A microbiological six-plate method for the identification of certain antibiotic groups in incurred kidney and muscle samples

A microbiological method was developed for group level identification of antibiotics in incurred kidney and muscle samples from cattle and pigs. The method was composed of six test bacterium-plate growth medium combinations and the result was recorded as a profile of growth inhibition zones. The sample profiles were compared to two sets of references: one constructed with standard antibiotic solution profiles, and the other with these combined with profiles of microbiologically and chemically identified residues from incurred samples. The algorithm employed in profile comparison located the minimal sum of absolute pairwise differences over the tests, with the addition of a number of experimentally observed intra-test criteria. Chemical identification and quantitation of incurred residues was based on liquid chromatography. The method identified penicillin G as a penicillinase sensitive penicillin, enrofloxacin and ciprofloxacin belonging to fluoroquinolone group, and oxytetracycline belonging to tetracycline group. Each of these residues was microbiologically identified below the Maximum Residue Limit (MRL) for kidney tissue. Combining sample profiles with the standard reference data set did not enhance the resolution. Microbiological and chemical identification test results were in good agreement. The results of this study show that a microbiological identification method is a useful tool in preliminary characterisation of antibiotic residues in animal tissues.     

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。     

Recent applications of surface plasmon resonance biosensors for analyzing residues and contaminants in food

Abstract  Optical biosensors based on surface plasmon resonance are increasingly used to detect and (semi)quantify residues and contaminants in food. This review provides an overview of the methods published in this field since the year 2003. Such biosensors have mostly been applied to veterinary drug residues, like aminoglycosides, beta-agonists, cephalexin, chloramphenicol, fluoroquinolones, levamisole, nicarbazin, nitroimidazoles, penicillins, ractopamine, sulfonamides, tetracyclines, and tylosin in milk, egg, honey, prawn, muscle, liver and kidney. Only a few methods have been published on pesticide residues, mycotoxins, phycotoxins, polychlorinated biphenyls and surfactants. These procedures are described with regard to biological recognition element, type of sensor chip, immobilisation procedure, sample extraction and clean-up, crossreactivity, nonspecific binding, matrix interference, chip regeneration, assay formats, calibration, validation and instrumentation. Graphical Abstract        

Recent applications of surface plasmon resonance biosensors for analyzing residues and contaminants in food

Abstract  

Optical biosensors based on surface plasmon resonance are increasingly used to detect and (semi)quantify residues and contaminants in food. This review provides an overview of the methods published in this field since the year 2003. Such biosensors have mostly been applied to veterinary drug residues, like aminoglycosides, beta-agonists, cephalexin, chloramphenicol, fluoroquinolones, levamisole, nicarbazin, nitroimidazoles, penicillins, ractopamine, sulfonamides, tetracyclines, and tylosin in milk, egg, honey, prawn, muscle, liver and kidney. Only a few methods have been published on pesticide residues, mycotoxins, phycotoxins, polychlorinated biphenyls and surfactants. These procedures are described with regard to biological recognition element, type of sensor chip, immobilisation procedure, sample extraction and clean-up, crossreactivity, nonspecific binding, matrix interference, chip regeneration, assay formats, calibration, validation and instrumentation.     

Detection of antibiotics in muscle tissue with microbiological inhibition tests: effects of the matrix.

The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended for muscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated with aqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solution without tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones of the beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels the differences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappeared completely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected in spiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These results indicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intact meat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initially intended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screening purposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitive for beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolones enrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.     

Bicarbonate as an interference in B. stearothermophilus assays.

The screening analysis of antibiotic residues in meat products is readily accomplished by a suite of bacterial inhibition assays. Inhibition of B. stearothermophilus is a standard assay noted for its sensitivity to penicillins. In New Zealand, a test kidney from slaughtered animals is accompanied by a urine sample. Generally, penicillins and tetracyclines are both richer in the urine sample, which is applied directly to wells cut in bacterially seeded agar plates. An interference in the assay for B. stearothermophilus has been traced to excessive amounts of bicarbonate in the urine. It is the bicarbonate, not the pH nor the ionic strength that causes the problem. Removal of the bicarbonate was by mild acetic acid treatment to pH 5.5 and vortex mixing to de-gas carbon dioxide. This resulted in complete eradication of the interference without affecting the ability to detect penicillin. The pH change was readily monitored by the indicator bromcresol purple and no complex equipment was needed.     

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.     

Analytical strategies to determine antibiotic residues in fish

The accelerated growth of aquaculture has resulted in a series of harmful effects to human health. The widespread and unrestricted use of antibiotics in this industry, to prevent bacterial infections, leads to remaining amounts in the aquatic environment. This has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments, in the increase in antibiotic resistance in fish pathogens as well as in the transfer of these resistance determinants to human pathogens. Moreover, the use of large amounts of antibiotics may lead to the presence of residual antibiotics in fish tissue and fish products. Fluoroquinolones, tetracyclines, penicillins, sulphonamides and other antibiotics, exhibiting activity against both Gram-positive and Gram-negative bacteria, are widely used for the treatment and prevention of diseases in fish. An extended and comprehensive review on the recent analytical methodologies concerning antibiotic residues in fish reported in the literature is provided in the present article. Emphasis is given on sample preparation regarding isolation and purification, chromatographic conditions and method validation according to legislation. Results of published assays are comparatively presented and criticised.     

Experiences with an identification and quantification program for inhibitor-positive milk samples

Beta-lactam antibiotics (penicillins, cephalosporins) are still the most commonly used antibiotics for dairy cows in Germany. In routine milk testing, according to the German milk quality regulation, a positive result obtained for bulk tank milk by microbiological inhibitor tests needs no further confirmation, but results in reduced milk payment of 0.05 euros kg(-1) for one month. In some cases, however, further identification of the causative agent can be of interest, either if antimicrobial drugs have not knowingly been used recently, or if improper use of such drugs is denied. As a service for milk producers, our laboratory offers further analyses of violative milk samples, aiming at the identification and quantification of the inhibitor(s). In this program, a panel of microbiological inhibitor tests, receptor tests, and enzyme immunoassays (EIA) is used in a step-by-step analysis, which primarily focusses on beta-lactams, but also includes other compounds such as sulfonamides or tetracyclines, respectively. Here we report results for violative milk samples (n=63) analysed between 2003 and 2005. In most cases (95%), beta-lactam antibiotics could be identified, although not always at levels exceeding the respective MRL values. Penicillin G (mostly together with benzylpenicilloyl metabolites) could be identified in 74.6% of all samples. Other compounds identified were, in decreasing order, ceftiofur (11%), ampicillin/amoxicillin (6.3%), isoxazolyl penicillins (3.2%), and sulfonamides (1.6%). The results indicate that penicillin G is still the predominant antibiotic responsible for violative bulk tank milk samples as detected during regulatory control.     

High-performance liquid chromatographic determination of penicillin G, penicillin V and cloxacillin in beef and pork tissues.

The objective was to develop confirmatory high-performance liquid chromatographic methods for penicillin residues in animal tissues with detection limits of less than or equal to 10 ng/g. A previously described procedure was modified by using a larger sample size and isocratic analysis. Tissues (15 g) were blended with 45 ml of water and 20 ml of homogenate were mixed with 40 ml acetonitrile and filtered. The filtrate (30 ml) was mixed with 10 ml of 0.2 M H3PO4 and extracted with methylene chloride. The combined methylene chloride layers were mixed with acetonitrile and hexane, washed with two 4-ml portions of water and then extracted with four 1-ml portions of 0.01 M phosphate buffer (pH 7). The combined buffer extracts were concentrated to 1 ml under reduced pressure. Analysis was isocratic during 0.01 M phosphate buffer (pH 7)-acetonitrile with proportions 85:15 (penicillin G), 82:18 (penicillin V) or 78:22 (cloxacillin). A polystyrene-divinylbenzene copolymer column, 150 x 4.6 mm I.D. (Polymer Labs. PLRP-S), was used with a flow-rate of 1 ml/min and detection at 210 nm. The presence of penicillins was confirmed by treating a duplicate sample with penicillinase. Recoveries were greater than 90% in most instances. Detection limits were 5 ng/g in muscle and higher in liver and kidney. The procedure is a simple and sensitive method for confirming the presence of penicillins in animal tissues.     

Determination of tetracyclines in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and clean-up

A novel, sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry) method with on-line extraction and clean-up for the screening and confirmation of residues of tetracyclines in kidney has been developed. After liquid extraction of homogenised kidney with McIlvain buffer, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization followed by multiple reaction monitoring. For each tetracycline the collisional decomposition of the protonated molecule to a unique, abundant fragment ion was monitored. The method has been validated for tetracycline, oxytetracycline, chlortetracycline and doxycycline. Calibration curves resulting from spiked blank kidney samples at the 100-1200 microgram/kg level showed good linear correlation. At the level of 600 microgram/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 7%. The limits of detection (LODs) for tetracycline, oxytetracycline, chlortetracycline and doxycyline were 18, 23, 24 and 21 microgram/kg, respectively. The limits of quantification (LOQs) for tetracycline, oxytetracycline, chlortetracycline and doxycyline were 36, 46, 47 and 42 microgram/kg, respectively. The recoveries ranged from 71 to 91%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of tetracyclines in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

Solid-phase extraction for multiresidue determination of sulfonamides, tetracyclines, and pyrimethamine in Bovine's milk

This paper describes a systematic approach to the development of a solid-phase extraction method for simultaneous extraction of 10 antibiotic residues in bovine milk, belonging to groups of sulfonamides, tetracyclines, and pyrimethamine. The sample preparation steps include acidic deproteinization of milk proteins followed by sample enrichment and cleanup using a polymer-based Oasis HLB solid-phase extraction cartridge. The analyses were carried out by using a method based on liquid chromatography-electrospray ionisation-mass spectrometry with positive ion mode. The parameters affecting the extraction efficiency such as sample loading pH, SPE wash solvent composition, and eluting solution pH were carefully investigated and optimized. The developed solid-phase extraction procedure coupled to multiresidue liquid chromatography-electrospray ionization-mass spectrometry method was applied for the analysis of 10 antibiotic residues in milk samples, and it proved to be simple, sensitive, and selective providing a recovery ranging from 70 to 106%.     

Simultaneous multiresidue determination of tetracyclines and fluoroquinolones in catfish muscle using high performance liquid chromatography with fluorescence detection

Efficient methods are needed for analysis of veterinary drug residues in food. A number of methods are available for single analytes. Multiresidue methods are now increasingly available. It is still rare, however, to find methods not involving mass spectrometry which allow for analysis of more than one class of drug residue. An efficient multiresidue method for the simultaneous determination of fluoroquinolones (FQs) and tetracyclines (TCs) in catfish muscle has now been developed. This method involves an extraction of the analytes with a mixture of acetonitrile and citrate buffer containing magnesium chloride. After centrifugation and evaporation of the supernatants, the residues are determined using high performance liquid chromatography with fluorescence detection. With this method, five fluoroquinolones and three tetracyclines were determined in fortified catfish muscle at levels of 20, 50, and 100 ng g(-1). Average recoveries for ciprofloxacin (CIP), sarafloxacin (SAR), danofloxacin (DANO), enrofloxacin (ENRO), difloxacin (DIF), oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were in the range of 60-92% with good relative standard deviations. The limits of quantitation ranged from 0.15 to 1.5 ng g(-1). Utilization of the method to successfully analyze catfish muscle samples incurred with enrofloxacin and with oxytetracycline is described.     

Highly sensitive detection of five typical fluoroquinolones in low‐fat milk by field‐enhanced sample injection‐based CE in bubble cell capillary

Fluoroquinolones are a group of synthetic antibiotics with a broad activity spectrum against mycoplasma, Gram‐positive, and Gram‐negative bacteria. Due to the extensive use of fluoroquinolones in farming and veterinary science, there is a constant need in the analytical methods able to efficiently monitor their residues in food products of animal origin, regulated by Commission Regulation (European Union) no. 37/2010. Herein, field‐enhanced sample injection for sample stacking prior the CZE separation was developed inside a bubble cell capillary for highly sensitive detection of five typical fluoroquinolones in bovine milk. Ethylenediamine was proposed as the main component of BGE for the antibiotics separation. The effect of BGE composition, injection parameters, and water plug length on the field‐enhanced sample injection‐based CE with UV detection was investigated. Under the optimized conditions, described field‐enhanced sample injection‐based CE‐UV analysis of fluoroquinolones provides LODs varying from 0.4 to 1.3 ng/mL. These LOD values are much lower (from 460 to 1500 times) than those obtained by a conventional CE in a standard capillary without bubble cell. The developed method was finally applied for the analysis of fluoroquinolones in low‐fat milk from a Swiss supermarket. Sample recovery values from 93.6 to 106.0% for different fluoroquinolones, and LODs from 0.7 to 2.5 μg/kg, were achieved. Moreover, the proposed ethylenediamine‐based BGE as volatile and compatible with MS system, enabled the coupling of the field‐enhanced sample injection‐based CE with a recently introduced electrostatic spray ionization MS via an iontophoretic fraction collection interface for qualitative fluoroquinolones identification.     

固相萃取-超高压液相色谱-串联质谱同时分析环境水样中四环素类和喹诺酮类抗生素

应用固相萃取及超高压液相色谱-质谱联用技术,建立了环境水样中4种四环素类和6种喹诺酮类抗生素的同时分析方法。样品经HLB固相萃取柱富集、净化后用甲醇洗脱,以超高压液相色谱-串联质谱仪多反应监测(MRM)离子模式定性、定量分析。以河水和海水为基质,卡巴氧为替代物进行回收率评价,4种四环素类抗生素在加标质量浓度分别为20.0 ng/L和100.0 ng/L时的回收率为94.0%～117.0%,相对标准偏差为2.0%～9.7%(n=4),方法的检出限均为20.0 ng/L;6种喹诺酮类抗生素在加标质量浓度分别为5.0 ng/L和20.0 ng/L时的回收率为63.6%～93.9%,相对标准偏差为1.6%～8.1%(n=4),方法的检出限为0.4 ng/L。结果表明,所建立的方法可成功地应用于近岸海域表层环境水样中目标抗生素残留的分析。     

Hydrolysis of furan and 5-nitrofuran penicillins by penicillinase Bacillus licheniformis

The enzymatic hydrolysis of various furan and 5-nitrofuran penicillins by penicillinaseBacillus licheniformis 749/c was investigated. The effect of various side-chain structural groupings on the rate of inactivation of these compounds was studied. The introduction of bulky substituents in the ortho or positions to the amide group and lengthening the side chain or increasing its degree of unsaturation sharply decrease the catalytic action of penicillinase and, consequently, the rate of cleavage of the-lactam ring of the antibiotic. The majority of the furan and 5-nitrofuran penicillins surpass benzylpenicillin in resistance to the enzyme but are inferior to methicillin and oxacillin.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 3, pp. 295–298, March, 1971     

Validation study for the determination of tetracycline residues in animal tissues.

An interlaboratory study of the liquid chromatographic (LC) determination of tetracyclines--oxytetracycline (OTC), tetracycline (TTC), and chlortetracycline (CTC)--in animal tissues was conducted. Isolation was performed with oxalic buffer followed by dechelation and deproteination with oxalic acid-acetonitrile. The extract was cleaned with a styrene-divinylbenzene cartridge. LC analysis was performed with a PLRP-S column and 0.01 M oxalic acid-acetonitrile (75 + 25, v/v) as mobile phase. Participants analyzed 2 control and 10 fortified porcine muscle and kidney samples. Additionally, porcine muscle samples containing incurred residues of tetracyclines were analyzed. Mean recoveries of fortified residues from porcine tissue ranged from 76.00 to 86.89%. Repeatabilities varied from 2.05% for OTC to 3.61% for TTC for muscle samples and from 6.75% for CTC to 8.74% for OTC for kidney samples. Reproducibilities ranged from 2.05 to 4.30% for muscle samples and from 15.77 to 18.81% for kidney samples.     

Introduction of the HPLC method for the determination of quinolone residues in various muscle tissues

For use in veterinary sanitary control of foodstuffs and raw materials of animal origin in Slovenia, we developed a routine and confirmation analytical method for determining the residues of enrofloxacin, ciprofloxacin and flumequine in the muscle tissue of cattle, pigs and poultry. For the muscle tissue of freshwater fish, the determination of the flumequine residues was introduced. The results obtained through simultaneous determination of the residues of enrofloxacin, ciprofloxacin and flumequine showed that the values for the examined antibiotics were up to 600 times lower than the prescribed maximum residue levels (MRL). Another advantage of this method is that it covers a wide range of different fluoroquinolones.     

Fluoroquinolones and sulfonamides: features of their determination in water. A review

Sulfonamides and fluoroquinolones are two group of antibiotics widely used in the treatment of bacterial infections. Monitoring these residues in live animals and animal products is commonly legislated; however, the environmental occurrence and fate is still sporadically assessed. The development of adequate analytical methods is a key issue to provide accurate data on concentrations of antimicrobial compounds and their residues in different organic matters. This review presents an overview of proposed methods published between 2008 and 2013 for analysis of fluoroquinolones and sulfonamides with special emphasis on sample preparation and detection systems employed. The coupling of high-performance liquid chromatography and mass spectrometry has been the most widely used method to determination of sulfonamides and fluoroquinolones, concomitantly to other antibiotics residues, due to high sensitivity and selectivity. The main drawbacks of this technique are the high cost and the equipment complexity. The coupling high-performance liquid chromatography and fluorescence detection has also been used to fluoroquinolones determination. Their fluorescent properties allowed the development of several methods with limits of detection in ng L^?1 range. Sample preparation is an important tool to reach lower detection limits and the online solid-phase extraction has a broader use. The hydrophilic–lipophilic balance polymeric are the mostly applied sorbents for sulfonamides and fluoroquinolones preconcentration. These sorbents have allowed reaching better recoveries and sensitivity improvement. Physico-chemical properties of these antibiotic groups in addition to trends on papers occurrence and frequency of analysis in different types of water (surface water, groundwater, drinking water and wastewater) are discussed.     

Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry

Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ß-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC–MS/MS. The method was validated at the current MRLs, which range from 25–300 µg kg^?1 in muscle and kidney to 4–30 µg kg^?1 in milk as well as at the target value of 100 µg kg^?1 chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94–113% (muscle), 83–111% (kidney) and 87–103% (milk) to 88–116% (animal feed). Intra-day precision (relative standard deviation (RSD)_r) ranged from 5–13% (muscle, n?=?18), 4–17% (kidney, n?=?7) and 5–18% (milk, n?=?7) to 11–32% (animal feed, n?=?18). Inter-day precision (RSD_RL, n?=?18) ranged from 6–23% (muscle) to 11–36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed.