62/2000 Protect the health of infants and young children! Wanyi ion chromatography determines seven types of human mil

Preface

Human milk oligosaccharides (HMOs) are a group of oligosaccharides with a degree of polymerization no higher than 3, naturally present in human milk; they are the third largest solid component in human milk, after lactose and fat. They regulate the intestinal flora—particularly promoting the colonization of Bifidobacteriaceae in the infant gut—while also maintaining intestinal health, supporting immune and brain development, and exerting anti-infective effects, playing an important role in infant growth. Since the first HMO-fortified infant formula was launched in the United States in 2015, a wide range of infant formulas, milk and dairy products, digestive-health supplements and functional foods containing HMOs have been sold worldwide. As of June 1, 2023, the US and EU have approved seven HMOs for use: 2'-fucosyllactose, 3'-fucosyllactose, difucosyllactose, lacto-N-neotetraose, lacto-N-tetraose, 3'-sialyllactose and 6'-sialyllactose.

Among common HMO detection methods, liquid chromatography requires a derivatization reaction before detection, involves complex sample preparation, and the derivatization reagents are often toxic; the heat-labile nature of HMOs also leads to low response and poor repeatability in LC-MS. In contrast, ion chromatography with amperometric detection for carbohydrates offers simple sample preparation, high response and good repeatability, giving it clear advantages for HMO analysis. This application note uses manually prepared sodium hydroxide, sodium acetate and ultrapure water as eluents, and employs the WAYEAL IC6300 ion chromatograph to determine HMOs in infant formula. The method features simple sample preparation, high sensitivity and good linearity, providing a useful reference for HMO detection in foods.

Keywords: ion chromatograph; human milk oligosaccharides; amperometric detector.

1. Instruments, Equipment and Reagents

1.1 Reagents and Standards

Table 1. List of reagents and standards

1.2 Laboratory Materials and Auxiliary Equipment

• 1.2.1 Analytical balance (0.1 mg readability);
• 1.2.2 Thermostatic water bath;
• 1.2.3 Centrifuge;
• 1.2.4 Aqueous polyethersulfone (PES) membrane filters (0.45 µm);
• 1.2.5 Ultrapure water;
• 1.2.6 Vacuum filtration device;
• 1.2.7 RP solid-phase extraction (SPE) pretreatment cartridges;
• 1.2.8 Disposable syringes (20 mL, 5 mL).

2. Experimental Method

2.1 Solution Preparation

• 2.1.1 200 mmol/L sodium hydroxide solution: Pipette 10.5 mL of 50% NaOH, dilute with water to 1000 mL, and filter under vacuum.
• 2.1.2 Mixed solution of 75 mmol/L NaOH and 250 mmol/L sodium acetate: Accurately weigh 20.51 g sodium acetate, dissolve in water and transfer to a 1000 mL volumetric flask; accurately pipette 3.95 mL of 50% NaOH, dilute to 1000 mL with ultrapure water, and filter under vacuum.
• 2.1.3 3% sulfosalicylic acid solution: Weigh 3.0 g sulfosalicylic acid, dissolve in water and dilute to 100 mL (prepare fresh).
• 2.1.4 Mixed standard stock solution: Accurately weigh 5 mg each of 2'-fucosyllactose, 3'-fucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose and lacto-N-neotetraose (to the nearest 0.1 mg), place in a 10 mL volumetric flask, dissolve in water and dilute to the mark to prepare a 500 mg/L mixed stock. Separately weigh 1 mg of difucosyllactose (to the nearest 0.1 mg), place in a 10 mL volumetric flask, dissolve and dilute to the mark to prepare a 100 mg/L stock.
• 2.1.5 Mixed standard working solutions: Accurately pipette the mixed stock solution (2.1.4) into volumetric flasks and dilute with water to prepare mixed working solutions of 2'-FL, 3'-FL, 3'-SL, 6'-SL, LNT, LNnT and DFL at 0.5, 2, 4, 6, 8 and 10 mg/L. Prepare fresh before use.

2.2 Sample Preparation

Weigh approximately 2.0 g sample into a small beaker, mix thoroughly with 50 mL warm water (about 50 °C), add 40 mL of 3% sulfosalicylic acid solution, transfer and dilute to 100 mL in a volumetric flask, and sonicate for 10 min. Filter through filter paper or centrifuge (12,000 r/min, 15 min). Pass the sample solution through an RP SPE cartridge and then a 0.45 µm aqueous membrane filter; discard the first 3 mL and collect the eluate for analysis. Before use, condition the RP SPE cartridge with 20 mL methanol followed by 15 mL water, and equilibrate for 30 min.

2.3 Experimental Conditions

2.3.1 Ion chromatography conditions

3. Results

3.1 Linear Range

An appropriate aliquot of each mixed standard working solution was analyzed. The linear range was 0.5–10 mg/L; deviations between measured and nominal concentrations were within the maximum allowable deviation, R² values were above 0.999, and all components showed good linearity.

Table 2. Linear ranges of the compounds

Figure 1. Overlaid calibration chromatograms of the seven HMO standards

3.2 Sample Test Results

Samples 1 and 2 were tested after 10-fold dilution. The content of each of the seven HMOs was calculated as follows: X is the content in the sample (mg/100g); c is the concentration in the sample solution from the calibration curve (mg/L); V is the constant volume (mL); m is the sample mass (g); f is the dilution factor. The calculated contents (mg/100g) are as follows:

Figure 2. Ion chromatogram of Sample 1 diluted 10-fold

Figure 3. Ion chromatogram of Sample 2 diluted 10-fold

Table 3. Sample test results

3.3 Precision

The working solution at the fourth calibration level was analyzed in six consecutive injections under the conditions in 2.3.1. The RSD of the seven HMO contents was calculated to evaluate repeatability. The results below indicate good method precision.

3.3.1 Standard Repeatability Precision

Test results:

Figure 4. Overlaid chromatograms of independent replicate injections of calibration standard S4

Table 4. Repeatability precision of the standard

3.4 Limits of Detection and Quantitation

LOD: Aliquots of the standard solution were diluted to a mixed solution containing 10 µg/L 3'-FL, 30 µg/L DFL, 30 µg/L 2'-FL, 22 µg/L LNnT, 67 µg/L LNT, 67 µg/L 6'-SL and 160 µg/L 3'-SL; 20 parallel samples were prepared and analyzed.

LOQ: Aliquots of the standard solution were diluted to a mixed solution containing 30 µg/L 3'-FL, 80 µg/L DFL, 90 µg/L 2'-FL, 65 µg/L LNnT, 200 µg/L LNT, 200 µg/L 6'-SL and 500 µg/L 3'-SL; six parallel samples were prepared and analyzed.

The test results are as follows:

Table 5. S/N results for LOD and LOQ of the seven HMOs

3.5 Specificity

Aliquots (100 µL each) of glucose, fructose, sucrose and maltose standard solutions were added to a 10 mL volumetric flask and diluted to the mark with Sample 2 solution diluted 67-fold. The solution was analyzed to check whether these sugars interfered with the peaks of the seven HMOs. As shown below, the four sugars showed no significant interference with the determination of the seven HMOs in the milk powder.

Figure 5. Overlaid chromatograms of calibration S6, Sample 2, and Sample 2 spiked with the four sugar standards

4. Conclusion

This validation used the WAYEAL ion chromatograph to verify the method for determining HMOs in foods. The data show that all chromatographic peaks are well-shaped without tailing; 2'-FL, 3'-FL, DFL, LNnT, LNT, 6'-SL and 3'-SL all elute within 60 min; sensitivity meets national standard requirements; and linear correlation coefficients are all greater than 0.999. Repeatability of both standards and samples is good. Except for the two sialyllactoses, the LOD and LOQ are at the µg/L level. The method has good specificity, with no interfering impurities found, and overall meets the standard requirements.