Abstract
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-α-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-α-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-α-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-α-tocopherol content and total α-tocopherol content are required for routine compliance testing.
Highlights
A robust and precise chomatographic method for the baseline resolution of RRR-α-tocopherol from its stereoisomers was acheived.
Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of immunoprotective, growth, and antimicrobial ...factors to the neonate.
To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005.
Following dilution of the sample in buffer, an automated, label-free, real-time optical biosensor immunoassay was used in a direct assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a 4-parameter calibration regression.
The analytical range (0-200 mg/hg), method detection limit (0.8 mg/hg), recovery (96.1-109.2%), and repeatability (1.0-5.3%) complied with the requirements given in the lactoferrin SMPR. The method was shown to be specific for native, intact lactoferrin; thermally denatured lactoferrin generated no measurable binding response.
The method described is suitable for the quantification of intact, undenatured lactoferrin in milk products, infant formulas (bovine milk protein-based, soy protein-based, and amino acid-based), and adult nutritionals and has been demonstrated to meet the performance requirements defined in SMPR 2020.005.
A single-laboratory validation of an automated biosensor immunoassay for the determination of intact, undenatured lactoferrin is described.
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Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with ...feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans.
A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated.
AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence.
The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81-112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4-12% RSD and intermediate precisions of 5-13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods.
A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose.
An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.
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Direct measurement of the bioavailable α-tocopherol content presents a significant analytical challenge and requires chiral separation of the α-tocopherol stereoisomers.
The objective of the study ...was to validate an analytical method for the analysis of α-tocopherol stereoisomers in infant formulas and dairy products.
Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the α-tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection.
The method was shown to be accurate, with spike recoveries of 91.9-108.8% for RRR-α-tocopherol and 90.1-104.7% for α-tocopherol, with no statistical bias against NIST 1849a certified reference material (p-value = 0.54) and an HPLC-UV analytical method (p-value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR-α-tocopherol and 4.6% RSDr (HorRat = 0.4) for α-tocopherol.
A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of α-tocopherol is described. In a single analytical run, the method can quantify: (i) the total α-tocopherol content; (ii) the nutritionally important RRR-α-tocopherol and/or 2R,4'-ambo,8'-ambo-α-tocopherol contents; (iii) the amount of all-rac-α-tocopherol, all-rac-α-tocopheryl acetate, or all-rac-α-tocopheryl succinate fortified into the product.
An accurate and precise chiral chromatographic method for the analysis of isomeric forms of α-tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.
Aflatoxin M1 (AFM1) is found in the milk of cows exposed to feed spoiled by Aspergillus fungi species. These fungi may produce the secondary metabolite aflatoxin B1, which is converted in the cow ...liver by hydroxylation to AFM1 and is then expressed in milk. AFM1 is regulated in milk and other dairy products because it can cause serious health issues, such as liver and kidney cancers, in humans and is an immunosuppressant.
To optimize the chromatographic protocol and to extend the matrix scope to include a wider range of dairy products: whey powder, whey protein concentrate, whey protein isolate, liquid milk, skim milk powder, whole milk powder, adult nutritional products, and yogurt.
AFM1 is extracted using 1% acetic acid in acetonitrile incorporating ionic salts. The AFM1 in the resulting extract is concentrated using an automated RIDA®CREST IMMUNOPREP® online cartridge coupled to quantification by HPLC-fluorescence.
The method was shown to be accurate, with acceptable recovery (81.2-97.1%) from spiked samples. Acceptable precision was confirmed, with a relative standard deviation (RSD) for repeatability of 6.6-11.2% and an RSD for intermediate precision of 7.5-16.7%. Method LOD and robustness experiments further demonstrated the suitability of this method for routine compliance testing. Analysis of an international proficiency trial sample generated results that were comparable with the value assigned from alternative independent methods.
A method with improved chromatography for high-throughput, routine testing of AFM1 in an extended range of dairy products is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit for purpose.
Single-laboratory validation of an automated online immunoaffinity cleanup fluorescence HPLC method for AFM1 in whey proteins, milk powders, nutritional products, liquid milk, and yogurt. Allows for high-throughput analysis of AFM1 with enhanced chromatographic performance. Method applicable to the analysis of AFM1 in an extended range of milk and milk-based products.
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A method for the simultaneous determination in milk of the 5′-mononucleotides adenosine 5′-monophosphate, cytidine 5′-monophosphate, guanosine 5′-monophosphate, inosine 5′-monophosphate and uridine ...5′-monophosphate and their corresponding nucleosides is described. Following deproteinisation, the sample extract was analysed by reversed-phase liquid chromatography, whereby chromatographic separation was achieved using a polymer grafted silica C
18 column, gradient elution with a simple binary mobile phase and UV detection by photodiode array. Performance parameters included recoveries of 95.5–105.2% and precision evaluated as 3.42–6.38% relative standard deviation. The described technique has been applied to the analysis of bovine and human milk, a range of commercial bovine milk-based infant and follow-on formulas, a seasonal study of skim milk powders and an assessment of alkaline phosphatase influence on nucleotide retention.
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Abstract Background Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective, and ...antimicrobial factors to the neonate. Objective To evaluate method reproducibility of AOAC First Action Official Method 2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. Methods Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct-assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a four-parameter calibration regression. Results After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable Horwitz Ratio (HorRatR) values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin cleanup LC–UV), showed negligible difference between results. Conclusion The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing (MLT) study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official MethodSM. Highlights A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described.
Infant formula is designed to provide the human infant with a sole source of nutrition and it is intended to imitate breast milk. In recent years, advances in the science of infant nutrition have led ...to an increasing number of novel ingredients that are supplemented into infant formula. As the list of these nutritionally important nutrients is lengthy, this review summarizes contemporary analytical methods that have been applied to a representative selection (lutein, carnitine, choline, nucleotides, inositol, taurine, sialic acid, gangliosides, triacylglycerides, oligosaccharides, α-lactalbumin, and lactoferrin).
Abstract
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 (NIST 1849a), showed no statistical bias (α = 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 (α = 0.05; thiamine, P = 0.399; pantothenic acid, P = 0.058). Acceptable precision was demonstrated with a repeatability of 7.2% repeatability relative standard deviation (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.
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An optical biosensor assay utilising folate-binding protein was developed for quantitation of the folate content in milk and milk-based paediatric and adult nutritional products. Samples extracted in ...reducing buffer were enzymatically deconjugated prior to biosensor analysis. The protein-binding assay was configured under inhibition conditions, utilising a sensor surface functionalised with folic acid, and was subjected to single laboratory validation. Cross-reactivity of folate-binding protein towards the dominant folate vitamers was demonstrated. The method was compared with both established microbiological and high performance liquid chromatographic methods for a range of products. As a rapid, simple and automated technique, the method proved to be a precise and accurate alternative to conventional techniques for application in routine compliance monitoring of milk-based nutritional products.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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