Multi-residue methods for confirmatory determination of antibiotics in milk

The prevalent use of antibiotics, e. g. sulphonamides, tetracyclines, beta-lactams, macrolides, aminoglycosides, etc., in dairy farming to prevent and treat microbial infections e. g. mastitis, can be a potential source of veterinary drug residues in milk. Antibiotic residues constitute a risk to human health, since they can cause allergic reactions in hypersensitive individuals or they may lead to the appearance of drug-resistant bacteria. Analysis of these residues plays a key role in ensuring food safety. Regulatory agencies in the European Union and in other countries have established maximum residue limits and requirements concerning the performance of analytical methods and the interpretation of the results. This review describes the state of the art in the analytical strategies concerning the multi-class as well as the multi-residue analysis of antibiotics in milk. Since milk is a complex matrix due to its high protein and fat content, which often interfere in analytical procedures, special focus has been placed on sample preparation: extraction and clean-up. Confirmation of antibiotic residues according to European Decision 657/2002/EC has been also discussed.     

Determination of tetracycline antibiotics in cow's milk by liquid chromatography/tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) multiresidue method for the simultaneous quantitative determination of oxytetracycline, 4-epi-oxytetracycline, tetracycline, 4-epi-tetracycline, chlortetracycline, 4-epi-chlortetracycline and doxycycline in milk has been developed. An extraction procedure consisting of a liquid extraction of the milk samples with trichloroacetic acid was performed. The extract was centrifuged and the supernatant was filtered. Solid-phase extraction (SPE) with an OASIS HLB SPE column was used to clean up the sample extracts. The samples were analysed by LC/MS/MS. The LC separation was performed on a reversed-phase C18 column using gradient elution with a mobile phase consisting of water and a mixture of methanol/acetonitrile. The tetracycline analytes were detected with a quadrupole mass spectrometer using positive ion electrospray ionisation. The confirmatory method has acceptable detection limits and the different tetracyclines can be detected at a residue concentration between 5 and 20 microg/L. The method is validated according to the European requirements for veterinary drug residues and all determined parameters were found to conform to the criteria. The recovery values ranged from 90.4 to 101.2% with relative standard deviations (RSDs) no larger than 9.7%. The overall or between-day precision of the analytical assay determined as repeatability at several residue concentrations and expressed as RSD ranged from 3.3 to 10%. This analytical assay is a useful tool within the Belgian monitoring programme for confirmation of samples which have been positively screened for residues of tetracyclines in raw farm cow's milk.     

Inhibitive detection of benzoic acid using a novel phenols biosensor based on polyaniline–polyacrylonitrile composite matrix

A novel sensitive and stable phenols amperometric biosensor, based on polyaniline–polyacrylonitrile composite matrix, was applied for determination of benzoic acid. The electrochemical biosensor functioning was based on the inhibition effect of benzoic acid on the biocatalytic activity of the polyphenol oxidase (PPO) to its substrate (catechol) in 0.1 M phosphate buffer solution (pH 6.5). A potential value of −50 mV versus SCE, and a constant catechol concentration of 20 μM were selective to carry out the amperometric inhibition measurement. The kinetic parameters Michaelis-Menten constant and maximum current (I_max) in the absence and in the presence of benzoic acid were also evaluated and the possible inhibition mechanism was deduced. The inhibiting action of benzoic acid on the polyphenol oxidase electrode was reversible and of the typical competitive type, with an apparent inhibition constant of 38 μM. This proposed biosensor detected levels of benzoic acid as low as 2 × 10⁻⁷ M in solution. In addition, the effects of temperature, pH value of solution on the inhibition and the interferences were investigated and discussed herein. Inhibition studies revealed that the proposed electrochemical biosensor was applicable for monitoring benzoic acid in real sample such as milk, yoghurt, sprite and cola.     

Chromatographic methods for tetracycline analysis in foods.

The tetracyclines have served for decades as an important class of antibiotics in food animal health and production. As such, they have also been a source of concern for residue monitoring authorities around the world. In response to this concern a number of microbial inhibition, immunoassay and bacterial receptor methods have evolved for the detection of this class of compounds in various foods of animal origin. However, these methods often lack specificity and are subject to false positive and false negative results. For these reasons a number of chromatographic methods for the separation and determination of the tetracyclines isolated from foods have been developed that are capable of identifying and quantifying individual tetracycline drugs. We present here an overview of tetracycline analytical methods, including microbial inhibition, immunoassay and receptor technologies for detection, techniques for isolation from food matrices, and thin-layer chromatographic, high-performance liquid chromatographic, gas chromatographic and mass spectrometric procedures for determination of this class of compounds. A discussion of the variables involved in such methodology and a review of method criteria are offered.     

Evaluation of a Microbiological Multi-Residue System on the detection of antibacterial substances in ewe milk

To protect both, public health and the dairy industry, from the presence of antibiotic residues in milk, control programmes have been established, which include the needed screening tests. This work focuses on the application of a Microbiological Multi-Residue System in ewe milk, a method based on the use of six different plates, each seeded with one of the following bacteria: Geobacillus stearothermophilus var. calidolactis (beta-lactams), Bacillus subtilis at pH 8.0 (aminoglycosides), Kocuria rhizophila (macrolides), Escherichia coli (quinolones), B. cereus (tetracyclines) and B. subtilis at pH 7.0 (sulphonamides), respectively. Twenty-three antimicrobial substances were analysed and a logistic regression was established for each substance assayed to relate the antibiotic concentration and the zone of microbial growth inhibition. Great linearity in the response was observed (regression coefficients of over 0.97). This fact suggests the possibility of establishing a decision level of antibiotic concentrations near to the Maximum Residue Limits (MRL). Zones of inhibition were suggested as proposed action levels for the different antimicrobial groups (diameters of inhibition of 18 mm for the aminoglycoside, beta-lactam and sulphonamide plates; 19 mm for the tetracycline plate, 21 mm for the macrolide plate, and 24 mm for the quinolone plate). Specificity and cross-reactivity were also assayed.     

Development of an Amperometric Microbial Biosensor Based on Thiobacillus thioparus Cells for Sulfide and Its Application to Detection of Sulfate‐Reducing Bacteria

This paper describes a novel electrochemical microbial biosensor based on Thiobacillus thioparus cells for sulfide detection. The morphology and electrochemical properties of the proposed biosensor were characterized by SEM and cyclic voltammetry, respectively. Working conditions of the microbial biosensor were optimized to obtain good electrochemical performances. Under the optimum conditions, analytical performances were evaluated, and the results suggested that the microbial biosensor could be used for selective detection of sulfide. The microbial biosensor was then successfully applied in detection of sulfate‐reducing bacteria by oxidizing its characteristic metabolite, sulfide, which was accumulated in culture media during bacterial growth.     

Biosensor analysis of penicillin G in milk based on the inhibition of carboxypeptidase activity

The β-lactam antibiotics, including penicillins, are the most important antimicrobial substances used for mastitis treatment. Consequently, this is also the most frequently occurring type of antibiotic residues in milk. Today, in addition to the traditional microbial inhibitor tests, rapid and sensitive receptor and immunoassays are used in residue control. Due to the limitations in throughput capacity of these tests, recent applications of automated biosensor technology in food analysis are of great interest.A surface plasmon resonance (SPR)-based biosensor (Biacore) was used to design an inhibition assay to detect β-lactam antibiotics in milk. A microbial receptor protein with carboxypeptidase activity was used as detection molecule. One advantage of using this receptor protein over antibodies that are more commonly used is that only the active, intact β-lactam structure is recognized, whereas most antibodies detect both active and inactive forms. In the presence of β-lactam antibiotics the formation of a stable complex between receptor protein and antibiotic inhibits the enzymatic activity of the protein. The decrease in enzymatic activity was measured using an antibody against the degraded substrate and penicillin G in milk samples was quantitatively determined. The limit of detection of the assay for penicillin G was determined to 2.6 μg kg⁻¹ for antibiotic-free producer milk, which is below the European maximum residue limit (MRL) of 4 μg kg⁻¹. The coefficient of variation at 4 μg kg⁻¹ penicillin G, ranged between 7.3 and 16% on three different days.     

Development of a novel strategy for preconcentration of antibiotic residues in milk and their quantitation by capillary electrophoresis

A novel analytical method based on capillary zone electrophoresis coupled with diode array detection is developed and validated for the identification and simultaneous quantitation of four antibiotics in bovine raw milk. The studied antibiotics belong to different groups: β-lactams, tetracyclines, quinolones, amphenicols and sulfonamides. An experimental design including both a factorial and a central composite design allowed a reduction in the number of optimization experiments. The multiple response criterion was successfully used to optimize the separation between chloramphenicol, ciprofloxacin, ampicillin, tetracycline and sulfamethoxazol, allowing the reduction of the analysis time with excellent peak resolutions and low capillary current. Different strategies for preconcentration and extraction of the studied antibiotics were applied, in order to remove potential interferences from the sample and to increase the sensitivity. Milk samples were prepared by a clean-up/extraction procedure based on protein precipitation with trichloroacetic acid followed by liquid-liquid extraction with dichloromethane combined with solid-phase extraction, and injection into the electrophoretic system hydrodynamically. The limits of detection and quantification (below 30 and 100 μg L⁻¹, respectively) were in all cases lower than the maximum residue limits tolerated for these compounds in milk. Accuracy was evaluated by computing recoveries for the target antibiotics which were between 93.08% and 102.89%.     

Development of an optical biosensor assay for detection of β-lactam antibiotics in milk using the penicillin-binding protein 2x*

An assay was developed for the detection of residues of penicillins and cephalosporins in milk using a surface plasmon resonance (SPR) biosensor. The assay was based on the inhibition of the binding of digoxigenin-labelled ampicillin (DIG-AMPI) to a soluble penicillin-binding protein 2x derivative (PBP 2x*) of Streptococcus pneumoniae. Samples were incubated with PBP 2x* in a first step, whereby β-lactams in positive samples would bind to the PBP 2x*. Non-complexed PBP 2x* was then allowed to form a complex with DIG-AMPI in a second incubation step. The formed DIG-AMPI/PBP 2x*-complexes were detected in a SPR-based biospecific interaction assay (BIA) for digoxigenin with an antibody against digoxigenin immobilised on the sensor chip. Although binding of matrix components to the sensor chip (non-specific binding) occurred, benzylpenicillin, ampicillin, amoxicillin, cloxacillin, cephalexin and cefoperazone could be detected in defatted bulk raw milk samples at concentrations corresponding to the maximum residue limits (MRL) set by the European Union. The influence of matrix components on the performance of the assay was examined in more detail by analysing individual raw milk samples from 19 cows. Compared to bulk raw milk samples, individual samples showed a higher level and variation of matrix interferences. Non-specific binding could be reduced to a lower and more constant level by a heat-treatment step, a centrifugation step and the addition of carboxymethylated dextran to the samples. With this sample preparation, benzylpenicillin could be detected at MRL (4 μg kg⁻¹) in individual raw milk samples. Thus, the assay could be the basis for a screening test for routine use.     

Simultaneous determination of different antibiotic residues in bovine and in porcine kidneys by solid-phase fluorescence immunoassay

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibiotic residue detection in milk, was used for analysis of bovine and porcine kidney tissue. Four tetracyclines, 2 broad-spectrum cephalosporins, 3 beta-lactam antibiotics, and cephapirin were detected in one run after minimal sample preparation. This commercially available test system is designed as cartridges, each with a combination of 1-4 tests. One cartridge can be used to detect 4 analytes in the same sample, or 1 or 2 analytes in different samples. The cartridge with the combination tetracyclines-ceftiofur-penicillin-cephapirin was selected because tetracyclines, beta-lactam antibiotics as well as cephalosporins, are registered for oral or parenteral use in bovines and pigs in Europe. The test is qualitative and is recommended only for screening. Tetracycline, oxytetracycline, chlortetracycline, and doxycycline were easily detected at 300 ppb with the tetracyclines channel; ceftiofur at 1000 ppb and cefquinome at 200 ppb with the ceftiofur channel; penicillin G, ampicillin, and amoxicillin at 50 ppb with the penicillin channel; and cephapirin at 100 ppb with the cephapirin channel. These levels are equal to or lower than the corresponding maximal residue limits in kidney tissue. Cephalexin was not detected. The SPFIA test can be used as an alternative to classical inhibition tests and for post-screening inhibitor- positive kidneys, because it detects 3 specific groups of antibiotics, which enables selection of specific confirmatory methods for identification and quantification.     

Multi-class confirmatory method for analyzing trace levels of tetracyline and quinolone antibiotics in pig tissues by ultra-performance liquid chromatography coupled with tandem mass spectrometry

An ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) method was developed to screen and confirm multi-class veterinary drug residues in pig tissues including pig kidney, liver and meat. Twenty-one drugs of two different classes including seven tetracyclines and four types of quinolones (quinoline, naphthyridine, pyridopyrimidine and cinoline) were determined simultaneously in a single run. The homogenized sample tissues were extracted with EDTA-McIlvaine buffer solution and further purified using a polymer-based Oasis HLB solid-phase extraction (SPE) cartridge. An ACQUITY UPLC BEH C18 column was used to separate the analytes followed by tandem mass spectrometry using an electrospray ionization source. MS data acquisition was performed in the positive ion multiple reaction monitoring mode, selecting two ion transitions for each target compound. Recovery studies were performed at different fortification levels. The overall average recoveries from pig muscle, kidney, and liver fortified with quinolones and tetracyclines at three levels ranged from 80.2 to 117.8% based on the use of matrix-fortified calibration with the coefficients of variation ranging from 2.1 to 17.8% (n=6). The limits of quantitation (LOQs) of quinolones and tetracyclines in different tissues ranged from 0.03-4.50 microg/kg and 0.16-10.00 microg/kg, respectively. The effects of the extraction solvent, SPE cartridge, elution solvent and sample matrix on the analyte recovery as well as the effects of the mobile phase composition and column temperature on the chromatographic behavior were also studied.     

Potentiality of IMAC as Sample Pretreatment Tool in Food Analysis for Veterinary Drugs

Immobilized metal ion affinity chromatography (IMAC) was evaluated as a sample pretreatment tool for residual veterinary drugs (VDs) in food. A test of six cations for the IMAC efficiency revealed that Cu²⁺ and Fe³⁺ were suitable in retaining tetracyclines, quinolones (QLs: fluoroquinolones, acid quinolones), macrolides, β-lactams, and aminoglycosides, but ineffective for sulfonamides and hormones. These results showed the IMAC had great potential for a multiclass multiresidual VDs analysis in a simple and selective way. This methodology was applied on milk analysis for eleven QLs, involving extraction with (1:1) acetonitrile/methanol and IMAC with Fe³⁺. Averaged recoveries ranged from 68 to 93% and limits of quantitation from 2 to 26 ng mL^?1. Ten commercial milk samples were found to be free from the QLs.     

多壁碳纳米管净化/超高效液相色谱串联质谱同时测定动物组织中四环素与喹诺酮多残留

采用超高效液相色谱-串联质谱(UPLC-MS/MS)在正离子模式下通过多反应监测(MRM)方式同时测定了猪肉和鸡肉中3种四环素和2种喹诺酮类药物的残留量.残留药物经Mcllvaine-Na2EDTA缓冲溶液(pH4.0)提取后,采用多壁碳纳米管(WMCNTs)固相萃取柱净化,甲酸-乙腈(体积比5:95)洗脱,UPLC-...     

高效液相色谱法同时检测蜂蜜中的5类抗生素残留

建立了蜂蜜中氯霉素类、硝基咪唑类、喹诺酮类、磺胺类、四环素类5类共15种抗生素残留同时检测的高效液相色谱分析方法.蜂蜜经pH 2.0的磷酸水溶液溶解后直接用PCX固相萃取小柱净化富集,以乙腈-磷酸溶液(pH 2.5)作流动相,梯度洗脱,紫外检测器在274、315 nm波长下进行检测.15种抗生素在0.2 ～20.0 m...     

液相色谱-串联质谱法测定牛奶中35种四环素类、磺胺类、青霉素类、大环内酯类、氯霉素类抗生素残留

建立了液相色谱-质谱测定牛奶中35种四环素类、磺胺类、青霉素类、大环内酯类、氯霉素类等5类抗生素残留的检测方法。样品经碱性乙腈-Mcllvaine缓冲液超声提取,用Eclipse XDB-C₈色谱柱(150 mm×2.1 mm, 3.5 μm)分离,梯度洗脱,流速0.25 mL/min,进样量10 μL,采用多反应监测正离子或负离子模式,可以一次性对牛奶中的目标化合物进行定性和定量测定。在优化条件下,35种化合物的检出限均低于10.0 μg/kg,方法回收率为70.1%~109.9%,相对标准偏差(RSD)为2.89%~9.99%。结果表明该方法适用于牛奶中抗生素残留的检测。采用所建立的检测方法对市售的50种不同乳品进行了检测,其中一个样品检出氯霉素含量为0.48 μg/kg。检测结果表明,中国市场上销售的乳品氯霉素污染的风险仍然存在。本研究建立的液相色谱-质谱联用方法实现了牛奶中35种四环素类、磺胺类、青霉素类、大环内酯类、氯霉素类等5类抗生素残留的测定。该方法简单、快捷、经济,可实现多种抗生素残留的快速测定。     

A preliminary study on electrochemical biosensors for the determination of total cholinesterase inhibitors in strawberries

Organophosphorous (OP) insecticides reveal acute toxicity because of their capability to affect the nervous system through the inhibition of acetyl cholinesterase function in regulating the neurotransmitter acetylcholine. The present work shows an example of an easy to be handled inhibition electrochemical biosensor, based on thick film technology for low cost production of screen printed electrodes. Anti-cholinesterase activity in specific fruits was determined measuring the inhibition of acetyl cholinesterase enzyme owing to the presence of OP pesticides. Paraoxon was taken as reference pesticide for each measurement. The main fluidic critical parameters were investigated under flow injection analysis, through the comparison of different enzymatic immobilisation methods. Analytical features were evaluated as a function of experimental parameters. The analytical detection was developed in a three step procedure and the pesticides content was measured in strawberries samples taken from the local market. The separation between the acetyl cholinesterase inhibition and the electrochemical detection with the choline oxidase biosensor decreases the total analysis time, allowing improvements in reproducibility and stability of the system. A comparison with reference materials and standard analytical procedures for pesticides will be required in the future for evaluating the reliability of the method.     

Multiresidue Screening of Veterinary Drugs in Meat,Milk, Egg,and Fish Using Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry

New approaches to veterinary drug screening based on liquid chromatography-mass spectrometry (LC-MS/MS) and time-of-flight mass spectrometry (ToF/MS) are rapid and have high selectivity and sensitivity. In this study, we developed a multiresidue method for screening over 100 veterinary drug residues using ion trap (IT)-ToF/MS. The screened compounds comprised major drug classes used in veterinary practice, representing the following: amphenicols, anthelmintics, benzimidazoles, β-lactams, coccidiostats, ionophores, macrolides, non-steroidal anti-inflammatory drugs, quinolones, sulfonamides, tetracyclines, and tranquilizers. The method was developed based on chromatographic retention time, specific accurate mass, isotope distribution, and fragment data. Each compound was validated at three levels, and the mass accuracy, accuracy, and repeatability were calculated. All parameters showed acceptable values and conformed to the Commission Decision 2002/657/EC criteria. This screening method can simultaneously analyze over 100 veterinary drugs in meat, milk, eggs, and fish in a single analytical run.     

Development of a quantitative multiclass/multiresidue method for 21 veterinary drugs in shrimp

A multiclass/multiresidue method has been developed and validated for the determination of 21 veterinary drug residues in shrimp, including sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine, and sulfaquinoxaline); tetracyclines (oxytetracycline, tetracycline, and chlortetracycline); (fluoro)quinolones (norfloxacin, ciprofloxacin, enrofloxacin, sarafloxacin, difloxacin, flumequine, oxolinic acid, and nalidixic acid); and cationic dyes (malachite green, gentian violet, leucomalachite green, and leucogentian violet), using HPLC/MS/MS. All drugs were quantifiable over a no less than 10-fold range with matrix-matched standards for linear external calibration, except for oxytetracycline, tetracycline, norfloxacin, and ciprofloxacin, for which norfloxacin-d5 was used as an internal standard. Two grams of preground shrimp sample was extracted twice with extractant at two different pH values. The combined supernatant was further diluted with an aqueous internal standard solution, and 50 microL extract was injected into the HPLC instrument. An online SPE system was set up for automated sample cleanup. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was operated in the multiple-reaction-monitoring mode to acquire data. The method has been validated at three levels within the designated linear ranges for each drug, with accuracies between 77 and 115%, and most CV values below 15%.     

Biosensor immunoassay for flumequine in broiler serum and muscle

Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity with other (fluoro)quinolones). This inhibition assay was based on a rabbit polyclonal anti-Flu serum and a CM5 biosensor chip coated with Flu which could be detected in the range of 15-800 ng mL(-1). For the detection of Flu in muscle, an easy extraction procedure in buffer was selected and the measuring range was from 24 to 4000 ng g(-1). Average recoveries of 66 till 75% were found with muscle samples spiked at 0.5, 1 and 2 times the maximum residue limit (MRL in muscle = 400 ng g(-1)) and the decision limit (CCalpha) and the detection capability (CCbeta) were determined as 500 and 600 ng g(-1), respectively. Incurred muscle samples were analysed by the BIA and by LC-MS/MS and a good correlation was found (R2 = 0.998). Serum and muscle samples from with Flu treated broilers were analysed and the concentrations found in serum were always higher than those found in muscle (average serum/muscle ratio was 3.5) and this proved the applicability of the BIA in serum as predictor of the Flu concentration in muscle.     

超高效液相色谱-串联质谱法同时测定鸡肝中残留的四环素类、磺胺类和喹诺酮类药物

采用超高效液相色谱-串联质谱(UPLC-MS/MS)在正离子模式下通过多反应监测(MRM)方式同时测定了鸡肝脏组织中3种四环素类药物、10种磺胺类药物以及8种喹诺酮类药物的残留。试样由McIlvaine缓冲液-乙腈(体积比为1:4)、乙腈提取,合并上清液并用氮气吹干,用0.05 mol/L磷酸三乙胺缓冲液-乙腈(体积比为85:15)溶解残余物,经正己烷脱脂后,采用UPLC-MS/MS进行定性、定量分析。该方法对测定的21种药物的检出限均为2 μg/kg,定量限均为5 μg/kg。在添加水平分别为5,10和50 μg/kg时,21种药物的加标回收率为66.8%～128.5%,日内测定的相对标准偏差(RSD)为0.8%～20.2%,日间测定的RSD为2.2%～15.3%。该方法可作为动物源性食品中这3类药物残留检测的确证方法。