Gentamicin Residues Research Articles

Boronic acid-functionalized magnetic covalent organic frameworks based solid-phase extraction coupled with hydrophilic interaction chromatography-tandem mass spectrometry for the determination of trace gentamicin residues in milk

Boric acid-functionalized magnetic covalent organic frameworks (Fe3O4-TpBD-B) with large surface area and high porosity were prepared and applied for magnetic solid-phase extraction adsorbent of gentamicin from milk before UPLC-MS/MS detection. By utilizing a new HILIC chromatographic column with zwitterionic sulfoalkyl betaine stationary phase based on ethyl bridged hybrid particles (BEH), isomers of gentamicin (C1, C1a, and C2+C2a components). The developed methods demonstrated good linearity (R2 > 0.99), acceptable accuracy and good precision (<10 %), and low limit of quantitation (1.59 ng mL⁻1 for C1, 1.52 ng mL⁻1 for C1a and 2.72 ng mL⁻1 for C2+C2a). In addition, this method has been effectively applied to the analysis of real milk samples.

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78 % to 96 %. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Evaluation of antibiotic use and analysis of ciprofloxacin and gentamicin residue in fish samples from farms in Lagos, Nigeria.

Antibiotic use in aquaculture for increase yield has been established over time; however, consumption of such fish may lead to inadvertent intake of sub therapeutic doses of antibiotics. Therefore, this study aimed to investigate the extent of antibiotic use in fish farming and ciprofloxacin and gentamicin residues in fish that are consumed in Lagos, Nigeria. It was conducted in two parts: a survey of 60 fish producers to assess their use of antibiotics and an analysis of ciprofloxacin and gentamicin residues in fish organs using high-performance liquid chromatography (HPLC). The survey found that twenty-nine (48%) of the farms used antibiotics, with oxytetracycline being the most common (20%), followed by ciprofloxacin (15%); gentamicin (5%); neomycin (8.33%); and probiotics (13%). HPLC analysis of fish liver and fillet samples showed that Lagos Island had the lowest residual ciprofloxacin dose (0.3014ug/g), while Lagos mainland had the greatest residual ciprofloxacin dosage (113.78765ug/g). The mean gentamicin residue in flesh of fish from farms in Lagos Island LGA was 0.37ug/g while that obtained for liver of fish samples from Surulere LGA was 2.12ug/g). The largest (5.3240ug/g) and lowest (0.2661ug/g) residual gentamicin antibiotic concentrations were found in fishes harvested from farms located in Surulere and Lagos Island, respectively. The residue levels were within the permitted range set by the WHO, but continuous regulatory surveillance is necessary to prevent antimicrobial resistance spread and improved food safety. The study has revealed that fish produced in the study area contained residues of the commonly used antibiotics hence farmers should be encouraged to consult veterinarians in cases of disease treatment for judicious use of antibiotics while self-medication and purchase of antibiotics from vendors should be discouraged.

Production of high affinity monoclonal antibody and development of indirect competitive chemiluminescence enzyme immunoassay for gentamicin residue in animal tissues

To determine gentamicin residues in animal tissues, a monoclonal antibody (Mab) was produced and a sensitive indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) was developed. At first, gentamicin was conjugated with bovine serum albumin as immunogens which were used to immunize BALB/c mice. Then, an anti-gentamicin Mab was prepared by hybridoma technology. Finally, a sensitive icCLEIA was developed with an 50% inhibition concentration (IC50) of 0.067 ng/mL for gentamicin. The limit of detection of the icCLEIA was 0.002 ng/mL. The cross reactivity of the Mab with structural analogues were<0.01%. The recoveries of gentamicin ranged from 80 to 101% and coefficient of variation was <6.4% in pork and fish samples. Samples were detected by UPLC-MS/MS for evaluating reliability of the icCLEIA. The results suggested that the prepared anti-gentamicin Mab can be used for rapid and convenient immunoassays to detect gentamicin residues in animal tissues.

Fate of bacterial community, antibiotic resistance genes and gentamicin residues in soil after three-year amendment using gentamicin fermentation waste

Over a three-year field trial, the impacts of composted and raw gentamicin fermentation waste (GFW) application to land on residual soil gentamicin levels, physicochemical properties, bacterial community composition, and antibiotic resistance genes (ARGs) were assessed. In the saline-alkali soil tested, GFW application decreased electrical conductivity (EC) and pH. Importantly, there was no measurable long-term accumulation of gentamicin as a result of GFW addition. Changes in the abundance of Bacillus was primarily associated with degradation of gentamicin in soil, whereas wider (i.e. more general) shifts in bacterial communities over the treatments was linked to alteration of soil physicochemical properties, particularly pH, total nitrogen, dissolved organic carbon, EC, NO3−-N and NH4+-N. Compared with other treatments, soils receiving composted GFW harbored more types of ARGs and significantly higher (P < 0.05) abundances of mobile genes elements (MGEs) (especially IncQ and Int1) and aminoglycoside ARGs (especially aminoglycoside phosphotransferases genes, APH). Finally, the abundances of ARGs in soils receiving raw and composted GFW were 59.60% and 50.26% higher than that in soils only receiving chemical fertilizer, respectively. Specifically, the abundances of APH, especially strB, were significantly higher than other kinds of ARGs (P < 0.05). The results of linear regression and partial least squares path model showed that MGEs, including plasmids, integrons, and transposons, along with soil properties (EC and NH4+-N) were the main factors associated with change in ARGs. Furthermore, different MGEs were involved in different transfer mechanisms of specific ARGs. Our findings demonstrated the potential risks of using raw and composted GFW as fertilizer, and suggest potential solutions to this problem.

Antibiotics use and gentamicin residues in commercial poultry and chicken eggs from Oyo and Lagos States, Nigeria

Nigeria poultry industry is fast growing to meet the demand of the increasing population with overdependence on antibiotics for production leading to consumer safety and public health concerns. The antibiotic use in poultry farms and presence of gentamicin residues in chicken eggs from city markets of Southwest Nigeria were investigated. A semi-structured questionnaire was administered to 45 poultry farmers to determine the patterns of antibiotic use, knowledge of withdrawal periods and food safety implications. Furthermore, 270 egg samples from six retail markets in Oyo and Lagos states were analyzed with ELISA for gentamicin residues. Residue concentrations were compared with Student’s t test and ANOVA (p &lt; 0.05). About 90% of the respondents reported they frequently administered antibiotics, 85% engaged in non-prescribed medication, and about 80% did not observe a withdrawal period before selling the eggs from treated chickens. In addition, 60% and 80% of pooled eggs from Oyo and Lagos states, respectively, contained gentamicin residues with means of 1461 ± 74 and 1350 ± 92 µg/kg, respectively. The mean residues obtained from the two states were higher than the maximum recommended residue limits. High levels of gentamicin residues, from unbridled use of antibiotics in poultry production, detected in retail eggs from markets rendered the eggs unsafe for human consumption. Therefore, regulatory control and veterinary supervision of antibiotic use are advocated to ensure Nigerian consumer protection.

Determination of gentamicin residues in chicken meat by high performance liquid chromatography

In the present study, the residual concentration of gentamicin was determined in chicken meat using ultra high performance liquid chromatography (UHPLC). In all 180 chicken meat samples including muscle, liver and kidney were analyzed. Out of 180 samples, 24 samples (13.33 per cent) were found positive for residual concentration of gentamicin in chicken meat. Positive meat samples of gentamicin were further analyzed for quantitative estimation. Six samples (25 per cent) were found gentamicin residue at higher levels while 18 samples (75 per cent) were found below maximum residue limits recommended by Commission Regulation of European Union for chlortetracycline in chicken muscle, liver and kidney samples. Positive meat samples were further subjected for boiling and results indicated a significant reduction in residual concentration of gentamicin.

Determination of gentamicin C components in fish tissues through SPE-Hypercarb-HPLC-MS/MS

In this work, an HPLC/MS/MS method for determination of gentamicin C components in fish tissues was developed based on strong cation exchange solid-phase extraction (SPE) purification coupled with Hypercarb chromatographic column in separation mode. Sample was extracted using trichloroacetic acid aqueous solution containing EDTA. Ion-pairing reagents were not needed because of the “graphite polarity retention effect” of the Hypercarb chromatographic column. HPLC-MS/MS was performed in multiple reaction monitoring (MRM) mode for simultaneous qualitative and quantitative analyses (using matrix external standard) of gentamicin C components in fish tissues. Good linearity was obtained for the target analytes within the concentration range from 0.0100 to 0.500 mg/L. The limits of quantification (LOQ) of this method were 10.0, 20.0, and 20.0 μg/kg for C1, C1a, and sum of C2 + C2a, respectively. The average recoveries of gentamicin C components were 80.0%–110% when spiked at three levels with the blank carp (Cyprinus carpio) matrix, and the relative standard deviations (RSD) were all less than 15% (n = 6). In addition, for the features of simple operation, high sensitivity and good reproducibility, the proposed method has been successfully applied for detection of gentamicin residues in fish tissues during actual breeding.

Gentamicin degradation and changes in fungal diversity and physicochemical properties during composting of gentamicin production residue

An indoor co-composting of gentamicin fermentation residues (GFR) and lovastatin fermentation residues (LFR) inoculated with gentamicin-degrading Aspergillus terreus FZC3 was conducted to remove gentamicin residues. The results showed that treatment MFZC3, consisting of a 10:1 blend of GFR and LFR (w/w), had the longest thermophilic phase (7days), quickest gentamicin degradation (t½=4.4days), and relatively higher gentamicin degradation percentage (96.7%) at the end of composting. Addition of Aspergillus terreus FZC3 affected fungal diversity of the compost and improved the removal of gentamicin during composting of the 15:1 GFR:LFR blend. By analyzing the variations of gentamicin and fungal community dynamics, it was speculated that Aspergillus terreus could accelerate gentamicin degradation. The microbial community and dynamic during composting were deeply affected by the physicochemical properties, and vice versa. In conclusion, co-composting of GFR with LFR could be a promising technology to solve the problem of gentamicin residue in GFR waste.

Intramammary treatment with gentamicin in lactating cows with clinical and subclinical mastitis

Abstract The study evaluated the microbiological profile of milk samples collected before and after mastitis treatment with gentamicin and investigated biofilms production and antimicrobial susceptibility of Staphylococcus spp. isolated. The presence of gentamicin residues in milk after the recommended withdrawal period was also evaluated. Antimicrobial residues were analyzed by Delvotest® SP NT over a period of 12 days beginning after 24 hours the last gentamicin application. Some of Staphylococcus spp. isolates were biofilm producers (19.05%). Staphylococcus spp. showed high levels of resistance to neomycin (16.95%), penicillin G (10.17%), and ampicillin (10.17%). Multidrug resistance to all antibiotics tested was observed in 1.69% of the Staphylococcus spp. isolates. Among 1440 mammary quarter milk samples 24.95% presented gentamicin residues after the withdrawal period. Gentamicin residues were also detected in 3.8% of samples from calibrated glass recorder jar (n=383) 4.1 days after treatment. The indiscriminate use of antibiotics may lead to the emergence of multidrug-resistant strains as well as increasing the risk of presence of residues of these drugs in milk. These problems affect the milk quality and may become a public health problem.

Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues

abstractGentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5th booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL−1 of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC50 value of anti-Gent IgY was 2.69 ng mL−1 and regression curve equation was y = −16.27x + 56.97 (R2 = 0.95, n = 3), confirming that, the detection limit (LOD, IC10 value) was 0.01 ng mL−1. Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.

Residue Depletion of Gentamicin in Swine Tissues after Intramuscular Administration

A sensitive and robust high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) was developed for the determination of gentamicin (GEN) residues in swine tissues. The limit of quantification (LOQ) of the method was 50 ng/g for muscle and 100 ng/g for liver and kidney. Mean recoveries at all fortification levels ranged from 82.34 to 93.20% with coefficient of variation (CV) below 5.39%. Residue depletion study of GEN in swine was performed after intramuscular injections twice daily at a dose of 4 mg/kg of bw with 12 h intervals for 5 consecutive days. The concentrations of GEN were determined in injection site, muscle, liver, and kidney by the HPLC-FLD method. The highest GEN concentration was measured in kidney, indicating that kidney was the primary target tissue for GEN residue. GEN concentrations in all examined tissues were below the maximum residue limit (MRL) recommended by the European Union (EU) at 50 days post-treatment.

Persistence of gentamicin residues in milk after the intramammary treatment of lactating cows for mastitis.

This study was designed to investigate persistence of gentamicin residues in milk after the intramammary treatment of lactating cows for mastitis. Milk samples were collected at a 1-d interval after the last administration from 34 individual cows that had received intramammary infusions of gentamicin. The doses and treatment times evaluated in this study represented those that have been applied by veterinarians in practice. The tetrazolium chloride assay was used to determine whether there were significant residues of the antibiotic in the samples. Persistence of detectable drug residues in milk from 33 cows (28 cows, <or=6 infusions at <or=0.7 g gentamicin; and 5 cows, 2 infusions at 0.8 g gentamicin) did not exceed 5 d; but 1 cow (5 infusions at 0.8 g gentamicin) had detectable residues in its milk for 9 d. Our results suggest that a 5-d milk withdrawal period might be insufficient to secure the clearance of the contamination of gentamicin, because treatment times and dosages contribute to the antibiotic clearance. A larger scale of samples are needed for further investigations.

Use of tissue–fluid correlations to estimate gentamicin residues in kidney tissue of Holstein steers

Gentamicin continues to be one of the most effective antibiotics for the treatment of gram-negative infections. Greater than 90% of the drug is rapidly eliminated from the body in <2 days, however, a small residue remains bound to the kidney cortex tissue for many months. In beef steers, the gentamicin residue is unacceptable and its presence is monitored by the FAST (Fast Antimicrobial Screen Test) applied to the kidney at the time of slaughter. The sensitivity of the FAST to gentamicin in the kidney cortex is reported to be 100 ng/g, therefore, this level of gentamicin defines the acceptable limit of gentamicin drug residue in the bovine kidney. In the present study, three doses of 4 mg/kg gentamicin was administered intramuscularly to eight steers. Gentamicin was allowed to deplete from the kidneys for a range of times from 7 to 10 months. At slaughter the level of gentamicin in the kidney cortex varied from 91 to 193 ng/g, but a total of 160 FAST tests performed on the kidneys were negative. Blood and urine samples were collected at varying times following the last dose of gentamicin. Kidney tissue samples were collected by laparoscopic surgery in the live steers as well as the final sample obtained at slaughter. Plasma levels of gentamicin declined rapidly to nondetectable within 3 days, while measurable urine persisted for 75 days before the concentration of gentamicin declined to levels too low to quantitate by the available liquid chromatography tandem mass spectrometry (LC/MS/MS) technique. An estimated correlation between an extrapolation of urine gentamicin concentration to the corresponding kidney tissue sample suggests a urine to kidney tissue relationship of 1:100. A test system sufficiently sensitive to a urine gentamicin concentration of 1 ng/mL will correlate with the estimated 100 ng/g gentamicin limit of the FAST applied to the fresh kidney of the recently slaughtered bovine.

Development of Competitive Direct Enzyme-linked Immunosorbent Assay for the Detection of Gentamicin Residues in the Plasma of Live Animals

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities (CR50) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/B0 ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/B0 ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life (t1/2) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 10 : 1498-1504)

Development of ELISA and Immunochromatographic Assay for the Detection of Gentamicin

Competitive direct enzyme-linked immunosorbent assay (ELISA) and the immunochromatographic assay were developed using a monoclonal antibody to detect gentamicin in the animal plasma and milk. No cross-reactivity of the antibody was observed with other aminoglycosides based on competitive direct ELISA, indicating that the antibody is highly specific for gentamicin. On the basis of the standard curves, the detection limits were determined to be 0.9 ng/mL in phosphate-buffered saline (PBS), 1.0 ng/mL in plasma, and 0.5 ng/mL in milk, respectively. Recoveries of gentamicin from spiked plasma and milk at levels of 25-100 ng/mL ranged from 85 to 112%. The concentration of intramuscularly injected gentamicin was successfully monitored in the rabbit plasma through competitive direct ELISA. The detection limits were estimated to be about 6 ng/mL of gentamicin in PBS, plasma, and milk using the colloidal gold-based immunochromatographic assay, which is suitable for the simple screening of gentamicin residues in the veterinary field. Observed positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could complement each other as well as veterinary field and laboratory findings.

Analytical method for the determination of the aminoglycoside gentamicin in hospital wastewater via liquid chromatography–electrospray-tandem mass spectrometry

A method for the determination of gentamicin residues in hospital wastewater has been developed using kanamycin as a surrogate standard. The method consists of solid-phase extraction (SPE) and detection by ion-pair chromatography with electrospray tandem mass spectrometry (LC–ES-tandem MS). The SPE was performed on a weak cation exchanger. Filtration should be avoided in the sample preparation, otherwise a significant loss of gentamicin occurs. Chromatographic separation on a C 18-column was achieved using a ternary eluent containing methanol, water and 20 mmol l −1 heptafluorobutyric acid solution. Mean relative recoveries of the analytes in hospital wastewater varied between 107 and 111%. The limit of quantification (LOQ) was 0.20 μg l −1 in hospital wastewater. Gentamicin was found in native hospital wastewater in a concentration range between 0.4 and 7.6 μg l −1.

A Direct (Non-Competitive) Immunoassay for Gentamicin Residues with an Optical Biosensor

Using sensor chip immobilized anti-gentamicin monoclonal antibodies in an optical biosensor (BIACORE 3000) resulted in the first single antibody based and label free noncompetitive immunoassay for the direct detection of residues of a low molecular weight compound in a food product. Calibration curves for gentamicin (Mw 466 Da) in buffer and milk showed 50% binding at 20 and 35 ng ml−1, respectively, which is well below the maximum residue limit of 100 ng ml−1.

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.

Dot‐Elisa for the rapid detection of gentamicin in milk

A dipstick dot-ELISA for the detection of gentamicin in milk of dairy cattle is reported for the first time. The test is based on a sandwich ELISA using high affinity monoclonal antibodies to gentamicin. Antibodies were adsorbed to nitrocellulose filters, blocked, dried, and stored for several weeks before use. The dipstick ELISA detected gentamicin at a concentration of 0.1 microgram/ml and produced strongly positive results at 0.2 microgram-0.3 microgram/ml. This ELISA is highly specific and no false positives were detected when tested against various aminoglycoside analogs including streptomycin, kanamycin, bekanamycin, amikacin, neomycin, and tobramycin. Further, the elimination in cow milk of gentamicin residues following intramammary administration of the drug was studied in two dairy cattle using dot-ELISA. Milk gentamicin levels were detected at post injection hours up to 120 hr in each of the two dairy cattle. It therefore, appears that gentamicin residues can still be detected in milk after 5 days using dot-ELISA. Based on the simplicity of performance and the economical nature of the test system, dipstick is recommended as a suitable method for wide scale use in field studies and diagnostic laboratories.