A rapid high performance liquid chromatographic method with evaporative light scattering detection (HPLC-ELSD), using a carbohydrate column, was developed for simultaneous determination of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) in dairy foods. Sample preparation was performed by precipitation using acetonitrile. The limits of detection were 2.097 mg/L for GlcNAc and 3.247 mg/L for GalNAc. The limits of quantification were 6.043 mg/L for GlcNAc and 9.125 mg/L for GalNAc. Accuracy ranged from 96.4 to 105.7% for GlcNAc and from 97.1 to 104.1% for GalNAc. The precision of the method was <1.7% for GlcNAc and <2.2% for GalNAc. The mean recovery of the method was measured by spiking samples with 30.0–120.0 mg/L GlcNAc or 12.5–50.0 mg/L GalNAc and was found to be 95.1–105.5% for GlcNAc and 99.5–105.9% for GalNAc. The stability test results of standard solutions stored at 4, 20, and 40°C were 96.2–104.7% for GlcNAc and 98.0–106.5% for GalNAc. This study determined GlcNAc and GalNAc in dairy foods using HPLC-ELSD method. This rapid, simultaneous quantitation method might be useful as a mean of convenient quality control of dairy foods.
N-Acetylgalactosamine (GalNAc) is known to be the most potent inhibitor of agglutination of trypsin-treated rabbit erythrocytes, being 6 times more inhibitory than galactose or
GlcNAc and GalNAc are kind of carbohydrates in milk [
GalNAc (Figure
Structure of GalNAc and GlcNAc: (a) GalNAc and (b) GlcNAc.
These previous methodologies were not simultaneous analysis for determining GlcNAc and GalNAc. It analyzed fructose, rhamnose, arabinose, galactinol, galactose, and so on which is one part of monosaccharides and more complex carbohydrates. Most of the analysis of carbohydrate studies was performed by complicated derivatization procedure [
In recent years, high-performance liquid chromatography- (HPLC-) ELSD has been developed for better quantitation of mono- and oligosaccharides in dairy foods [
The aim of this study was to develop a no derivatization procedure for the simultaneous analysis of dairy foods. The developed method may be employed as a valuable tool for quality control of dairy products analysis of GlcNAc and GalNAc.
Analytical reagent grade methanol (MeOH) and acetonitrile (ACN) were obtained from Fisher scientific (Fair Lawn, NJ, USA). Water was purchased from J. T. Baker (Phillipsburg, NJ, USA). GlcNAc (99%) and GalNAc (99%) standards, Zinc sulfate heptahydrate (99%), and Potassium hexacyanoferrate trihydrate (98.5~102.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Samples (infant formula, yogurt, UHT milk, and raw milk) were immediately frozen and stored at −70°C. Infant formula, yogurt, and UHT milk (Namyang Dairy, Gongju, South Korea) were purchased at a local grocery store in Daejeon city (South Korea). The raw milk samples were collected from Southwestern cattle ranches in South Korea Gongju city. About 1 g of infant formula was weighted and then diluted with 10 mL water. The sample was prewarmed in a 40°C water bath, homogenized by a homogenizer (Omnimacro, model 17505, GA, USA) at 3000 rpm for 5 minutes, and combined with 10 mL ACN. The solution was sonicated at room temperature for 10 minutes and then centrifuged at 13,000 rpm for 10 min at 4°C (Kendro, Hanau, Germany). 1 mL of supernatant was taken and concentrated with N2 and then re-dissolved with 1 mL of mobile phase and injected into the chromatographic system. About 2 g of yogurt sample was weighted and then diluted with 2 mL water. The solution was treated using the method described above, infant formula, and then 6 mL of ACN was added. Raw milk and UHT milk were also treated with the same method, but raw milk and UHT milk were not diluted with water. According to the content of total solids in dairy foods, there are a few differences on sample procedures. The crude fat and proteins were precipitated with using Carrez solutions I (2.7 g of Potassium hexacyanoferrate (II) trihydrate in 100 mL water) and II (5.5 g of Zinc sulfate heptahydrate in 100 mL water) and the samples were centrifuged at 13000 rpm for 15 min at 4°C in order to remove fats [
The HPLC (Agilent 1100, Palo Alto, CA, USA) equipped with ELSD (Alltech 2000AS, Deerfield, IL, USA) was used to separate and detect GlcNAc and GalNAc. A column, Prevail Carbohydrate ES (250 mm × 4.6 mm, 5
The validation was conducted according to the International Union of Pure and Applied Chemistry (IUPAC) 2002 guidelines and International Conference on Harmonisation (ICH) 2005 [
The stock standard solution of each compound was prepared as follows: an accurately weighed amount of GlcNAc and GalNAc (20 mg and 16 mg, resp.) was placed into a 100 mL volumetric flask and brought up to volume with a mixture of ACN : water : MeOH (60 : 20 : 20, v/v). The final concentration of GlcNAc was 200
Matrix-matched standard solutions were prepared using raw milk, UHT milk, yogurt, and infant formula. Ranges of 10–200
A calibration curve was used when determining precision, accuracy, recovery, and dynamic range. The standard curve was composed of ten points. We used a third degree polynomial equation of the form
To evaluate the selectivity of the method, matrix samples of raw milk, UHT milk, yogurt, and infant formula were prepared according to the method (Section
Precision studies were carried out by determining the interday and intraday reproducibility of the peak areas. Intraday tests were carried out using four determinations at concentration levels corresponding to 25–150
The limit of detection (LOD) is defined as the lowest concentration of an analyte in a sample that can be positively identified against background. The limit of quantification (LOQ) is the lowest concentration of an analyte that can be accurately measured with acceptable precision under the operational conditions of the method. The LOD is defined as the concentration when the signal to noise ratio (S/N ratio) is 3. The LOQ is the analyte concentration at which the S/N ratio is equal to 10.
Recovery tests were performed by spiking infant formula, yogurt, UHT milk, and raw milk. The concentrations of GlcNAc were 30, 60, and 120
To determine the stability of standard solutions samples were stored at 4, 20, and 40°C for 10 days. The individual stock solutions of GlcNAc and GalNAc were prepared in mobile phase and the concentration of 25, 150 mg/L for GlcNAc and 10, 100 mg/L for GalNAc was diluted with the same solvent composition. In order to gain stability of samples, infant formula, yogurt, UHT milk, and raw milk also were stored at 4, 20, and 40°C for 10 days. Samples were performed in triplicate for three-temperature level to determine stability. The standard solution and samples stored at 4°C were kept in a refrigerator, and the samples stored at 20 and 40°C were stored in a microorganism incubator (Tuttlingen, BINDER GmbH, Germany).
GlcNAc and GalNAc were extracted from the dairy foods, using ACN or Carrez solution. Highest resolution was achieved using extraction by ACN. The crude fat content was 2.0 mg/100 mL using Carrez solution and 12.7 mg/100 mL using ACN solution. The crude protein content was 9.1 mg/100 mL using Carrez solution and 24.5 mg/100 mL using ACN solution. The ACN extracted solution was reduced crude fat and proteins in dairy foods (infant formula, yogurt, UHT milk, and raw milk). In the case of crude fat and protein, the Carrez method showed the lowest content of crude fat. However, when samples were extracted via the Carrez method, it was found that GalNAc remained unseparated and the sensitivity to GlcNAc was low. In the case of extraction with ACN, the ratio of elimination in crude fat and crude protein was lower than that of the Carrez method but the peaks resolved the most clearly with ACN method and showed high yielding results for GlcNAc and GalNAc. The yields obtained in the extraction step for Carrez solution and ACN were 67.2~75.3% and 95.1~105.9%, respectively. Moreover, the ACN method was very easy to utilize for sample handling and accurate analysis was possible. Therefore, ACN was selected as the extraction solvent.
Calibration curve for GlcNAc and GalNAc was evaluated using ten-pointed calibration on four dairy foods (infant formula, yogurt, UHT milk, and raw milk). The determination coefficient (
Parameters of regression equations for calibration curves and range.
Compound |
|
|
|
|
|
Range ( |
---|---|---|---|---|---|---|
GlcNAc | −7.353 |
4.489 |
12.327 | 79.609 | 0.9999 | 25–200 |
GalNAc | −1.922 |
6.089 |
−4.936 | 252.349 | 0.9997 | 10–160 |
For each curve the equation is
Table
Comparison of results obtained by standard solution-based and matrix-matched calibration.
Matrix | FC |
Recovery (%) | |||
---|---|---|---|---|---|
GlcNAc | GalNAc | ||||
S |
M |
S |
M | ||
Raw milk | 25 | 101 | 100 | 98 | 101 |
50 | 101 | 97 | 97 | 100 | |
100 | 102 | 100 | 99 | 102 | |
|
|||||
UHT milk | 25 | 98 | 100 | 100 | 100 |
50 | 99 | 102 | 101 | 101 | |
100 | 102 | 101 | 99 | 100 | |
|
|||||
Yogurt | 25 | 103 | 103 | 98 | 97 |
50 | 99 | 99 | 99 | 98 | |
100 | 99 | 101 | 100 | 99 | |
|
|||||
Infant formula | 25 | 99 | 100 | 100 | 100 |
50 | 103 | 102 | 101 | 103 | |
100 | 102 | 102 | 102 | 102 |
The mixture of GlcNAc and GalNAc was successfully separated by the optimum chromatographic conditions (Section
HPLC-ELSD chromatograms of undiluted samples of (a) infant formula, (b) yogurt, (c) raw milk, and (d) UHT milk. Experimental conditions as in Section
The precision of the chromatographic system was tested by performing five independent intra- and interday replicate measurements of a standard solution containing GlcNAc and GalNAc and then checking the RSD of the peak areas. Five independent replicates were performed each day for five consecutive days. Table
Intra- and interday precision and accuracy data of GlcNAc and GalNAc determination (
Compound | FC |
Intraday | Interday | ||||
---|---|---|---|---|---|---|---|
MAC |
RSD |
AC |
MAC |
RSD |
AC | ||
GlcNAc | 25 | 24.11 | 1.2 | 96.4 | 25.10 | 1.7 | 100.4 |
75 | 75.10 | 0.4 | 100.1 | 74.49 | 1.6 | 99.3 | |
120 | 121.78 | 0.7 | 101.5 | 121.68 | 1.0 | 101.4 | |
150 | 158.48 | 0.3 | 105.7 | 153.17 | 1.3 | 102.1 | |
|
|||||||
GalNAc | 10 | 10.31 | 2.2 | 103.1 | 10.37 | 1.7 | 103.7 |
25 | 24.55 | 2.0 | 98.2 | 24.91 | 1.8 | 99.6 | |
50 | 48.54 | 1.7 | 97.1 | 50.13 | 1.6 | 100.3 | |
100 | 104.07 | 0.7 | 104.1 | 101.95 | 1.1 | 101.9 |
Within-laboratory precision, expressed as relative standard deviation for different working days (RSD), for four different matrices fortified with GlcNAc and GalNAc at the same concentration as was originally detected (
Recovery (%) | ||||||||
---|---|---|---|---|---|---|---|---|
Operator | Raw milk | UHT milk | Yogurt | Infant formula | ||||
Recovery | RSD |
Recovery | RSD |
Recovery | RSD |
Recovery | RSD | |
GlcNAc | ||||||||
A |
101.7 | 0.5 | 101.7 | 0.7 | 102.1 | 0.8 | 97.7 | 1.1 |
B |
101.8 | 1.0 | 101.6 | 0.1 | 98.6 | 1.2 | 100.9 | 0.5 |
C |
102.4 | 0.6 | 103.8 | 0.5 | 100.0 | 1.0 | 102.0 | 1.0 |
|
||||||||
GalNAc | ||||||||
A |
102.5 | 12 | 98.5 | 0.2 | 101.6 | 1.2 | 101.8 | 2.3 |
B |
101.8 | 0.1 | 101.2 | 0.7 | 101.0 | 0.5 | 100.0 | 0.5 |
C |
98.4 | 1.3 | 99.4 | 1.3 | 101.2 | 0.4 | 101.3 | 0.5 |
LOD and LOQ under the present chromatographic condition were determined on the basis of response and slope of each regression equation at a signal to noise ratio (S/N) of 3 and 10, respectively. Matrix spiked method was used for LOD and LOQ calculation. The LOD and LOQ ranged from 2.097 to 6.043
The recovery rates were close to 100% in almost all cases. The recovery of the method was satisfactory with accuracy ranging from 95.12% to 105.88%, respectively. Considering the results of the recovery test, this method can be considered accurate. The detailed data is shown in Table
Recovery and endogenous concentration data of GlcNAc and GalNAc from four different dairy foods (
Matrix | GlcNAc | GalNAc | ||||||
---|---|---|---|---|---|---|---|---|
EC |
FC |
Recovery (%) | RSD |
EC |
FC |
Recovery (%) | RSD | |
Raw milk | 152.3 ± 3.4 | 30.0 | 102.06 | 1.94 | 189.3 ± 12.1 | 12.5 | 100.50 | 2.46 |
60.0 | 101.41 | 1.18 | 25.0 | 99.92 | 1.37 | |||
120.0 | 95.12 | 0.59 | 50.0 | 103.30 | 2.08 | |||
|
||||||||
UHT milk | 150.2 ± 5.4 | 30.0 | 102.39 | 3.21 | 115.0 ± 1.1 | 12.5 | 103.34 | 1.81 |
60.0 | 97.02 | 0.42 | 25.0 | 105.88 | 0.84 | |||
120.0 | 101.06 | 2.99 | 50.0 | 102.70 | 0.57 | |||
|
||||||||
Yoghurt | 151.2 ± 11.1 | 30.0 | 101.20 | 0.66 | 157.0 ± 5.5 | 12.5 | 99.39 | 0.91 |
60.0 | 100.20 | 0.79 | 25.0 | 100.58 | 0.92 | |||
120.0 | 100.81 | 1.03 | 50.0 | 99.79 | 0.62 | |||
|
||||||||
Infant formula | 720.0 ± 1.6 | 30.0 | 105.45 | 0.68 | 1118.4 ± 12.3 | 12.5 | 100.56 | 1.38 |
60.0 | 103.95 | 2.90 | 25.0 | 102.17 | 0.81 | |||
120.0 | 103.26 | 1.25 | 50.0 | 99.51 | 1.10 |
Endogenous concentration of the GlcNAc and GalNAc from dairy foods were determined by constructing a five-point calibration curve using HPLC-ELSD conditions identical to those used for the test materials. Analyte concentrations were calculated according to the following:
The contents of GlcNAc and GalNAc found in dairy foods are shown in Table
The stability of standard solution was tested at 4, 20, and 40°C for 10 days. The stability of standard solution was satisfactory with accuracy ranging from 96.20 to 106.50%. We conclude that standard solutions are stable for at least 10 days at three different temperatures. The detailed data is shown in Table
Stability results for GlcNAc and GalNAc standard solutions after ten days at 4, 20, and 40°C (
Compound | ST |
FC |
Stability ( | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Day 1 | Day 2 | Day 3 | Day 7 | Day 10 | ||||||||
AS |
RSD |
AS |
RSD |
AS |
RSD |
AS |
RSD |
AS |
RSD | |||
GlcNAc | 4 | 25 | 100.16 | 0.04 | 98.32 | 0.28 | 102.16 | 0.77 | 104.72 | 0.52 | 104.20 | 1.54 |
150 | 99.72 | 1.04 | 101.84 | 0.27 | 101.59 | 1.25 | 100.33 | 0.28 | 102.00 | 0.80 | ||
20 | 25 | 98.32 | 3.32 | 98.76 | 3.60 | 99.64 | 2.92 | 102.64 | 1.85 | 96.20 | 3.99 | |
150 | 99.72 | 1.11 | 99.69 | 0.83 | 101.79 | 0.79 | 101.49 | 1.29 | 102.21 | 0.75 | ||
40 | 25 | 102.36 | 3.27 | 103.44 | 2.65 | 101.72 | 3.73 | 104.56 | 0.66 | 104.16 | 1.74 | |
150 | 101.99 | 1.12 | 102.88 | 0.46 | 102.30 | 0.15 | 101.84 | 0.43 | 97.99 | 1.34 | ||
|
||||||||||||
GalNAc | 4 | 10 | 103.50 | 1.81 | 102.50 | 1.23 | 103.70 | 2.79 | 105.70 | 0.20 | 103.10 | 4.64 |
100 | 98.58 | 1.11 | 98.54 | 0.42 | 105.04 | 0.38 | 104.86 | 0.53 | 101.25 | 0.49 | ||
20 | 10 | 98.00 | 1.75 | 103.40 | 3.95 | 105.08 | 3.54 | 102.50 | 1.27 | 105.70 | 3.63 | |
100 | 102.36 | 1.05 | 103.53 | 1.08 | 103.05 | 0.78 | 104.24 | 1.14 | 98.68 | 0.37 | ||
40 | 10 | 99.00 | 1.08 | 106.50 | 3.83 | 102.10 | 4.98 | 100.90 | 3.84 | 98.40 | 4.37 | |
100 | 101.58 | 0.21 | 100.77 | 2.16 | 101.66 | 3.34 | 100.24 | 2.96 | 100.46 | 0.17 |
In this paper we report a simple, rapid, and sensitive HPLC-ELSD method for the simultaneous quantification of GlcNAc and GalNAc in dairy foods. The developed method could be employed as a valuable tool during routine analysis for GlcNAc and GalNAc in dairy foods, as the ability to simultaneously determine the levels of these components in dairy foods will play an important role in research.
Validation experiments were performed using assays with standard solutions, samples, and spiked samples to evaluate the quality of the data and ensure the reliability of the method. Method linearity, accuracy, precision, stability, and selectivity were evaluated, as were limits of detection and quantification. The simultaneous method described here offers a convenient means for quality control of dairy foods.
The authors declare that there is no conflict of interests regarding the publication of this paper.