Differential Thermal Isomerization: Its Role in the Analysis of Vitamin D3 in Foods.

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.

Lipid oxidation and vitamin D3 degradation in simulated whole milk powder as influenced by processing and storage.

Vitamin D3 levels are known to sometimes decline in fortified products, which could be due to its degradation, although the exact mechanism is unknown. In this study, the influence of processing and storage conditions on lipid oxidation and vitamin D3 degradation were studied. Simulated whole milk powders with and without heat treatment were stored for 12 months at two different storage temperatures (20 °C and 40 °C). Stored samples without heat treatment showed higher lipid oxidation products analyzed by PV and TBARS values compared to those with heat treatment. Higher storage temperature also resulted in higher levels of lipid oxidation products. The concentration of vitamin D3 was also analyzed using UHPLC-MS/MS after PTAD derivatization in stored samples. An inverse relationship was observed between lipid oxidation products and vitamin D3 content. Finally, previtamin D3 and vitamin D3 oxidation products were quantified in stored samples using MRM analysis.

Identification of Vitamin D3 Oxidation Products Using High-Resolution and Tandem Mass Spectrometry.

In a successful fortification program, the stability of micronutrients added to the food is one of the most important factors. The added vitamin D3 is known to sometimes decline during storage of fortified milks, and oxidation through fatty acid lipoxidation could be suspected as the likely cause. Identification of vitamin D3 oxidation products (VDOPs) in natural foods is a challenge due to the low amount of their contents and their possible transformation to other compounds during analysis. The main objective of this study was to find a method to extract VDOPs in simulated whole milk powder and to identify these products using LTQ-ion trap, Q-Exactive Orbitrap and triple quadrupole mass spectrometry. The multistage mass spectrometry (MSn) spectra can help to propose plausible schemes for unknown compounds and their fragmentations. With the growth of combinatorial libraries, mass spectrometry (MS) has become an important analytical technique because of its speed of analysis, sensitivity, and accuracy. This study was focused on identifying the fragmentation rules for some VDOPs by incorporating MS data with in silico calculated MS fragmentation pathways. Diels-Alder derivatization was used to enhance the sensitivity and selectivity for the VDOPs' identification. Finally, the confirmed PTAD-derivatized target compounds were separated and analyzed using ESI(+)-UHPLC-MS/MS in multiple reaction monitoring (MRM) mode. Graphical Abstract ᅟ.

Investigation of concentration of thiocyanate ion in raw cow's milk from China, New Zealand and the Netherlands.

Thiocyanate ion is a natural component of cow's milk (hereinafter as milk) which may be artificially augmented to activate the lactoperoxidase milk preservation system. This study presents a survey of thiocyanate levels in raw milk and proposes a naturally occurring baseline concentration of thiocyanate in milk, which is the basis for market supervision. 1669 raw milk samples from China, 270 samples from New Zealand and 120 from the Netherlands were collected in the survey. 65% of the samples contained thiocyanate above the detection limit. The average concentration of thiocyanate was 2.11mg/kg (0.10-16.20mg/kg). Differences in the concentrations of thiocyanate were found among three countries, the 12 selected provinces in China, and between summer and winter. The baseline concentration of thiocyanate found in raw cow's milk was statistically calculated and rounded to 9.0mg/kg. Thiocyanate in milk at this level does not present a food safety concern.

Degradation studies of cholecalciferol (vitamin D3) using HPLC-DAD, UHPLC-MS/MS and chemical derivatization.

In any food fortification program, the stability of added micronutrients is an important factor. Cholecalciferol or vitamin D3 is known to isomerise under various conditions, thereby making its analysis challenging. In the current study, the effects of different parameters, such as temperature, iodine, acidic conditions, and oxidation, on the isomerisation of vitamin D3 were studied using HPLC-DAD and UHPLC-MS/MS. Vitamin D3 thermally and reversibly transforms to pre-vitamin D3 type isomers. In the presence of iodine, cis/trans isomerisation of both cholecalciferol and pre-vitamin D3 takes place to form trans-vitamin D3 and tachysterol, respectively. Another isomer, isotachysterol, was formed under acidic conditions. The different rates of reaction of these products with a dienophile through the Diels-Alder reaction confirmed the formation of vitamin D3 isomerisation products. The derivatization enhanced the ionisation efficiency of vitamin D3 and its isomers in UHPLC-MS/MS and improved the separation and fragmentation enabling sensitive detection.

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. The vitamin D adduct is then re-extracted into a small volume of acetonitrile and analyzed by RPLC. Detection is by tandem MS, using multiple reaction monitoring. Stable isotope-labeled vitamin D2 and vitamin D3 internal standards are used for quantitation to correct for losses in extraction and any variation in derivatization and ionization efficiencies. A single-laboratory validation of the method using AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) kit samples was performed and compared with parameters defined according to the vitamin D Standard Method Performance Requirements (SMPR(®)). Linearity was demonstrated over the range specified in the SMPR, with the LOD being estimated at below that required. Method spike recovery (vitamin D2, 97.0-99.2%; and vitamin D3, 96.0-101.0%) and RSDr (vitamin D3, 1.5-5.2%) were evaluated and compared favorably with limits in the vitamin D SMPR. Acceptable bias for vitamin D3 was demonstrated against both the certified value for National Institute of Standards and Technology 1849a Standard Reference material (P(α = 0.05) = 0.25) and AOAC INTERNATIONAL reference method 2002.05 (P(α = 0.05) = 0.09). The method was demonstrated to meet the requirements of the vitamin D SMPR as defined by SPIFAN, and was recently approved for Official First Action status by the AOAC Expert Review Panel on SPIFAN Nutrient Methods.

Generation of semicarbazide from natural azine development in foods, followed by reaction with urea compounds.

This paper proposes a mechanism to explain the trace levels of natural semicarbazide occasionally observed in foods. The analytical derivative of semicarbazide, 2-nitrobenzaldehyde semicarbazone, is often measured as a metabolite marker to detect the widely banned antibiotic nitrofurazone. However, this marker is not specific as semicarbazide may be present in foods for several reasons other than exposure to nitrofurazone. In some cases, an entirely natural origin of semicarbazide is suspected, although up until now there was no explanation about how semicarbazide could occur naturally. In this work, semicarbazide is proposed as being generated from natural food compounds via an azine intermediate. Hydrazine, in the form of azines or hydrazones, may be generated in dilute aqueous solution from the natural food compounds ammonia, hydrogen peroxide and acetone, following known oxidation chemistry. When this mixture was prepared in the presence of ureas such as allantoin, urea, biuret or hydroxyurea, and then analysed by the standard method for the determination of semicarbazide, 2-nitrobenzaldehyde semicarbazone was detected. 2-Nitrobenzaldehyde aldazine was also found, and it may be a general marker for azines in foods. This proposal, that azine formation is central to semicarbazide development, provides a convergence of the published mechanisms for semicarbazide. The reaction starts with hydrogen peroxide, peracetic acid, atmospheric oxygen or hypochlorite; generates hydrazine either by an oxaziridine intermediate or via the chlorination of ammonia; and then either route may converge on azine formation, followed by reaction with a urea compound. Additionally, carbamate ion may speculatively generate semicarbazide by reaction with hydrazine, which might be a significant route in the case of the hypochlorite treatment of foods or food contact surfaces. Significantly, detection of 2-nitrobenzaldehyde semicarbazone may be somewhat artefactual because semicarbazide can form during the acid conditions of analysis, which can free hydrazine in the presence of urea compounds.

Lactose semicarbazone as a marker for semicarbazide adulteration in milk.

A liquid chromatography-mass spectrometry method to detect semicarbazide and lactose semicarbazone in milk was developed as part of a programme to set up methods for detecting the economically motivated adulteration of raw milk with nitrogen-containing compounds. The detection of semicarbazide was hampered by that fact that this compound tended to give broad, poor intensity peaks in the hydrophobic interaction chromatographic method employed. When spiked into milk at levels of 20-200 ppm, semicarbazide either partially or completely reacted with the matrix, which both increased the limit of detection of the method and made the setting of a threshold by using low level spikes almost impossible. Thus using lactose semicarbazone as a marker for semicarbazide addition to milk was investigated. Lactose semicarbazone was detected in semicarbazide-spiked milk, and its identity was confirmed by fragmentation analysis and comparison with the synthesised compound. The level of lactose semicarbazone correlated with the amount of semicarbazide added to the milk, and the acidic conditions employed in the extraction method appeared to enhance the sensitivity of detection by driving the semicarbazone-forming reaction towards completion. Thus lactose semicarbazone can be used as a marker for the addition of semicarbazide to milk; however, both compounds should be monitored during surveys looking for the semicarbazide adulteration of milk.

Rapid detection of economic adulterants in fresh milk by liquid chromatography-tandem mass spectrometry.

A method to aid in the detection of the economically driven adulteration of fresh milk with a range of small, nitrogen containing compounds, including melamine, ammeline, ammelide, cyanuric acid, allantoin, thiourea, urea, biuret, triuret, semicarbazide, aminotriazine, 3- and 4-aminotriazole, cyanamide, dicyandiamide, guanidine, choline, hydroxyproline, nitrate, and a range of amino acids, has been developed. (15)N2-Urea is used as an internal standard. The adulteration of milk with exogenous urea has previously been difficult to detect because of the variation in the naturally occurring levels of urea in milk. However, by monitoring the contaminants biuret and triuret, which comprise up to 1% of synthetic urea, the adulteration of milk with urea-based fertilizer can be detected. We estimate that to be economically viable, adulteration of the order of 90-4000ppm of the above adulterants would need to be added to fresh milk. For most of the compounds, an arbitrary detection threshold of 2ppm is therefore more than sufficient. For biuret, a lower detection threshold, better than 0.5ppm, is desirable and the sensitivity for biuret and triuret can be improved by the post-column addition of lithium to create lithium adducts under electrospray ionisation. Sample handling involves a two-step solvent precipitation method that is deployed in a 96-well plate format, and the hydrophilic interaction liquid chromatography uses a rapid gradient (1.2min). Three separate injections, to detect the positively charged compounds, the negatively charged compounds and amino acids and finally the lithium adducts, are used. This rapid and qualitative survey method may be deployed as a second tier screening method to quickly reduce sample numbers indicated as irregular by an FTIR based screening system, and to direct analysis to appropriate quantification methods.

Detection of 3-amino-1,2,4-triazine adulteration in milk using an oxidation product 3-amino-1,2,4-triazin-5(2H)-one.

A rapid liquid chromatography-mass spectrometry method to detect 3-amino-1,2,4-triazine (ATZ) in milk was developed as part of a programme to set up methods for detecting the economically motivated adulteration of raw milk with nitrogen-containing compounds. When ATZ was added to unpasteurised or pasteurised milk at levels of 10-1000 ppm, the levels declined over a period of a few days and in some cases declined below the limit of detection of the analytical method (1 ppm). ATZ did not degrade in deproteinised milk extracts, in aqueous standards or in aqueous (non-milk) controls, suggesting that degradation was mediated by a pasteurisation-resistant enzymatic or microbial process. An oxidation product of ATZ was detected by mass spectrometry, and a tentative structure for this compound (3-amino-1,2,4-triazin-5-one, ATZO) was determined by fragmentation analysis and high resolution mass spectrometry. The accumulation of this oxidation product correlated with the loss of ATZ in milk samples. It was concluded that the detection of ATZO could be used as a marker for the addition of ATZ and that both compounds should be monitored during surveys looking for the ATZ adulteration of milk.

A rapid analytical method for cholecalciferol (vitamin D3) in fortified infant formula, milk and milk powder using Diels-Alder derivatisation and liquid chromatography-tandem mass spectrometric detection.

A method for analysing vitamin D(3) (VD3, cholecalciferol) has been established and validated. This method is rapid and cost effective and is intended for use in quality control in the manufacture of fortified infant formulae and milk powders. Milk or reconstituted milk powder was solubilised in methanol and extracted in one step into isooctane, which was separated by centrifugation. A portion of the isooctane layer was then transferred, and an aliquot of 4-phenyl-1,2,4-triazoline-3,5-dione was added to derivatise VD3. The analyte was then re-extracted into a small volume of acetonitrile and analysed by reverse-phase chromatography. Detection was by triple quadrupole mass spectrometer using a selective transition, m/z 560 → 298. An internal standard, deuterium-labelled VD3, was used to correct for losses in extraction and any variation in derivatisation and ionisation efficiencies. The method has been subjected to a single-laboratory validation and has been found to be linear, highly selective and accurate with respect to National Institute of Standards and Technology Standard Reference Material 1849, analyte spiking experiments and comparison with an LC-UV-based method. The repeatability standard deviation was 4.23 %. Significantly for routine laboratories, the method returns results within 2 h, generates minimal waste and minimises health and safety concerns to the analyst.

Determination of immunoglobulin G in bovine colostrum and milk powders, and in dietary supplements of bovine origin by protein G affinity liquid chromatography: collaborative study.

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.