Development of Pretreatment Method for Analysis of Vitamin B₁₂ in Cereal Infant Formula using Immunoaffinity Chromatography and High-Performance Liquid Chromatography

The powdered milk used in this study was purchased from a local market and kept at 4°C for further use. An powdered milk standard reference material, SRM 1849a (National Institute of Standard and Technology, Gaithersburg, MD, USA), which is a certified reference material, was used in the recovery tests. Vitamin B₁₂ content in SRM 1849a was 48.2±8.5 μg/kg. Sodium acetate was purchased from Junsei Chemical (Tokyo, Japan), while the enzyme, amylase, was purchased from ANKOM (catalogue TAHTL-NC24). HPLC grade water and acetonitrile were purchased from Merck (Darmstadt, Germany).

Vitamin B₁₂ in the form of cyanocobalamin (Cat. No. 1152009), with a purity of 1.04% (10.4 μg/mg), was bought from US Pharmacopeial Convention (USP, North Bethesda, MD, USA) to be used as the reference standard. The standard material (100 mg) was dissolved in water in a 100 mL volumetric flask to prepare a 10 mg/L stock solution. This stock solution was serially diluted with water to prepare 25, 50, 100, 250, and 500 μg/L working solutions.

A previously reported sample preparation method (Kirchner et al., 2012; Moon et al., 2018) was used to remove protein, fat, and starch from the sample, after a slight modification of the method. Five grams of the cereal infant formula sample was placed on a 55 mL screw cap tube and dissolved in 49 mL of 0.2 M sodium acetate. The pH of the sample solution was adjusted to 4.0 to remove casein, which comprises ~80% of the milk protein fraction. Lowering the pH beyond 4.0 (isoelectric point of casein) resulted in isoelectric precipitation. Following this, 0.5 mL of 1% sodium cyanide was added and mixed, and the sample was extracted ultrasonically at 25°C for 10 min. After the addition of 0.5 mL of α-amylase, the sample was incubated for 30 min at 40°C and then for 30 min at 100°C in an incubator to initiate the reaction. Next, 20 mL of the above solution was filtered by a Whatman paper and transferred to an immunoaffinity column (Easi-Ex-tract Vitamin B₁₂, r-Biopharm, Glasgow, UK). The column was washed with 10 mL water and injected with 40 mL of air by syringe to dry it. The loaded sample was eluted with 3 mL of methanol. The eluate was volatilized to dryness and then reconstituted in 0.5 mL of water. This was used as the test sample.

Chromatographic conditions were determined based on previously reported analogous methods that used LC–UV. A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) equipped with a Shiseido Capcell Pak C18 UG 120 column (4.6 mm×250 nm, 5 μm) was used for the analysis of vitamin B₁₂. Water and acetonitrile were used as the mobile phases for gradient elution. A flow rate of 1.0 mL/min and a column temperature of 35°C were maintained, and the injection volume was 50 μL. HPLC grade solvents were filtered through a 0.45-μm membrane and ultrasonically degassed prior to use. The specific chromatography conditions are A (water): B (acetonitrile) gradient system 0–3.4 min (100:0), 3.5–10.9 min (75:25), 11.0–18.9 (65:35), 19–20 min (90:10), and 20–26 min (100:0) (Table 1).

Selectivity for vitamin B₁₂ detection was determined by comparing the chromatographic peaks of the test sample with those of the standard solutions. Linearity was assessed by injecting 25 to 500 μg/L of vitamin B₁₂ solutions in duplicate. Qualitative parameters were determined by comparing the retention times of the standard solution with those of the samples. The analyte was quantified from the calibration plot equations calculated by the least-squares method. Precision was calculated in terms of intra-day (n=3) and inter-day repeatabilities (n=3) by analyzing spiked cereal infant formula samples and was evaluated by calculating the relative standard deviation (RSD). Accuracy of the method was determined by calculating the recovery and appropriate standard deviation (SD) in cereal infant formula samples spiked with different amounts of vitamin B₁₂. Detection limits were assessed in terms of limit of detection [LOD, signal-to-noise ratio (S/N)=3] and limit of quantitation (LOQ, S/N=10).

Analysis using the current method proposed by the MFDS is complex; moreover, it does not yield a desirable peak resolution in the analysis of powdered milk samples. In addition, the MFDS has not yet provided an appropriate method for the analysis of powder milk containing starch. Although the reason for the low peak resolution is not clear, the unstable nature of starch, proteins, and fats during sample treatment has been assumed to be a limitation in this conventional method. In addition, it is difficult to detect vitamin B₁₂ in some food samples using only one pretreatment method (http://foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=324). Since vitamin B₁₂ exists in different forms at very low concentrations in powdered milk containing cereal, the sample preparation methodology is extremely crucial (Lee et al., 2015). In this study, individual pretreatment methods were developed by modifying the Association of Official Analytical Chemists (AOAC, 2002) method to detect vitamin B₁₂ in powdered milk containing starch in high amounts. In the modified AOAC method, samples were purified using an immunoaffinity column and then subjected to HPLC to quantitate vitamin B₁₂ in the samples. Sodium cyanide and α-amylase were used to remove starch, as mentioned in the experimental section. The pre-treatment involving clean-up and concentration using an immunoaffinity column enabled the efficient separation of trace amounts of vitamin B₁₂ from powdered milk samples. As a result of the sample pretreatment, vitamin B₁₂ was eluted at 9.3 min in the HPLC run, suggesting its efficient separation from the degradation products. This method allowed the separation and detection of vitamin B₁₂ within 10 min (Fig. 1). Detection using this approach under the described experimental conditions was slightly more rapid compared to that under the experimental conditions employed in a previous study (Heudi et al., 2006).

Fig. 1. Chromatogram of vitamin B₁₂. (A) Standard of vitamin B₁₂, (B) Powdered milk containing starch.

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The specificity of the proposed technique was ensured by employing the well-established method of using highly selective immunoaffinity column for sample preparation (Anatol et al., 2019; Nakos et al., 2017). Detection limit and powdered milk containing starch recovery were determined by quantitative analysis, and certified reference material, SRM 1849a, was used to validate our analytical HPLC method. The amount of vitamin B₁₂ recovered in the SRM 1849a reference was 54.10 μg/kg. Compared with value of 48.20 μg/kg (given SRM 1843a certified value), the test represented recovery of the authentication value of 112.24%. The external calibration curve of vitamin B₁₂ standard solutions was linear in the range of 25–500 μg/L, with r²>0.9999. The equation of the calibration curve was y = 53.806x − 150.44, where y represents the peak area of the curve obtained through UV detection, and x is the concentration (μg/L) of vitamin B₁₂. It is evident that the correlation coefficients were greater than 0.9999, which indicated a good correlation between the concentration and peak area of the investigated compounds. The LOD and LOQ were 2.71 and 8.21 μg/L, respectively (Table 2). Accuracy was assessed by adding a known amount of the analyte, followed by calculating the recovery using standards. Accuracy of the method was satisfactory, ranging from 83.41% to 106.57%, which was well within the recovery range reported for other food matrices (Chamlagain et al., 2015; Zironi et al., 2013). Intra-day and inter day variations were used to determine the precision of the established method. As shown Table 3, RSD of intra-day and inter-day variations for compound was less than 5.31% and 3.90%, respectively. These results suggest that the HPLC method involving sample pretreatment, immunoaffinity column separation is precise, accurate and sensitive for quantitative determination of active compounds in powdered milk containing starch.

Four different powdered milk containing starch samples, of which two were manufactured in Korea and two were manufactured in USA, were analyzed using the method developed in this study. The sample pre-treatment was repeated three times for each sample; the results are presented in Table 2. It is evident from Table 2 that the vitamin B₁₂ content in powdered milk products was in the range of 11.03–42.18 μg/kg. As determined from the HPLC analysis, all the products contained trace nutrients that were higher than those displayed on the content labels. Therefore, the vitamin B₁₂ content displayed in powdered milk packaging available in the Korean markets was well verified.