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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%.
761
НАУЧНИ ТРУДОВЕ SCIENTIFIC WORKS
ТОМ LX VOLUME LX
“ХРАНИТЕЛНА НАУКА, ТЕХНИКА И „FOOD SCIENCE, ENGINEERING AND
ТЕХНОЛОГИИ – 2013“ TECHNOLOGY 2013“
18-19 октомври 2013, Пловдив 18-19 October 2013, Plovdiv
fructose, sucrose, maltose and lactose in foodstuffs. The and water in ratio 75:25 and 80:20; flow rate 1.4
developed method must be simple, successfully validated in ml/min and 2.0 ml/min; Agilent Zorbax
everyday laboratory practice, suitable for a routine sugar Carbohydrate Analysis (4.6 ID × 250 mm) and (4.6
analysis and applicable for a wide range of foods. 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
Materials and Methods injector. The baseline stability and separation of the
All used reagents and solvents were analytical sugars was observed. The detectors noise was
grade. HPLC grade water (Lichrosolve Merck) and measured by integrating 15 peaks from baseline of
acetonitrile (Lab-Scan Analitycal Science) were used each injected sample.
for preparation of the mobile phase. Linearity of the HPLC method. The detector
Instrumentation: Chromatographic separation linearity was tested by injecting 5 sugars solutions at
was performed on HPLC apparatus Hewlet Packard 0.2, 0.5, 1.0, 2.5 and 5 mg/mL. HPLC separation was
1100 Series consisted of degasser Iso pump, performed using as the mobile phase
automatic sample injector and associated with IBM- acetonitrile/water (80:20), at a flow rate of 2
compatible computer. Refractive index detector mL/min. The column and the refractive index
(RID) Hewlet Packard 1100 Series was used for detector were maintained at 30 °C. The injection
sugars detection and quantification. The HPLC volume was 5 μL.
separation of sugars was performed on columns Determination of Limit of Detection (LOD)
Agilent Zorbax Carbohydrate Analysis – and Limit of quantification of instrument (LID)
aminopropyl column (stationary phase silica LOD is calculated by measuring signal-to-noise
spherical particles, 5 µm in diameter, which is (S/N) of the baseline and multiplying this value by 3.
reacted with 3-aminopropylsilane), as two colum LOQ is determinate by measuring signal-to-noise
were tested for the method development: one with (S/N) and multiplying this value by 10 [8, 9].
dimension 4.6 mm × 250 mm and 4.6 mm × 150 Precision
mm; guard column Agilent Zorbax NH2, 4.6 mm × Repeatability and reproducibility of the
12.5 mm. The temperature of the column was instrument. For evaluation of repeatability of the
remained constant by a thermostat Agilent 1100. The instrument standard solution with concentration 2.5
control of the system, data acquisition and data mg/ml of each sugar was injected 10 times in HPLC
analysis were under the control of the software apparatus in single day. For checking of
program Agilent Chem Station. The used mobile reproducibility standard solution with the same
phase was acetonitrile-water in ratio 75/25 and concentration was injected in once per day in the
80:20. Sugars concentration was calculated based on HPLC apparatus for a period of 10 days [2, 8, 9] .
peak area measurements. Test for Interference of Sodium Chloride in
the Biscuit Samples. The solution of 1mg/ml
Method development sodium chloride with concentration was injected in
Sugar standards were dried at 60 °C in vaccum the HPLC under mention above HPLC condition.
oven overnight and then dissolved in acetonitrile- The obtained HPLC chromatogram was overlaid
water 50/50. The stock solutions with sugar (glucose, with sample chromatogram.
fructose, sucrose, maltose and lactose) concentration Sample preraration. Biscuit sample was finely
0.5 mg/ml and 0.1 mg/ml rhamnose were injected ground in laboratory homogenizer (Ika, Germany).
into HPLC column Agilent Zorbax Carbohydrate Five grams biscuits were weighed in 250 ml
Analysis with size 4.6 ID × 250 mm with guard Erlenmeyer flask, 100 ml 60 % (v/v) EtOH were
column, flow rate 1.4 ml/min, isocratic mobile phase added to them. Three samples were prepared at the
acetonitrile/water in ratio 75:25, temperature of same time. The samples were measured on a balance,
detector and column 30ºC, volume of injection 5 μl cover with Parafim “M” and were placed in shaking
without needle wash. Under the same conditions six water bath at 83°C for 20 min. Then the samples
stock solutions with concentration 10 mg/ml were were cooled to the room and their weigh were
injected to observed the order of elution of the sugars brought to the initial by 60 % EtOH addition. The
and their retention times. samples were filtered through Whatman N4 paper.
Optimization of the HPLC method. The Additional sample clean-up was performed on the
standard solutions of sugar with concentration 5 filtrate by sequentially filtering through a Sep-Pak
mg/ml, 2.5 mg/ml, 1 mg/ml and 0.5 mg/ml were C18 cartridge (Waters, Milford, MA) and a 0.22 µm
injected in the HPLC apparatus under different membrane filter (Sterivex™-GS (Millipore®) before
combination of conditions: Mobile phase acetonitrile
762
НАУЧНИ ТРУДОВЕ SCIENTIFIC WORKS
ТОМ LX VOLUME LX
“ХРАНИТЕЛНА НАУКА, ТЕХНИКА И „FOOD SCIENCE, ENGINEERING AND
ТЕХНОЛОГИИ – 2013“ TECHNOLOGY 2013“
18-19 октомври 2013, Пловдив 18-19 October 2013, Plovdiv
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).
Table 1
Baseline noise of RI detector under different HPLC conditions
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.
763
НАУЧНИ ТРУДОВЕ SCIENTIFIC WORKS
ТОМ LX VOLUME LX
“ХРАНИТЕЛНА НАУКА, ТЕХНИКА И „FOOD SCIENCE, ENGINEERING AND
ТЕХНОЛОГИИ – 2013“ TECHNOLOGY 2013“
18-19 октомври 2013, Пловдив 18-19 October 2013, Plovdiv
Table 2
Linearity of calibration curve (n=5), limit of detection and limit of quantification for sugar analysis
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)
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
The developed HPLC method showed satisfactory recovery of the three levels of added sugars (Table 5).
Table 5
Result from recovery of the sugars
764
НАУЧНИ ТРУДОВЕ SCIENTIFIC WORKS
ТОМ LX VOLUME LX
“ХРАНИТЕЛНА НАУКА, ТЕХНИКА И „FOOD SCIENCE, ENGINEERING AND
ТЕХНОЛОГИИ – 2013“ TECHNOLOGY 2013“
18-19 октомври 2013, Пловдив 18-19 October 2013, Plovdiv
[11] Nielson, Suzanne S., ”Food Analysis”, 3th edition,
chapter 28, Kluwer Academic/Plenum, Springer, New
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[12] Nollet, Leo M.L., ”Food Analysis by HPLC”, 2nd
A simple and reproducible HPLC method with edition, 7 HPLC Determination of carbohydrates in
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glucose, fructose, sucrose, maltose and lactose in [13] Richmond, M.L et al., ”Separation of Carbohydrates
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validated in laboratory practice. This method is Chromatography”, J Dairy Sci 65, 1982, pp. 1394-
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the sugars content in foods, possible adulteration and Analysis’’, Agilent Technologies Inc, Printed in USA,
2002., pp. 86-90
stability in the sugar fraction. The method provides
[15] Sesta, G., “Determination of Sugars in Royal Jelly by
satisfactory precision, recovery and sensitivity. HPLC“, Apidology, 37, 2006
[16] Sims A., “HPLC analysis of Sugars in Foods
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The authors are grateful to Institute Meurice 377-380.
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HPLC equipment. J., “Practical HPLC Method“, 2nd edition, John Wiley
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