Residues In Raw Milk Research Articles

Validation of HPLC-UV Multi-Residue Method for the Simultaneous Determination of Tetracycline, Oxytetracycline, Spiramycin and Neospiramycin in Raw Milk

The use of antimicrobial agents in livestock breeding is inevitable for prophylactic and treatment purposes, in order to maintain health and improve production of food products. However, this practice resulted in development of community resistance to antimicrobials due to residues found in livestock products. Milk is a key nutritional product that’s widely consumed for its high nutritional value. Maximum residue limits (MRL) for antimicrobials were established to minimize their consumption and increase the safety of food products. Although several research papers were validated for multi-class, multi-residue determination of antimicrobials, they were mainly LC coupled with MS/MS detection. This detection technique is expensive, so it doesn’t consider the economic aspects in low- and middle-income countries’ small regulatory laboratories. The proposed method enables the simultaneous determination of the residues of three widely used veterinary antimicrobials: Oxytetracycline (OTC), tetracycline (TTC) and spiramycin (SPI) besides its active metabolite neo-spiramycin (NSPI) in raw milk. Separation was performed on RP-C18 column at flow rate 1.0 mL min−1 and UV detection at 232 nm. Isocratic elution was done using mobile phase composed of 0.05 M phosphate buffer, pH 2.8 and acetonitrile, (8:2, v/v), respectively. Drug residues in raw milk were simply extracted using acetonitrile followed by centrifugation. The method was found linear across the ranges of 20–1000 ng mL−1 for TTC and OTC, and 100–1000 ng mL−1 for SPI and NSPI. The proposed method was found sensitive to low detection and quantification limits covering the MRL of studied drugs with LOD of 5.0 ng mL−1 for OTC and TTC and 16.0 ng mL−1 for SPI and NSPI.

Oxytetracycline and beta- lactam residues in raw milk of different species marketed in Alexandria city, Egypt

Antibiotic residues in milk are of great public health concern since milk is being widely consumed by infants, youngster and adults throughout the globe. Therefore, eighty samples of different species including (cow, buffalo, camel and sheep) 20 of each type were collected from different markets and dairy shops located in Alexandria governorate, Egypt for determination antibiotic residues using HPLC. The obtained results were revealed that oxytetracycline, penicillin G and cloxacillin residues were detected at incidence rate (30, 30, 15, & 30%), (25, 20, 10, & 25%), and (10, 10, 0, &15%) with a mean values of (97.9±28.1, 85.5± 25.9, 48.4±28.0 & 122.9±29.2 ppb); (8.86 ± 3.60, 7.583± 4.73, 1.95± 0.95 &10.2 ± 3.40 ppb), and (39.3 ± 7.40, 21.5 ± 9.05, 0 & 37.6 ± 9.91 ppb) in examined cow, buffalo , camel and sheep’s milk samples, respectively. Positive samples for antibiotic residues were compared with the maximum residual limits established by international standards. In addition, calculated daily intake for each antibiotic residues were compared with acceptable daily intake. One sample of camel and sheep milk may contain three antibiotic residues and these may cause public health hazards for consumers. Therefore, Legislations for monitoring and control antibiotics treatment of food producing animals should be adopted.

Survey on the presence of antibiotic residues in raw milk samples from six sites of the dairy pool of Niamey, Niger.

Background and Aim:Antibiotics are widely used in animal production for treating the diseases and for preventing or increasing animal growth. The presence of antibiotic residues in milk is a public health problem. The aim of this study was to assess the use of antibiotic residues in raw milk from the dairy pool of Niamey in three farms (Toukounous, Kirkissoye, and Niamey) and three collection centers (Hamdallaye, Kollo, and Say).Materials and Methods:A direct interview (questionnaire) was used to collect data regarding the mode of use of antibiotics, the level of knowledge of farmers according to the withdrawal period, and a cross-sectional study was conducted on 192 samples of raw milk. The Delvotest® T was used to monitor antibiotic residues in milk. The data were analyzed using SAS and R software.Results:The most commonly used antibiotics were those from the family of tetracycline (86.7%) and from the family of beta-lactams (13.3%). Regarding the statements of farmers, the reasons why the farmers use antibiotics were the following: About 47% in case of prevention and treatment, 29% for treatment, 12% for prevention, and 12% for increase dairy production. Moreover, the farmers lacked the necessary information about withdrawal period. Screening of antibiotic residues was performed using a standardized biological test kit, the Delvotest®. In total, from 192 samples of raw milk, 19 (9.9%) were positive including ten from collection centers and nine from farms. This could lead to a risk of exposure when a consumer drinks locally produced raw milk.Conclusion:Raw milk supplied from the area of the study has a level of antibiotic residues, and the breeders have a low level of knowledge about the withdrawal period.

The Effect of Boiling on Stability of Oxytetracycline and Sulfamethazine Residues in Raw Milk using HPLC Method

Two hundred raw milk samples (250 ml of each) were collected from small dairy farms, street peddlers and dairy shops in Mansoura Governorate. These samples were screened using Bacillus Subtilis Diffusion Assay for qualitative detection of antibiotics residues; where the percentage of suspected positive samples was 12.5%. High Performance Liquid Chromatography – Ultra Violet detector (HPLC- UV) method was developed and validated to determine the amount of oxytetracycline (OTC) and sulfamethazine (SMZ) residues in raw milk before and after boiling. The results revealed that 8.5 % of the raw milk samples were containing (OTC) residues (6.5 % of them exceed MRL) while, (SMZ) was detected in4 % of the raw milk samples (3 % of them exceed MRL). Upon applying heat treatment, the reduction in the (OTC) content in milk boiled for 2 minutes was 30.5% but boiling for 5 minutes was accompanied with 54.1% reduction. On the other hand, the percentage of (SMZ) reduction was 1.7% and 9.5% in milk boiled for 2 and 5minutes respectively which could be attributed to the low heat stability of (OTC) and high stability of (SMZ).

The Effect of Boiling on Stability of Oxytetracycline and Sulfamethazine Residues in Raw Milk using HPLC Method

Two hundred raw milk samples (250 ml of each) were collected from small dairy farms, street peddlers and dairy shops in Mansoura Governorate. These samples were screened using Bacillus Subtilis Diffusion Assay for qualitative detection of antibiotics residues; where the percentage of suspected positive samples was 12.5%. High Performance Liquid chromatography – Ultra Violet detector (HPLC- UV) method was developed and validated to determine the amount of oxytetracycline (OTC) and sulfamethazine (SMZ) residues in raw milk before and after boiling. The results revealed that 8.5 % of the raw milk samples were containing (OTC) residues (6.5 % of them exceed MRL) while, (SMZ) was detected in4 % of the raw milk samples (3 % of them exceed MRL). Upon applying heat treatment, the reduction in the (OTC) content in milk boiled for 2 minutes was 30.5% but boiling for 5 minutes was accompanied with 54.1% reduction. On the other hand, the percentage of (SMZ) reduction was 1.7% and 9.5% in milk boiled for 2 and 5minutes respectivelywhich could be attributed to the low heat stability of (OTC) and high stability of (SMZ).

Farm Test for Rapid Identification of Antibiotic Residues in Raw Milk

Farm Test for Rapid Identification of Antibiotic Residues in Raw Milk

Monitoring of some pesticides residues in raw milk in Alexandria province, Egypt

Pesticides are used worldwide to increase the production but many persistent residues of pesticides cause problems in environment contamination and human health. Therefore, the present study was conducted to monitor the concentrations of some organochlorines pesticides (OC), organophosphorus (OP) pesticide and synthetic pyrethroids using Gas chromatography in raw milk samples collected from different three districts at Alexandria province, Egypt. The obtained results revealed that some of organochlorines pesticides DDT, Dieldrin, Endrin and Lindane were monitored respectively at percent of 10,10, 2.5, and 5% in district A; 6.66, 6.66, 3.33 and 0% in district B and 10, 3.33, 3.33 and 6.66 % in district C. Concerning organophosphorus pesticides including (malathion and Chlorpyrifos) were detected at percent 5 and 2.5% in district A; 6.66 and 3.33 % in district B and district C were detect in 3.33 and 3.33% of examined raw milk samples. Cyhalothrin and Deltamethrin synthetic pyrethroids were detected in district A, B and C at percent 5 and 7.5%; 3.33 and 6.66%; 3.33 and 3.33%, respectively in examined positive raw milk samples. This study illustrated the risks of pesticides residues exposure that threaten consumer health. Thus, monitoring survey of pesticide residues in milk is necessary to safeguard consumer health.

RESÍDUOS DE ANTIMICROBIANOS EM LEITE BOVINO CRU NO ESTADO DO RIO GRANDE DO NORTE

Resumo O descumprimento do período de carência dos medicamentos aplicados em vacas leiteiras leva à presença de substâncias químicas em limites inadequados no leite. Com o intuito de avaliar a presença de resíduos de antimicrobianos no leite bovino cru produzido no Rio Grande do Norte (RN), foram coletadas 112 amostras de leite cru oriundas de seis municípios do Estado do RN. A pesquisa de resíduos foi realizada com o uso do kit ECLIPSE 50®. Foi observado que seis (6,72%) amostras apresentaram resultado positivo para detecção de resíduos. Desta forma, o leite comercializado no Estado pode gerar riscos à saúde da população consumidora.

Survey of Veterinary Drug Residues in Raw Milk in Hebei Province, China

The objective of this study was to investigate the occurrence of veterinary drug residues in raw milk from Hebei, the second-largest dairy production province in the People's Republic of China. A total of 192 raw milk samples were collected from 64 milk stations in seven districts. Twenty-eight veterinary drug residues were analyzed by ultraperformance liquid chromatography with tandem mass spectrometry based on a China National Standard. Raw milk samples with multiple residues of veterinary drugs were not found in the present study. Residues of four veterinary drugs, penicillin G, sulfacetamide, trimethoprim, and lincomycin, were detected in 12 (6.25%) raw milk samples, with detection ratios of 1.04, 0.52, 3.13, and 1.56%, respectively. All veterinary drug residues detected were under the maximum residue levels as regulated by China, the European Union, the United States, and the Codex Alimentarius Commission. In general, raw milk from Hebei province was considered relatively safe for human consumption because of the low prevalence of veterinary drug residues. However, stringent control measurements for veterinary drug residues in raw milk are required because some veterinary drugs were detected in milk from some areas of Hebei province.

Simultaneous Determination of Erythromycin, Tetracycline, and Chloramphenicol Residue in Raw Milk by Molecularly Imprinted Polymer Mixed with Solid-Phase Extraction

A rapid, simple, and efficient method was described for the simultaneous determination of three common antibiotic residues including erythromycin, tetracycline, and chloramphenicol in raw milk. A molecular imprinting technique combined with solid-phase extraction (SPE) was used to pretreat the test samples and then simultaneously detected with HPLC. The whole process, only including one step of pretreatment, was able to detect the entire target molecule using specific enrichment and analysis method. The detection was verified by comparison with Chinese standard methods of GB/T 22988-2008, GB/T 22990-2008, and GB/T 29688-2013. The testing lines, accuracy, reproducibility, specificity, and recoveries were evaluated, and calculated values were in line with established standards. The recoveries of erythromycin, tetracycline, and chloramphenicol were 77.82~87.08, 81.02~88.17, and 72.94~83.57%, respectively. By using of this novel solid-phase extraction substrate, the detection limits for erythromycin, tetracycline, and chloramphenicol were 10, 20, and 10 μg kg−1, respectively. The method was suitable for routine analysis, and the experimental procedure was simplified, with the detection time greatly reduced. This method showed broad application prospects for the detection of antibiotic residues in raw milk.

Climate conditions associated with the occurrence of antimicrobial and macrocyclic lactone residues in bulk tank milk

ABSTRACT The present study aimed to identify the climate condition parameters that are associated with the occurrence of antimicrobial and macrocyclic lactone residues in bulk tank milk using a multivariate principal components analysis (PCA). A total of 132 raw milk samples were collected at dairy farms in Minas Gerais State in Brazil and analyzed for 35 analytes, comprising macrocyclic lactones and antibacterials, using liquid chromatography coupled with mass spectrometry in tandem mode spectrometry. Of the 132 samples, 34 (25.76%) bulk tank milk samples were positive for at least one analyte. PCA showed that antimicrobial residues in bulk tank milk occurred less frequently on days with a higher average temperature, maximum temperature and temperature-humidity index. In contrast, relative humidity was inversely associated with antimicrobial residues in raw milk. The PCA showed that daily milk production was also related to macrocyclic lactone residues, while rainfall showed an inverse association. Thus, some climate conditions, such as average temperature, maximum temperature and temperature-humidity index, can predict the moments with lower risk of occurrence of antimicrobial residues in bulk tank milk, in contrast to relative humidity. Furthermore, the risk of macrocyclic lactone residues in bulk tank milk was higher in months with less rainfall.

Simultaneous screening for marbofloxacin and ofloxacin residues in animal-derived foods using an indirect competitive immunoassay

ABSTRACTA broad-specificity immunoassay was established to monitor marbofloxacin (MBF) and ofloxacin (OFX) residues in raw milk and porcine muscle samples. The main influential parameters in the assay buffer were investigated to improve the assay performance. The cross-reactivities (CRs) were lower than 0.25%, except for OFX (41.38%). The quantitative working ranges for MBF and OFX based on fortified matrices ranged from 0.11 to 30.85 ng mL−1, with a 50% inhibitory concentration of 0.76±0.12 ng mL−1 for MBF and 2.70±0.28 ng mL−1 for OFX. The detection limits of enzyme-linked immunosorbent assay (ELISA) ranged from 0.053 to 0.358 ng mL−1 in spiked samples. The mean recoveries were in the range of 80.52–95.46%, less than 12.06% of the coefficient of variation. A good correlation between the ELISA results and those from instrument methods demonstrated the practical application of the novel ELISA. Therefore, the immunoassay proposed is feasible for MBF and OFX analysis in animal-derived foods.

Simultaneous Determination of Phenobarbital, Pentobarbital, Amobarbital and Secobarbital in Raw Milk via Liquid Chromatography with Electron Spray Ionization Tandem Mass Spectrometry.

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of four barbiturates (phenobarbital, pentobarbital, amobarbital and secobarbital) in raw milk. The barbiturates were extracted using liquid-liquid extraction, ultrasonication and centrifugation, and purified on an SPE column. Analytes were separated by HPLC on a CSH C18 column eluted using an acetonitrile-water system with a linear gradient dilution programme, and detected by MS/MS. The recoveries of the barbiturates were 85.0-113.5%, and the intra- and inter-assay RSDs were less than 9.8% and 7.3%, respectively. The limit of detection was 5 ng/mL for all four of the barbiturates. The analytical method exhibited good linearity from 10 to 1000 ng/mL; the correlation coefficient (r2) was greater than 0.9950 for each barbiturate. This method was also applied to the determination of barbiturates in real milk samples and was found to be suitable for the determination of veterinary drug residues in raw milk.

Determination of Penicillins in Milk by a Dual-Optrode Biosensor

ABSTRACTPenicillins are the most frequently found antibiotic residues in milk, as they are commonly used for the treatment of bacterial infections in cows. In the present study, we introduce a method for the rapid detection of penicillin residues in raw milk based on the determination of glucose concentration in milk with a dual flow-through biosensor. The molar concentration of glucose in milk is typically over 500 times lower than the concentration of lactose and is highly dependent on the rate of lactose hydrolysis, which is catalyzed by β-galactosidase. Glucose concentrations in milk change with variation in the β-galactosidase activity. β-Galactosidase is an enzyme produced in the microbiota in milk and its activity is inhibited by benzylpenicillin. Spiking milk with benzylpenicillin lowers glucose concentrations in comparison to high-quality milk after short storage intervals. The presence of penicillin in the milk of treated animals resulted in decreased glucose concentrations in comparison with high-quality milk that contained no antibiotics. The glucose concentration in milk samples was followed by the system enabling the elimination of the effects of bacterial respiration in the output with reliable results in less than 1 min.

Effect of various storage conditions on the stability of quinolones in raw milk

ABSTRACTResearch on the storage stability of antibiotic residues in milk is important for method development or validation, milk quality control and risk assessment during screening, confirmation, qualitative or quantitative analysis. This study was conducted using UPLC-MS/MS to determine the stability of six quinolones – ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), difloxacin (DIF) and flumequine (FLU) – in raw milk stored under various conditions to investigate if quinolones degrade during storage of milk, and finally to determine optimal storage conditions for analysis and scientific risk assessment of quinolone residues in raw milk. The storage conditions included different temperatures and durations (4°C for 4, 8, 24 and 48 h; –20°C for 1, 7 and 30 days; –80°C for 1, 7 and 30 days), thawing temperatures (25, 40 and 60°C), freeze–thaw cycles (1–5), and the addition of different preservatives (sodium thiocyanate, sodium azide, potassium dichromate, bronopol and methanal). Most quinolones exhibited high stability at 4°C for up to 24 h, but began to degrade after 48 h. In addition, no degradation of quinolones was seen when milk samples were stored at –20°C for up to 7 days; however, 30 days of storage at –20°C resulted in a small amount of degradation (about 30%). Similar results were seen when samples were stored at –80°C. Moreover, no losses were observed when frozen milk samples were thawed at 25, 40 or 60°C. All the quinolones of interest, except sarafloxacin, were stable when milk samples were thawed at 40°C once and three times, but unstable after five freeze–thaw cycles. Preservatives affected the stability of quinolones, but the effects differed depending on the preservative and quinolone. The results of this study indicate optimum storage protocols for milk samples, so that residue levels reflect those at the time of initial sample analysis, and should improve surveillance programmes for quinolones in raw milk.

Influence of Preservative Concentration, pH Value and Fat Content in Raw Milk at Detection Limit of Microbial Inhibitor Tests (Delvotest® Accelerator) for Amoxicillin and Oxytetracycline

The presence of antibiotic residues in raw milk is a big threat not only to the dairy industry but also to public health. Therefore, it is very important to detect antibiotic residues in raw milk. There are many reliable antibiotic test used in dairy laboratories. Unfortunately, some factors can affect the results causing, so-called false-positive results. The aims of this paper are to check the detection limits of microbiological inhibitor test with test organisms Geobacillus stearothermophilus var. calidolactis, which are provided on Delvotest® Accelerator instrument (DSM, The Netherlands) for antibiotics amoxicillin and oxytetracycline and to examine the influence of variations of milk fat contents, pH values and azidiol concentrations in raw milk on detection limits, e.g., test results. The obtained results showed that detection limit for amoxicillin is 3 μg/L and oxytetracycline 250 μg/L; concentration of preservative azidiol of 0.1 mL per 40 mL of milk does not affect the detection limits of the test; microbiological inhibitor test is extremely sensitive to low pH value of samples so, it is recommended to avoid testing milk samples with low pH value, or to adjust the sample’s pH value by addition of some base; and that fat content in raw milk up to 5 % does not affect the detection limit of the methods for amoxicillin and for oxytetracycline.

Performance of Eclipse Farm Test Coupled to e-Reader for Antibiotic Residues Detection in Raw Milk

A new system for the analysis of antimicrobial residues in raw milk was optimized by combining a microbial inhibitor tube test (Eclipse Farm) and a device (e-Reader) that integrates incubation at 65 °C and colour change continuous monitoring. The system determines automatically the end point of the assay and interprets the results in an objective way. Thus, the new method is adapted for the use at farms and small dairies since it simplifies the analysis and increases the accuracy of the results. The performance of a new system was validated according to the European Commission Decision 2002/657/EC. Limits of detection of the new system were firstly evaluated for 30 molecules from several families of antimicrobials and were found to be close to the European maximum residue limits (MRLs). Detection capabilities were also determined for six molecules representing the main antimicrobial groups used in dairy husbandry (penicillins, cephalosporins, tetracyclines, sulphonamides, macrolides and aminoglycosides). All molecules were detected at or below the MRL level. Ruggedness was also studied, demonstrating that the new method was unaffected by reasonable changes in the procedure (sample volume, incubation temperature, test batch). Applicability was demonstrated in raw cow milk regardless fat and protein content or high somatic cell counts. Eclipse Farm coupled to e-Reader has proved to be a valuable tool for the screening of a broad-spectrum of antimicrobial residues in raw milk.

Simultaneous determination of 38 veterinary antibiotic residues in raw milk by UPLC–MS/MS

A selective and rapid method has been developed to determine, simultaneously, 38 veterinary antibiotic residues in raw milk by ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). One milliliter of raw milk was diluted with 0.5mL water and 3mL acetonitrile, then purified using an Oasis HLB cartridge. The eluates were evaporated by nitrogen drying and then reconstituted to 4mL with water/acetonitrile (8:1) before being injected into the UPLC–MS/MS system. The results indicated recoveries of 68–118% for 14 β-lactams, 79–118% for eight quinolones, 71–106% for eight sulfonamides, 76–116% for four tetracyclines, 78–106% for three macrolides, and 88–103% for one lincosamides, with coefficients of variation less than 15% for intraday and interday precisions. The limit of quantification for all antibiotics was 0.03–10μgkg−1. This methodology was then applied to field-collected real raw milk samples and trace levels of four antibiotics were detected.

Identification of Antibiotic Residues in Raw Milk Samples Coming from the Metropolitan Area of Bucharest

The study aimed at evaluating antibiotic residues in raw milk coming from two categories of providers: dairy farmers and processed milk producers. The analyses were carried out in a private laboratory at the request of the providers. 210 samples, collected monthly during 2012 (January to December) were analyzed (n = 210). Of the 210 samples of raw milk, 45.71% (n = 96) came from dairy farmers and 54.29% (n = 114) came from processed milk producers. The milk samples were tested using the Beta Star Combo method based on chemical reactions with reagents attached to gold particles. All milk samples had a pH ranging between 6.6-6.9. The presence of tetracycline and beta-lactam antibiotic was analyzed as these are the most used antibiotics in the treatment of mammary glands diseases in dairy cows. Of all the 210 samples, antibiotic residues were detected in 66 samples of which 42 contaminated samples came from dairy farmers. Beta-lactam antibiotics residues have been detected in 12 samples and tetracycline in 30 samples. In the contaminated samples coming from the processed milk producers, only traces of tetracycline were found. These results are a warning signal showing antibiotics entering the human food chain in this way, this being even more alarming if we take into consideration the fact that milk, in general, is a product mainly consumed by children and the elderly.

Fast extraction of amphenicols residues from raw milk using novel fabric phase sorptive extraction followed by high-performance liquid chromatography-diode array detection

A simple, sensitive, reliable, and fast analytical method was developed for the simultaneous determination of amphenicols residues in raw milk by combining fabric phase sorptive extraction (FPSE) and high-performance liquid chromatography-diode array detection. FPSE, a new generation green sample preparation technique, efficiently incorporates the advanced and tunable material properties of sol–gel derived microextraction sorbents with the rich surface chemistry of a cellulose fabric substrate, resulting in a flexible, highly sensitive, and fast microextraction device capable of extracting target analytes directly from complicated sample matrices. Due to the strong chemical bonding between the sol–gel sorbent and substrate, the microextraction device demonstrates a very high chemical and solvent stability. Therefore, any organic solvent/solvent mixture can be used as the eluent/back-extraction solvent.Herein, a highly polar polymer coated FPSE media was created using short-chain poly(ethylene glycol) (PEG) and the applicability of this novel microextraction device to extract highly polar amphenicol antibiotics from raw milk was investigated. Due to the intense affinity of amphenicols towards the strongly polar sol–gel PEG-coated FPSE device, absolute recovery of the selected antibiotics residues were found to be 44% for thiamphenicol, 66.4% for florfenicol, and 81.4% for chloramphenicol. The developed method was validated in terms of sensitivity, linearity, accuracy, precision, and selectivity according to European Decision 657/2002/EC. Decision limit (CCα) values were 52.49μgkg−1 for thiamphenicol, 55.23μgkg−1 for florfenicol, and 53.8μgkg−1 for chloramphenicol, while the corresponding results for detection capability (CCβ) were 56.8μgkg−1, 58.99μgkg−1, and 55.9μgkg−1, respectively.