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HPLC ANALYSIS OF MONO-AND DISACCHARIDES IN FOOD PRODUCTS

Conference Paper · October 2013

DOI: 10.13140/RG.2.1.1139.1840

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 НАУЧНИ ТРУДОВЕ SCIENTIFIC WORKS
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18-19 октомври 2013, Пловдив 18-19 October 2013, Plovdiv

HPLC ANALYSIS OF MONO- AND DISACCHARIDES IN FOOD PRODUCTS

Petkova, Tr., Nadezhda1, Brabant A. Pascal2, Masson Annick2, Denev P. Panteley1
1
Departament of Organic Chemistry, University of Food Technology, 26 Maritza Blvd, Plovdiv, 4002,
Bulgaria
2
Institute Meurice, Campus CERIA, Batiment 4C 5ème étage, Avenue Émile Gryzon, 1, 1070,
Brussels, Belgium

Abstract
A simple and reproducible high-performance liquid chromatography (HPLC) with refractive index (RI) method for the
qualitative and quantitative analysis of five mono- and disaccharides (fructose, glucose, sucrose, maltose and lactose) in
food products has been developed and validated. The best separation of sugars was achieved with mobile phase
acetonitile-water (80:20) and flow rate 2 ml/min. HPLC method showed good linearity with determination coefficients
exceeding 0.998. The limits of detection (DL) in these sugars were 0.13, 0.13, 0.21, 0.26 and 0.36 mg/ml, respectively; and
the limits of quantification (QL) - 0.44, 0.47, 0.51, 0.69 and 0.70 mg/ml. Recoveries in all sugars were between 81 and
121%.

Keywords: HPLC-RID, sugars, food product, method validation

Although chromatographic methods may currently be

Introduction
The term “sugars” has been defined to include a sum of
monosaccharides and disaccharides [6, 19]. According to
the Food and Drug Administration’s new policy their
analysis in foods is required when sugars presence is greater
than 1 % [14].
The determination of mono- and disaccharides is one of
the most frequently used in the food analysis and has
considerable application in nutritional and biochemical
studies [5]. For the labeling of foodstuffs, the European law
requires details of composition in sugars. [19]. The
monitoring is important; because frequent consumption of
sugar-containing foods and sugar-sweetened beverages can
increase risk of dental caries, contribute to weight gain and
diabetes type II. Sugars in food are analyzed for regulatory
and quality monitoring purposes, including: monitoring
food-labeling claims in calorie reduced foods, determining
food energy content, testing fruit juice quality or
adulteration [12], measuring lactose content in milk, low
lactose or lactose-free foods [3], monitoring sugar beet,
cane molasses, regular and high-fructose corn syrups [18].
The mono- and disaccharide composition of foods requires
determination of some or all of the following: glucose,
fructose, sucrose, lactose, and maltose. Analysis for these
sugars must be applicable to a wide range of foods,
including cereal products, dairy products, fruits, vegetables,
preserves and confectionery, and beverages [5].
The choice of methods in the sugar analysis must be
based on knowledge of composition of mixture of sugars in
the food. Most analytical methods require the removal of
interfering materials prior to analysis [12, 18].
regarded as the methods of choice, physical, chemical, and
biochemical methods still play a role in the analysis of
carbohydrates [5]. Most AOAC approved methods for
sugar determination are indirect physical, enzymatic or
semi-empirical chemical methods. [1, 7, 19].
Because of the high specificity and the capability for
simultaneous determination of several sugars, HPLC and
gas-liquid chromatography (GLC) are advantageous and
have been actively developed over the past two decades.
Both methods have been used for sugar determination, but
HPLC is considered the more advantageous method for
routine analysis [4]. HPLC has become the preferred
method for quantitating simple sugars in variety of food
products [18]. It is the most appropriate technique for
accuracy, precision and practicality for nutritional labeling
purposes. HPLC often offers direct injection of a sample
with little pretreatment and simple interpretation of
chromatograms. Sugars can be determined using a variety
of chromatographic modes in conjunction with RI, ELSD
or PAD detection [3, 4, 11, 12].
The analysts choice depends on sugars of interest, the
sample matrix, and the cost of equipment. For routine
HPLC determination of common food sugars, a simple
isocratic system incorporating an aminopropyl–silica
column and a refractive index detector is adequate [10, 12,
13, 15]. In some HPLC methods the amino column can be
replaced by a calcium or Pb2+ form resin column, for
analysis of foods or control of biotechnology process [5,
20].
The object of the current study was to develop and
validate a rapid HPLC-RID method for analysis of glucose,

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fructose, sucrose, maltose and lactose in foodstuffs. The
developed method must be simple, successfully validated in
everyday laboratory practice, suitable for a routine sugar
analysis and applicable for a wide range of foods.
Materials and Methods
All used reagents and solvents were analytical
grade. HPLC grade water (Lichrosolve Merck) and
acetonitrile (Lab-Scan Analitycal Science) were used
for preparation of the mobile phase.
Instrumentation: Chromatographic separation
was performed on HPLC apparatus Hewlet Packard
1100 Series consisted of degasser Iso pump,
automatic sample injector and associated with IBM-
compatible computer. Refractive index detector
(RID) Hewlet Packard 1100 Series was used for
sugars detection and quantification. The HPLC
separation of sugars was performed on columns
Agilent Zorbax Carbohydrate Analysis – and Limit of quantification of instrument (LID)
aminopropyl column (stationary phase silica
spherical particles, 5 µm in diameter, which is
reacted with 3-aminopropylsilane), as two colum
were tested for the method development: one with
dimension 4.6 mm × 250 mm and 4.6 mm × 150
mm; guard column Agilent Zorbax NH2, 4.6 mm ×
12.5 mm. The temperature of the column was
remained constant by a thermostat Agilent 1100. The
control of the system, data acquisition and data
analysis were under the control of the software
program Agilent Chem Station. The used mobile
phase was acetonitrile-water in ratio 75/25 and
80:20. Sugars concentration was calculated based on
peak area measurements.
Method development
Sugar standards were dried at 60 °C in vaccum
oven overnight and then dissolved in acetonitrile-
water 50/50. The stock solutions with sugar (glucose,
fructose, sucrose, maltose and lactose) concentration
0.5 mg/ml and 0.1 mg/ml rhamnose were injected
into HPLC column Agilent Zorbax Carbohydrate
Analysis with size 4.6 ID × 250 mm with guard
column, flow rate 1.4 ml/min, isocratic mobile phase
acetonitrile/water in ratio 75:25, temperature of
detector and column 30ºC, volume of injection 5 μl
without needle wash. Under the same conditions six
stock solutions with concentration 10 mg/ml were
injected to observed the order of elution of the sugars
and their retention times.
Optimization of the HPLC method. The
standard solutions of sugar with concentration 5
mg/ml, 2.5 mg/ml, 1 mg/ml and 0.5 mg/ml were
injected in the HPLC apparatus under different
combination of conditions: Mobile phase acetonitrile
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and water in ratio 75:25 and 80:20; flow rate 1.4
ml/min and 2.0 ml/min; Agilent Zorbax
Carbohydrate Analysis (4.6 ID × 250 mm) and (4.6
id × 150 mm), column temperature 35 and 30°C,
temperature of RID 35 and 30°C, injection sample: 5
and 15 µl, with and without needle wash of the
injector. The baseline stability and separation of the
sugars was observed. The detectors noise was
measured by integrating 15 peaks from baseline of
each injected sample.
Linearity of the HPLC method. The detector
linearity was tested by injecting 5 sugars solutions at
0.2, 0.5, 1.0, 2.5 and 5 mg/mL. HPLC separation was
performed using as the mobile phase
acetonitrile/water (80:20), at a flow rate of 2
mL/min. The column and the refractive index
detector were maintained at 30 °C. The injection
volume was 5 μL.
Determination of Limit of Detection (LOD)
LOD is calculated by measuring signal-to-noise
(S/N) of the baseline and multiplying this value by 3.
LOQ is determinate by measuring signal-to-noise
(S/N) and multiplying this value by 10 [8, 9].
Precision
Repeatability and reproducibility of the
instrument. For evaluation of repeatability of the
instrument standard solution with concentration 2.5
mg/ml of each sugar was injected 10 times in HPLC
apparatus in single day. For checking of
reproducibility standard solution with the same
concentration was injected in once per day in the
HPLC apparatus for a period of 10 days [2, 8, 9] .
Test for Interference of Sodium Chloride in
the Biscuit Samples. The solution of 1mg/ml
sodium chloride with concentration was injected in
the HPLC under mention above HPLC condition.
The obtained HPLC chromatogram was overlaid
with sample chromatogram.
Sample preraration. Biscuit sample was finely
ground in laboratory homogenizer (Ika, Germany).
Five grams biscuits were weighed in 250 ml
Erlenmeyer flask, 100 ml 60 % (v/v) EtOH were
added to them. Three samples were prepared at the
same time. The samples were measured on a balance,
cover with Parafim “M” and were placed in shaking
water bath at 83°C for 20 min. Then the samples
were cooled to the room and their weigh were
brought to the initial by 60 % EtOH addition. The
samples were filtered through Whatman N4 paper.
Additional sample clean-up was performed on the
filtrate by sequentially filtering through a Sep-Pak
C18 cartridge (Waters, Milford, MA) and a 0.22 µm
membrane filter (Sterivex™-GS (Millipore®) before
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injection in HPLC [13]. Duplicate analysis was 4.sucrose, 5. maltose and 6. lactose all in
performed on the individual extractions. concentration 0.5 mg/ml in 50/50 ACN/Water
Precision of the method. For repeatability test
ten samples with 1.25 g were extracted and analyzed
on the same day. For reproducibility one sample per 1 2 3 4 5 6
day was extracted and analysed on HPLC for 10
days.
Recovery. For evaluation of the recovery,
standard addition method was used. The extraction
procedure was carried out with 3 g sample. The first Figures 2. HPLC Chromatogram: 1. rhamnose,
sample contained only biscuit. The standard addition 0.1mg/ml, 2. fructose, 3. glucose, 4. sucrose, 5.
method was used for the other three samples, as 0.1 g maltose and 6. lactose all in concentration 0.5
of each sugar - glucose, fructose, sucrose, maltose mg/ml in 50/50 ACN/Water
and lactose were added, 0.2 g -to the third sample
and to the forth – 0,3 g was added. Each sample was The best separating HPLC conditions was shown in
extracted and analyzed in replication of 5 times [2]. Table 1. The lower value of baseline noise was
obtained at 2 ml/min, column and RID temperature
30ºC, 5 μl sample. The HPLC chromatogram was
Results, Discussion shown on Fig 2. Under these HPLC conditions the
last sugar was eluted around 15 minutes from the
Initial work on HPLC system for method analysis
development used a mobile phase consisting of
acetonitrile/water (75:25) at flow rate 1.4 ml/min.
The run time for resolution of fructose, glucose,
sucrose, maltose and lactose 16 min, but the tailing
and not good separated peaks of maltose and lactose
were observed (Fig.1).

Figure 3. HPLC chromatograms obtained after test

Figure 1. First HPLC Chromatogram: 1.
rhamnose, 0.1mg/ml, 2. fructose, 3. glucose,
for interference with NaCl solution.
The presence of NaCl in sample didn`t interfered in
the sample analysis [16]. The resolution was
adequate for the target sugars chromatograms show
no significant interferences

Table 1
Baseline noise of RI detector under different HPLC conditions

Mobile Phase column Flow rate, Column RID Injection BaselineNoise

ACN/H2O mg/ml T,ºC T, ºC volume, μl Area[nRIU*s] Height, [nRIU]
75:25 long 1.4 30 30 5 1104,61 634,59
80:20 long 1.4 35 35 5 1865,51 146,72
80:20 long 2.0 30 30 5 925,72 78,21
80:20 long 2.0 35 35 5 2299,38 170,82
80:20 long 2.0 30 30 5 308,81 38,22
80:20 short 2.0 30 30 5 619,26 58,61
80:20 long 2.0 30 30 15 1940,29 185,73

The linear correlation coefficient R2 was 0.999 for all five analysed sugars as the calibration curves were
linear in the concentration range 0.5 to 5 mg/ml. Low LOD and LOQ values (Table 2) were defined for all
analysed carbohydrate standards The obtained results indicated the satisfactory sensitivity of the proposed
HPLC method.

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Table 2
Linearity of calibration curve (n=5), limit of detection and limit of quantification for sugar analysis

Sugars equation R2a IDLb, mg/ml LOQc, mg/ml

Fructose y=20681x-7.11 0.999 0.13 0.45
Glucose y=19414+145.52 0.999 0.14 0.47
Sucrose y=21692x-1966.6 0.999 0.22 0.52
Maltose y=15358x-1332.8 0.999 0.27 0.69
Lactose y=18748x-3969.1 0.999 0.36 0.71
a
correlation coefficient, blimit of detection of the instrument, climit of, x-concentration (mg/ml); y-peak area.

The precision of the presented method was evaluated by the repeatability expressed by the repeatability and
reproducibility. Using the peak area it was defined that RSD was in range 3.6-10 %. (Table 3). Data from the
analysis demonstrated good precision.

Table 3
Precision of the instrument (n=10)

Sugars repeatability reproducibility

Mean±SD, mg/ml RSD, % Mean±SD, mg/ml RSD, %
Fructose 2.5±0.1 3.9 2.6±0.1 3.7
Glucose 2.5±0.1 3.9 2.6±0.1 4.1
Sucrose 2.6±0.2 6.8 2.5±0.1 1.5
Maltose 2.4±0.2 10.5 2.5±0.2 6.6
Lactose 2.4±0.2 8.6 2.6±0.2 8.7
SD- standard deviation, RSD - relative standard deviation

To evaluate the repeatability of the method, ten replicate determinations were carried out on the same day.
For reproducibility, ten determinations with the same biscuit sample on different days were done. The standard
deviations and relative standard deviations (R.S.D.s) show good precision (Table 4) within the limits of
acceptable variability in methods of analysis.

Table 4
Repeatability and reproducibility of the method

Sugars Repeatability Reproducibility

Mean±SD, mg/ml RSD, % Mean±SD, mg/ml RSD, %
Fructose 4.1±0.5 12.5 6.2±0.6 9.9
Glucose 4.2±0.5 13.3 6.0±0.9 14.5
Sucrose 10.8±0.5 4.8 9.5±0.7 7.6
Maltose 0.9±0.1 9.5 0.6±0.1 9.6
Lactose 1.2±0.1 7.5 0.7±0.1 5.9
SD- standard deviation, RSD - relative standard deviation

The developed HPLC method showed satisfactory recovery of the three levels of added sugars (Table 5).
Table 5
Result from recovery of the sugars

Added sugars, g/100g Fructose Glucose Sucrose Maltose Lactose

3.3 83±19 94±24 94±18 107 ±26 84±16
6.6 96±5 107±6 106±5 116±8 93±12
10 103±6 118±19 108±4 121±12 100±11

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