
2.4. Method validation
For comparative purposes, all HPLC methods were validated using
the standard validation practice in compliance with the International
Conference on Harmonization (ICH) Q2(R1) guidelines (ICH, 1995). The
assessment included linearity, sensitivity, accuracy, and precision of the
methods.
2.4.1. Linearity
The standard sugars were analyzed in triplicate at concentrations of
0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg/mL to obtain
the mean and standard deviation and construct calibration curves by
plotting the peak area against the known sugar concentrations. Linear
regression analysis was used to measure the slopes and coefficients of
determination (R2) of the best-fitting lines.
2.4.2. Sensitivity
The sensitivity of the method was measured using a linear regression
approach in which the limit of detection (LOD) and the limit of quan­
tification (LOQ) were calculated as follows:
σ
LOD = 3.3 ×
S
LOQ = 10 ×
σ
S
where σ is the standard deviation of the y-intercept and S is the standard
curve slope.
2.4.3. Accuracy and precision
Accuracy and precision were measured using the standard sugar
solutions at high, medium, and low concentrations of 10, 5, and 0.625
mg/mL, respectively. Accuracy was expressed as percent recovery (%
recovery) by comparing the obtained value with the theoretical value,
while, precision was expressed as percent relative standard deviation (%
RSD) to measure the closeness of five replicates (Rao, 2018). Two pre­
cision procedures were considered: repeatability (inter-day precision
analysis) and intermediate precision (inter-day precision analysis). The
%recovery and %RSD were calculated as follows:
Measuredconcentration
%recovery = × 100
Actualpreparedconcentration
%RSD =
Standarddeviationofthemeasuredconcentration
× 100
Meanofthemeasuredconcentration
2.4.4. Robustness
Robustness was tested using a full factorial experimental design to
see how small chromatographic changes in the experimental conditions
affected the quantification and system compatibility. Three parameters
(flow rate, column temperature, and solvent composition) were tested at
two levels: low (− 1) and high (1), as well as one center point (0),
generating nine random experiments (Supplementary Table S1). The
experiments were carried out sequentially using the fructose sample
solution (10 mg/mL), and the resulted peak area and retention time
were computed and analyzed by Minitab® 20.2 statistical software
(Minitab Inc., Pennsylvania, USA).
2.5. Sugar content in the honey sample
The sugar content in the honey sample was measured in two ways.
First, the linear equation from the calibration curve was used, with the
sugar concentrations determined by substituting the peak area value at
the respective elution time of the sugar in the honey sample to the linear
equation. Second, the amount of sugar in the honey was calculated using
an equation proposed by the KFDA to validate the sugar composition of
honey (Food Safety Korea, 2020):
I. Jalaludin and J. Kim Food Chemistry 365 (2021) 130514

AU V W’ are typically composed of carbon–carbon (C–C) single bonds, hydroxyl

%SC = × × × 100
AU’ V’ W
Here, AU and AU′ are the peak area of the sample and the standard, V
and V′ are the volume of the sample and the standard, and W and W′ are
the weight of the sample and the standard, respectively. The calculated
result was expressed as a percentage, and the sugar concentration (%SC)
was measured using the 10 mg/mL sugar standard solution.
3. Results and discussion
3.1. HPLC-RI and UV analyses of sugars using isocratic elution
(OH) groups, and ether groups. The λmax of C–C bonds was approxi­
mately 160 nm, while the λmax of the OH groups was about 150 nm
(Higashi, Ikehata, & Ozaki, 2007; Tachibana et al., 2011). The cyclic
ether in the tetrahydrofuran of fructose and sucrose, and the tetrahy­
dropyran of glucose, sucrose, and maltose (Fig. 1d insert) have absorp­
tion peaks at about 58 × 103 cm− 1 (~172 nm) and 57 × 103 cm− 1
(~175 nm), respectively (Pickett, Hoeflich, & Liu, 1951). The λmax for
both fructose and glucose has been reported to be 178 nm, while that of
sucrose is 175 nm (Uchiho et al., 2015). These observations indicate that
UV absorption by fructose and glucose is dependent on cyclic ether
groups, while that of sucrose is dependent on C–C bonds or OH groups.

Fructose, glucose, sucrose, and maltose are commonly observed in

3.2. HPLC-UV analysis of sugars using gradient elution
honey (Lee et al., 2020). Isocratic separation of the fructose, glucose,
sucrose, and maltose mixture was performed using an acetonitrile:water
One of the main advantages of UV detection over RI detection is that
(75:25, v/v) mobile phase, with sequential detection using RI and UV
both isocratic and gradient elution can be used in UV detection, while
detectors. A PDA detector was used to obtain the UV absorbance of the
only isocratic elution is possible in RI detection. Stability is the primary
sugars in the wavelength range from 190 to 400 nm. All four sugars were
reason that RI detection is undesirable for application in gradient elution
measured successfully using both detectors. All four sugar peaks in the
because temperature and pressure must be precisely regulated to
RI chromatogram appeared at a similar height (Fig. 1a). However, in the
maintain a stable baseline (Hirata et al., 1996).
UV chromatogram at 190 nm, the fructose peak was approximately five
Gradient elution was attempted and optimized for the HPLC-UV
times higher than the other peaks (Fig. 1b). The higher absorbance of
detection of sugars to shorten the analysis time while ensuring that
fructose than glucose or sucrose at 190 nm has been reported previously
the sugar peaks did not overlap. Supplementary Fig. S2 shows 12
(Uchiho et al., 2015). The UV contour map in Fig. 1c shows the fructose
different gradient elution profiles and their corresponding chromato­
UV absorption at wavelengths higher than 190 nm.
grams from the investigation. The optimized gradient profile alongside
The wavelength of maximum absorbance (λmax) for each sugar was
the chromatogram is shown in Supplementary Fig. S2L, where solvent B
below 190 nm, with the peaks increasing continuously at the extreme
(water) began at 35% for 4 min and decreased linearly to 20% from 4 to
left of the UV spectra, as shown in Fig. 1d. However, due to the limi­
10 min (Supplementary Fig. S1). The solvent composition with a
tations of typical commercial UV detectors, 190 nm is the lowest
stronger elution strength (solvent B) should be gradually increased in a
available wavelength. The low λmax values are related to the distinctive
conventional gradient elution procedure. However, the composition of
configurations of fructose and the other sugars studied. Neutral sugars
solvent B had to decrease to obtain better separation of the sugars in this

Fig. 1. Chromatograms and spectra of fructose, glucose, sucrose, and maltose: (a) RI chromatogram, (b) UV isocratic chromatogram at 190 nm, (c) UV contour
spectrum, and (d) UV absorption spectra.

3
I. Jalaludin and J. Kim
study.
Fig. 2 shows the UV chromatogram and the UV contour spectrum of
the mixture of four sugars. Gradient elution reduced the total analysis
time from 15 min in the isocratic elution, as shown in Fig. 1, to less than
10 min. In general, all sugar peaks were shifted to earlier retention times
with gradient elution compared to isocratic elution. Furthermore, the
baseline (Fig. 2a) of gradient elution was slightly wavier than that of the
isocratic method (Fig. 1b), but this did not affect the consistency of the
resulting peaks in terms of their areas and retention times.
Peak symmetry and resolution are critical considerations that ensure
the gradient profile is optimized. The peaks in Fig. 2a are symmetric,
with no tailing or fronting. The resolution was determined based on the
difference in elution time between the peaks and their widths. The
resolution was calculated as follows:
Δtr
Resolution =
wav
Fig. 2. (a) HPLC-UV gradient chromatogram at 190 nm and (b) UV contour spectrum of fructose, glucose, sucrose, and maltose. Gradient elution shortened the
analysis time without compromising the quantitative and qualitative details of sugars.
4
Food Chemistry 365 (2021) 130514
Here, Δtr is the distance between the peaks and wav is the average width
of the two peaks in their respective units (both were calculated in time
units) (Harris, 2003). The resolution values obtained using the two
neighboring peaks were equivalent to 1. Supplementary Table S2 shows
the resolution values for the three methods (HPLC-RI isocratic, HPLC-UV
isocratic, and HPLC-UV gradient).
The performance and effectiveness of columns can be expressed
using the number of theoretical plates. The column used in this study
was the same as that used in many previous sugar analysis experiments
(Barreira, Pereira, Oliveira, & Ferreira, 2010; Ma, Sun, Chen, Zhang, &
Zhu, 2014). It was important to know how efficient the column was to
ensure that it was compatible with our research. The more theoretical
plates in a column, the more likely it is to achieve an equilibrium be­
tween the stationary and mobile phases and the higher the separation
efficiency is. The number of theoretical plates was determined as
follows:
I. Jalaludin and J. Kim Food Chemistry 365 (2021) 130514

( )2
tr Table 1
Numberoftheoreticalplates = 5.54
w0.5 The LOD, LOQ range, and R2 for the HPLC-RI, HPLC-UV isocratic, and HPLC-UV
gradient methods.
Here, tr is the retention time of the peaks, and w0.5 is the width at half of Methods Compounds LOD [mg/ Range (from LOQ) R2 a

the peak height. Supplementary Table S3 shows the number of theo­ mL] [mg/mL]
retical plates measured at the fructose peak for all three methods. The HPLC-RI Fructose 0.111 0.337–10 1.000
largest theoretical plate number was 2732, which was obtained from the Glucose 0.021 0.064–10 0.999
HPLC-UV gradient, where the value was almost double that of the Sucrose 0.019 0.058–10 0.999
Maltose 0.016 0.047–10 0.999
isocratic approaches.
HPLC-UV Fructose 0.044 0.134–10 1.000
isocratic Glucose 0.063 0.190–10 0.999
Sucrose 0.326 0.988–10 0.995
3.3. Preparation of the calibration curves
Maltose 0.475 1.440–10 0.994

To ensure that all three methods could be used on the actual sample,
calibration curves were prepared to determine the linear relationship
between sugar concentration and response unit (Fig. 3). The y-axis of
each calibration curve was set as a peak area unit instead of the corre­
sponding detectors’ responding units (RIU for RI and AU for UV) to
allow the comparison to be more visible. The RI detection calibration
curve (Fig. 3a) indicated a similar response for all sugars. In contrast, UV
detection for both the isocratic and gradient modes revealed very similar
profiles (Fig. 3b and c). The profiles revealed that fructose had a slope
nearly four times steeper than those of the other sugars.
There were significant deviations at the lower sugar concentrations
(0.625–0.0391 mg/mL) in all methods, particularly for RI detection
(Fig. 3a insert). The peak area variations were most likely due to the
occasionally inconsistent baseline. In addition, the Waters Empower
software’s automated peak area estimation can lead to fluctuations for
diluted samples. Therefore, if the sample concentration is close to the
quantitative limit, a manual assessment of the peak area is advised.
3.4. Method validation
The sensitivity of all three methods tested was expressed in terms of
the LOD and LOQ. Table 1 displays LOD, LOQ, and R2 values for each
sugar. The LOD and LOQ values of the RI detector for all sugars were
generally comparable to those obtained previously using the same
detection method (Barreira et al., 2010); for instance, the LOD values of
fructose, glucose, and sucrose were 0.05, 0.08, and 0.06 mg/mL in the
report and 0.111, 0.021, and 0.019 mg/mL in the current study,
respectively.
In contrast, LOD and LOQ values of the UV detector showed com­
parable values to the RI detector for only the two monosaccharides. For
the disaccharides, the LOD and LOQ values using UV detection were not
comparable to those using RI detection; in particular, the LOQ for
maltose was highest for the HPLC-UV isocratic and gradient methods,
1.440 and 1.581 mg/mL, respectively, and only 0.047 mg/mL for the
HPLC-RI method. The LOD and LOQ values for the comparison of UV
and RI detectors were closely related based on R2, where only the su­
crose and maltose analyzed by the UV detector gave R2 < 0.996.
HPLC-UV Fructose 0.021 0.065–10 0.999
gradient Glucose 0.087 0.263–10 0.996
Sucrose 0.125 0.378–10 0.990
Maltose 0.522 1.581–10 0.998
a
R2 was measured within the calibration curve range in Fig. 3 (0.039–10 mg/
mL).
3.5. Repeatability, intermediate precision, and recovery
Supplementary Table S4 presents the precision and accuracy datasets
for the HPLC-RI isocratic, HPLC-UV isocratic, and HPLC-UV gradient
methods. All %RSD values in the repeatability test (intra-day precision)
for the high (10 mg/mL), medium (5 mg/mL), and low (0.625 mg/mL)
sugar concentrations were less than 2.5%, except for HPLC-UV mea­
surements of maltose with gradient elution at the low sugar concen­
tration, was found to be greater than 3.5% RSD. All three methods
generally had better repeatability for high and medium concentrations
than the low concentration. The inter-day precision %RSD values were
higher (range: 0.15–3.53%) than the intra-day precision %RSD values
(range: 0.33–5.86%).
In terms of accuracy, all three methods had %recovery values close to
100%. In particular, the HPLC-RI isocratic method collectively had the
least deviation from 100%, except for the 5 mg/mL fructose sample,
which had a value of 109.7%. Because the isocratic elution for the RI and
UV detectors always operated simultaneously, which will have reduced
the disparity in the analyte environment, any inconsistency between the
two HPLC methods could only be due to the detectors.
3.6. Robustness
Supplementary Fig. S3 summarizes the degree of chromatographic
variances caused by minor changes in three parameters: flow rate, col­
umn temperature, and solvent composition. The parameters were
selected because they are more likely to vary when the method is
transferred across laboratories or when different operators handle the
experiments (Vander Heyden, Nijhuis, Smeyers-Verbeke, Vandeginste,
& Massart, 2001). As its number of variables was small, a two-level full
factorial design was the preferred technique for evaluating robustness

Fig. 3. Calibration curves generated from serial dilution of standard working solutions for the (a) HPLC-RI, (b) HPLC-UV isocratic, and (c) HPLC-UV gradient
methods. All calibration curves had an acceptable linear range for all sugars (0.039–10 mg/mL) analyzed.

5
I. Jalaludin and J. Kim
(Ferreira et al., 2017). Consequently, the peak area and retention time
were measured, as recovery and separation quality are the primary foci
of this investigation.
In response to the peak area, the flow rate displays a greater effect
than the other factors (Supplementary Fig. S3a). This is most likely
owing to a longer period for the sugar to be maintained on the stationary
phase when using a low flow rate, which causes the peak’s base width to
widen. As a result, the peak area corresponding to the concentration of
sugar increases, and the recovery might be substantially impacted. On
the other hand, the retention time was considerably impacted by two
factors: flow rate and solvent composition (Supplementary Fig. S3b).
Thus, the two aspects (flow rate and solvent composition) must be
carefully evaluated to achieve a high quality of separation.
3.7. Sugar content in a honey sample
Simple sugars such as fructose and glucose are the main components
of honey, with the remaining 17–20% being water (Kamal & Klein,
2011). To analyze the saccharides in the honey sample, a commercial
honey sample was diluted 100-fold with water before the HPLC analyses
to lessen its viscosity so that the friction between the column packed
particles and samples decreased, allowing the flow rate to be sustained
without putting much pressure on the system.
The three methods evaluated in this study were able to measure
fructose, glucose, and sucrose in the honey sample because the three
sugars were within a quantifiable range (Fig. 4). Fig. 5 shows the sugar
contents in the honey using the three methods, which were very similar
as a consequence of the calculation method used. The peaks at 6.5–7.5
min in Fig. 4d might be disaccharide variants, such as trehalose and
cellobiose, as reported previously (De La Fuente, Ruiz-Matute, Valencia-
Barrera, Sanz, & Martínez Castro, 2011). However, because the peaks
were smaller than the quantification limit used in this analysis, they
Fig. 4. Chromatograms and spectra of honey: (a) HPLC-RI chromatogram, (b) HPLC-UV isocratic chromatogram at 190 nm, (c) isocratic UV contour spectrum, (d)
HPLC-UV gradient chromatogram at 190 nm, and (e) gradient UV contour spectrum. a Concentration was estimated from three replications using the calibration
curve of the respective standard. b Percentage in honey was estimated from three replications using the KFDA formula based on the respective 10 mg/mL standards.
6
Food Chemistry 365 (2021) 130514
were too ambiguous to be defined; therefore, we did not include them in
the final estimation.
The sugar concentrations in Fig. 5 were estimated from the calibra­
tion curve and expressed in mg/mL. To evaluate the differences among
the three methods, the %RSD values of the three methods for fructose,
glucose, and sucrose were calculated (Supplementary Table S5). Fruc­
tose and glucose had a low variation of 0.02–0.03% RSD, while
considerable variation was apparent in sucrose, with a value of 0.3%
RSD (Supplementary Table S5).
3.8. Advantages of UV detection over RI detection
Compared to RI detection, UV detection enabled the use of gradient
elution, which provided the greatest benefits of decreasing the operating
time and cost of analysis. In addition, UV detection had greater baseline
stability, with less time required to achieve a stable baseline. This was
apparent from the calibration curve, where large deviations were found
at low sugar concentrations (0.625–0.0391 mg/mL) for RI detection
(Fig. 3a insert).
UV detection is usually considered to have greater sensitivity than RI
detection for compounds with a UV chromophore, with a detection
capability in the nanomole range (Swartz, 2010). However, the use of
UV detection may be inefficient for non-UV-chromophore compounds,
such as sugars. Regardless, our results supported HPLC-UV analysis of
sugar as an alternative because the sensitivity of the two detectors was
comparable for all sugars tested. If a more reliable detection is required
to enhance sensitivity, labeling methods, such as 1-phenyl-3-methyl-5-
pyrazolone labeling, are recommended to narrow the detection to the
picomole range (Honda et al., 1989). However, an unlabeled analysis
with UV-based detection is adequate for sugar analysis in the micromole
range.
As indicated by previous studies, RI detection is the gold standard for
I. Jalaludin and J. Kim
Fig. 5. Estimates of the sugar content in a honey sample using the three methods validated in this study.
detecting sugars (Chávez-Servín, Castellote, & López-Sabater, 2004).
However, the current study showed that the UV detection of mono­
saccharides using both isocratic and gradient methods provided com­
parable results to RI detection in terms of the LOD and LOQ. For the
monosaccharides tested, the peak areas between the RI and UV detectors
were similar, although the two monosaccharides do not have chromo­
phores. Satisfactory sensitivity results were not obtained for the di­
saccharides using HPLC-UV methods. However, HPLC-UV detection
methods may be useful for measuring high concentrations of
disaccharides.
4. Conclusions
The HPLC-RI and HPLC-UV analyses of sugars, particularly mono­
saccharides, were comparable in terms of performance, even though UV
detection is primarily used to identify compounds with a UV chromo­
phore. It is important to note that the two detection methods for the
isocratic mode were reasonably validated by many previous studies and
have been used widely to determine the composition of sugars in foods.
We compared these methods with our newly developed gradient elution
method for UV detection to confirm that all three methods could be used
for sugar analysis. The significantly increased number of plates in the
HPLC-UV gradient method suggested that more complex sugar samples
could be successfully analyzed. Although RI detection is useful for sugar
quantification, direct UV analysis (with no pretreatment and no sample
labeling) may also be a practical option given its versatility and time-
and cost-effectiveness when paired with gradient elution.
CRediT authorship contribution statement
Iqbal Jalaludin: Writing – original draft, Resources, Investigation,
Data curation, Visualization. Jeongkwon Kim: Writing – review &
editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was supported by Basic Science Research Program
7
Food Chemistry 365 (2021) 130514
through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education (2016R1D1A1B02008854) and by Basic Science
Research Capacity Enhancement Project through the Korea Basic Sci­
ence Institute (National Research Facilities and Equipment Center) grant
funded by the Ministry of Education (Grant No. 2019R1A6C1010030).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130514.
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