Determination of Vitamin B<sub>12</sub> in Milk and Dairy Products by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry

Huang, Baifen; Zhang, Jingshun; Wang, Mengli; Cai, Zengxuan

doi:https://doi.org/10.1155/2022/7649228

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Volume 2022 | Article ID 7649228 | https://doi.org/10.1155/2022/7649228

Determination of Vitamin B₁₂ in Milk and Dairy Products by Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry

Baifen Huang,¹Jingshun Zhang,¹Mengli Wang,¹and Zengxuan Cai¹

Academic Editor: Ying Hu

Received13 Apr 2022

Accepted23 Jul 2022

Published12 Aug 2022

Abstract

An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B₁₂ in milk and dairy products. The samples were spiked with stable isotope-labeled vitamin B₁₂ and digested by pepsin and amylase. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by an immunoaffinity SPE cartridge and then measured in multiple reaction monitoring mode (MRM). The linear correlation coefficient (R²) of this method was greater than 0.999 in the range of 2–100 ng/mL. The detection limit and the quantification limit were 0.5 μg/kg and 1.0 μg/kg, respectively. The spiked recoveries ranged from 92.0% to 99.4% at the three spiked levels with the relative standard deviation (RSD) between 1.89% and 4.51%. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. The method is simple, rapid, accurate, and sensitive, and it is suitable for the determination of vitamin B₁₂ in different types of milk and dairy products such as whey powder, whole milk powder, pure milk, fermented milk, infant formula, and prescription food for special medical purposes.

1. Introduction

Vitamin B₁₂ (VB₁₂) is also known as cobalamin or cobamide, and it has at least five chemical variants with similar molecular structure [1] (Figure 1). The naturally occurring forms of VB₁₂ in food are hydroxocobalamin, 5′-deoxyadenosylcobalamin, methylcobalamin, sulphitocobalamin, and a small amount of cyanocobalamin. Among them, cyanocobalamin has the most stable chemical structure, and other chemical variants of VB₁₂ are photolabile [2, 3]. The amount of VB₁₂ required by the human body is very small. However, VB₁₂ is an essential nutrient for humans, which plays an important role in the formation of normal red blood cells and the maintenance of the normal function of myelinated nerve cells in the human body. VB₁₂ deficiency can cause anemia, nervous system disorders, and other symptoms [4–6]. The recommended intake for adults is 2 μg/d and the adequate intake (AI) for infants at 0–6 months and 6–12 months is 0.6 μg/d [7]. VB₁₂ cannot be synthesized by humans de novo and it must be acquired through dietary intake of animal-based foods rich in VB₁₂ such as milk and dairy products [8]. VB₁₂ is usually presented as a protein-bound form in milk and dairy products, whereas cyanocobalamin is the main form of VB₁₂ used in human dietary supplements. The low content of VB₁₂ in food usually has various photolabile forms, which make it difficult for quantitative detection. It is urgent to establish an effective detection method for the determination of VB₁₂ in various foods including milk and dairy products.

The existing reported methods for determination of VB₁₂ in infant formula mainly include microbiological assays [9, 10], high-performance liquid chromatography (HPLC) [11–19], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [20–26], and liquid chromatography and inductively coupled plasma-mass spectrometry (LC-ICP-MS) [27–29]. The microbiological assays usually have a long measurement time and complicated operation. The strains need to be frequently resurrected and stored until analysis. Multiple dilutions need to be prepared to optimize a suitable linear range for the determination of samples with unknown contents. It is difficult for unskilled inspectors to carry out the inspection tests. HPLC methods for determination VB₁₂ usually employ immunoaffinity purification and online solid phase extraction (SPE) followed by column switching techniques. Although HPLC methods have the advantages of simple operation, efficient separation, and high degree of automation, the methods based on UV detectors are not sensitive enough to detect VB₁₂ in non-fortified food. It takes a long time to transform VB₁₂ to fluorescent compounds by derivatization reaction using a fluorescence detector as VB₁₂ itself does not emit fluorescence. In recent years, LC-MS/MS has been used to detect the content of VB₁₂ in food. The trace level VB₁₂ content in food can be detected by LC-MS/MS due to its high selectivity and sensitivity. However, the sample preparation using pepsin and potassium cyanide has not been comprehensively optimized in the existing reports. Based on our research of vitamins [30–32], in this study, an isotope-dilution ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to detect VB₁₂ content in milk and dairy products. This study focused on the effects of pepsin and potassium cyanide solution during sample preparation on the determination results of VB₁₂ in milk and dairy products. The sample preparation procedure, quality control, and chromatographic and mass spectrometry conditions were systematically optimized for the first time. The immunoaffinity SPE cartridge and stable isotope-labeled VB₁₂ were applied for sample pretreatment and MS analysis to ensure high specificity and good accuracy of the developed method. The method has the characteristics of high sensitivity, good repeatability, and high accuracy, and it can be applied and popularized in food testing laboratories.

2. Materials and Methods

2.1. Chemical Reagents

HPLC grades of acetonitrile (ACN), ethanol (EOH), acetic acid, and ammonium acetate were purchased from Fisher Chemical (Canada). Analytical grade sodium acetate and sodium hydroxide were purchased from Shanghai Lingfeng Chemical Reagent Co. Ltd. (China). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, USA). Vitamin B₁₂ (cyanocobalamin, 1.000 ± 0.006 mg/mL) was purchased from SIGMA (USA). [¹³C₇]-vitamin B₁₂ (0.995 ± 0.030 μg/mL) was purchased from Beijing Manhage Biotechnology Co. Ltd. (China). Immunoaffinity SPE cartridge was purchased from R-Biopharm (Germany). Pepsin from porcine gastric mucosa (250 U/mg, 400 U/mg, 600–1800 U/mg, 2500 U/mg) and Takadiastase (100 U/mg) were purchased from SIGMA (USA). Milk and dairy products were purchased from local supermarkets in Hangzhou, China.

2.2. Analytical Instrumentation

The analyte chromatographic separation was performed on an Waters BEH C₁₈ column (10 cm × 2.1 mm i. d.; 1.7 μm), and the mobile phase consisted of water containing 2.5 mmol/L ammonium acetate (A) and water/acetonitrile (10 : 90 V/V) (B), for a total run time of 7 min and column temperature of 40°C, with a sample injection volume of 2 μL. The chromatographic gradient was operated at a flow rate of 0.4 mL/min starting from 0 min: 2% B; 0–1 min: 2% B; 1–4 min: 2%–90% B; 4–5 min: 90% B; 5–7 min: 2% B.

The mass spectrometer was XEVO TQ-S, a triple quadrupole instrument equipped with an ESI ionization source (Waters Corp.). All analyses were conducted in positive ESI mode using multiple reaction monitoring on the 2 main product ions; the optimized mass spectrometer conditions for both analytes are reported in Table 1. The capillary voltage was 3.0 kV, source temperature was 150°C, and desolvation temperature was 650°C. Nitrogen was used as desolvation gas (1000 L/h) and cone gas (50 L/h), whereas the collision gas was argon (flow rate of 0.25 mL/min). Data acquisition processing was performed using MassLynx 4.1 software (Waters Corp.).

2.3. Preparation of Standard Solutions

Vitamin B₁₂ intermediate standard (20.0 μg/mL) was prepared by diluting 1 mL of vitamin B₁₂ stock standard solution (cyanobalamine, 1.000 ± 0.006 mg/mL) to 50 mL with water. Vitamin B₁₂ working standard (200 ng/mL) was prepared by diluting 1 mL of vitamin B₁₂ intermediate standard (20.0 μg/mL) to 100 mL with water. [¹³C₇]-vitamin B₁₂ working standard (50 ng/mL) was prepared by diluting 2.5 mL of [¹³C₇]-vitamin B₁₂ stock standard solution (0.995 ± 0.030 μg/mL) to 50 mL with water. A set of standard solutions containing vitamin B₁₂ in the concentration range of 2–100 ng/mL was prepared, containing 5 ng/mL of [¹³C₇]-vitamin B₁₂.

2.4. Sample Preparation-Extraction and Purification

Weigh, to the nearest 0.01 g, about 30 g of solid milk and dairy products into a 500 mL flask, dissolve the sample in 180 g of warm water (40°C to 45°C) and mix until homogeneous. The liquid milk can be weighed directly after shaking. Reconstituted milk or liquid milk was accurately weighed into a 50 mL centrifuge tube to withstand high temperatures of 100°C. 100 μL of [¹³C₇]-vitamin B₁₂ working standard solution (50 ng/mL), 25 mL of sodium acetate solution, 10 mg of Takadiastase, and 1 mL of potassium cyanide (1%) were added, respectively, under agitation and the solution was incubated at 37°C for 30 min in thermostatic oscillator. The hydrolysates were transferred to a water bath at 100°C for 30 min. Cooling down to room temperature, the solution was shaken fully and centrifuged at 8000 r/min for 10 min. The supernatant was filtered through a 1.6 μm glass fiber filter paper before purification.

All filtrate was loaded onto an immunoaffinity column, using a suitable glass adapter. The column was washed with 10 mL of water and then completely dried by passing through at least 10 mL of air. Vitamin B₁₂ was eluted into a 10 mL glass tube with 1 mL of methanol for 3 times by complete denaturation of the antibody. The eluate was concentrated to dryness at 60°C under slow nitrogen gas flow and reconstituted in 1 mL of mobile phase and filtered through a 0.22 μm membrane filter before HPLC analysis.

3. Results and Discussion

3.1. Optimization of the MS/MS Conditions

Structural information to confirm the identity of analytes and the optimization of the instrumental sensitivity were obtained by performing a preliminary fragmentation study and MS/MS experiments. Full scans under the positive ESI mode for vitamin B₁₂ and [¹³C₇]-vitamin B₁₂ were acquired for the selection of the precursor ions according to the relative intensities of multiple charged ions. [M + 2H]²⁺ at m/z 678.1 and 681.8 were the precursor ions selected for vitamin B₁₂ and [¹³C₇]-vitamin B₁₂ as it gave the most intense peak in the mass spectrum. Product ion scan mass spectra are shown in Figure 2. Collision-induced dissociation (CID) of vitamin B₁₂ produced two product ions at m/z 147 and 359, and [¹³C₇]-vitamin B₁₂ produced two product ions at m/z 154 and 366, also shown by the fragmentation pattern in Figure 2. For each analyte, two among all available MRM transitions were chosen on the basis of the best chromatographic signal-to-noise (S/N) ratio, to perform quantitative and confirmative analysis on the selected food matrices.

(a)

(b)

(c)

(d)

3.2. Optimization of the LC Separation Conditions

Acetonitrile was selected as an organic modifier for its chromatographic selectivity, and ammonium acetate was added to the mobile phase, it could provide the best ionization contions. The best compromise in terms of sensitivity and analyte separation was afforded by the 2.5 mmol/L concentration of ammonium acetate as mobile phase A and water/acetonitrile (10 : 90 V/V) as mobile phase B.

3.3. Optimization of Extraction Solvent

Vitamin B₁₂ can dissolve in strong polar solvents, such as water, methanol, and ethanol, but not in organic solvents with medium polarity, such as acetone, chloroform, and ether. Thus, strong polar solvent was suitable for extraction in the sample process. There are other kinds of naturally occurring cobalamin in infant formula, prepared milk powder, and instant grain food including fortified cyanocobalamin. These cobalamins coordinated with the protein to form stable large molecular compounds. The cobalamin must dissociate from combined compounds and transform into cyanobalamin prior to determination, and the reaction was proceeded in the condition of aqueous solution. Enzymatic hydrolysis of diastase can also react in aqueous solution. Therefore, water was chosen as the extraction solvent.

3.4. Necessity of Potassium Cyanide

There are many speciations of vitamin B₁₂ with physiological activity, such as cyanocobalamin, aquacobalamin, adenosylcobalamin, and mecobalamin in natural food. Cyanocobalamine was the most stable speciation among them, and other speciation of vitamin B₁₂ must be transformed into cyanobalamin prior to determination. In this study, measured results of 9 milk and dairy products were compared by adding potassium cyanide or not. As shown in Table 2, there was a significant difference between the two groups, most of the p values were less than 0.05, excepting sample of NIST SRM 1849a, wich was 0.814. The measured values adding potassium cyanide are 1.1–14.9 times more than the results without potassium cyanide. Measured values would significantly lower if potassium cyanide was not used in the determination of vitamin B₁₂ in milk and dairy products and would cause determination deviation.

3.5. Optimization of Enzymatic Hydrolysis Conditions

Pepsin is often used coupled with diastase in the determination of vitamin B₁₂ [11–14]; in the meanwhile, there are background values of vitamin B₁₂ in pepsin. To further evaluate the influence of background value, several pepsins were purchased with different activities of 250 U/mg, 400 U/mg, 600 U/mg, and 2500 U/mg, respectively. The concentration of vitamin B₁₂ was determined following the optimization method in 1 g sample with 0.2 g pepsin and 0.05 g diastase added. As shown in Figure 3, the background value of vitamin B₁₂ in pepsin increased with the activity of pepsin. The background value of vitamin B₁₂ in pepsin cannot be neglected.

3.6. Choice of Internal Standard

In most cases, the concentrations of vitamin B₁₂ are not very high in foods, although it widely exists in dairy products. It is necessary to purify and concentrate in the determination to achieve ideal detection sensitivity. Two studies [21, 22] reviewed vitamin B₁₂ loss during cooking treatment with contradictory results. The absolute recovery of immunoaffinity SPE cartridge for vitamin B₁₂ is not very well; meanwhile, quality differences between batches would influence the accuracy of determination. Luo [24] developed an analytical method for the determined of vitamin B₁₂ in food products and multivitamin multimineral tablets by HPLC-ESI-MS, and ginsenoside Re was used as an internal standard (I.S.). Ginsenoside Re is not a perfect internal standard of vitamin B₁₂ for the obvious contrast between the molecular structure of ginsenoside Re and vitamin B₁₂. In this study, to achieve the purpose of accurate quantification, [¹³C₇]-vitamin B₁₂ isotope-labeled vitamin B₁₂ was used as an internal standard in the whole analysis.

3.7. Matrix Effect

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a “gold standard” in many fields of analytical chemistry. One of the most common interference types in the case of LC-MS/MS analyses is the alteration of ionization efficiency (usually ionization suppression) due to co-eluting compounds, called matrix effect (ME). Matrix effect results in differing analyte peak areas while comparing the sample and standard with the same analyte concentration. There are many fats, proteins, and sugars naturally present in milk, which would produce interfering substances after enzymatic hydrolysis and heating process. These interfering substances would cause certain matrix effects in the LC-MS/MS analyses if purification process was less efficient. In common, matrix effect was calculated as a ratio of the slope of the matrix matching the standard curve and the slope of the solvent standard curve. There is no obvious matrix effect if the ratio is between 85% and 115%. It is difficult to find a blank matrix of vitamin B₁₂ for vitamin B₁₂ is widespread in milk and dairy products, so the isotope of vitamin B₁₂ was used to evaluate the matrix effect of infant formula, non-fat milk powder, fermented milk, and cheese samples instead of vitamin B₁₂ in this study. The matrix effect (ME%) can be quantitatively expressed by (1), where A_matrix and A_standard are the peak areas of the equal amount of [¹³C₇]-vitamin B₁₂, respectively, in presence and in the absence of possibly interfering compounds.

This technique is also called post-extraction spiking: the analytical signal of a blank sample extract spiked with [¹³C₇]-vitamin B₁₂ (A_matrix) is compared with the signal of the equal amount of [¹³C₇]-vitamin B₁₂ in pure solvent (A_standard).

ME% (n = 6) of infant formula, non-fat milk powder, fermented milk, and cheese was 99.51% ± 1.71, 102.51% ± 6.12, 102.15% ± 4.65, and 102.18% ± 1.66, respectively. The results have shown that the purification effect of immunoaffinity SPE cartridge was efficient, and the interfering substance almost can be removed after purification treatment with immunoaffinity SPE cartridge.

3.8. Method Validation

The standard series solutions with the concentration of 2, 5, 10, 25, 50, and 100 ng/mL were prepared, and the standard curves were drawn according to the corresponding peak areas. The correlation coefficients were all greater than 0.999, and the standard curves had good linearity. The limit of quantitation (LOQ) and the limit of detection (LOD) were investigated according to 10 times and 3 times of signal-to-noise ratio, respectively. When 1 g of infant formula was taken for determination, the limit of quantitation and the limit of detection were 1.0 μg/kg and 0.5 μg/kg, respectively.

The whole milk powder (the background value was 16.7 μg/kg ± 1.8 μg/kg) was selected as the sample. Each standard addition concentration was determined 6 times in parallel. The recoveries of vitamin B₁₂ were 92.0%∼99.4%. The RSD of six repeated determination was 3.03%∼5.76%.

For further confirmation of the accuracy of the established method, two standard reference materials (SRMs), NIST SRM1849a and NIST SRM1869 infant formula, were selected as verified objects. The results are shown in Table 3.

The present method was also applied to determine vitamin B₁₂ in the infant formula of FAPAS Proficiency Test in 2021. The measured value was 19.9 ± 1.0 μg/kg, and Z value was 0.8, and the result is satisfied.

4. Concluding Remarks

An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B₁₂ in milk and dairy products. The sample preparation procedure using pepsin and potassium cyanide, quality control, and chromatographic and mass spectrometry conditions were systematically optimized for the first time. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by immunoaffinity SPE cartridge before UPLC-MS/MS. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. All validation results showed that the method is simple, accurate, and sensitive, and it is suitable for the determination of vitamin B₁₂ in different types of milk and dairy products.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

References

F. Watanabe and T. Bito, “Determination of cobalamin and related compounds in foods,” Journal of AOAC International, vol. 101, no. 5, pp. 1308–1313, 2018.
View at: Publisher Site | Google Scholar
T. Bito and F. Watanabe, “Biochemistry, function, and deficiency of vitamin B₁₂ in Caenorhabditis elegans,” Experimental Biology and Medicine, vol. 241, no. 15, pp. 1663–1668, 2016.
View at: Publisher Site | Google Scholar
S. S. Kumar, R. S. Chouhan, and M. S. Thakur, “Trends in analysis of vitamin B₁₂,” Analytical Biochemistry, vol. 398, no. 2, pp. 139–149, 2010.
View at: Publisher Site | Google Scholar
F. O’Leary and S. Samman, “Vitamin B₁₂ in health and disease,” Nutrients, vol. 2, no. 3, pp. 299–316, 2010.
View at: Publisher Site | Google Scholar
Z. Schneider and A. Stroinski, Comprehensive B₁₂: Chemistry, Biochemistry, Nutrition, Ecology, Medicine, De Gruyter, Berlin, Germany, 1987.
I. Ortigues-Marty, D. Micol, S. Prache, D. Dozias, and C. L. Girard, “Nutritional value of meat: the influence of nutrition and physical activity on vitamin B₁₂ concentrations in ruminant tissues,” Reproduction, Nutrition, Development, vol. 45, no. 4, pp. 453–467, 2005.
View at: Publisher Site | Google Scholar
C. Nutrition Society, Chinese Dietary Reference Intakes, Science Press, Beijing, China, 2013.
E. M. Brouwer-Brolsma, R. A. M. Dhonukshe-Rutten, J. P. Van Wijngaarden, N. Zwaluw, N. Velde, and L. de Groot, “Dietary sources of vitamin B-12 and their association with vitamin B-12 status markers in healthy older adults in the B-PROOF study,” Nutrients, vol. 7, no. 9, pp. 7781–7797, 2015.
View at: Publisher Site | Google Scholar
L. H. Zhang, K. X. Wang, and M. Wang, “Application and discussion of microbiological method for assay of vitamin B₁₂ in milk powder,” China Dairy Industry, vol. 36, no. 10, pp. 58–60, 2008.
View at: Google Scholar
M. Berth, C. Bonroy, K. Guerti, W. Uyttenbroeck, and M. Uytterhoeven, “Comparison of five commercially available ELISA kits for the determination of intrinsic factor antibodies in a vitamin B12 deficient adult population,” The International Journal of Literary Humanities, vol. 38, no. 1, pp. e12–e14, 2016.
View at: Publisher Site | Google Scholar
D. Y. Fan, Y. Z. Zhang, and H. P. Wu, “Development of a simple and sensitive HPLC-DAD method for quantification of vitamin B12 fortified in infant food,” Analytical Methods, vol. 13, no. 41, pp. 4920–4925, 2021.
View at: Publisher Site | Google Scholar
E. C. Marley, E. Mackay, and G. Young, “Characterisation of vitamin B12 immunoaffinity columns and method development for determination of vitamin B12 in a range of foods, juices and pharmaceutical products using immunoaffinity clean-up and high performance liquid chromatography with UV detection,” Food Additives & Contaminants: Part A, vol. 26, no. 3, pp. 282–288, 2009.
View at: Publisher Site | Google Scholar
K. Schimpf, R. Spiegel, L. Thompson, and D. Dowell, “Determination of vitamin B₁₂ in infant formula and adult nutritionals by HPLC: first Action 2011.10,” Journal of AOAC International, vol. 95, no. 2, pp. 313–318, 2012.
View at: Publisher Site | Google Scholar
X. Qiu, H. S. Zhang, Y. H. Yin et al., “Determination of active vitamin B12 (cobalamin) in dietary supplements and ingredients by reversed-phase liquid chromatography: single-laboratory validation,” Food Chemistry, vol. 298, Article ID 125010, 2019.
View at: Publisher Site | Google Scholar
B. Nshime, J. Koedam, B. Stanton, Q. Tran, and P. Chen, “Liquid chromatography method for the simultaneous quantification of biotin and vitamin B12 in vitamin B supplements,” Journal of AOAC International, vol. 102, no. 2, pp. 445–450, 2019.
View at: Publisher Site | Google Scholar
International Organization for Standardization, ISO 20634:2015(E) Infant formula and adult nutritionals-Determination of vitamin B12 by reversed phase high performance liquid chromatography, Vernier, Geneva, Switzenland, 2015.
L. D. Butler-Thompson, W. A. Jacobs, K. J. Schimpf et al., “Determination of Vitamin B₁₂ in infant, adult, and pediatric formulas by HPLC-UV and column switching: collaborative study, final action 2011.10,” Journal of AOAC International, vol. 98, no. 6, pp. 1655–1665, 2015.
View at: Publisher Site | Google Scholar
A. S. Fayed, M. A. M. Hegazy, and N. S. A. Wahab, “Chromatographic analysis of a multicomponent mixture of B1, B6, B12, benfotiamine, and diclofenac; Part I: HPLC and UPLC methods for the simultaneous quantification of these five components in tablets and capsules,” Journal of AOAC International, vol. 99, no. 6, pp. 1513–1521, 2016.
View at: Publisher Site | Google Scholar
W. J. Lee, Y. B. Lee, M. H. Huh, and J. K. Choi, “Determination of the chemical stability of cyanocobalamin in medical food by a validated immunoaffinity column-linked HPLC method,” Journal of Food Quality, vol. 2022, Article ID 1619936, 8 pages, 2022.
View at: Publisher Site | Google Scholar
A. Repossi, E. Zironi, T. Gazzotti, A. Serraino, and G. Pagliuca, “Vitamin B12 determination in milk, whey and different by-products of ricotta cheese production by ultra performance liquid chromatography coupled with tandem mass spectrometry,” Italian Journal of Food Safety, vol. 6, no. 4, pp. 6795–7155, 2017.
View at: Publisher Site | Google Scholar
M. L. Wang, S. Asam, J. Q. Chen, and M. Rychlik, “Development of stable isotope dilution assays for the analysis of natural forms of vitamin B12 in meat,” Journal of Agricultural and Food Chemistry, vol. 69, no. 36, pp. 10722–10730, 2021.
View at: Publisher Site | Google Scholar
E. Zironi, T. Gazzotti, A. Barbarossa, C. Devicienti, M. Scardilli, and G. Pagliuca, “Technical note: development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B₁₂ concentrations in milk and dairy products,” Journal of Dairy Science, vol. 96, no. 5, pp. 2832–2836, 2013.
View at: Publisher Site | Google Scholar
E. Zironi, T. Gazzotti, A. Barbarossa, F. Farabegoli, A. Serraino, and G. Pagliuca, “Determination of vitamin B₁₂ in dairy products by ultra performance liquid chromatography-tandem mass spectrometry,” Italian Journal of Food Safety, vol. 3, no. 4, pp. 4513–5255, 2014.
View at: Publisher Site | Google Scholar
J. H. Lee, J. H. Shin, J. M. Park et al., “Analytical determination of vitamin B₁₂ content in infant and toddler milk formulas by liquid chromatography tandem mass spectrometry (LC-MS/MS),” Korean Journal for Food Science of Animal Resources, vol. 35, no. 6, pp. 765–771, 2015.
View at: Publisher Site | Google Scholar
J. Li, W. J. Lin, and Q. W. Cao, “Determination of total vitamin B₁₂ in milk powder by ultra-performance liquid chromatography-tandem mass spectrometry after purifiication on an immunoaffinity cartridge,” Acta Nutrimenta Sinica, vol. 42, no. 1, pp. 72–77, 2020.
View at: Google Scholar
L. D’Ulivo, L. Yang, J. F. Ding et al., “Determination of cyanocobalamin by isotope dilution LC-MS/MS,” Analytica Chimica Acta, vol. 990, pp. 103–109, 2017.
View at: Publisher Site | Google Scholar
S. Dubascoux, J. Richoz Payot, P. Sylvain, M. Nicolas, and E. Campos Gimenez, “Vitamin B12 quantification in human milk-Beyond current limitations using liquid chromatography and inductively coupled plasma-Mass spectrometry,” Food Chemistry, vol. 362, Article ID 130197, 2021.
View at: Publisher Site | Google Scholar
A. Knoop, P. Planitz, B. Wust, and M. Thevis, “Analysis of cobalt for human sports drug testing purposes using ICP-and LC-ICP-MS,” Drug Testing and Analysis, vol. 12, no. 11-12, pp. 1666–1672, 2020.
View at: Publisher Site | Google Scholar
R. Wenzel, D. Major, and K. Harly, “Determination of vitamin B₁₂ in equine urine by liquid chromatography-inductively coupled-plasma mass spectrometry,” Journal of Trace Elements in Medicine & Biology, vol. 50, pp. 634–639, 2018.
View at: Google Scholar
B. F. Huang, X. D. Pan, J. S. Zhang, J. J. Xu, and Z. X. Cai, “Determination of vitamins D₂ and D₃ in edible fungus by reversed-phase two-dimensional liquid chromatography,” Journal of Food Quality, vol. 2020, Article ID 8869279, 6 pages, 2020.
View at: Publisher Site | Google Scholar
B. F. Huang, Z. X. Cai, J. S. Zhang, and J. Xu, “An efficient solid-phase extraction-based liquid chromatography method to simultaneously determine diastereomers α-tocopherol, other tocols, and retinol isomers in infant formula,” Journal of Food Quality, vol. 2021, Article ID 5591620, pp. 1–8, 2021.
View at: Publisher Site | Google Scholar
B. Y. Lu, Y. P. Ren, B. F. Huang, W. Liao, Z. Cai, and X. Tie, “Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry,” Journal of Chromatographic Science, vol. 46, no. 3, pp. 225–232, 2008.
View at: Publisher Site | Google Scholar

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Copyright © 2022 Baifen Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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