Estimation of udder emptying based on milk constituents of strip samples after milking.

Milk ejection disorders were induced by oxytocin receptor blockade. We tested the hypothesis that the degree of udder emptying at incomplete milk ejection can be estimated based on the concentration of various milk constituents in different milk fraction samples. To induce different levels of spontaneous udder emptying (SUE) 10 Holstein dairy cows were milked either with or without i.v. injection of the oxytocin receptor blocking agent atosiban (ATO). In ATOearly, 12 µg/kg BW ATO was injected immediately before and in ATOlate directly after a 1-min manual udder preparation. The normal milking routine served as the control treatment. In all 3 treatments the udder was completely emptied by the i.v. injection of 10 IU oxytocin (OT) at the end of spontaneous milk flow. During all experimental milkings 4 milk samples were taken in all treatments: at the start of udder preparation (foremilk; FM), immediately after cessation of spontaneous milk flow and cluster detachment by hand stripping (strip milk; SM), from spontaneous removed milk in bucket 1 (milk before OT; MBOT) and from the milk obtained after OT injection in bucket 2 (milk after OT; MAOT). Fat, protein, lactose, and electrolytes (Na, Cl, and K) were measured in each milk sample. In addition, electrical conductivity (EC) was determined in parallel to continuous milk flow recording. The treatments induced individual degrees of SUE; therefore, the final evaluations of data were based on SUE classes instead of treatments. The most pronounced differences of milk constituents at different degrees of SUE were found for the milk fat content. The fat content of SM and MBOT remained almost unchanged up to 60% SUE, but was considerably higher if >80% of the milk was spontaneously removed. The concentrations of Na and Cl were highest and of K lowest if less than 20% of milk was received in the different samples. The EC was higher in SM and MBOT if <20% of milk was received. In conclusion, the blockade of the OT effect influences primarily the fat content, which confirmed an OT-induced fat secretion during milking. Similar effects are likely found in situations of disturbed milk ejections, caused by a lack of or reduced release of OT in response to different degrees of tactile udder stimulation. Our results show that the measurement of fat content and the EC in SM samples collected after cluster detachment can be used to estimate the completeness of udder emptying.

A Novel Design Eco-friendly Microwave-assisted Cu-N@CQDs Sensor for the Quantification of Eravacycline via Spectrofluorimetric Method; Application to Greenness Assessments, Dosage Form and Biological Samples.

Community-acquired pneumonia is one of the most common infectious diseases and a substantial cause of mortality and morbidity worldwide. Therefore eravacycline (ERV) was approved by the FDA in 2018 for the treatment of acute bacterial skin infections, GIT infections, and community-acquired bacterial pneumonia caused by susceptible bacteria. Hence, a green highly sensitive, cost-effective, fast, and selective fluorimetric approach was developed for the estimation of ERV in milk, dosage form, content uniformity, and human plasma. The selective method is based on the utilization of plum juice and copper sulphate for the synthesis of green copper and nitrogen carbon dots (Cu-N@CDs) with high quantum yield. The quantum dots' fluorescence was enhanced after the addition of ERV. The calibration range was found to be in the range 1.0 - 80.0 ng mL-1 with LOQ equal to 0.14 ng mL-1 and LOD was found to be 0.05 ng mL-1. The creative method is simple to deploy in clinical labs and therapeutic drug health monitoring system. The current approach has been bioanalytically validated using US-FDA and validated ICH criteria. High-resolution transmission electron microscopy (HR-TEM), X-ray photon spectroscopy (XPS), Zeta potential measurements, fluorescence, UV-VIS, and FTIR spectroscopy have all been used to fully characterize the Cu-N@CQDs. The Cu-N@CQDs were effectively applied in human plasma and milk samples with a high percentage of recovery ranging from 97.00 to 98.80%.

Estimation of the performance of two real-time polymerase chain reaction assays for detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in pooled milk samples in a field study.

The early detection of major mastitis pathogens is crucial for the udder health management of dairy herds. Testing of pooled milk samples, either individual test-day cow samples (TDCS) or aseptically collected pre-milk quarter samples (PMQS) may provide an easy to use and cost-effective group level screening tool. Therefore, the aim of this study was (1) to evaluate the sensitivity (Se) and specificity (Sp) of 2 commercial multiplex real-time PCR test kits applied to pooled milk samples using a Bayesian latent class analysis and (2) to estimate the probability of detection in relation to the pool size and the number of cows positively tested by bacteriological culture (BC) within a pool. Pools of 10, 20 and 50 cows were assembled from 1,912 test-day samples and 7,336 PMQS collected from a total of 2,045 cows from 2 commercial dairy farms. Two commercial quantitative real-time PCR kits were applied to detect Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae in the pooled samples, and a BC was applied to PMQS yielding a cumulative pool result. A pool was considered BC-positive if it contained at least one BC-positive PMQS. Pathogens were more frequently detected in the PMQS pools than in the TDCS pools. Pools of 10 cows showed the highest probability of detection irrespective of sample type or type of PCR kit compared with larger pool sizes. Estimation with a Bayesian latent class analysis resulted in a median Se in PMQS pools of 10 cows for Staph. aureus of 63.3% for PCR kit I, 78.1% for PCR kit II, and 95.5% for BC; the Sp values were 97.0%, 97.6%, and 89.1%, respectively. The estimated median Se for Strep. species for PCR kits ranged between 77.5 and 85.6% and for BC between 73.7% and 79.2%; the median Sp values ranged between 93.6 and 99.2% for PCR kits, and between 96.9% and 97.4% for BC. In addition, the probability of detection increased with an increasing number of BC-positive cows per pool. To achieve a probability of detection of 90%, the estimated number of positive cows in PMQS pools of 10 cows for kit I was 4.1 for Staph. aureus, 1.5 for Strep. agalactiae, and 1.3 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 6.9, 1.9, and 2.0 positive cows were required, respectively. For Kit II and PMQS pools, the number of positive cows required was 2.8 for Staph. aureus, 1.4 for Strep. agalactiae, and 1.2 for Strep. dysgalactiae; for the equivalent TDCS pools and pathogens, 5.3, 1.8, and 2.0 positive cows were required, respectively. In conclusion, the type of samples used for pooling, the pool size and the number of infected cows per pool determine the probability of detecting an infection with major mastitis pathogens within a pool by PCR testing.

A Carboxyl Group-Functionalized Ionic Liquid Hybrid Adsorbent for Solid-Phase Extraction and Determination of Trace Diclofenac Sodium in Milk Samples.

A simple and efficient sample pretreatment technology is very important for the accurate determination of trace drug residues in foods to ensure food safety. Herein, we report a new carboxyl group-functionalized ionic liquid hybrid solid- phase adsorbent (PS-IL-COOH) for the highly efficient extraction and quantitative determination of diclofenac sodium (DS) residue in milk samples. It was found that the adsorption efficiency of PS-IL-COOH for the ppb level of DS was greater than 93.0%, the adsorption capacity was 934.1 mg/g, and the enrichment factor was 620.0, which surpass most of the previously reported values for DS adsorbents. The high concentration of salts did not interfere with the adsorption of DS. Importantly, the recovery of DS was above 90% after 16 adsorption--regeneration cycles. The synergistic effect of the multiple interactions was found to be the main factor for the high efficiency of DS adsorption. The proposed method was applied to the extraction and detection of DS in milk samples, with the relative recovery ranging from 88.2 to 103.0%.

A Fast Method for Determination of Seven Bisphenols in Human Breast Milk Samples with the Use of HPLC-FLD.

Plastic pollution, where bisphenol A (BPA) is widely used in its production, has gained popularity. BPA omnipresence and toxicity, especially for infants, has led food safety authorities to place restrictions on BPA usage. It has led to the introduction of the marked 'BPA-free'-labelled products, where BPA is often replaced by other bisphenols (BPs) which are suspected of being similar or even more toxic than BPA. Moreover, the free forms of BPs are more dangerous than their conjugated forms and the conjugation of BPs is less effective in infants than in adults. Considering that human breast milk is the main source of nutrition for infants, the constant biomonitoring not only of BPA, but the wider group of BPs in such crucial matrices seems to be vital. In this study, a fast, simple, 'green' and cost-effective DLLME-based extraction technique combined with HPLC-FLD was optimized for the determination of seven selected bisphenols simultaneously. The procedure has satisfactory recovery values of 67-110% with the most RSD% at 17%. The LODs and LOQs ranged from 0.5 ng/mL to 2.1 ng/mL and 1.4 ng/mL to 6.3 ng/mL, respectively. The procedure was successfully applied to the biomonitoring of free forms of BPs in 10 real human breast milk samples.

Arsenic level in groundwater and biological samples in Khanewal, Pakistan.

Groundwater is the most valuable natural source in our earth's planet, being contaminated in various regions worldwide. Despite considerable research, there are scarce data regarding arsenic (As) levels in groundwater and its build-up in biological samples in Pakistan. The current investigation analyzed As contamination in four tehsils of District Khanewal (Kabirwala tehsil, Jahaniyan tehsil, Mian Channu tehsil, and Khanewal tehsil). For that, 123 groundwater samples, 19 animal milk samples, 20 human nails, and 20 human hair samples were collected from the study area. Arsenic concentration in groundwater was up to 51.8 µg/L with an average value of 7.2 µg/L. About 28 water samples (23%) had As contents > WHO limit and 38 samples (31%) > DEP-NJ limit. Low levels of As were detected in biological samples. Average As levels were 23 µg/L in the milk samples and 298 µg/kg in human hair. Arsenic contents were not detected in nail samples, except in one sample from Kabirwala tehsil. The maximum values of hazard quotient and cancer risk in District Khanewal were 4.9 and 0.0022, respectively. It is anticipated that long-term use of As-containing water may led to poisoning of humans in the study area, especially in Kabirwala. Therefore, it is necessary to monitor As contamination in the groundwater of Kabirwala tehsil to reduce the potential health hazards.

Fabrication of magnetic covalent organic framework for efficient extraction and determination of phthalate esters in milk samples.

Using two monomers of 4,4″-diamino-p-terphenyl and 1,3,5-triformylphloroglucinol, a co-precipitation structured magnetic covalent organic framework adsorbent was fabricated. After that, a high efficient vortex-assisted magnetic solid-phase extraction method was developed prior to gas chromatography-tandem mass spectrometry analysis for the determination of phthalate esters in milk samples. The fabricated magnetic adsorbent was facilely fabricated, fully characterized, and exhibited high extraction efficiency, which can be attributed to its larger pore size as well as strong hydrophobic and π-π stacking interactions between adsorbent and phthalate esters. Key parameters affecting extraction efficiency were investigated. Under the optimized conditions, the proposed method possessed good linearity (3.0-1000 µg/L), high sensitivity (0.8-2.1 µg/L for limits of detection), and satisfactory recoveries (76.8-99.2%). The relative standard deviations for intra-day was 3.1-4.5% and inter-day was 3.3-6.1%. This work is suitable for high efficient separation/preconcentration of phthalate esters in milk samples.

Characterization of telomere length in Agerolese cattle breed, correlating blood and milk samples.

Studies into telomere length in cattle are relatively recent and have focused mainly on the Holstein Friesian cattle breed, making it arduous to evaluate the correlation with ageing due to the early age of culling in this breed. Telomere length provides information about the productive lifespan and the quality of farm management, complying with the 'One Health' approach. This study evaluated telomere length in Agerolese cattle, an autochthonous dairy breed characterized by a long productive lifespan (13 years). Multiplex quantitative PCR estimated telomere length in DNA extracted from blood and milk matrices. Interestingly, the results showed longer telomeres in Agerolese (compared to the Holstein Friesian cattle control group), with a negative correlation between telomere length and increasing age and a synchronous trend between blood and milk samples, with a positive correlation between them.

Electrochemical Biosensor Designed to Distinguish Tetracyclines Derivatives by ssDNA Aptamer Labelled with Ferrocene.

Controlling food safety and preventing the growing spread of antibiotics into food products have been challenging problems for the protection of human health. Hence, the development of easy-to-use, fast, and sensitive analytical methods for the detection of antibiotics in food products has become one of the priorities in the food industry. In this paper, an electrochemical platform based on the ssDNA aptamer for the selective detection of tetracycline has been proposed. The aptasensor is based on a thiolated aptamer, labelled with ferrocene, which has been covalently co-immobilized onto a gold electrode surface with 6-mercaptohexan-1-ol. The changes in the redox activity of ferrocene observed on the aptamer-antibiotics interactions have been the basis of analytical signal generation registered by square-wave voltammetry. Furthermore, the detection of tetracycline-spiked cow milk samples has been successfully demonstrated. The limits of detection (LODs) have been obtained of 0.16 nM and 0.20 nM in the buffer and spiked cow milk, respectively, which exceed the maximum residue level (225 nM) more than 1000 times. The proposed aptasensor offers high selectivity for tetracycline against other structurally related tetracycline derivatives. The developed biosensor characterized by simplicity, a low detection limit, and high reliability shows practical potential for the detection of tetracycline in animal-origin milk.

Application of d-SPE before SPE and HPLC-FLD to Analyze Bisphenols in Human Breast Milk Samples.

In this study, we propose a simple, cost-effective, and sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the simultaneous determination of seven bisphenols (bisphenol F (BPF), bisphenol E (BPE), bisphenol B (BPB), BADGE (bisphenol A diglycidyl ether), BADGE∙2H2O, BADGE∙H2O, BADGE∙2HCl) in human breast milk samples. The dispersive solid phase extraction (d-SPE) coupled with solid phase extraction (SPE) procedure performed well for the majority of the analytes with recoveries in the range 57-88% and relative standard deviations (RSD%) of less than 9.4%. During the d-SPE stage, no significant matrix effect was observed thanks to the application of different pairs of salts such as zirconium-dioxide-based sorbents (Z-Sep or Z-Sep +) and primary secondary amine (PSA) or QuEChERS Enhanced Matrix Removal-Lipid (EMR-Lipid) and PSA. The method limits of quantification (mLOQs) for all investigated analytes were set at satisfactory low values in the range 171.89-235.11 ng mL-1. Analyte concentrations were determined as the average value from human breast milk matrix samples. The results show that the d-SPE/SPE procedure, especially with the application of EMR-Lipid and PSA, could be used for further bisphenol analyses in human breast milk samples.

Prevalence of contamination of aflatoxin M1 in milk: a retrospective analysis of studies conducted in Pakistan.

Aflatoxins, produced by multiple fungal species, are present in several kinds of food items and animal feed. Several studies conducted in Pakistan have reported the presence of aflatoxin M1 (AFM1) in milk. Hence, owing to the public health concern and absence of general statistics regarding the prevalence of AFM1 contamination, current study was aimed to investigate the prevalence of AFM1 in milk in Pakistan. For this study, various databases were searched from 2007 to 2020. A random effect model was applied for analytical purpose and heterogeneity of selected studies was investigated with an I2 index. Comprehensive meta-analysis (version 3) was used for analysis of data. According to the results, prevalence of AFM1 in milk was 84.4% (95% CI 75.0-90.7%). Regarding the heterogeneity based on meta-regression, it has been observed that there was a significant difference between the effect of year of study and sample size with prevalence of AFM1 in animal milk. These results suggest that AFM1 contamination in animal milk is high in Pakistan. Hence, continuous monitoring of AFM1 in animal milk requires utmost attention from the respective food and drug regulatory authorities of Pakistan so that the strict actions and preventive measures should be taken to prevent the prevalence of exposure of AFM1 in animal milk.

Organochlorine pesticide residues in raw milk samples collected from dairy farms and urban areas of Lahore district, Pakistan.

The current study was employed to investigate the organochlorine pesticides (OCP) concentrations in milk, as the milk we consume, has residues of these notorious pesticides. The residual concentrations of OCP in milk have numerous harmful effects on health especially the children. Therefore, milk was analyzed using gas chromatography equipped with µECD for seven OCP residues, namely α-endosulfan, ß-endosulfan, Endosulfan-sulphate, DDE, γ-HCH, Dieldrin, and DDT. Three hundred and sixty raw milk samples were collected from urban areas (10 areas of Lahore N = 300) and Dairy Farms (10 farms in Lahore N = 60) from September 2012 to September 2013. Samples were collected after an interval of 2 months, for 12 months. Mean values of OCPs in milk samples from urban areas were reported as α-endosulfan, ß-endosulfan, Endosulfan-sulphate, DDE, γ-HCH, and Dieldrin with concentration of 17.44 ± 3.99, 35.74 ± 7.50, 20.28 ± 3.95, 2.51 ± 0.55, 0.93 ± 0.16 and 1.12 ± 0.18 µg kg-1, respectively, while the milk samples from dairy farms with concentration of 26.94 ± 4.63, 59.88 ± 6.76, 32.07 ± 4.51, 4.64 ± 0.48, 1.20 ± 0.17 and 1.93 ± 0.18 µg kg-1, respectively. None of the samples analyzed were found positive for the presence of DDT, just as none of the samples from area milk shops exceeded the Maximum Residual Limits (MRLs). γ-HCH and ß-endosulfan were found higher in dairy farm milk samples than the MRLs. Conclusively, these pesticide residues are present in milk available in Lahore in enough quantity (some exceeding the MRLs) to threaten human health, particularly the infant and children.

Use of a magnetic covalent organic framework material with a large specific surface area as an effective adsorbent for the extraction and determination of six fluoroquinolone antibiotics by HPLC in milk sample.

A magnetic covalent organic framework material was synthesized with a core-shell structure using a simple solvothermal method. It was prepared with Fe3 O4 as the magnetic core, covalent organic framework as the shell, which synthesized from 1,3,5-triformylphloroglucinol and p-phenylenediamine by Schiff base reaction. Transmission electron microscopy, Fourier transform infrared spectroscopy, powder X-ray diffraction, vibrating sample magnetometry, and nitrogen adsorption-desorption were used to characterize magnetic adsorbent. It has showed a large specific surface area (505.6 m2 /g), which can provide many adsorption sites. Moreover, the saturation magnetization value was 48.4 emu/g enough to be separated by external magnet. Six kinds of fluoroquinolones (enoxacin, fleroxacin, ofloxacin, norfloxacin, pefloxacin, and lomefloxacin) were extracted by magnetic solid phase extraction with the magnetic adsorbent. High-performance liquid chromatography detects the entire adsorption and desorption process to further evaluate the optimal extraction and desorption conditions. Under the optimal chromatographic conditions, this method showed a low detection limit (0.05 to 0.20 µg/L), good linearity in the range of 0.5 to 200 µg/L, and the enrichment factor reaches 115.5-127.3. The spiked recovery of the fluoroquinolones in milk sample ranged from 90.4 to 101.2%.

Determination of content of camel milk in adulterated milk samples by normalized real-time polymerase chain reaction system based on single-copy nuclear genes.

BACKGROUND: Compared with the traditional qualitative polymerase chain reaction (PCR), which only identifies the category of species, the quantitative PCR method provides a value, which is very important for appropriate penalty enforcement according to the extent of adulteration. However, most of the current quantitative PCR methods are based on mitochondrial genes, expressing different copy numbers in different cells and reducing the accuracy of quantitative results. In this study, single-copy nuclear housekeeping genes, instead of multicopy mitochondrial genes, were selected as both camel species-specific and reference genes to develop a novel normalized PCR system. RESULTS: This system had an excellent linear correlation (R2 = 0.9614) between camel milk content and Ct ratio (specific/reference genes), and allowed quantitative determination of the content of camel milk in adulterated milk samples. The accuracy was effectively validated using simulated adulterated samples with recoveries ranging from 90% to 120% and coefficient of variation less than 10%, exhibiting sufficient parameters of trueness and precision. CONCLUSIONS: The normalized PCR system based on single-copy nuclear genes is a simple, rapid and reliable method for the determination of the content of camel milk in adulterated milk samples, and also provides technical support for appropriate penalty enforcement. © 2020 Society of Chemical Industry.

Comparison of quantitative PCR and MALDI-TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis.

AIMS: The objective of this study was to compare qualitatively and quantitatively the results of identification of the bacteria present in milk samples from cows with subclinical mastitis using multiplex qPCR assay and matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS® ) after bacteriological growth. METHODS AND RESULTS: A total of 182 samples were aseptically collected from 119 cows with high somatic cell counts (>2·105 SCC per ml) on 11 farms in Belgium in 2014. The mutiplex qPCR assay was carried out on 350 µl of milk with the PathoProof® Complete-16kit. Ten microlitre of milk was streaked on Columbia blood agar and three selective agar plates. Growing colonies were identified by MALDI-TOF MS. Of the 182 samples, 90 gave positive results with either or both tests for one or two bacterial species/genera. Total qualitative agreement of the bacteria identified was observed in 41 mono- or bi-bacterial samples (46%) and partial agreement in 19 bi-bacterial samples at both or either tests (21%). The results of both tests on those mono- and bi-bacterial samples were not significantly different (McNemar test; P = 0·395) with a fair agreement (Cohen's kappa test; k = 0·375; P = 0·055). Moreover, quantitative correlation between the qPCR intensity and the numbers of growing colonies was observed in half of the 60 samples with qualitative matching results. CONCLUSIONS: Both methods give identical qualitative and quantitative results with approximately a half and a quarter of the mono- and bi-bacterial samples respectively. Several reasons can explain the differences. The multiplex qPCR assay only targets the most important mammary gland pathogens and can detect DNA of bacteria both alive and dead. Conversely, bacteria only grow when alive and the MALDI-TOF MS databases do not include all bovine milk-associated bacterial species yet. SIGNIFICANCE AND IMPACT OF THE STUDY: This study further highlights the limitations and complementarity of the genetic and phenotypic tests for the identification of bacteria present in milk samples.

An impedimetric aptasensor for ultrasensitive detection of Penicillin G based on the use of reduced graphene oxide and gold nanoparticles.

The authors describe an impedimetric aptasensor for Penicillin G (PEN) which is an important antibiotic. The method is based on the use of a pencil graphite electrode (PGE) modified with reduced graphene oxide (RGO) and gold nanoparticles (GNPs) for ultrasensitive detection of PEN. The morphology of a bare PGE, RGO/PGE, and GNP/RGO/PGE, and the functional groups on graphene oxide (GO) and RGO were studied using scanning electron microscopy and Fourier transform infrared spectroscopy. Electrochemical impedance spectroscopy was used for detection of PEN by measuring the charge transfer resistance (Rct). Also, cyclic voltammetry was recorded at potential range of 0.30 to +0.70 V for PGE treatment. This aptamer-based assay has a wide linear range that extends from 1.0 fM to 10 µM, and a limit of detection as low as 0.8 fM. The method was applied to the determination of PEN in spiked milk from cow, sheep, goat and water buffalo. Recoveries ranged from 92% to 104%. The assay is fast, ultrasensitive, high reproducible, and selective over antibiotics such as streptomycin, tetracycline, and sulfadiazine. Graphical abstract Schematic presentation of an impedimetric aptasensor for Penicillin G antibiotic using a pencil graphite electrode (PGE) modified with reduced graphene oxide (RGO) and gold nanoparticles (GNPs). This aptamer based assay has limit of detection as low as 0.8 fM.

Method Development for Selected Bisphenols Analysis in Sweetened Condensed Milk from a Can and Breast Milk Samples by HPLC-DAD and HPLC-QqQ-MS: Comparison of Sorbents (Z-SEP, Z-SEP Plus, PSA, C18, Chitin and EMR-Lipid) for Clean-Up of QuEChERS Extract.

Background: Identification and quantitative determination of analytes released from the packaging material is undoubtedly a difficult and tricky task, requiring the chemical analyst to develop an individual approach to obtain reliable analytical information. Unfortunately, it is still challenging for scientists to determine bisphenols at trace or even ultra-trace levels in samples characterized by a very complex, and often variable, matrix composition. Objective: Optimization and application of QuEChERS/d-SPE coupled with HPLC-DAD (and LC-QqQ-MS) method for the simultaneous determination of bisphenols (A, S, F, B, BADGE and derivatives) in milk samples from a can and breast milk samples have been performed. Methods: Concerning the analysis of unconjugated analytes, after the thawing and shaking the sample (5 mL breast milk or 10 mL milk samples from a can), it was transferred into a 50 mL polypropylene centrifuge tube. For the analysis of the total amount of analytes, prior to the extraction with acetonitrile, a deconjugation step was implemented in a tube by adding to sample, the an Isotopically Labelled Internal Standard (IS) solution (50 ng/mL) and 1 mL of the enzymatic solution with the ß-Glucuronidase (3500 U/mL). The mix was homogenized and incubated for 16-18 h at 37 °C. Next, 10 mL of acetonitrile, and a QuEChERS salt packet (4 g anhydrous MgSO4, 1 g NaCl) were added. After shaking and centrifugation, the total acetonitrile layer was isolated in a polypropylene tube evaporate to dryness, and reconstitute in 1.2 mL acetonitrile. During d-SPE step the extract was transferred into a 15 mL polypropylene tube with Z-Sep and primary secondary amine (PSA). Next, shake the tube, store in fridge, and centrifuge for 15 min. The acetonitrile supernatant was obtained with a pipette and evaporated to dryness. Mixture MeOH: water (20:80, v/v) were added to the dry residue and the extract was reconstitute in 200 µL and analyzed by HPLC-DAD and HPLC-QqQ-MS equipment. Conclusion: Six different salts during d-SPE step were evaluated such as: zirconium dioxide-based sorbent (Z-Sep, Z-Sep Plus), primary secondary amine (PSA), octadecyl (C18), EMR-Lipid, Chitin and also their mixtures. Negligible matrix interference was observed for most of the analytes due to application of Z-Sep and PSA in dispersive-solid phase extraction clean-up step. Extraction of target analytes was performed using QuEChERS/d-SPE cleanup, and presents good performance for selected analytes with recoveries in the range of 15-103% and relative standard deviations (RSD) less than 10% in breast milk samples.

Fabrication of a highly sensitive electrochemical sensor based on electropolymerized molecularly imprinted polymer hybrid nanocomposites for the determination of 4-nonylphenol in packaged milk samples.

Herein, an imprinted electrochemical sensor based on graphene-Au nanoparticles incorporated with molecularly imprinted polymer (MIP) was fabricated for determination of 4-nonylphenol (4-NP). Grafted MIP electropolymerized on nanoscale multilayer films electrode was achieved using 4-NP as a template and P-aminothiophenol as a functional monomer. The electrochemical properties of the MIP nanoscale multilayer membrances were characterized and measured by cyclic voltammetry and differential pulse voltammetry techniques; using ferrocyanide/ferricyanide-redox marker. Several important parameters were optimized and investigated to improve the performance of the sensor. Under the optimized conditions, the developed sensor showed an excellent linear response over the concentration ranges of 50-500 ng mL-1 (4-NP) with a detection limit of 0.01 ng mL-1(S/N = 3). The developed sensor showed a good selective recognition of 4-NP compared with structural analogue, exhibited a good reproducibility and accuracy with long-term stability. At last, the feasibility of the proposed methodology was successfully applied fordetection of 4-NP in milk and its packaging materials.

Fluorescent aptasensor for detection of four tetracycline veterinary drugs in milk based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

Based on a novel signal amplification strategy by catalytic hairpin assembly and displacement of G-quadruplex DNA, an enzyme-free, non-label fluorescent aptasensing approach was established for sensitive detection of four tetracycline veterinary drugs in milk. The network consisted of a pair of partially complementary DNA hairpins (HP1 and HP2). The DNA aptamer of four tetracycline veterinary drugs was located at the sticky end of the HP1. The ring region of HP1 rich in G and C could form a stable G-quadruplex structure, which could emit specific fluorescence signal after binding with the fluorescent dye and N-methylmesoporphyrin IX (NMM). When presented in the system, the target analytes would be repeatedly used to trigger a recycling procedure between the hairpins, generating numerous HP1-HP2 duplex complexes and displacing G-quadruplex DNA. Thus, the sensitive detection of target analytes was achieved in a wide linear range (0-1000 µg/L) with the detection limit of 4.6 µg/L. Moreover, this proposed method showed high discrimination efficiency towards target analytes against other common mismatched veterinary drugs, and could be successfully applied to the analysis of milk samples. Graphical abstract Schematic of target analyte detection based on catalytic hairpin assembly reaction and displacement of G-quadruplex.

Removal of antibiotics from water and waste milk by ozonation: kinetics, byproducts, and antimicrobial activity.

The use of antibiotics in the dairy farming for curing and growth promotion results in the production of massive quantities of non-recyclable wastewater by the conventional purification techniques. Additionally, waste milk is produced during the drug withholding periods, which is not suitable for human or animal consumption and cause huge economic loss as well as present serious environmental waste. This study was designed to investigate the decomposition of various antibiotic compounds in un-buffered aqueous solutions and milk samples by ozonation process. Commonly administered broad-spectrum antibiotics such as amoxicillin, doxycycline, ciprofloxacin, and sulphadiazine were selected as model examples in the current investigation. Gradual exposure of these antibiotics to increasing ozone gas concentration induced increasing removal percentages of the antibiotics in spiked water and milk samples. The removal reached 95% across all the tested treated antibiotics with ozone dose as low as 75 mg L-1. It was noted that the removal of antibiotics in milk samples is more efficient with faster rate constants. This was attributed to the self-buffering characteristic of milk that maintains the neutral pH, keeping the amine groups un-protonated and more reactive towards the electrophilic attack by the molecular ozone. 1H NMR as well as HPLC experiments support the near complete removal of antibiotics and indicated the break down to simpler and more soluble fragments of acidic nature. Bacterial growth experiments, conducted with E. coli, and milk ageing experiments provided clear evidences that the resulting decomposition byproducts lack both toxicity effect and antimicrobial activity. This study provides a viable route to remove hazardous materials, which contribute to a growing issue of antibiotic resistance of pathogenic bacteria.