Vitamin B12 plays a vital role in human metabolism and is an essential vitamin obtained predominantly from food of animal origin. Amongst all animal products, naturally occurring vitamin B12 in milk has the highest bioavailability and dairy products are a broad-access source, especially for vegetarian individuals. The dairy industry requires an accurate and highly sensitive detection method for vitamin B12, however, the extremely low concentration and instability of vitamin B12 creates challenges in analysis. This review discusses the application of modern instrumental techniques for analysis of vitamin B12 in milk as well as a variety of sample preparations, together with their respective advantages and drawbacks.
An automated optical biosensor-based immunoassay exploiting surface plasmon resonance detection for the quantitation of aflatoxin M1 (AFM1) in milk and milk powders is described. A monoclonal antibody and an immobilized protein–AFM1 conjugate are utilized in a simple inhibition format following aqueous extraction and immunoaffinity clean-up of the sample, thereby avoiding the need for signal amplification techniques. The sensor surface is stable over multiple regeneration cycles, and the technique yields a method detection limit of 0.1 ng/g, which is five times lower than the European Commission maximum residue limit. The described antibody-based biosensor technique provides the advantages of quantitative data, automation, and real-time and non-labeled detection of AFM1. The method therefore facilitates routine quantitative threshold-level screening for the identification of potential non-compliance of AFM1 content prior to confirmatory analysis by reference chromatographic methods and may be considered to complement the enzyme-linked immunosorbent assay technique.
Background: For nutritional purposes, the measurement of vitamin D3 (defined as the sum of vitamin D3 and previtamin D3) is required to obtain an accurate and reliable estimate of its content in foods. An often neglected aspect in the development of methods for the analysis of vitamin D3 is accounting for any potential analytical bias in the results associated with differential thermal isomerization between previtamin D and vitamin D. Conclusions: For LC‑UV methods using a vitamin D2 internal standard, cold saponification or direct lipid extraction techniques should be avoided, unless chromatographic separation of vitamin D2, vitamin D3, and their previtamin forms is achieved so that UV absorbance corrections can be made. For both LC‑UV and LC‑MS methods using calciferol internal standards, the simplest solution to avoid analytical bias due to the presence of previtamin D is to utilize heating conditions (typically during saponification) such that previtamin D and vitamin D in the sample and the internal standard reach an equivalent equilibrium state prior to instrumental analysis. Only under such circumstances is the integration of previtamin D unnecessary to obtain accurate results for vitamin D3. Highlights: A detailed discussion of the quantitation of vitamin D3 in food with concise recommendations for avoiding measurement bias as a consequence of differential thermal isomerization.
Background: Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. Objective: A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. Method: Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography-tandem mass spectrometry (LC‑MS/MS), with quantitation accomplished by internal standard technique. Results: The method was shown to be accurate, with acceptable recovery (thiamine, 99.3‑101.1%; pantothenic acid, 99.2‑108.6%). A certified reference material (NIST1849a), showed no statistical bias (a = 0.05) for thiamine (p = 0.64); although a statistically significant bias (p < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC‑MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (a = 0.05; thiamine, p = 0.399; pantothenic acid, p = 0.058). Acceptable precision was demonstrated, with a repeatability of 7.2% RSDr (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. Conclusions: This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.
Background: Sorbic acid (E, E-2, 4-hexadienoic acid) is added as a preservative to cheese because of its fungistatic and antimicrobial activity. Objective: A facile method for the analysis of sorbic acid that is applicable to sliced processed cheese and grated cheese products. Method: A cheese sample and dry-ice mixture was blended and sorbic acid was extracted with methanol and analyzed by HPLC-ultraviolet with external standardization. A large sample size was used to overcome sample inhomogeneity due to imprecise sorbic acid addition techniques during production and sorbic acid migration through the fat over time. Results: The method was shown to be accurate for both processed cheese and grated Cheddar cheese, with acceptable spike recovery (93.7, 103.7%, respectively), and no bias (α = 0.05) against an international reference method (p = 0.59, p = 0.13, respectively) was found. Acceptable precision was confirmed for both processed cheese slices and grated Cheddar cheese, with repeatability of 5.3% and 4.3% relative standard deviation, respectively, and intermediate precision Horwitz ratio values of 1.3 and 1.7 for processed cheese slices and grated Cheddar cheese, respectively. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. Conclusions: A method for high-throughput, routine testing of sorbic acid is described. The method was subjected to single laboratory validation and was found to be accurate, precise, and fit-for-purpose.
Background: α‑Tocopherol can exist as eight possible stereoisomers due to the presence of three chiral carbons. Regulations and industry guidelines necessitate that dietary vitamin E intakes be based on the vitamin E activity of RRR‑tocopherol. Food products fortified with synthetic all‑rac‑α‑tocopherol or all‑rac‑α‑tocopheryl acetate during manufacturing will require chiral separation of the α‑tocopherol stereoisomers for accurate estimation of vitamin E activity. Objective: The development of an HPLC method utilizing a chiral column for the chromatographic separation of RRR‑tocopherol from other α‑tocopherol stereoisomers. Method: Normal phase liquid chromatographic separation using a polysaccharide-based chiral column with fluorescence detection of α‑tocopherol stereoisomers. Results: The described chromatographic method achieves baseline resolution of RRR‑tocopherol from its stereoisomers. Method selectivity, precision, and robustness were evaluated and acceptable performance was achieved. Conclusions: The chromatographic method was found to be suitable for application where both RRR‑tocopherol content and total a-tocopherol content are required for routine compliance testing. Highlights: A robust and precise chromatographic method for the baseline resolution of RRR‑tocopherol from its stereoisomers was achieved.
Received: 30 May 2019; Accepted: 25 July 2019; Published: 8 August 2019
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Vitamin B12 plays a vital role in human metabolism and is an essential vitamin obtained predominantly from food of animal origin. Amongst all animal products, naturally occurring vitamin B12 in milk has the highest bioavailability and dairy products are a broad-access source, especially for vegetarian individuals. The dairy industry requires an accurate and highly sensitive detection method for vitamin B12, however, the extremely low concentration and instability of vitamin B12 creates challenges in analysis. This review discusses the application of modern instrumental techniques for analysis of vitamin B12 in milk as well as a variety of sample preparations, together with their respective advantages and drawbacks.
Received: 15 April 2019; Accepted: 31 July 2019; Published: 10 August 2019
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An automated optical biosensor-based immunoassay exploiting surface plasmon resonance detection for the quantitation of aflatoxin M1 (AFM1) in milk and milk powders is described. A monoclonal antibody and an immobilized protein–AFM1 conjugate are utilized in a simple inhibition format following aqueous extraction and immunoaffinity clean-up of the sample, thereby avoiding the need for signal amplification techniques. The sensor surface is stable over multiple regeneration cycles, and the technique yields a method detection limit of 0.1 ng/g, which is five times lower than the European Commission maximum residue limit. The described antibody-based biosensor technique provides the advantages of quantitative data, automation, and real-time and non-labeled detection of AFM1. The method therefore facilitates routine quantitative threshold-level screening for the identification of potential non-compliance of AFM1 content prior to confirmatory analysis by reference chromatographic methods and may be considered to complement the enzyme-linked immunosorbent assay technique.
Received: 11 July 2019; Accepted: 23 October 2019; Published: 12 March 2020
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Background: For nutritional purposes, the measurement of vitamin D3 (defined as the sum of vitamin D3 and previtamin D3) is required to obtain an accurate and reliable estimate of its content in foods. An often neglected aspect in the development of methods for the analysis of vitamin D3 is accounting for any potential analytical bias in the results associated with differential thermal isomerization between previtamin D and vitamin D. Conclusions: For LC‑UV methods using a vitamin D2 internal standard, cold saponification or direct lipid extraction techniques should be avoided, unless chromatographic separation of vitamin D2, vitamin D3, and their previtamin forms is achieved so that UV absorbance corrections can be made. For both LC‑UV and LC‑MS methods using calciferol internal standards, the simplest solution to avoid analytical bias due to the presence of previtamin D is to utilize heating conditions (typically during saponification) such that previtamin D and vitamin D in the sample and the internal standard reach an equivalent equilibrium state prior to instrumental analysis. Only under such circumstances is the integration of previtamin D unnecessary to obtain accurate results for vitamin D3. Highlights: A detailed discussion of the quantitation of vitamin D3 in food with concise recommendations for avoiding measurement bias as a consequence of differential thermal isomerization.
Received: 16 September 2019; Accepted: 02 December 2019; Published: 12 March 2020
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Background: Thiamine and pantothenic acid play a critical role in numerous metabolic reactions and are typically supplemented in infant and adult nutritional formulas as thiamine chloride hydrochloride and calcium pantothenate salts. Objective: A rapid compliance method for the analysis of thiamine and pantothenic acid applicable to infant formula and milk-based nutritional products is described. Method: Proteins are removed by centrifugal ultrafiltration, followed by analysis by reversed-phase liquid chromatography-tandem mass spectrometry (LC‑MS/MS), with quantitation accomplished by internal standard technique. Results: The method was shown to be accurate, with acceptable recovery (thiamine, 99.3‑101.1%; pantothenic acid, 99.2‑108.6%). A certified reference material (NIST1849a), showed no statistical bias (a = 0.05) for thiamine (p = 0.64); although a statistically significant bias (p < 0.01) for pantothenic acid was found, the nominal bias was only 4.7% (mean = 7.1 mg/hg; certified value = 6.8 mg/hg). A comparison of results by LC‑MS/MS and current methods showed negligible bias (mean bias: thiamine, 0.01 mg/hg; pantothenic acid, 0.17 mg/hg) and no statistical significance (a = 0.05; thiamine, p = 0.399; pantothenic acid, p = 0.058). Acceptable precision was demonstrated, with a repeatability of 7.2% RSDr (HorRat: 0.6) and an intermediate precision of 7.0% RSD for thiamine, and a repeatability of 5.7% RSDr (HorRat: 0.5) and an intermediate precision of 6.1% RSD for pantothenic acid. Conclusions: This rapid method is intended for use in high-throughput laboratories as part of routine product compliance release testing of thiamine and pantothenic acid in manufactured infant and milk-based nutritional products.
Received: 18 September 2019; Accepted: 13 December 2019; Published: 17 February 2020
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Background: Sorbic acid (E, E-2, 4-hexadienoic acid) is added as a preservative to cheese because of its fungistatic and antimicrobial activity. Objective: A facile method for the analysis of sorbic acid that is applicable to sliced processed cheese and grated cheese products. Method: A cheese sample and dry-ice mixture was blended and sorbic acid was extracted with methanol and analyzed by HPLC-ultraviolet with external standardization. A large sample size was used to overcome sample inhomogeneity due to imprecise sorbic acid addition techniques during production and sorbic acid migration through the fat over time. Results: The method was shown to be accurate for both processed cheese and grated Cheddar cheese, with acceptable spike recovery (93.7, 103.7%, respectively), and no bias (α = 0.05) against an international reference method (p = 0.59, p = 0.13, respectively) was found. Acceptable precision was confirmed for both processed cheese slices and grated Cheddar cheese, with repeatability of 5.3% and 4.3% relative standard deviation, respectively, and intermediate precision Horwitz ratio values of 1.3 and 1.7 for processed cheese slices and grated Cheddar cheese, respectively. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. Conclusions: A method for high-throughput, routine testing of sorbic acid is described. The method was subjected to single laboratory validation and was found to be accurate, precise, and fit-for-purpose.
Received: 12 February 2020; Accepted: 07 April 2020; Published: 20 April 2020
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Background: α‑Tocopherol can exist as eight possible stereoisomers due to the presence of three chiral carbons. Regulations and industry guidelines necessitate that dietary vitamin E intakes be based on the vitamin E activity of RRR‑tocopherol. Food products fortified with synthetic all‑rac‑α‑tocopherol or all‑rac‑α‑tocopheryl acetate during manufacturing will require chiral separation of the α‑tocopherol stereoisomers for accurate estimation of vitamin E activity. Objective: The development of an HPLC method utilizing a chiral column for the chromatographic separation of RRR‑tocopherol from other α‑tocopherol stereoisomers. Method: Normal phase liquid chromatographic separation using a polysaccharide-based chiral column with fluorescence detection of α‑tocopherol stereoisomers. Results: The described chromatographic method achieves baseline resolution of RRR‑tocopherol from its stereoisomers. Method selectivity, precision, and robustness were evaluated and acceptable performance was achieved. Conclusions: The chromatographic method was found to be suitable for application where both RRR‑tocopherol content and total a-tocopherol content are required for routine compliance testing. Highlights: A robust and precise chromatographic method for the baseline resolution of RRR‑tocopherol from its stereoisomers was achieved.