International Journal of Food Engineering and Technology

2018; 2(2): 36-40

http://www.sciencepublishinggroup.com/j/ijfet
doi: 10.11648/j.ijfet.20180202.14
ISSN: 2640-1576 (Print); ISSN: 2640-1584 (Online)

HPLC Determination of Fructo-Oligosaccharides in Dairy

Products
Zhen Zhao, Guoyan Wen*, Cuizhi Li, Peng Wan, Zhiyong Lv
Quality Management Department, Inner Mongolia Yili Industrial Group Co. Ltd, Hohhot, China

Email address:

*
Corresponding author

To cite this article:

Zhen Zhao, Guoyan Wen, Cuizhi Li, Peng Wan, Zhiyong Lv. HPLC Determination of Fructo-Oligosaccharides in Dairy Products.
International Journal of Food Engineering and Technology Vol. 2, No. 2, 2018, pp. 36-40. doi: 10.11648/j.ijfet.20180202.14

Received: November 7, 2018; Accepted: December 4, 2018; Published: January 11, 2019

Abstract: Fructo-oligosaccharides is a natural active substance with excellent physiological functions such as improving the
gastrointestinal tract, regulating intestinal flora, etc. It is an important nutrient in infant dairy products. Therefore, it is extremely
important to accurately detect fructo-oligosaccharides in infant dairy products. Nowadays, the main methods for detection of
fructo-oligosaccharides at home and abroad include high performance liquid chromatography, ion exchange chromatography,
mass spectrometry, etc. In this article, a method for the determination of fructo-oligosaccharides in modified milk and infant
formula milk powder by high performance liquid chromatography (HPLC) was developed. Fructo-oligosaccharides was
extracted by water. Protein was precipitated by acetonitrile. The method was performed on NH2 column, the detector was
refractive index detector (RID) and the mobile phase was 70% acetonitrile. Fructo-oligosaccharides content of modified milk
was in the range of 20-500mg/100g, the recovery was 80.2%-107.1%. The content of Fructo-oligosaccharides in infant formula
milk powder was in the range of 100-500mg/100g, the recovery was 71.9%-95.3%. The quantitative detection limit was
20mg/100g. This method was easily to be operated with a high accuracy and the results indicated that it was suitable for the
analysis of fructo-oligosaccharides in modified milk and infant formula milk powder.

Keywords: HPLC, Fructo-Oligosaccharides, Modified Milk, Infant Formula Milk Powder

Currently, the main oligofructose quantification methods

1. Introduction include thin layer chromatography, gas chromatography mass
Fructo-oligosaccharides (FOS), also known as spectrometry, ion chromatography and liquid chromatography.
oligofructose or oligosaccharides belongs to sugarcane [8-20] Among them, Thin layer chromatography has a low
trisaccharide, is a naturally occurring non-reducing sugar in sensitivity, gas chromatography mass spectrometry is
nature which is mainly composed of 1-Estose (GF2), Nystose complicated in pretreatment and expensive, ion
(GF3), 1F-Fructofuranosylnystose (GF4). [1] Oligofructose chromatography is susceptible to interference from the
has the functions of promoting the growth of beneficial complex substrate, so these methods are difficult to apply in
bacteria in the intestine, improving digestive system function, practical detection. [21, 22] Wheresa HPLC method has the
preventing diarrhea and constipation, promoting absorption of characteristics of high penetration rate and rapid detection,
calcium, preventing dental caries as well as other making it the main method for detecting oligofructose.
physiological function. [2-5] Thus, oligofructose is However, at present, the HPLC method for detecting fructo-
increasingly widely used in a variety of dairy products, infant oligosaccharides in dairy products has the problems such as
foods, sweets and beverages, baking and leisure food and low resolution, the target peak is easily interfered by lactose
health care products. [6, 7] So monitoring the amount of and the result is inaccurate, etc. To solve the above problems,
oligofructose added in food is of great significance for the HPLC treatment of fructo-oligosaccharides in dairy
examining the efficacy of the product and monitoring the products and the conditions of the machine were studied.
quality of the product.
37 Zhen Zhao et al.: HPLC Determination of Fructo-Oligosaccharides in Dairy Products

2. Materials and Experiments According to the sensitivity and the content of the FOS of
the product, dilute the above standard solution with the mobile
2.1. Reagent phase to a series of mixed standard working solution with
Acetonitrile was chromatographically pure; 1-Kestose, suitable concentrations before used.
Nystose, 1F-Fructofuranosylnystose standard products were 2.3.3. Sample Processing
purchased from Japanese Wako company; modified milk Weighed 2.5g of the liquid sample or 1.0g solid sample into
product and infant formula milk powder product of Inner a 50mL volumetric flask, accurate to 0.0001g, added 12.5mL
Mongolia Yili Group. of water (ultra-pure water) at 45°C, dissolved and mixed the
2.2. Equipment sample fully, ultrasonic extraction lasted for 10min at room
temperature, then the sample was diluted to the constant
HPLC (1260, with refractive index detector), Agilent, USA; volume with acetonitrile, continue extracted the sample by
analytical balance (AB265-S), METTLER-DOLEDO ultrasonic extraction for another 10 minutes, filtered the
company, Switzerland; ultrasonic cleaner (GE013), Kunshan solution through filter paper after 30min standing, the filtrate
Ultrasonic Instrument Co., Ltd; high speed refrigerated was filtered through a 0.45µm filter membrane, if the filtered
centrifuge (3K15), Sigma, Germany. liquid is not clear, centrifugation will be carried out at
10000r/min for 5min at 4°C, the supernatant is filtered
2.3. Methods through a 0.45µm filter membrane. Performed the filtrate
2.3.1. Chromatographic Conditions (testing solution) by liquid chromatography.
Column: High performance carbohydrate cartridge (Waters, 2.3.4. Results Calculation
250mm×4.6 mm, 4µm); flow rate 1mL/min; injection volume
10µL; column temperature 35°C; mobile phase acetonitrile: c × V × 100
x= (1)
water = 70:30 (V/V). m × 1000
2.3.2. Standard Solution Preparation Here: x - the content of GF2 (GF3/GF4) in the sample
1-Kestose, Nystose, 1F-fructofuranosylnystose standard (g/100g);
solution (1mg/mL): weigh 0.025g (accurate to 0.0001g) of c - the concentration of GF2 (GF3/GF4) in the testing
these three standard product separately in three 25mL solution (mg/mL);
volumetric flasks, dissolve them with water to the constant V - the volume of the constant volume (mL);
volume and store the solution in a refrigerator at 4°C. m - mass of the sample (g).
C(FOS,g/100g)=C(1-Kestose,g/100g)+C(Nystose,g/100g)+C(1F-Fructofuranosylnystose,g/100g) (2)

repeatedly, the relative standard deviations (RSD) of the

3. Results retention time and the peak area are shown in Table 1 and 2.
3.1. Instrument Repeatability, Standard Curve, Linear The RSD of the retention time of GF2, GF3 and GF4 are
Range and Detection Limit 0.048%, 0.060%, 0.058%. The RSD of the peak area of GF2,
GF3 and GF4 are 1.19%, 2.72%, 0.68%.
Performed the mixed standard solution for six times
Table 1. Instrument repeatability of the retention time.

FOS Retention time/min RSD/%

GF2 7.006, 7.004, 7.001, 7.005, 7.001, 6.999 0.048
GF3 8.635, 8.634, 8.625, 8.634, 8.628, 8.623 0.060
GF4 10.724, 10.725, 10.718, 10.721, 10.713, 10.706 0.058

Table 2. Instrument repeatability of the peak area.

FOS Peak area/nRIU RSD/%

GF2 7820, 7752, 8088, 7893, 7687, 8241 1.19
GF3 8038, 7437, 7729, 8087, 7758, 7904 2.72
GF4 7566, 7604, 7444, 7438, 7760, 7896 0.68

The mixed standard working solution of were determined drawn, the linear equations and correlation coefficient were
under the above chromatographic conditions. The mass obtained (Table 3). The linear range was 20-500mg/100g.
concentration of GF2, GF3 and GF4 were the abscissa (X) and According to the signal-to-noise ratio (RSN)=10, the quantitative
the peak area was the ordinate (Y). Three standard curves were detection limit of the three oligofructose was 20mg/100g.
 International Journal of Food Engineering and Technology 2018; 2(2): 36-40 38

Table 3. Linear equations and correlation coefficients of the fructo-oligosaccharides.

FOS Linear equation Correlation coefficient

GF2 y = 88.4 x - 112.22 0.9996
GF3 y = 82.2 x - 34.96 0.9990
GF4 y = 75.94 x + 106.33 0.9980

Figure 1. HPLC chromatogram of the mixed standard solution of fructo-oligosaccharides.

The chromatograms of the mixed standard solution and retention time of them from the modified milk are 7.560min
samples are shown in Figure 1, 2 and 3. It can be seen from for GF2, 9.478min for GF3, and GF4 was not detected. As can
Figure 1 that the retention time of 1-Kestose, Nystose, be seen from Figure 3, the retention time of them from the
1F-Fructofuranosylnystose are 7.502min, 9.419min, and infant formula milk powder are 7.546min, 9.461min and
11.916min, respectively. As can be seen from Figure 2, the 11.931min, respectively.

Figure 2. HPLC chromatograms of modified milk.

Figure 3. HPLC chromatograms of infant formula milk powder.

39 Zhen Zhao et al.: HPLC Determination of Fructo-Oligosaccharides in Dairy Products

3.2. Method Recovery and Precision

Different concentrations of GF2, GF3 and GF4 were added 4. Discussion

to the blank-modulated milk samples, and the recovery 4.1. Sample Pretreatment Optimization
experiments were carried out, the determination was
performed in parallel six times. As shown in Table 4 and 5, The modified milk sample contains a lot of protein,
when the added amount were 20, 100, 200, 500mg per 100g precipitated the protein with lead acetate, methanol and
for modified milk and 100mg per 100g for infant formula milk acetonitrile respectively. The results showed that all these
powder, the average recovery rate were 83.3%-104.3% for three substances can precipitate the protein in the sample, but
GF2, 80.2%-107.1% for GF3, 81.7%-101.6% for GF4, because the method used a refractive index detector, it has the
respectively for modified milk and 71.9% for GF2, 95.3% for specificity of reference. Therefore, the sample treated with
GF3, 90.0% for GF4 respectively for infant formula milk lead acetate and methanol showed unstable peaks and poor
powder. Six replicate experiments were performed on samples peak shape. When acetonitrile was used as a precipitant and
containing 30mg/100g GF2, GF3 and GF4. The relative the amount of acetonitrile was adjusted to be close to the flow
standard deviations were 3.19%, 1.67%, and 2.04%, ratio, the precipitation effect was good, the retention time of
respectively (Table 6). the peaks were stable, and the peaks shape were better. Well,
acetonitrile was the final choice as a precipitant, the ration was
Table 4. Recovery of blank sample for modified milk. 70% of the constant volume.
The average recovery rate/%
Add amount/mg/100 g 4.2. Selection of Chromatographic Conditions
GF2 GF3 GF4
20 83.3 80.2 81.7
100 88.8 89.5 90.4 When 65% acetonitrile solution was selected as the mobile
200 95.6 100.5 97.1 phase, the peaks of the kestose and lactose in the sample were
500 104.3 107.1 101.6 partially overlapped, and the resolution was not good enough
for detection. When the acetonitrile content was adjusted to
Table 5. Recovery of blank sample for infant formula milk powder. 75%, the kestose and the lactose were able to be separated.
FOS amount/mg/100 g recovery /% However the retention time of 1F-fructofuranosylnystose was
GF2 100 71.9 delayed to 20 min with a broadened peak shape, which was
GF3 100 95.3 not conducive to accurate quantification. The selection of 70%
GF4 100 90.0 acetonitrile was able to completely separate the kestose and
lactose in the sample, and the retention time of
Table 6. Precision of the modified milk.
1F-fructofuranosylnystose was also within 20 min. Moreover,
FOS Contents/mg/100 g RSD/% the peaks shape were good, which can be accurately and
GF2 25.2, 26.9, 24.7, 25.4, 26.1, 26.4 3.19 quantitatively detected. The 70% acetonitrile solution was
GF3 25.3, 24.8, 25.6, 26.0, 25.8, 25.7 1.67
GF4 26.1, 25.7, 25.3, 25.0, 24.8, 26.2 2.04
finally used as the mobile phase.
4.3. Selection of Column Temperature
3.3. Products Results
Performed the testing solution under 25°C, 30°C, 35°C and
Determined modified milk products and infant formula
40°C column temperature conditions respectively, compared
milk powder products by this method, the results of 1-Kestose,
to other spectrums, the spectrum of 35°C column temperature
Nystose, 1F-Fructofuranosylnystose and
showed the best peak shape, a high resolution as well as most
fructo-oligosaccharides are shown in Table 7 and 8.
number of plates, thus 35°C column temperature was chosen
Table 7. Results of modified milk products. to be the final column temperature for the method.
GF2/ GF3/ GF4/ FOS/
Sample
g/100g g/100g g/100g g/100g 5. Conclusion
S1 0.0498 0.0370 — 0.0868
S2 0.0450 0.0256 — 0.0706 The method for detecting fructo-oligosaccharides in dairy
S3 0.0520 0.0312 — 0.0832 products was established by using acetonitrile to precipitate
protein for the purpose of removing impurities,
Table 8. Results of infant formula milk powder products. fructo-oligosaccharides was extracted by water under the
condition of ultrasonic extraction, 70% acetonitrile was used
GF2/ GF3/ GF4/ FOS/
Sample as mobile phase, the fructo-oligosaccharides was separated by
g/100g g/100g g/100g g/100g
J1 0.0732 0.1506 0.0434 0.2672 high performance carbohydrate cartridge and detected by
J2 0.0338 0.0636 0.0116 0.1090 refractive index detector, 1-Estose, Nystose and
J3 0.0750 0.2030 0.0365 0.3145 1F-Fructofuranosylnystose were well separated. The
quantitative detection limit of the method is 20mg/100g. It has
a good linear relationship in the range of 20-500mg/100g. The
 International Journal of Food Engineering and Technology 2018; 2(2): 36-40 40

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