Collaborative trial of the Penzym assay: a rapid method for the detection of β-lactam antibiotics in milk

The Penzym assay is a rapid enzymic method for the detection of β-lactam antibiotics in milk in 20 minutes. Two collaborative trials of the Penzym assay were conducted between 11 laboratories using raw whole milk samples containing levels of 0.005 iu/ml, 0.015 iu/ml and 0.025 iu/ml penicillin G and were tested at 0.01 iu/ml and 0.02 iu/ml pass/fail levels. Trial 1 indicated a degree of operator dependency with a tendency to give false passes. Trial 2 confirmed the operator dependency, and both false passes and false failures were obtained. These results make the test unacceptable as a rejection test for detecting β-lactam antibiotics in dairy laboratories. However, given the speed of testing and level of accuracy (92%), the assay may still be valuable as a screening test .     

乳品中β-内酰胺类抗生素快速检测试纸条研制

通过β-内酰胺类抗生素-载体蛋白偶联物的合成,单克隆抗体的制备与纯化,胶体金标记物的制备,β-内酰胺类抗生素单克隆抗体-胶体金标记物的制备并冻干到微孔试剂,样品吸收垫和反应膜的制备等研究过程,研制出乳制品中β-内酰胺类抗生素的快速检测试纸条。检测限分别为:青霉素G2μg/L、氨苄青霉素4μg/L、阿莫西林5μg/L、苯唑青霉素/邻青霉素/双青霉素均为6μg/L、头孢洛宁10μg/L、萘夫西林20μg/L、头孢喹肟20μg/L、头孢曲松25μg/L、头孢哌酮50μg/L、头孢噻呋90μg/L;本试纸条特异性好、假阳性率不高于3%、假阴性率为0,检测时间不超过15min,检测限量达到了我国和欧盟的要求,适用于乳品流通环节乳品中β-内酰胺类抗生素残留的检测。     

An evaluation of the Penzym® assay: a rapid method for the detection of β-lactam antibiotics in milk

The Penzym® method, an enzymatic method which allows the detection of β-lactam antibiotics within 20 min, was evaluated using raw whole milk to establish its repeatability and reproducibility for the determination of penicillin G. Three screening thresholds, 0.005, 0.010 and 0.020 iu/ml, were tested by two operators from different laboratories.
Chi-squared(χ²) analysis of the results obtained by each operator for 10 replicate tests of eight penicillin G standards for each screening threshold showed the repeatability of the assay to be satisfactory. Similarly, χ² analysis of the results obtained by each operator for duplicate tests on the same standards assayed on five consecutive days showed that reproducibility for the assay was also satisfactory. It was shown that by altering the volume of milk assayed the sensitivity of the test can be adjusted. The sensitivity of the method to other β-lactam antibiotics used in bovine therapy requires investigation, and a more extensive examination of the repeatability and reproducibility of the assay in several laboratories is needed before it can be recommended for routine use.     

荧光免疫层析法快速测定牛奶中头孢氨苄残留量

采用荧光免疫层析法结合现场检测仪建立牛奶中头孢氨苄残留的快速定量检测方法。方法 利用反向微乳技术合成稀土铕荧光纳米颗粒,与头孢氨苄单克隆抗体结合制备荧光检测探针,以头孢氨苄卵清蛋白全抗原和羊抗小鼠抗体分别作为检测线和质控线制备免疫层析试纸条,结合现场检测仪建立牛奶中头孢氨苄残留的快速定量检测方法。结果 试验结果表明,该方法对头孢氨苄的检测限为0.16 ng/ml,半数抑制浓度(IC₅₀)为0.6 ng/ml,线性范围为0.16～5 ng/ml,标准添加回收率为100%～115%之间,与头孢菌素类及青霉素等其他12种抗生素的交叉反应率均＜0.01%,与ELISA方法比较,测定结果相关性良好。结论 本试验所建立的荧光免疫层析法快速检测牛奶中头孢氨苄残留,具有简便、快速、灵敏、直观的优点,可用于抗生素残留的筛查,极具推广和应用价值。     

牛奶中β-内酰胺类和三聚氰胺检测方法的建立

建立一种快速、简便,同时针对牛奶中β-内酰胺类抗生素和三聚氰胺残留的检测方法。应用竞争抑制免疫层析原理,研制β-内酰胺类和三聚氰胺胶体金试纸条。该试纸条检测限分别为青霉素G2μg/L、氨苄青霉素3μg/L、阿莫西林3μg/L、苯唑青霉素6μg/L、邻氯青霉素6μg/L、双氯青霉素6μg/L、萘夫西林20μg/L、头孢喹肟20μg/L、头孢哌酮40μg/L、头孢曲松50μg/L、头孢噻呋90μg/L、头孢洛宁10μg/L、三聚氰胺50μg/L,假阳性率低于5%、假阴性率为0,检测时间不超过15min。该方法操作简便、灵敏度高、特异性强,适用于乳品流通环节中β-内酰胺类抗生素和三聚氰胺残留的快速检测。     

β-lactam antibiotics residues analysis in bovine milk by LC-ESI-MS/MS: a simple and fast liquid-liquid extraction method

This study presents the development and validation of a simple method for the detection and quantification of six β-lactam antibiotics residues (ceftiofur, penicillin G, penicillin V, oxacillin, cloxacillin and dicloxacillin) in bovine milk using a fast liquid-liquid extraction (LLE) for sample preparation, followed by liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS). LLE consisted of the addition of acetonitrile to the sample, followed by addition of sodium chloride, centrifugation and direct injection of an aliquot into the LC-MS/MS system. Separation was performed in a C(18) column, using acetonitrile and water, both with 0.1% of formic acid, as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Limits of detection ranged from 0.4 (penicillin G and penicillin V) to 10.0 ng ml(-1) (ceftiofur), and linearity was achieved. The decision limit (CCα), detection capability (CCβ), accuracy, inter- and intra-day repeatability of the method are reported.     

酸度计法快速检测牛奶中残留的β-内酰胺酶

为了快速、准确地检测牛奶制品中残留的β- 内酰胺酶的含量,利用β- 内酰胺酶分解青霉素产酸使牛奶pH 值下降的原理,对人工添加了β- 内酰胺酶的牛奶制品与青霉素反应后pH 值的下降规律进行研究。得到牛奶中残留的β- 内酰胺酶与青霉素反应的较适宜条件,从而获得快速检测牛奶制品中残留的β- 内酰胺酶的方法。结果确定牛奶制品中残留的β- 内酰胺酶与青霉素反应的适宜条件为温度33℃、底物质量浓度10mg/mL,检测时间仅为60min。此方法对液态纯牛奶中β- 内酰胺酶的最低检出限为8.92U/mL。     

Determination of β-lactam residues in foodstuffs of animal origin using supported liquid membrane extraction and liquid chromatography–mass spectrometry

β-Lactams have been used extensively both in human and veterinary medicine practices. In veterinary medicine, they are used mainly as growth promoters as well as chemotherapeutic and prophylactic agents. The occurrence of β-lactam residues in foodstuffs is a serious health hazard. In this work, a sample purification and enrichment technique involving the use of supported liquid membrane have been developed for β-lactams in milk, kidney and liver tissues. The liquid membrane made of n-undecane:di-n-hexyl ether (1:1) was used to enrich a mixture of four β-lactams namely, ampicillin, cloxacillin, penicillin V and penicillin G. Separation and detection of the enriched β-lactam extracts were performed using a high performance liquid chromatography coupled to a mass spectrometer operating under positive ion electrospray mode (LC–PI–ESI–MS). The detection limits (DLs) obtained were found to be 1 ng/kg for penicillin G and penicillin V in kidney and liver tissues and 0.7 μg/L in milk. For ampicillin, the DLs were found to be 1.4 μg/kg in kidney and liver tissues and 1.7 μg/L in milk. Limits of quantification based on a minimal value of the signal-to-noise ratio of 10, were estimated to be 1.1 and 0.01 for penicillin G in kidney and liver tissues, respectively, while for penicillin V it was 0.4 and 0.01 for kidney and liver tissues, respectively. The DL values obtained using this approach were found to be 2–3 order of magnitudes lower than the stipulated tolerance levels as set by the EU and FDA.     

High throughput detection of antibiotic residues in milk by time-resolved fluorescence immunochromatography based on QR code

ABSTRACT

Herein, we have successfully established a novel, rapid, and simple lateral-flow immunoassay based on time-resolved fluorescence and biotin-streptavidin to detect the residues of various antibiotics in milk. The fluorescence signal and sensitivity of immunochromatography were enhanced through biotinylated antibody coupled with streptavidin europium microspheres. Moreover, due to the use of a QR Code and fluorescent reader, quantitative detection and real-time data uploading can be achieved. Under the optimal conditions, the various antibiotic residues were detected in the milk samples. The results showed that the limits of detection of tylosin, lincomycin and doxycycline were 0.10, 0.06, and 0.27 ng/mL, respectively. The recoveries of the spiked milk samples were 88.9%~127%, with coefficients of variation less than 11%, and the test strip can be stored at room temperature for 12 months. This study shows that the proposed time-resolved fluorescence immunoassay is sensitive, rapid and reliable, and has the potential to be used for detection of veterinary antibiotic residues in food safety fields.     

Performance of a rapid ELISA for penicillin G in milk

A rapid enzyme-linked immunosorbent assay (ELISA) for the detection of penicillin G in milk at concentrations of 6 ng/ml (0.01 IU/ml) was used to screen 1651 off-farm milk samples previously reported as containing no detectable levels of antimicrobial substances. Using a single-well per test format and a percent inhibition cut-off for the determination of positive/negative endpoint, 3.1% positives were obtained. Comparison with intra-assay standards lowered the numbers of positives to 0.42%. Analysis of 170 milk samples positive for antimicrobials confirmed 92.4% as containing penicillin G using the prescribed cut-off level and 87.6% when compared to standards. The ELISA, now available commercially in kit form, analyses between 1 and 48 samples in 15 minutes. It will detect 0.01 IU/ml to a level of confidence of 99% and 0.005 IU/ml with between 75 and 95% confidence.     

An Enhanced Direct Competitive Immunoassay for the Detection of Kanamycin and Tobramycin in Milk Using Multienzyme-Particle Amplification

Kanamycin (Kan) and tobramycin (Tob) are widely found in many foods of animal origin, including milk. More rapid, simple, and sensitive methods are urgently needed to monitor antibiotic residues in milk. An enhanced direct competitive enzyme-linked immunosorbent assay (dcELISA) based on gold nanoparticles (AuNPs)/horse radish peroxidase-Kan (HRP-Kan) was developed. A monoclonal antibody (Mab) against Kan was developed by classic hybridoma technology. The Mab had higher cross-reactivity with Tob (99.07%) and no cross-reactivity with other related antibiotics (<?0.5%). A novel multienzyme probe was synthesized based on AuNPs modified using HRP-Kan. The Mab against Kan, fixed by a goat anti-mouse antibody, was competitively bound by AuNPs/HRP-Kan and Kan in samples. After optimization, the limit of detection of the enhanced dcELISA was 0.022 ng/mL, representing a fivefold improvement when compared to that of conventional dcELISA (0.13 ng/mL). The recoveries of Kan and Tob in milk samples varied from 81.0 to 121.0% and 86.4 to 123.9%, respectively. Kan or Tob was found to be present at concentrations of 0.352–0.548 ng/mL in five milk samples from local markets. The results by the enhanced ELISA and UPLC-MS/MS had good correlation. It was suggested that the enhanced dcELISA, based on AuNPs/HRP-Kan, has higher sensitivity and reliable reproducibility, and thus, this could be used to detect trace contaminants.     

DETECTION OF ANTIMICROBIAL DRUG RESIDUES IN KENYAN MILK

The improved Dutch tube diffusion test was used to study the occurrence of inhibitory substances in raw bulk milk samples within the Nakuru District in Kenya. Initially the detection limits of the method were verified using milk standards spiked with selected antibiotics. Addition of penicillinase to inhibitor-positive samples was used for preliminary identification of penicillin G-type antibiotics and residue levels were estimated against a standard curve constructed by means of a B. stearothermophilus disc assay. The two-tube test was used to screen 1109 field samples of which 229 (21%) were suspect positive. The identification procedure confirmed 165 samples (14.9%) to contain penicillin G-type residues of which 118 contained levels exceeding the established EU MRL for penicillin G (4 μg/kg). This study indicates that antibiotic residues are prevalent in milk within the Nakuru district of Kenya. It suggests that the improved tube diffusion test in combination with a multiplate system could be useful for qualitative and quantitative identification of antimicrobial drug residues in milk.     

Effect of heat treatments on stability of β-lactams in milk

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on β-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of β-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 β-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of β-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15 s), the degradation of β-lactam in milk did not exceed 1% for the 10 antibiotics tested.     

Detection limits of beta-lactam antibiotics in ewe milk by penzym enzymatic test.

The Penzym is an enzymatic test widely used for the detection of beta-lactam antibiotic residuals in milk. It is a specific method with good sensitivity to this group of antibiotics and enables results to be obtained within a short time. In the present work, the detection limits of 10 beta-lactam antibiotics were determined in ewe milk, given the lack of previous studies of the Penzym test in ovine milk. For each antibiotic, eight concentrations were tested on 20 ewe milk samples proceeding from individual ewes (160 analyses per drug). The limits of the Penzym test were determined by means of logistic regression models, as follows: 5 microg/kg amoxicillin, 4 microg/kg ampicillin, 33 microg/kg cloxacillin, 3 microg/kg penicillin "G," 43 microg/kg cephadroxil. 10 microg/kg cephalosporin "C," 16 microg/kg cephalexin, 900 microg/kg cephoperazone, 120 microg/kg Ceftiofur, and 77 microg/kg cephuroxime. The percentages of positive results for those antibiotics at the maximum residue limit (MRL) concentration established by the European Union (EU) were: 100% (penicillin "G"), 93.3% (ampicillin), 93.3% (cloxacillin), 56.7% (Ceftiofur), and 56.7% (amoxicillin).     

Potential for milk containing penicillin G or amoxicillin to cause residues in calves

The potential for antibiotic residues in calves from consuming milk containing penicillin G or amoxicillin was investigated. Six calves were fed milk replacer, 6% body weight twice daily, containing 0.293, 2.92, or 5.85 microg of penicillin/ml (ppm) G or 0.25, 1.0, or 2.0 microg of amoxicillin/ml for three consecutive feedings. Urine and blood samples were collected after each feeding. Serum and urine samples were tested with a microbial receptor assay and a microbial growth inhibition assay to indicate potential drug residues. Penicillin G and amoxicillin were detected in the serum and urine of several calves 3 h after drinking spiked milk replacer. Possible violative drug residues in the calves were detected by the microbial growth inhibition assay up to 15 h after drinking spiked milk replacer. Penicillin G, but not amoxicillin, could be detected in urine 24 h after the final feeding of spiked milk replacer. Subsequently, six calves were fed milk replacer containing 11.7 microg of penicillin G/ml (ppm) twice daily, 6% body weight per feeding. Calves were slaughtered 3 h after the final feeding. Mean (+/-SD) concentrations of penicillin G measured by high-pressure liquid chromatography in liver, kidney, muscle, and serum were 0.409 (+/-0.167) microg/g, 0.031 (+/-0.012) microg/g 0.008 (+/-0.002) microg/g, and 0.013 (+/-0.006) mg/ml, respectively. This study indicates that calves fed milk with amoxicillin or penicillin G could possibly have violative residues if slaughtered within 24 h after feeding. Violative drug residues in liver tissue were found in calves slaughtered 3 h after consuming milk replacer containing 11.7 microg of penicillin G/ml (ppm).     

Single-Step Multiresidue Determination of β-Lactam Antibiotics and β-Agonists in Porcine Muscle by Liquid Chromatography-Tandem Mass Spectrometry

A multiresidue method has been optimized and validated for the rapid determination of 12 veterinary drugs belonging to β-agonists and β-lactam antibiotics in porcine muscle. It has been based on liquid chromatography-tandem mass spectrometry and a simple extraction procedure using acetonitrile/H₂O. The extract was centrifuged, and the supernatant was directly analyzed by LC-MS/MS in positive ion mode with multiple reaction monitoring (MRM). The linearity of each analyte was almost the coefficient of determination (r) > 0.99. Mean recoveries for all analytes ranged from 74.6 to 115.3% with intra-day RSD ≤ 17.2% and inter-day RSD ≤18.1%. Limits of detection (LODs) and limits of quantification (LOQs) ranged from 0.4 to 2.0 and 1.0 to 8.0 ng/g, respectively. Finally, the validated method was successfully applied to the quantitative analysis of real samples, and clorprenaline was detected in one out of 15 pork samples.     

Short communication: Selection of extended-spectrum β-lactamase-producing Escherichia coli in dairy calves associated with antibiotic dry cow therapy—A cohort study

Antimicrobial residues in milk have been discussed as a possible selector for Enterobacteriaceae that produce extended-spectrum β-lactamases (ESBL) in dairy herds. Such residues are found in waste milk after antibiotic treatment of mastitis, but antibiotic dry cow therapy might also lead to antibiotic residues in colostrum and in milk during early lactation. While it is known that feeding of waste milk selects ESBL bacteria in calves, this was not investigated for colostrum yet, which is supposed to contain much lower antibiotic concentrations than waste milk. In this observational prospective case study on 2 farms, we hypothesized that blanket dry cow treatment with β-lactams would have more selective (here: increasing) effects on ESBL concentrations than selective (here: individually chosen) antibiotic dry cow therapy. Thus, we compared concentrations of ESBL-producing Enterobacteriaceae in feces of calves (n = 50) at 2 dairy farms with different management of antibiotic dry cow therapy. Considerably higher concentrations of ESBL-producing Escherichia coli were observed in blanket antibiotic dry cow therapy on d 3 of the calf's life (7.6 vs. 5.3 log cfu/g of calf feces). Both farms used narrow-spectrum penicillin combined with aminoglycosides for drying off, and the majority of ESBL isolates (93%) were co-resistant to aminoglycosides. No waste milk was fed to calves and no calf was treated with β-lactam antibiotics or aminoglycosides during the first 3 d of life, thus differences were most likely associated with different frequency of antibiotic dry cow therapy on farms (19 of 25 mother cows on farm A, 9 of 25 on farm B). Even though the presumable selection effect of antibiotics used for drying off decreased within the next 3 wk, this result further emphasizes the need for the reduction and prudent use of antibiotic dry cow therapy on farms.     

Detection limits of four antimicrobial residue screening tests for β-lactams in goat's milk

This study was conducted to compare the detection limits (DL) of several antibiotic residue screening tests with the maximum residue limits (MRL) authorized by the EU according to the guidance for the standardized evaluation of microbial inhibitor tests of the International Dairy Federation. Composite antibiotic-free milk samples from 30 primiparous Murciano-Granadina goats in good health condition were used to prepare test samples spiked with different concentrations of each antimicrobial. In total, 5,760 analytical determinations of 10 β-lactam antibiotics (penicillin-G, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin, cefadroxyl, cefalexin, cefoperazone, and cefuroxime) were performed using 4 antibiotic residue screening tests: the brilliant black reduction test BRT AiM (AiM-Analytik in Milch Produktions-und Vertriebs GmbH, München, Germany), Delvotest MCS (DSM Food Specialties, Delft, the Netherlands), Eclipse 100 (ZEU-Inmunotec SL, Zaragoza, Spain), and the Copan Milk Test (CMT; Copan Italia SpA, Brescia, Italy). For each method, we estimated the detection limits of the antimicrobial agents using a logistic regression model. Using the CMT and Delvotest on samples spiked with the 8 antibiotics for which MRL were available, DL were at or below the MRL. The BRT test provided DL at or below the MRL for all of the agents except cefalexin, whereas the Eclipse 100 method failed to detect 4 antibiotics (ampicillin, amoxicillin, cloxacillin, and cefoperazone) at MRL or below. Logistic regression-determined levels of agreement were highest for the CMT method (98.6 to 100%) and lowest for Eclipse 100 (66.3 to 100%). In general, agreement levels indicated good correlation between observed results and those predicted by logistic regression. The lowest b values (closely related to test sensitivity) were recorded for the cephalosporins (0.074 to 0.430) and highest for penicillin G, ampicillin, and amoxicillin (11.270 to 11.504). Delvotest and CMT best fulfilled IDF criteria for the ideal test for detecting antibiotic residues in milk.     

High-throughput method for the determination of residues of β-lactam antibiotics in bovine milk by LC-MS/MS

This study describes the development and validation procedures for scope extension of a method for the determination of β-lactam antibiotic residues (ampicillin, amoxicillin, penicillin G, penicillin V, oxacillin, cloxacillin, dicloxacillin, nafcillin, ceftiofur, cefquinome, cefoperazone, cephapirine, cefalexin and cephalonium) in bovine milk. Sample preparation was performed by liquid-liquid extraction (LLE) followed by two clean-up steps, including low temperature purification (LTP) and a solid phase dispersion clean-up. Extracts were analysed using a liquid chromatography-electrospray-tandem mass spectrometry system (LC-ESI-MS/MS). Chromatographic separation was performed in a C18 column, using methanol and water (both with 0.1% of formic acid) as mobile phase. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Main validation parameters such as linearity, limit of detection, decision limit (CCα), detection capability (CCβ), accuracy, and repeatability were determined and were shown to be adequate. The method was applied to real samples (more than 250) and two milk samples had levels above maximum residues limits (MRLs) for cloxacillin – CLX and cefapirin – CFAP.     

Development of Sensitive,Rapid, and Effective Immunoassays for the Detection of Vitamin B<Subscript>12</Subscript> in Fortified Food and Nutritional Supplements

An indirect competitive enzyme-linked immunosorbent assay (Ic-ELISA) method and lateral-flow immunochromatographic (ICA) strip assay method were developed for the detection of vitamin B₁₂ (four major forms, cyanocobalamin, hydroxocobalamin, adenosylcobalamin, and methylcobalamin) in different food products. The limit of detection and 50 % inhibitory concentration for the Ic-ELISA method were 0.065 and 0.43 ng/mL, respectively. The visual limit of detection and cutoff value for the lateral-flow ICA strip assay method were 1 and 4 ng/mL, respectively. For the detection of fortified food and nutritional supplements in vitamin tablets, energy drink, and infant milk powder samples, the recovery rates were in the range of 81 to 122 % for the Ic-ELISA method, and even the lowest content in infant milk powder was identified by the lateral-flow ICA strip assay. Therefore, both of these methods are sensitive, rapid, and effective and are suitable for the on-site detection and rapid screening of mass samples.