Study of the bioavailability of selenium in cows’ milk after a supplementation of cow feed with different forms of selenium

The purpose of the work described in this paper was to develop an easy and quick in-vitro method for comparing the bioavailability of selenium in cows’ milk after different cow feed. The study focuses on bioavailability differences resulting from the use of different selenium species (organic selenium as selenised yeast and sodium selenite) for supplementation of forage. A procedure for determination of selenium in cows’ milk and dialysates, by hydride-generation atomic-fluorescence spectrometry (HG-AFS) after microwave-assisted acid digestion, was optimised. The results show it is possible to obtain cows’ milk enriched with selenium at different concentration without altering the original composition of the milk. The bioavailability was statistically greater for cows’ milk obtained after supplementation of forage with organic selenium at levels of 0.4 and 0.5 μg Se g⁻¹ than for that obtained after supplementation with inorganic and organic selenium at levels of 0.2 and 0.3 μg Se g⁻¹.     

Presence of pesticides in breast milk and infants’ formulae in Himachal Pradesh,India

This study documents the levels of pesticide residues in milk samples of mothers from Himachal Pradesh, India, and time trend comparison of pesticide load based on various studies conducted around the world. The regional difference in xenobiotic levels of breast milk varied with demographic characteristics of mothers and altitudinal variations. The single or multiple pesticides contamination of p,p′-DDE, p,p′-DDT and chlorpyrifos was revealed in 27.45% mothers’ milk samples. Among these p,p′-DDE was the major contaminant found in 26.79% samples followed by p,p′-DDT (1.31%) and chlorpyrifos (0.65%). However, residues of other 26 pesticides comprising organochlorines, organophosphorus and synthetic pyrethroids included in this study were below detectable limit (BDL). The determination of a low DDT/DDE ratio (0.050) indicated past exposure of mothers to DDT from the environment. The pesticide residues level in samples drawn from 14 branded infant formulae was BDL. The calculated infants’ daily intake (DI) of DDT was 0.0015 mg kg^?1 body weight per day compared with a decade-old study (0.021 mg kg^?1 body weight per day) suggesting a sharp decline in the residue levels of these pesticides in the Himalayan region. The trend comparison with past studies conducted around the world indicate a decline in the levels of organochlorine pesticide residues in mothers’ milk and further drop of DI in infants. However, such comparisons confer very limited utilisation of data generated on pesticide load in mothers’ milk and simultaneous infants’ DI due to lack of proper research protocol.     

Comprehensive screening and quantification of veterinary drugs in milk using UPLC–ToF-MS

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC–ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC–ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80–120% for 88% of the compounds) were satisfactory. Evaluation of the CCβ values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 μg/l) that tested positive for this compound. UPLC–ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.     

Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe⁺), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe⁺ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled ¹³CD₃⁸²SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe⁺, the standard addition method was applied. Quality control for the accurate quantitation of TMSe⁺ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L⁻¹. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L⁻¹ for TMSe⁺, 5.6 μg Se L⁻¹ for SeMet, and 0.1 μg Se L⁻¹ for both SeGalNAc and SeGluNAc.     

Quantification of cow’s milk percentage in dairy products – a myth?

The substitution of ewe's and goat's milk for cheaper cow's milk is still a fraudulent practice in the dairy industry. Moreover, soy-based products (e.g., soy milk, yoghurt) have to be checked for cow's milk as they are an alternative for people suffering from an allergy against bovine milk proteins. This work reports the evaluation of different protein-based electrophoretic methods and DNA-based techniques for the qualitative detection as well as the quantitative determination of cow's milk percentage in dairy and soy milk products. Isoelectric focusing (IEF) of γ-caseins using an optimized pH gradient was appropriate not only for the detection of cow's milk, but also for an estimation of cow's milk percentage in mixed-milk cheese varieties. Urea-polyacrylamide gel electrophoresis (PAGE) proved the method of choice to detect cow's milk in soy milk products, whereas IEF and SDS-PAGE of proteins were not applicable due to false-positive results. Polymerase chain reaction (PCR) analysis was used to confirm the results of protein-based electrophoretic methods. Problems inherent in quantitative analysis of cow's milk percentage using protein-based techniques and even more using DNA-based methods were emphasized. Applicability of quantitative real-time PCR for the determination of cow's milk percentage in mixed-milk cheese was shown to be hampered by several factors (e.g., somatic cell count of milk; technological parameters influencing the final DNA concentration in ripened commercial cheese samples). The implementation of certified reference standards (of major relevant cheese groups) containing 50% cow's milk was urgently recommended to enable at least a yes/no decision in commercial mixed-milk cheese samples.     

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and ¹H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.     

Melt–vapor phase transition in the lead–selenium system at atmospheric and low pressure

The boiling temperature and the corresponding vapor phase composition in the existence domain of liquid solutions were calculated from the partial pressures of saturated vapor of the components and lead selenide over liquid melts in the lead–selenium system. The phase diagram was complemented with the liquid–vapor phase transition at atmospheric pressure and in vacuum of 100 Pa, which allowed us to judge the behavior of the components during the distillation separation.     

Identification of selenium species in urine by ion-pairing HPLC–ICP–MS using laboratory-synthesized standards

This study focused on the detection/identification of possible selenium metabolites in human urine. Organoselenium compounds not commercially unavailable were synthesized and characterized by electrospray mass spectrometry. Separation of selenomethionine, methylselenomethionine, trimethylselonium, selenoethionine, and selenoadenosylmethionine was achieved by ion-pairing HPLC with a mobile phase of 2 mmol L^–1 hexanesulfonic acid, 0.4% acetic acid, 0.2% triethanolamine (pH 2.5), and 5% methanol. The column effluent was introduced on-line to inductively coupled plasma–mass spectrometry for selenium-specific detection (⁷⁷Se and ⁷⁸Se). For selenium speciation in urine, solid-phase extraction was carried out using C₁₈ cartridges modified with hexanesulfonic acid. Selective retention of cationic species was observed from acidified urine (perchloric acid, pH 2.0). After elution with methanol, evaporation, and dissolution in the mobile phase, the sample was introduced to the HPLC–ICP–MS system and the chromatographic peaks were assigned by adding standards. The species identified in urine were selenomethionine, trimethylselonium ion, and selenoadenosylmethionine. The last species was detected for the first time and our results suggest that selenomethionine might enter the metabolic pathway of its sulfur analog in the activated methylation cycle.Kazimierz Wrobel and Katarzyna Wrobel are on the leave from the Institute of Scientific Research, University of Guanajuato, L. de Retana No. 5, 36000 Guanajuato, Gto., Mexico     

Determination of phthalate monoesters in human milk,consumer milk,and infant formula by tandem mass spectrometry (LC–MS–MS)

Daily exposure of humans to phthalates may be a health risk because animal experiments have shown these compounds can affect the differentiation and function of the reproductive system. Because milk is the main source of nutrition for infants, knowledge of phthalate levels is important for exposure and risk assessment. Here we describe the development and validation of a quantitative analytical procedure for determination of phthalate metabolites in human milk. The phthalate monoesters investigated were: monomethyl phthalate (mMP), monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mono-(2-ethylhexyl) phthalate (mEHP), and monoisononyl phthalate (mNP). The method is based on liquid extraction with a mixture of ethyl acetate and cyclohexane (95:5) followed by two-step solid-phase extraction (SPE). Detection and quantification of the phthalate monoesters were accomplished by high-pressure liquid chromatography using a Betasil phenyl column (100 mm×2.1 mm×3 m) and triple tandem mass spectrometry (LC–MS–MS). Detection limits were in the range 0.01 to 0.5 g L^–1 and method variation was from 5 to 15%. Analysis of 36 milk samples showed that all these phthalates were present, albeit at different concentrations. Median values (g L^–1) obtained were 0.11 (mMP), 0.95 (mEP), 3.5 (mBP), 0.8 (mBzP), 9.5 (mEHP), and 101 (mNP). We also analysed seven samples of consumer milk and ten samples of infant formula. Only mBP and mEHP were detected in these samples, in the ranges 0.6–3.9 g L^–1 (mBP) and 5.6–9.9 g L^–1 (mEHP).     

The effect of heteroatom substitution of sulfur for selenium in glucosidase inhibitors on intestinal α-glucosidase activities

The synthesis of selenium analogues of de-O-sulfonated ponkoranol, a naturally occurring sulfonium-ion glucosidase inhibitor isolated from Salacia reticulata, and their evaluation as glucosidase inhibitors against two recombinant intestinal enzymes maltase glucoamylase (MGAM) and sucrase isomaltase (SI) are described.     

Determination of chlormequat in pig serum and sow milk by LC–MS/MS

Chlormequat is a plant growth regulator widely used on cereals, and there is general concern that it may impair human fertility. A LC–MS/MS method for the analysis of chlormequat in milk and serum was developed and validated in connection with an investigation on the effect of chlormequat on pig reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol–water–acetic acid, centrifuged, filtrated and determined by LC–MS/MS. The mean recoveries were in the range 80–110%, and the LOD was 0.2 ng/g for serum and 0.3 ng/g for milk. The values for repeatability and reproducibility were within 2/3 of the limits given by the Horwitz equation. Samples of pig serum (59) and sow milk (27) were analyzed using the method. Chlormequat was determined in four milk samples in the range of 0.4 ng/g to 1.2 ng/g and in all serum samples in the range of 0.2 ng/g−4.0 ng/g.     

Unsaturated five-membered selenium–germanium containing heterocycles based on the reactions of selenium di- and tetrahalides with diorganyl diethynyl germanes

Regio- and stereoselective reactions of selenium dihalides with diorganyl diethynyl germanes in CHCl₃ afford in yields from preparative to quantitative the first representatives of a new class of selenium–germanium containing heterocycles - 3,6-dihalogen-4,4-diorganyl-1,4-selenagermafulvenes. The reaction of selenium tetrachloride under the same conditions leads to the first representatives of a new class of selenium–germanium containing cyclopentene heterocycles - 2-dichloromethyl-2,4-dichloro-3,3-diorganyl-1-selena-3-germacyclopentenes-4 as well as to the unknown 3,6,6-trichloro-4,4-dialkyl-1,4-selenagermafulvenes formed as a result of a spontaneous dehydrochlorination of the cyclopentene heterocycles. In a case of SeBr₄ the process of dehydrobromination is dominating. The structures of the heterocycles were proved by multinuclear (¹H, ¹³C, ⁷⁷Se) spectroscopy and mass-spectrometry. In the ¹H NMR spectra of Z-isomers of 1,4-selenagermafulvenes a long-range spin–spin interaction between exo- and endocyclic olefinic protons through five bonds is revealed lacking in the E-isomers.     

Determination of selenomethionine, selenocysteine, and inorganic selenium in eggs by HPLC–inductively coupled plasma mass spectrometry

A method for the simultaneous determination of selenomethionine (SeMet), selenocysteine (SeCys), and selenite [Se(IV)] in chicken eggs was developed. A sample preparation protocol including defatting, protein denaturation, and carbamidomethylation was optimized in order to achieve complete protein digestion and to avoid SeCys losses. Quantification was carried out by reversed-phase HPLC–inductively coupled plasma mass spectrometry (ICP MS) after quantitative isolation of the selenium-containing fraction by size-exclusion liquid chromatography. The detection limits were 0.06, 0.003, and 0.01 μg g⁻¹ (dry weight) for SeCys, Se(IV) and SeMet, respectively, and the precision was 5–10%. The end products of carbamidomethylation of the different selenium species were identified for the first time by electrospray QTOF MS after custom-designed 2D HPLC purification. Differences in selenium speciation in egg yolk and white were highlighted, the yolk containing more SeCys and the white more SeMet. An insight into selenium bioaccessibility in eggs was obtained by digestion with simulated gastric and gastrointestinal juices and size-exclusion HPLC-ICP MS.     

Ether-compatible lithium sulfur batteries with robust performance via selenium doping

The increasing demand of the green energy storage system encourages us to develop a higher energy storage system to take the place of the present lithium-ion batteries with limited energy/power densities[1,2].Among the diverse candidates。     

Determination of non-steroidal anti-inflammatory drugs and their metabolites in milk by liquid chromatography–tandem mass spectrometry

Non-steroidal anti-inflammatory drugs are widely used for treatment of animals. According to Council Directive 96/23/EC, residues of these drugs must be monitored because of the potential risk they pose to the consumers' health. For this reason an LC-MS-MS method was developed for detection of wide range of NSAIDs, including both "acidic" NSAIDs (carprofen, diclofenac, flunixin, meloxicam, phenylbutazone, oxyphenbutazone, tolfenamic acid, mefenamic acid, naproxen, ketoprofen, ibuprofen, firocoxib, rofecoxib, and celecoxib) and "basic" NSAIDs (four metamizole metabolites). Analytes were extracted from milk samples with acetonitrile in the presence of ammonium acetate. One portion of the extract was directly analyzed for the presence of metamizole metabolites; a second portion was cleaned with an amino cartridge. All NSAIDs were separated on a Phenomenex Luna C8(2) column and analyzed by LC-MS-MS in negative (acidic NSAIDs) and positive (metamizole metabolites) ion modes. The method was validated in accordance with the requirements of Commission Decision 2002/657/EC. Within-laboratory reproducibility was in the range 7-28%, and accuracy was in the range 71-116%. The method enabled detection of all the analytes with the expected sensitivity, below the recommended concentrations. The method fulfills the criteria for confirmatory methods and, because of its efficiency, may also be used for screening purposes. The procedure was also successfully verified in the proficiency test organized by EU-RL in 2010. As far as the authors are aware, this is one of the first methods capable of detecting diclofenac residues below the MRL in milk (0.1 μg kg(-1)). An additional advantage is the possibility of simultaneous determination of "acidic" NSAIDs and metamizole metabolites.     

Assessment of natural and artificial radioactivity in infants’ powdered milk and their associated radiological health risks

Journal of Radioanalytical and Nuclear Chemistry - A high-resolution gamma-ray spectrometer was applied to measure the activity concentration of natural and artificial radionuclides 226Ra, 232Th,...     

Speciation of trace elements in foods,with special reference to cadmium and selenium: is it necessary?

Studies on the speciation of trace elements in foods are required to validate existing risk assessment procedures, e.g. those used for Cd which are based on inorganic Cd salts, and to better understand how the absorption and bioavailability of elements can be reduced (Cd) or improved (Se). The methodology for speciation studies is very much in the research and development phase, with few reference materials or standard procedures. The use of stable isotopes with chromatographic separation techniques and improved instrumentation for ICP-MS and MS in general offers a way forward in this area.     

Aerated milk protein emulsions — new microstructural aspects

The formation of stable aerated products based on milk protein emulsions, such as whipped creams, depends on (i) the physical properties of the emulsion before the whipping step; and (ii) how the air is incorporated and the created air bubbles are stabilised. The final texture of the product is determined by the arrangement of the microstructural entities and the interaction among them. Advances are being made mainly in the better understanding of the destabilisation of the oil–water interface of the emulsion, which is a key point in the formulation process. However, the connection to the ‘real product’ has still to be made. Further investigations, especially with regard to the foaming behaviour, have to be encouraged in order to predict and control the physical quality and stability of aerated milk protein emulsions.     

Studies in hydride generation atomic fluorescence determination of selenium and tellurium. Part 2 — effect of thiourea and thiols

In determination of selenium and tellurium by continuous flow hydride generation atomic fluorescence spectrometry, the effect of thiourea and thiols was investigated in view of their potential to achieve mild reaction conditions and as masking agents of interference from foreign elements. The effect of thiourea and thiols was first tested in the absence of interfering species and using different addition modes to reaction system. In the absence of interfering species, thiols negatively influenced the hydride evolution of both selenium and tellurium and, in general, they did not produce the desired effects. Thiourea was well tolerated in the determination of both elements by appropriate choice of experimental conditions. Possible mechanisms producing the depressive effect of thiourea and thiols were also investigated and are discussed later. Compromise reaction conditions were identified by using on-line addition of a neutral thiourea solution to acidified sample, combined with KI addition to NaBH₄. Mild reaction conditions can be achieved by decreasing the NaBH₄ concentration but at the expense of a reduced linear dynamic range. In the presence of foreign elements, thiourea allowed good control of interferences generated by Cu(II), Co(II), Ni(II), Au(III), Ag(I) and Bi(III). Tolerance limits could be improved by factors in the range of 7–2000, for both selenium and tellurium determination. The method has been successfully applied in the determination of traces of tellurium and selenium in copper, lead and molybdenum ores, stainless steel and pure copper metal without any additional steps other than sample dissolution.     

Ultra-high-pressure liquid chromatography–tandem mass spectrometry for the analysis of six resorcylic acid lactones in bovine milk

This work reports a rapid, reliable and sensitive multi-residue method for the simultaneous determination of six resorcylic acid lactones in bovine milk by ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The resorcylic acid lactones were extracted, purified, and concentrated from milk samples in one step using a solid-phase extraction (SPE) cartridge that contained a polymeric mixed-mode anion-exchange sorbent. The analysis was performed on a Waters Acquity BEH C₁₈ column utilizing a gradient elution profile. Each LC run was completed in 3.5 min. The analytes were detected by multiple reaction monitoring (MRM) using electrospray ionization (ESI) negative mode. Mean recoveries from fortified samples ranged from 92.6% to 112.5%, with relative standard deviations lower than 11.4%. Using 5 mL bovine milk, the limits of detection and quantification for resorcylic acid lactones were in the ranges of 0.01–0.05 and 0.05–0.2 μg/L, respectively. The application of this newly developed method was demonstrated by analyzing bovine milk samples from markets.