Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: Collaborative Study, Final Action 2021.07.

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.

Limitations of current analytical reference methods to determine vitamins in foods: Challenges to support regulatory compliance and nutritional composition data.

Foods are analysed for their vitamin content to support the verification of regulatory compliance or to generate food composition data. Many international reference methods for the analysis of vitamins in foods originate from the 1990s. Advances in nutrition science and analytical technology and the continuing evolution of statutory regulations necessitate the need of new or supplementary regulatory standards.

Enhanced Automated Online Immunoaffinity Liquid Chromatography-Fluorescence Method for the Determination of Aflatoxin M1 in Dairy Products.

Background: 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.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: First Action 2021.07.

Background: 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.

Objective: To evaluate the analytical performance of an optical biosensor immunoassay for compliance with the method performance requirements described in SMPR 2020.005.

Assessment of Regulatory Compliance Testing for Vitamin D in Infant Formula-Impact of Delegated Regulation (EU) 2019/828.

Background: Since the publication of Standard Method Performance Requirements (SMPR®) for vitamin D in infant formula (SMPR 2011.004) by AOAC INTERNATIONAL, revised vitamin D limits have been recommended by the European Food Safety Authority (EFSA) for infant formula and adopted in Commission Delegated Regulation (EU) 2019/828. The vitamin D range introduced, 2-2.

Analysis of α-Tocopherol Stereoisomers in Fortified Infant Formula by Chiral Chromatography.

Background: Direct measurement of the bioavailable α-tocopherol content presents a significant analytical challenge and requires chiral separation of the α-tocopherol stereoisomers.

Objective: The objective of the study was to validate an analytical method for the analysis of α-tocopherol stereoisomers in infant formulas and dairy products.

Method: 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.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection.

Background: 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.

Comparison of LC-MS/MS and Enzymatic Methods for the Determination of Total Choline and Total Carnitine in Infant Formula and Milk Products.

Background: Choline and l-carnitine are classified as pseudo-vitamins because of their conditionally essential status. As they are involved in multiple physiological metabolic pathways in the human body, they are routinely fortified in infant and adult nutritional formulas.

Objective: The performance of an LC-MS/MS method for the analysis of choline and carnitine, compared with enzymatic methods in routine use for the analysis of total carnitine and total choline, is described.

Separation of RRR-α-Tocopherol by Chiral Chromatography.

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.

Rapid Method for the Determination of Thiamine and Pantothenic Acid in Infant Formula and Milk-Based Nutritional Products by Liquid Chromatography‒Tandem Mass Spectrometry.

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.

Determination of Sorbic Acid in Cheese by High Performance Liquid Chromatography.

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 Rapid Method for the Determination of Biotin and Folic Acid in Liquid Milk, Milk Powders, Infant Formula, and Milk-Based Nutritional Products by Liquid Chromatography-Tandem Mass Spectrometry.

Background: Biotin and folate are B-group vitamins that play a critical role in numerous metabolic reactions, and they are supplemented to infant and adult nutritional formulas as free biotin and folic acid.

Objective: We describe a rapid method for the analysis of biotin and folic acid that is applicable to liquid milk, milk powders, infant formula, and milk-based nutritional products.

Methods: Samples are autoclaved, centrifuged, filtered, and analyzed by HPLC-MS/MS, with quantitation accomplished by the internal standard technique.

Analysis of Vitamin D₂ and Vitamin D₃ in Infant and Adult Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: A Multilaboratory Testing Study.

A multilaboratory testing study was conducted on AOAC First Action Method 2016.05 "Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders, Infant Formulas, and Adult/Pediatric Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry." Nine laboratories participated in the analysis of duplicate samples of 20 nutritional products.

Analysis of Vitamin D2 and Vitamin D3 in Fortified Milk Powders and Infant and Nutritional Formulas by Liquid Chromatography-Tandem Mass Spectrometry: Single-Laboratory Validation, First Action 2016.05.

A method for the determination of vitamin D2 and vitamin D3 in fortified milk powders and infant and adult nutritional formulas is described. Samples are saponified at high temperature and lipid-soluble components are extracted into isooctane. A portion of the isooctane layer is transferred and washed, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione is added to derivatize the vitamin D to form a high-molecular-mass, easily ionizable adduct.

Current Methods for the Analysis of Selected Novel Nutrients in Infant Formulas and Adult Nutritionals.

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).

Determination of vitamin A and vitamin E esters in infant formulae and fortified milk powders by HPLC: Use of internal standardisation.

An HPLC method is described using normal phase conditions with an unbonded silica column to determine concentrations of supplementary vitamin A and vitamin E esters and β-carotene in infant formulae. The method utilises selective dual-channel fluoresence for vitamins A and E and visible absorbance for β-carotene. An attribute of the method is the use of retinol propionate, α-tocopheryl propionate and all-E-β-apo-8'-carotenoic acid ethyl ester internal standards to compensate for analytical variations associated with these labile vitamins.

Analysis of Nucleotide 5'-Monophosphates in Infant Formulas by HPLC-UV: Collaborative Study, Final Action 2011.20.

A collaborative study was conducted on AOAC First Action Method 2011.20: 5'-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula. After the successful analysis of National Institute of Standards and Technology (NIST) 1849a Standard Reference Material (SRM) as a practice sample, 12 laboratories participated in the analysis of duplicate samples of six different infant formula products.

A rapid method for the determination of vitamin D3 in milk and infant formula by liquid chromatography/tandem mass spectrometry.

A rapid method for the determination of vitamin D3 applicable to milk and infant formula products is described. Samples are saponified at high temperature, and lipophilic components are extracted into isooctane in a single tube. Vitamin D3 is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to form a Diels-Alder adduct, which is re-extracted into a small volume of acetonitrile and analyzed by UHPLC-MS/MS with quantification accomplished by an internal standard technique utilizing deuterium-labeled vitamin D3.

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography-tandem mass spectrometry.

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration.

Analysis of 5'-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First Action 2011.20.

A method for the routine determination of 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92-101%) and repeatability (1.

Determination of folate in infant formula and adult/pediatric nutritional formula by optical biosensor assay: First Action 2011.05.

After a review of data from a single-laboratory validation (SLV) study published in the International Dairy Journal 21, 783-789 (2011), a method for folate in infant formula and adult/pediatric nutritional formula was submitted for consideration of adoption by AOAC as an automated assay that is rapid and simple. The method uses an optical biosensor assay to quantitate total folate content in milk and milk-based pediatric and adult nutritional products. The assay uses folate binding protein and a functionalized sensor surface.