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Vitamin K is a dietary component that, as a cofactor, has critically important physiological roles in the body. As it is present in milk at very low levels, it is fortified at higher levels in paediatric milk-based formulae. Currently,... more
Vitamin K is a dietary component that, as a cofactor, has critically important physiological roles in the body. As it is present in milk at very low levels, it is fortified at higher levels in paediatric milk-based formulae. Currently, the predominant analytical strategy for both food database surveys and routine compliance testing is high-performance liquid chromatography with post-column zinc reduction and fluorescence detection. This study presents a single-laboratory validation of a pre-column reduction modification that provides enhanced sensitivity (> 2×) and increased analytical throughput. Accuracy was confirmed by analysis of a certified reference infant formula and milk proficiency samples, and by comparison against the reference method for a wide range of dairy products containing vitamin K1 at both natural and supplemented levels. The method offers several performance advantages and is suitable for routine product compliance release testing.
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A simplified, simultaneous determination of vitamins B1, B2, B3, and B6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified... more
A simplified, simultaneous determination of vitamins B1, B2, B3, and B6 in supplemented infant formulas was developed from a single deproteinized sample extract, with analysis by reversed-phase, ion-pair chromatography with an acidified methanol–water mobile phase. The dioctylsulfosuccinate counter-ion facilitates unique retention of the pyridine-based vitamins (niacinamide and pyridoxine) and allows for concurrent measurement of both the pyridoxal and riboflavin 5′-phosphate endogenous components of milk. Other naturally occurring undetected vitamin congeners have minimal analytical significance. UV detection is used for niacinamide, and programmed fluorescence detection is used for riboflavin and the B6 vitamins. Thiamine is routinely determined sequentially under modified elution conditions.
Research Interests: Chemistry, Analytical Chemistry, Chromatography, Vitamins, Medicine, and 15 moreQuantitative analysis, Food Analysis, Humans, Infant, High Performance Liquid Chromatography, High Pressure Liquid Chromatography, Pyridoxine, Pyridoxal, Liquid Chromatography, Food Sciences, Quantitative Analysis, Thiamine, Riboflavin, Infant food, and Niacinamide
Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion,... more
Vitamin K1 was determined in a variety of foods by using reversed-phase liquid chromatography with a C30 column followed by post-column reduction to the fluorescent hydroquinone derivatives. Lipids were removed by lipase digestion, followed by single extraction into hydrocarbon, and the protocol was extended to selected natural and processed foods. Biologically active trans- and inactive cis-vitamin K1 isomers were measured individually to evaluate the true nutritional status of the products. Method performance parameters confirmed the validity of the technique. The use of the triacontylbonded C30 phase for selective phylloquinone isomer measurement extends previously validated AOAC Method 999.15 for vitamin K1 in milk and infant formula to a wider range of foods important in the human diet. The cis-vitamin K1 isomer contributes up to about 15% of total phylloquinone in certain foods.
Research Interests: Chemistry, Analytical Chemistry, Chromatography, Medicine, Quality Control, and 11 moreQuantitative analysis, Food Analysis, High Performance Liquid Chromatography, EXTRACTION, High Pressure Liquid Chromatography, Vitamin, Liquid Chromatography, Food Sciences, Quantitative Analysis, Hydroquinone, and Vitamin K
A collaborative study was conducted on a coupled enzymatic–spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the... more
A collaborative study was conducted on a coupled enzymatic–spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45–175 mg/100 g sample. After the combined acid hydrolysis–phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26–6.34%) with a HORRAT value of 0.91 (range, 0.76–1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42–0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.
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Biomolecular interaction analysis was evaluated for the automated determination of vitamin B12 in a range of foods. The analytical technique was configured as a biosensor-based, nonlabeled inhibition protein-binding assay using... more
Biomolecular interaction analysis was evaluated for the automated determination of vitamin B12 in a range of foods. The analytical technique was configured as a biosensor-based, nonlabeled inhibition protein-binding assay using nonintrinsic R-protein. Sample extraction conditions were optimized, and both ligand specificity and nonspecific binding considerations were evaluated. Performance parameters included a quantitation range of 0.08–2.40 ng/mL, recoveries of 89–106%, agreement against assigned reference values for 3 independent certified food reference materials, and a mean between-laboratory reproducibility relative standard deviation of 4.9%. The proposed method was compared with reference microbiological and radioisotope protein-binding methods for a range of food samples. A wide selection of milks, infant formulas, meats, and liver were evaluated for their vitamin B12 content. The influence of season was studied in herd milk, early lactation was followed for a single animal,...
Research Interests: Chemistry, Analytical Chemistry, Chromatography, Medicine, Food Microbiology, and 15 moreQuantitative analysis, Calibration, Female, Animals, Cobalamin, Milk, Goats, Cattle, Reproducibility, Dairy Products, Food Sciences, Quantitative Analysis, Protein Binding, Biosensing Techniques, and Certified Reference materials
An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The... more
An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15–10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSDR) of 0.47%, mean between-run RSDR of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during ...
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A interlaboratory study was conducted to evaluate a liquid chromatographic (LC) procedure for the determination of total vitamin C in foods at levels of 5–60 mg/100 g. Emphasis was placed on fruit juices, although selected foods were also... more
A interlaboratory study was conducted to evaluate a liquid chromatographic (LC) procedure for the determination of total vitamin C in foods at levels of 5–60 mg/100 g. Emphasis was placed on fruit juices, although selected foods were also included in the study. Following dissolution of sample in water, endogenous dehydroascorbic acid was converted to ascorbic acid by precolumn reduction with dithiothreitol at neutral pH. Total ascorbate was determined by C18 reversed-phase LC with a phosphate eluent at pH 2.5, incorporating dithiothreitol to maintain vitamin C in the reduced form, and UV detection at 254 nm. Seven types of fruit juices and foods were tested by 19 collaborators in 7 countries. Three duplicate juices and foods met the criteria for Youden pairs and yielded repeatability relative standard deviation of 5.80–14.66%. Reproducibility relative standard deviation ranged from 6.36 to 35.54% (n = 10) with HORRAT values of 0.82–4.04. The LC method is suitable for routine use in ...
Research Interests: Chemistry, Analytical Chemistry, Chromatography, Medicine, Quantitative analysis, and 12 morevitamin C, Fruit, High Performance Liquid Chromatography, High Pressure Liquid Chromatography, Beverages, Ascorbic Acid, Repeatability, Liquid Chromatography, Solutions, Food Sciences, Quantitative Analysis, and Reference standards
Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using... more
Biomolecular interaction analysis was evaluated for the automated analysis of biotin- and folate-supplemented infant formulas and milk powders. The technique was configured as a biosensor-based, nonlabeled inhibition immunoassay using monoclonal antibodies raised against analyte-conjugate. Sample extraction conditions were optimized and antibodies were evaluated for cross-reactivity. Performance parameters included a quantitation range of 2–70 ng/mL, recoveries of 86–102%, agreement against assigned reference values for National Institute of Standards and Technology Standard Reference Material 1846, between-laboratory reproducibility relative standard deviation of 9.1% for biotin and 8.1% for folate, respectively, and equivalence against reference microbiological assay methods for both analytes.
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Abstract Conventional methods using HPLC with UV detection have used vitamin D2 as an internal standard with the expectation that this fully compensates for the heat-dependent equilibrium of vitamin D3 with its previtamin. Previtamin D... more
Abstract Conventional methods using HPLC with UV detection have used vitamin D2 as an internal standard with the expectation that this fully compensates for the heat-dependent equilibrium of vitamin D3 with its previtamin. Previtamin D has a different spectral absorptivity from vitamin D and may be present in different proportions in samples and standards. Therefore, vitamin D2 and vitamin D3 and their previtamin forms must be chromatographically resolved to achieve accurate quantitation of total vitamin D. This study identified four chromatographic columns (ACE C18, ACE C18 AR, Vydac 201 TP C18 and Polaris C18-Ether) with adequate selectivity that should be applied for food testing and further confirmed that both parent vitamins isomerise at the same rate under thermal conditions.
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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... more
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).
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Milk from domesticated livestock has been a significant part of the human diet for millennia, with milk from the cow (genus Bos) dominant in terms of volume produced. Modern process technologies produce a very wide range of consumer... more
Milk from domesticated livestock has been a significant part of the human diet for millennia, with milk from the cow (genus Bos) dominant in terms of volume produced. Modern process technologies produce a very wide range of consumer products based on raw bovine milk. Nitrates and nitrites are reactive forms of nitrogen that are found in the environment and therefore are commonly present in foods destined for the human diet. Although raw bovine milk is not a significant source of nitrate and nitrite, post-secretory processing can result in elevated levels. Dietary consumption of these reactive nitrogen species has generally been regarded with concern, although recent research suggests a potential role in health. This article discusses these topics and also provides a modern review of analytical methods used, for regulatory purposes, to quantitate the levels of nitrate and nitrite in milk products.
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Biotin is a member of the B group of water-soluble vitamins. This chapter summarises its chemistry, its biological and nutritional significance in the human diet and its distribution in foods, and describes methods for its quantitative... more
Biotin is a member of the B group of water-soluble vitamins. This chapter summarises its chemistry, its biological and nutritional significance in the human diet and its distribution in foods, and describes methods for its quantitative determination in foods, with emphasis on dairy products. The various analytical strategies currently available are briefly reviewed and include microbiological, biological, chromatographic and ligand-binding principles.
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ABSTRACT A gas chromatography-flame ionisation detection method was developed to establish the content of myo-inositol in milk powders and infant formulations subsequent to formation of the volatile trialkylsilyl derivative. Samples were... more
ABSTRACT A gas chromatography-flame ionisation detection method was developed to establish the content of myo-inositol in milk powders and infant formulations subsequent to formation of the volatile trialkylsilyl derivative. Samples were prepared by acid digestion, thereby releasing inositol from its multiple bound forms. A digestion time of 4 h at 114 °C was sufficient to hydrolyse potentially interfering carbohydrates and quantitatively recover bound inositol from milk-based products. Single laboratory method validation showed a within-day relative standard deviation (RSDr) of 5.9% and between-day relative standard deviation (RSDiR) of 9.6%. A between-laboratory (n=7) collaborative study yielded an average reproducibility (RSDR) of 12.8%, considered fit-for-purpose as a quality control method for milk-based infant formulations. Soy-based products, containing significant inositol hexaphosphate (IP6, phytic acid), required 32 h for release of all inositol. Although current regulations do not specify the inclusion of IP6 in an inositol measurement, the current method allows for its discrimination if present.
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Research Interests: Chemistry, Chromatography, Ion Chromatography, Nitrate, Nitrite, and 14 moreDairy, Ion Exchange Chromatography, Clinical Sciences, Ion, Validation Studies, Liquid Chromatography, Centrifugation, Dairy Products, Food Sciences, Molecular weight, Ion Exchange, Ions, Biochemistry and cell biology, and Infant Formula
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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... more
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 sa...
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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... more
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. Method Powder samples were reconstituted, with release of carnitine and choline facilitated by both acid and alkaline hydrolysis and the extract analyzed by LC-MS/MS. Quantitation was by internal standard technique using deuterium-labeled carnitine and deuterium-labeled choline. Results Method range, specificity, sensitivity, precision, recovery, accuracy, and ruggedness were assessed for milk powders, infant formulas, and soy- and milk-based nutritional products. Spike recoveries of 94.0–108.4% were obtained f...
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Bovine colostrum and milk samples were collected from two herds over the course of the first month post-partum, pooled for each herd by stage of lactation and total potentially available nucleosides were determined. Sample analysis... more
Bovine colostrum and milk samples were collected from two herds over the course of the first month post-partum, pooled for each herd by stage of lactation and total potentially available nucleosides were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate clean-up, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the available