Monitoring of melamine contamination in fat watery milk by the photoluminescence analysis

A photoluminescence method to detect the toxic melamine contamination in fat watery milk has been proposed. Despite the quite different luminescence origins of milk and melamine patterns, their wide emission spectra under UV excitation are similar and in the range of 2.2-3.5 eV. The complex milk photoluminescence spectrum composed of riboflavin, furosine, lactulose, Vitamin E and tryptophan emitting species can be modified if milk pattern is undergone by acid treatment (for example, in vinegar). At the same time the melamine emission is not subjected to any modification in vinegar. It allows quantitatively discriminating the melamine contamination in milk in linear range, at least, 0.05-7 g/l from different photoluminescence spectra of milk (water) with and without melamine. Limit of melamine detection achieves 0.01 g/l.     

Speciation of Se, Fe and Zn in Human Milk Whey: The use of Instrumental Neutron Activation Analysis (INAA) to corroborate element profiles measured with Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES)

In human milk trace elements normally exist in a form which can easily be absorbed by the newborn infant. For investigations of the bioavailability of trace elements it is necessary to carry out a speciation analysis of the elements of interest. An independent analytical method has been used for the quality control of the shape of the element profiles obtained from the speciation analysis of Se, Fe and Zn in individual samples to human milk whey after chromatographic separation. For the element detection in the untreated milk fractions Instrumental Neutron Activation Analysis (INAA) was chosen as the reference method. Element distribution patterns have been obtained with Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) using on-line digestion of the fraction, reduction and hydride formation of Se (T-mode). Prior results are compared with those obtained by means of element detection in the untreated fractions (U-mode) with ICP-AES. The elution profiles of Se, Fe and Zn in human milk whey obtained by means of ICP-AES detection using the T-mode show good agreement with the distribution patterns obtained with INAA. Recoveries of 103% for Fe, 86% for Zn and 87% for Se were obtained. The element distribution patterns of Fe and Zn obtained with ICP-AES speciation using the T-mode also show good agreement with those obtained by means of ICP-AES using the U-mode.Dedicated to Professor Dr. Peter Brätter on the occasion of his 60th birthday.     

Improved determination of taurine by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD)

The advantages of the high selectivity of high-performance anion-exchange chromatography (HPAEC) and the sensitive response of taurine at a gold electrode with integrated pulsed amperometric detection (IPAD) have been combined, in order to establish a new analytical method for its determination in real matrices. Potential-time settings of the potential waveform were optimized in order to get the highest amperometric response. The separation of taurine in milk samples was achieved using an alkaline eluent (100 mM NaOH) containing 1 mM Ba(OAc)₂ and a column temperature of 15 °C. The inherent merits of using a barium-modified eluent, in terms of taurine separation and detection, are demonstrated. The enhancement in sensitivity under these experimental conditions makes it suitable for taurine determination in milk. Indeed, this method allows high recovery of taurine and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity with a detection limit of 50 nmol/L, which corresponds to 2.5 pmol. The taurine content in milk samples of some common mammals was evaluated, including human milk. In goats milk, the taurine content ranged from 46 to 91 mg/L, whereas human and buffalo milk samples exhibited an average content of 18 mg/L and 23 mg/L, respectively.     

Rapid Determination of Lactulose in Milk Using Seliwanoff's Reaction

ABSTRACT

A rapid method based on Seliwanoff's reaction to distinguish between UHT and sterilised milk was proposed. Lactulose was determined directly in milk with no treatment. Analysis of raw milk and its carbohydrates showed that the only interference was the relatively high concentration of lactose, which imposes the lower limit of detection. Small variations of lactulose (9mg dL^?) during heat treatment could be observed when the absorbance was measured against pretreated milk (blank). This method showed a linear range between 17–170mg dL^? and a detection limit lower than that of the official HPLC method. This novel procedure was compared with a commercially available enzymatic method and the results obtained correlated well.     

TIRF-based biosensor for sensitive detection of progesterone in milk based on ultra-sensitive progesterone detection in water

We report on recent advances of our immunoassay for the hormone progesterone in cows milk. Detection is based on total internal reflectance fluorescence (TIRF), the binding-inhibition assay with an immobilized progesterone derivative, and a commercially available monoclonal antibody to progesterone as biological recognition element. The fully automated River Analyzer (RIANA) biosensor for unattended, cost-effective, and continuous monitoring of environmental pollution therefore was adapted for sensitive determination of progesterone in milk. First, the sensitivity and robustness of the existing progesterone assay for water analysis were improved, resulting in a detection limit (LOD) of only 0.2 pg mL^–1 and a quantification limit (LOQ) of only 2.0 pg mL^–1. These extraordinary results are the lowest detection and quantification limits for progesterone determination using biosensors yet reported in the literature. Second, the accurate indicator of ovulation was calibrated and detected in three different types of milk (UHT milk, fresh milk, and raw milk). For commercial milk and randomly procured raw milk nominal levels of progesterone are typically in the range 5–15 ng mL^–1. Limits of detection (LOD) achieved for added progesterone (i.e. spiked samples) were between 45.5 and 56.1 pg mL^–1 depending on milk type. Having in mind the 1:10 dilution factor, these results are still a success. For the first time a commercially available antibody was incorporated into an immunoassay for progesterone detection in bovine milk, giving a detection limit below 1 ng mL^–1 for a fully automated biosensor. Thus the outstanding progress made with this biosensor in environmental monitoring and water analysis has now been successfully adapted to milk analysis for use in the field of reproduction management.Dedicated to the memory of Wilhelm Fresenius.     

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

Determination of total selenium and selenium distribution in the milk phases in commercial cow’s milk by HG-AAS

A procedure has been developed for determining the selenium in cows milk using hydride generation–atomic absorption spectrometry (HG-AAS) following microwave-assisted acid digestion. The selenium distributions in milk whey, fat and micellar casein phases were studied after separating the different phases by ultracentrifugation and determining the selenium in all of them. The detection limits obtained by HG-AAS for the whole milk, milk whey and micellar casein were 0.074, 0.065 and 0.075 g l^–1, respectively. The accuracy for the whole milk was checked by using a Certified Reference Material CRM 8435 whole milk powder from NIST, and the analytical recoveries for the milk whey and casein micelles were 100.9 and 96.9%, respectively. A mass balance study of the determination of selenium in the different milk phases was carried out, obtaining values of 95.5–100.8%. The total content of selenium was determined in 37 milk samples from 15 different manufacturers, 19 whole milk samples and 18 skimmed milk samples. The selenium levels found were within the 8.5–21 g l^–1 range. The selenium distributions in the different milk phases were studied in 14 whole milk samples, and the highest selenium levels were found in milk whey (47.2–73.6%), while the lowest level was found for the fat phase (4.8–16.2%). A strong correlation was found between the selenium levels in whole milk and the selenium levels in the milk components.     

Simple and Rapid Method for Determination of Selenium in Breast Milk by HG-AFS

A digestion procedure using H₂SO₄/HNO₃/H₂O₂ was found to be effective for destruction of human milk samples. In conjunction with a sensitive hydride generation atomic fluorescence spectrometry detection system, it is suitable for determination of selenium in those samples where the available mass of breast milk and the low selenium concentration are limiting factors. Only 1g of milk sample is needed. The procedure is simple, rapid and of low contamination potential since it is performed in the same Teflon tube from weighing to measurement. The digestion of 20 samples is completed in three hours. The detection limit is 0.25±0.04ngg^–1 of a measured solution of sample or 2.5ngg^–1 of milk. The relative uncertainty is 10% (coverage factor of 2.3, 95% probability). Because of these advantages the method is particularly suitable for epidemiological studies. The mean concentration of selenium in 62 samples of human milk from lactating women residing in the North East of Italy was 12±3ngg^–1, which is in the range of reference data.     

Rapid determination of lactulose in milk by microdialysis and biosensors

A simple and rapid flow system for the determination of lactulose in milk samples was developed. It is based on the hydrolysis of lactulose to galactose and fructose by the enzyme beta-galactosidase immobilised in a reactor. The amount of fructose produced was measured with an electrochemical biosensor based on the fructose dehydrogenase enzyme, K3[Fe(CN)6] as mediator and a platinum based electrochemical transducer. Parameters such as the enzyme immobilisation in the reactor and under the electrode surface, the lifetime of the beta-galactosidase reactor and of the dehydrogenase biosensor and the flow parameters were studied and optimised. Fructose was determined in the range 1 x 10(-6)-5 x 10(-3) mol l-1 with an RSD of about 2% and a detection limit of 5 x 10(-7) mol l-1. The use of a microdialysis probe as the sampling system permitted the direct measurement of lactulose in milk samples without pre-treatment in the range 1 x 10(-5)-5 x 10(-3) mol l-1. The sensitivity of the procedure allowed pasteurised, UHT and in-container sterilised milk to be distinguished.     

Fluorometric determination of the antibiotic kanamycin by aptamer-induced FRET quenching and recovery between MoS<Subscript>2</Subscript> nanosheets and carbon dots

A rapid and sensitive aptamer-based assay is described for kanamycin, a veterinary antibiotic with neurotoxic side effects. It is based on a novel FRET pair consisting of fluorescent carbon dots and layered MoS₂. This donor-acceptor pair (operated at excitation/emission wavelengths of 380/440 nm) shows fluorescence recovery efficiencies reaching 93 %. By taking advantages of aptamer-induced fluorescence quenching and recovery, kanamycin can be quantified in the of 4–25 μM concentration range, with a detection limit of 1.1 μM. The method displays good specificity and was applied to the determination of kanamycin in spiked milk where it gave recoveries ranging from 85 % to 102 %, demonstrating that the method serves as a promising tool for the rapid detection of kanamycin in milk and other animal-derived foodstuff.

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Graphical Abstract A fluorometric aptasensor was developed for the determination of kanamycin. It is based on a novel FRET pair of carbon dots and layered MoS₂. The fluorescence recovery efficiency reached 93 % with a good sensitivity, specificity and recoveries in spiked milk.

     

Molecularly imprinted electrochemical aptasensor for the attomolar detection of bisphenol A

Gold nanoparticles (AuNPs) were electrodeposited on the surface of a glassy carbon electrode (GCE) and then treated with a mixture of a thiolated DNA sequence (p-63; with high affinity for bisphenol A) and free bisphenol A (BPÀ). Pyrrole was then electropolymerizaed on the surface of the GCE to entrap the BPA@p-63 complex. BPA is then extracted with acetic acid solution to obtain MIP cavities where the embedded DNA sequence acts as the binding site for BPA. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were employed to characterize the surface of the modified GCE. Under the optimum conditions, the assay has a dynamic range that covers the 0.5 fM to 5 pM BPA concentration range and an 80 aM detection limit. It was applied to the quantitation of BPA in (spiked) milk, milk powder and water samples and gave acceptable recoveries.

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Graphical Abstract Schematic of the procedure for aptamer-based detection of BPA using unique features of the aptamer-based modified electrodes and MIP-based sensors. This assay has high sensitivity and good selectivity. It can presumably be transferred to other detection schemes for small molecules.

     

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

A Lactulose Bienzyme Biosensor Based on Self‐Assembled Monolayer Modified Electrodes

A bienzyme biosensor in which the enzymes β‐galactosidase (β‐Gal), fructose dehydrogenase (FDH), and the mediator tetrathiafulvalene (TTF) were coimmobilized by cross‐linking with glutaraldehyde atop a 3‐mercaptopropionic acid (MPA) self‐assembled monolayer on a gold disk electrode, is reported. The working conditions selected were E_app=+0.10 V and (25±1) °C. The useful lifetime of one single TTF‐β‐Gal‐FDH‐MPA‐AuE was surprisingly long, 81 days. A linear calibration plot was obtained for lactulose over the 3.0×10^?5–1.0×10^?3 mol L^?1 concentration range, with a limit of detection of 9.6×10^?6 mol L^?1. The effect of potential interferents (lactose, glucose, galactose, sucrose, and ascorbic acid) on the biosensor response was evaluated. The behavior of the SAM‐based biosensor in flow‐injection systems in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining lactulose in a pharmaceutical preparation containing a high lactulose concentration, and in different types of milk. Finally, the analytical characteristics of the TTF‐β‐Gal‐FDH‐MPA‐AuE are critically compared with those reported for other recent enzymatic determinations of lactulose.     

Determination of sulfamethoxazole in milk using molecularly imprinted polymer monolith microextraction coupled to HPLC

We report on a new method for the selective extraction of the antibiotic sulfamethoxazole (SMO) in milk that is making use of a molecularly imprinted polymer (MIP) monolith as the sorbent. The monolith was synthesized in the tip of a micropipette using SMO as the template and a combination of acrylamide and 4-vinylpyridine as the co-functional monomers. The monolith was connected to syringes in different sizes and used for microextraction without any other treatment and showed high selectivity and enrichment ability for SMO. It was applied to the selective extraction and sensitive determination of SMO in milk. The linear range is from 5–600?μg?L^?1, the correlation coefficient (r²) is 0.9984, and the detection limit (at S/N?=?3) is 1?μg?L^?1. Recoveries range from 93.6 to 101.7?%, with relative standard deviations of <6.1?%.

Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk

To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL^?1 milk and 0.4 ng mL^?1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n?=?8) or non-transgenic (n?=?7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.

SYNTHESIS OF GLYCOSYLAMINES: IDENTIFICATION AND QUANTIFICATION OF SIDE PRODUCTS

The synthesis of some glycosylamines (1-amino-1-deoxy D-glucose, 1-amino-1-deoxy-D-galactose and 1-amino-1-deoxylactose) was carried out by treatment of the corresponding reducing sugars with ammonium hydrogencarbonate in concentrated ammonia. The reaction mixture was first analyzed by capillary electrophoresis with indirect absorbance detection and high performance anion-exchange chromatography with pulsed amperometric detection. Beside glycosylcarbamate, a known reaction by-product, fructose and lactulose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-amino-1-deoxylactose, respectively. Quantification of glycosylamines was carried out by micellar electrokinetic chromatography with UV detection of their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmocglycosylamines were easily purified from the other components of the crude synthesis mixtures.     

Development of a molecularly imprinted polymer-matrix solid-phase dispersion method for selective determination of β-estradiol as anabolic growth promoter in goat milk

A simple, fast, and sensitive method for determination of 17 β-estradiol (E2) in goat milk samples has been developed by combining selective molecularly imprinted matrix solid-phase dispersion (MIP–MSPD) and liquid chromatography with diode-array detection (DAD). The molecularly imprinted polymer was synthesized by use of 17β-estradiol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker monomer, azobisisobutyronitrile as initiator, and acetonitrile as porogen, and was used as selective solid support for matrix solid-phase dispersion. The selected dispersant had high affinity for E2 in the goat milk matrix and the extract obtained was sufficiently clean for direct injection for HPLC analysis without any interferences from the matrix. The proposed MIP–MSPD method was validated for linearity, precision, accuracy, decision limit (CCα) and detection capability (CCβ), in accordance with European Commission Decision 2002/657/EC criteria. Linearity ranged from 0.3–10 μg g^?1 (correlation coefficient r ²?>?0.999). Mean recovery of E2 from goat milk samples at different spiked levels was between 89.5 and 92.2%, with RSD values within 1.3–2%. CCα and CCβ values were 0.36 and 0.39 μg g^?1, respectively. The developed MIP–MSPD method was successfully applied to direct determination of E2 in goat milk samples.

A highly sensitive europium nanoparticle-based lateral flow immunoassay for detection of chloramphenicol residue

A europium nanoparticle-based lateral flow immunoassay for highly sensitive detection of chloramphenicol residue was developed. The detection result could be either qualitatively resolved with naked eye or quantitatively analyzed with the assistance of a digital camera. In the qualitative mode, the limit of detection (LOD) was found to be 0.25 ng/mL. In the quantitative mode, the half-maximal inhibition concentration (IC50) was determined to be 0.45 ng/mL and the LOD can reach an ultralow level of 0.03 ng/mL, which is ~100 times lower than that of the conventional colloidal gold-based lateral flow immunoassay. Potential application of the established method was demonstrated by analyzing representative cow milk samples.

Magnetic cobalt-nitrogen-doped carbon microspheres for the preconcentration of phthalate esters from beverage and milk samples

The authors describe the preparation of magnetic cobalt/nitrogen-doped carbon microspheres (Co-N/Cs) by combining a hydrothermal procedure with a carbonization process. The textures of the Co-N/Cs were investigated by powder X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, nitrogen adsorption-desorption isotherms and vibration sample magnetometry. The Co-N/Cs possess a high surface area and strong magnetism. This results in good adsorption capability and enables magnetic separation. The Co-N/Cs are shown to be an effective magnetic solid-phase extraction adsorbent for the enrichment of various phthalate esters (diethyl phthalate, diallyl phthalate and diisobutyl phthalate) from sport beverages and milk samples prior to their determination by HPLC. The limits of detection (at an S/N ratio of 3) are between 0.1–0.2 and 0.08–0.3 ng mL^?1 for sport beverages and milk samples, respectively. The recoveries when extracting all the spiked samples varied from 80.3% to 116.2%.

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Graphical abstract Magnetic cobalt/nitrogen-doped carbon microspheres (Co-N/Cs) were prepared and used as an effective magnetic solid phase extraction adsorbent for the enrichment of phthalate esters from beverages and milk samples.