Trace Tetracycline Research Articles

Preparation of hydrophilic molecularly imprinted solid‐phase microextraction fiber for the selective removal and extraction of trace tetracyclines residues in animal derived foods

The present work reported a novel hydrophilic and selective solid-phase microextraction fiber by improved multiple co-polymerization method immobilization of tetracycline molecularly imprinted polymer on a stainless steel wire and directly coupled with high-performance liquid chromatography for sensitive determination of trace tetracyclines residues in animal derived foods. The developed molecularly imprinted polymer coated solid-phase microextraction fibers were characterized through scanning electron microscopy, Fourier transfer infrared spectroscopy, thermogravimetric analysis, and adsorption experiments, the fiber with cross-linked and porous structure was observed and high thermal and chemical stability. The maximum adsorption capacity of this fiber with good selectivity reached 2.35µg/mg in aqueous matrices, and showed good repeatability (relative standard deviation≤6.6%, n=5) and satisfying reproducibility between fiber to fiber (relative standard deviation ≤7.8%, n=5). Under the optimized solid-phase microextraction conditions, satisfactory linearity (5-1000µg/L) and detection limits (0.38-0.72µg/kg, S/N=3) for all the tetracyclines were obtained. The practicality of this method was proved by adding tetracycline, oxytetracycline at three levels to milk, chicken, and fish samples with good recoveries of 77.3-104.4%.

Gold nanostars-enhanced Raman fingerprint strip for rapid detection of trace tetracycline in water samples.

Rapid and accurate detection of antibiotics at trace levels in food represents a great challenge. Tetracycline (TC), as a sort of broad-spectrum antibiotic, has been extensively used in animal infection therapy and animal husbandry as growth promoters. Large amounts of TC residues in animal-derived foods affect food quality and safety, and cause undesirable side effects such as allergic reactions and bacterial antibiotic resistance. Here, a Raman fingerprint strip sensor was reported based on surface-enhanced Raman scattering technology and demonstrated for ultrasensitive detection of TC. In this approach, 4-aminothiophenol (4-ATP) modified gold nanostars (GNSs) were used as a strong Raman reporter, which was coated with anti-TC monoclonal antibody serving as a biorecognition to acquire both visual and Raman signals on the test line. To demonstrate the performance of this strip, TC standard solutions with concentrations ranging from 0.5 to 50ng/mL was detected, the limit of the detection (LOD) for the Raman signal was 0.04ng/mL, which was 100 times more sensitive than those of color intensity quantifications. The other analogues, oxytetracycline, and chlortetracycline were detected using this method, making them suitable for the samples with TC analogues screening.

Novel methods for the rapid detection of trace tetracyclines based on the fluorescence behaviours of Maillard reaction fluorescent nanoparticles.

The Maillard reaction and its fluorescent products have attracted widespread attention in the field of food safety and biology. Herein, the novel Maillard reaction fluorescent nanoparticles (MRFNs) as a fluorescent probe were synthesized via a “green” method with simple technical processes. In addition, the effects of tetracycline (TC) and chlorotetracycline (CTC) representing certain properties of tetracyclines (TCs) on the fluorescence behaviour of MRFNs were studied, respectively. The present study showed that the fluorescence intensity of MRFNs greatly enhanced with a linear increase in the CTC concentration. However, with the gradual increase in the TC concentration, the intensity of MRFNs tended to significantly decrease linearly. Based on this, novel fluorescence analysis methods for the simple and rapid detection of TC and CTC in water bodies were established, respectively. Significantly, the proposed detection methods were successfully adopted for detecting TC and CTC in some environmental water samples. Besides, the possible mechanisms for TC-induced fluorescence quenching and CTC-induced fluorescence enhancement of MRFNs were also discussed, respectively.

Determination and validation of Tetracycline residues in Poultry by High Performance Liquid Chromatography - Diode Array Detector Technique

The aim of this research is separation and determination of trace Tetracycline residues in Poultry chest, thigh and liver using High Performance Liquid Chromatography - Diode Array Detector (HPLC-DAD), with a mobile phase mixture consisting of acetonitrile: methanol: oxalic acid (0.01M) (25:15:60) and chromatographic column C8. The study was done on 32 live poultry individuals. All individuals were injected in the chest by 1m of Tetracycline standard solutions, then slaughter for analysis throughout four successive days. The injection with 10×103 ppb of Tetracycline showed that the traces of Tetracycline residues (according to the Codex Alimentarius Commission) exceeded the value of the maximum residue limit (MRL = 200 ppb) in the thigh and chest meat at the 1st day and the 1st & 2nd days of slay respectively, and exceeded the value of (MRL = 600 ppb) in the liver at the 3rd and 4th days of slay. limit of detection was LOD = 0.451 ppb, limit of quantification LOQ = 1.502 ppb, and recovery percentages of Tetracycline at a concentration of 200.0 ppb for 20 sample Rec.% = (88.966 - 91.055%), (84.623 - 87.667%), (82.198 - 83.688%) for Poultry chest, thigh and liver respectively with a percentage relative standard deviations (RSD%) of < 1 %.

Dynamics of microbial community and tetracycline resistance genes in biological nutrient removal process

The occurrence of antibiotics in wastewater has become a serious concern due to the possible development of antibiotic resistant bacteria in wastewater treatment process. In order to understand the dynamics of microbial community and tetracycline resistance genes in biological nutrient removal (BNR) process, three lab-scale sequencing batch reactors (SBRs) were operated under the stress of tetracycline. Results indicated that microbial community structure was altered, and tetracycline efflux pump genes were enhanced over 150-day operation in the presence of trace tetracycline of 20 and 50 μg L−1, respectively. Furthermore, when the initial tetracycline concentrations were increased to 2 and 5 mg L−1, substantial enhancement of tetracycline resistance was observed, accompanied with a sharp shift in microbial community structure. In this study, horizontal gene transfer was found to be the main mechanism for the development of tetracycline resistance genes under the long-terms stress of trace tetracycline. About 90.34% of the observed variations in tetracycline resistance genes could be explained by the dynamics of potential hosts of tetracycline resistance genes and class 1 integron. It should be noticed that the functional bacteria (e.g. Nitrospira, Dechloromonas, Rhodobacter and Candidatus_Accumulibacter) responsible for nutrient removal were positively correlated with tetracycline resistance, which might promote the prevalence of tetracycline resistance during biological wastewater treatment. Consequently, this study provided in-depth insights into the occurrence and prevalence of tetracycline resistance genes and their microbial hosts in BNR process.

Magnetic stir cake sorptive extraction of trace tetracycline antibiotics in food samples: preparation of metal-organic framework-embedded polyHIPE monolithic composites, validation and application.

In this work, a novel Fe3O4@Cu3(btc)2-embedded polymerized high internal phase emulsion (Fe3O4@HKUST-1-polyHIPE) monolithic cake was synthesized, characterized and used as an adsorbent in the magnetic stir cake sorptive extraction (MSCSE) and determination of tetracycline antibiotics (TCs) in food samples by a combination of with high-performance liquid chromatography-fluorescence detection (HPLC-FLD). The prepared Fe3O4@HKUST-1-polyHIPE monolithic composites displayed a strong extraction ability and high column capacity due to enhanced interactions such as π-π interactions, hydrogen bonding, and electrostatic interactions. The extraction and desorption conditions were evaluated, and the calibration curves of four spiked TCs were linear (R2 ≥ 0.9991) in the range from 20 to 800ngmL-1 for milk and egg samples, and 20 to 800ngg-1 for chicken muscle and kidney samples. The limits of detection and the limits of quantification of the four TCs by using the proposed MSCSE-HPLC-FLD method were in the range of 1.9-4.6 and 5.5-13.9ngmL-1 for milk and egg samples, and 1.8-3.7 and 5.3-13.0ngg-1 for chicken muscle and kidney samples, respectively. The recoveries of the target TCs from spiked food samples were in the range from 86.6 to 110.7% with relative standard deviations lower than 7.0%. The proposed method was successfully applied for the determination of these four TCs in milk, egg, chicken muscle, and kidney samples.

Application of a novel diol-based porous organic polymer to the determination of trace-level tetracyclines in water

A novel diol-based porous organic polymer (NTdiol-POP) was used as a new solid phase extraction material to improve extraction efficiency of trace tetracyclines in water before HPLC-MS/MS analysis.

Effect of tetracycline on microbial community structure associated with enhanced biological N&P removal in sequencing batch reactor

The presence of antibiotics in wastewater has raised serious concerns about its potential impacts on biological nitrogen and phosphorus (N&P) removal. This study investigated the long-term process performance and microbial structures in response to tetracycline in the enhanced N&P removal process. Results showed that trace tetracycline (20 and 50 μg/L) had no obvious effect on the N&P removal, while the relative abundances of Nitrospira and poly-phosphate accumulating organisms (PAOs) were found to increase. Moreover, the decreased abundance of ammonia oxidizing bacteria (AOB) was observed. When the tetracycline concentrations were further increased to 2 and 5 mg/L, initially the N&P removal was seriously inhibited, but gradually recovered with the restored abundances of Nitrospira and PAOs. However, AOB, Nitrobacter and denitrifiers were found to be more vulnerable to high-concentration tetracycline with slow activity recovery. Consequently, this study offered useful information about long-term microbial responses to tetracycline in enhanced biological nutrient removal process.

Bifunctional monomer magnetic imprinted nanomaterials for selective separation of tetracyclines directly from milk samples

Novel magnetic molecularly imprinted nanomaterials (DA + BSA-MMIPs) were prepared adopting bovine serum albumin (BSA) and dopamine as bifunctional monomers for the first time. Besides the role of assistant functional monomer, BSA can exclude the proteins with like charges and promote low molecular weight tetracyclines to be adsorbed. Thus, the DA + BSA-MMIPs could fulfil the selective separation of tetracyclines directly from milk samples. The characteristics, polymerization conditions, and adsorption performances of the resultant nanomaterials were investigated in detail. In addition of uniform imprinting layers, stable crystalline phase, and good magnetism of the DA + BSA-MMIPs, they have rapid binding kinetic, high adsorption capacity, and favorable reusability. The imprinted nanomaterials were coupled with HPLC to selectively extract and determine trace tetracyclines from untreated milk samples. The recoveries of tetracyclines are in the range of 84.1–95.8% with relative standard deviations of less than 6.7%. The developed method is especially suitable for the selective enrichment and detection of target compounds directly from a complex sample with proteins.

Detection of trace tetracycline in fish via synchronous fluorescence quenching with carbon quantum dots coated with molecularly imprinted silica

A novel fluorescence-based sensor combining synchronous fluorescence spectroscopy (SFS) with molecularly imprinted polymers (MIPs) was fabricated with reverse microemulsion method. Tetracycline (TC), (3-aminopropyl) triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and carbon quantum dots (CDs) were used as template, functional monomer, cross-linker and signal sources respectively in the probe preparation. A synchronous fluorescence emission (λem) at 355nm was observed for the prepared MIP-coated CDs (MIP@CDs) particles when the wavelength interval (Δλ) was set as 70nm, and the synchronous fluorescence intensity could be rapidly and efficiently quenched by TC based on inner filter effect (IFE). The quenching efficiencies of synchronous fluorescence intensity was linearly fitted with tetracycline (TC) concentrations ranging from 0.1 to 50μmolL−1 with a detection limit (DL) of 9nmolL−1 (3σ, n=9). The MIP@CDs was used as a probe to detect TC in fish samples with the recoveries ranging from 98.4% to 103.1% and the relative standard deviation less than 6.0%. The results illustrated that the as-prepared MIP@CDs could be applied to the detection of trace TC in fish samples with rapidity, high sensitivity and accuracy.

Magnetic solid phase extraction based on graphene oxide/nanoscale zero-valent iron for the determination of tetracyclines in water and milk by using HPLC-MS/MS

A magnetic solid-phase extraction (MSPE) based on graphene oxide/nanoscale zero-valent iron (GO/nZVI) coupled with HPLC-MS/MS was proposed for the determination of trace tetracycline antibiotics (TCs) in water and milk.

Development and validation of polymerized high internal phase emulsion monoliths coupled with HPLC and fluorescence detection for the determination of trace tetracycline antibiotics in environmental water samples.

A polymerized high internal phase emulsion monolith was used as a novel sorbent for solid-phase extraction coupled with high-performance liquid chromatography and fluorescence detection for the determination of oxytetracycline, tetracycline, doxycycline, and chlorotetracycline in environmental water samples. The polymerized high internal phase emulsion monolithic column was prepared by the in situ polymerization of the continuous phase of a high internal phase emulsion containing glycidyl methacrylate, styrene, and divinylbenzene in pipette tips, and then functionalized with iminodiacetic acid. The resulting monolith exhibited highly interconnected porosity and large surface areas, making it an excellent candidate as an solid-phase extraction sorbent for the enrichment of trace tetracycline antibiotics. Several factors affecting the extraction performance of polymerized high internal phase emulsion monoliths, including the pH of sample solution, the eluting solvents, the sample loading flow rate and volume, were investigated, respectively. Under the optimized conditions, the mean recoveries of oxytetracycline, tetracycline, doxycycline, and chlorotetracycline spiked in pond and farm wastewater samples ranged from 78.1 to 119.3% with relative standard deviation less than 15%. The detection limits (S/N = 3) of the proposed method were in the range of 51-137 pg/mL. This study demonstrated that the monolithic polymerized high internal phase emulsion would be promising solid-phase extraction sorbents in the extraction and proconcentration of trace analytes from complex samples.

Novel silver nanoparticle-enhanced fluorometric determination of trace tetracyclines in aqueous solutions

Metal-enhanced fluorescence (MEF) has exhibited promise for applications in fluorometric assays. The effects of silver nanoparticles (AgNP) on the fluorescence behaviours of tetracycline hydrochloride (TCH) and chlortetracycline hydrochloride (CTC) in aqueous solutions were investigated. The experimental results demonstrated that the fluorescence intensities of each tetracycline in water solutions were greatly enhanced by AgNP through the MEF effect. In addition, a novel silver nanoparticle-enhanced fluorometric method was established for the direct determination of TCH and CTC in aqueous solutions. Under optimum experimental conditions, the linear dynamic ranges for the determination of TCH and CTC in aqueous solutions varied from 0.10 to 6.0mgL−1 and 0.050 to 3.0mgL−1 with detection limits of 0.63µgL−1 and 0.19µgL−1, respectively, and with the relative standard deviation of less than 1.9% (n=9). The experimental recovery results for the determination of TCH and CTC in aqueous solutions ranged from 93–106% and 95–104%, respectively. Compared with the established method without the addition of AgNP, the limits of quantitation of the silver nanoparticle-enhanced fluorometric method were approximately 5-fold lower for TCH and 3-fold lower for CTC. Moreover, the newly established silver nanoparticle-enhanced fluorometric method was successfully applied to the direct determination of TCH and CTC in pharmaceutical preparations.

Photoresponsive molecularly imprinted hydrogel casting membrane for the determination of trace tetracycline in milk.

This study aimed to develop a photoresponsive molecularly imprinted hydrogel (MIH) casting membrane for the determination of trace tetracycline (TC) in milk. This MIH casting membrane combined the specificity of MIHs, the photoresponsive properties of azobenzene, and the portable properties of a membrane. Photoresponsive TC-imprinted MIHs were initially fabricated and then cast on sodium dodecyl sulfonate polyacrylamide gel. After TC removal, a photoresponsive MIH casting membrane was obtained. The photoresponsive properties of the MIH casting membrane were robust, and no obvious photodegradation was observed after 20 cycles. The MIH casting membrane displayed specific affinity to TC upon alternate irradiation at 365 and 440 nm; it could quantitatively uptake and release TC. The TC concentration (0.0-2.0 × 10(-4) mol l(-1)) in aqueous solution displayed a linear relationship with the photoisomerization rate constant of azobenzene within the MIH casting membrane. As such, a quick detection method for trace TC in aqueous foodstuff samples was established. The recovery of this method for TC in milk was investigated with a simple pretreatment of milk, and a high recovery of 100.54-106.35% was obtained. Therefore, the fabricated membrane can be used as a portable molecular sensor that can be easily recycled.

Extraction of Trace Tetracycline by Phase-Separated Sublation

Extraction of Trace Tetracycline by Phase-Separated Sublation

Comparison of tetracycline rejection in reclaimed water by three kinds of forward osmosis membranes

Comparison of the rejection of trace tetracycline (TC) in the secondary effluent of an anoxic–oxic (AO) system by forward osmosis (FO) was investigated at the mini-lab scale. The effects of different operating conditions including membrane orientation, flow velocity, and draw solution concentration and solute on TC rejection were also explored. The results revealed that TC rejection increased with increasing water flux, but high reverse salt flux may reduce TC rejection through enhanced solution ionic strength. Membranes with a nonwoven structure could reduce TC rejection via increased flow velocity. In the pressure retarded osmosis (PRO) mode, rejection of charged TC was higher than in the FO mode because of enhanced electrostatic repulsion. In addition, the reverse draw solute could hinder the forward diffusion of TC, thus leading to a decline in TC rejection. The cellulose triacetate with embedded polyester screen (CTA-ES) membrane displayed consistently higher TC rejection than that of the cellulose triacetate with a cast nonwoven (CTA-NW) membrane, and the rejection of TC by a thin-film composite with embedded polyester screen (TFC-ES) membrane was relatively lower compared to CTA membranes. These findings provide further insight into TC rejection mechanisms and potentially can be useful for FO process operation in wastewater reclamation.

Correlation among extracellular polymeric substances, tetracycline resistant bacteria and tetracycline resistance genes under trace tetracycline

Antibiotic resistance occurrences and proliferation in activated sludge have attracted more and more attention nowadays. However, the role which extracellular polymeric substance (EPS) plays on the antibiotic resistance is not clear. The changes and correlation among EPS, tetracycline (TC) resistant bacteria (TRB) and TC resistance genes (TRGs) of sequencing batch reactors (SBRs) were investigated. Performance of SBR without TC was compared with two other SBRs to which different amounts of TC were added. Total average EPS contents were found to increase significantly from 66mgg−1 VSS to 181mgg−1 VSS as the TC concentrations increased from 0 to 100μgL−1. As the EPS content increased, TRB in sludge of the three SBRs increased significantly from 105 to 106 colony forming unit mL−1 after being exposed to TC. In addition, the concentrations of three groups of TRGs (copies mL−1) were determined by real-time fluorescence quantitative polymerase chain reaction and followed the order: efflux pump genes>ribosome protected genes>degradation enzyme genes. The numbers of TRGs in the idle stage were larger than those in the aeration sludge. Correlation coefficients (R2) between EPS and TRB in sludge were 0.823 (p<0.01) while the correlation between EPS and total TRGs was poor (R2=0.463, p>0.05). But it showed the same tendency that EPS and TRGs in sludge increased with the increasing of TC.

Removal performance and changes in the microbial communities of SBRs under aerobic and anoxic conditions with trace tetracycline pressure

The reactor performance and microbial community composition of sequencing batch reactor (SBR) under aerobic and anoxic conditions were investigated in this study. The experimental results showed high chemical oxygen demand (COD) removal efficiency. The tetracycline (TC) removal efficiencies were not obviously affected by aerobic and anoxic conditions, and were 64–97 and 60–87%, respectively. Aerobic condition was observed to be more suitable for decreasing tetracycline‐resistant bacteria (TRB) than anoxic condition in synthetic and real wastewater. Denaturing gradient gel electrophoresis (DGGE) and clone library analysis revealed that Chlorobaculumthiosulfatiphilum was the dominant species in the tested SBR systems. TC significantly influenced the relative numbers of TRB‐ and TC‐resistant genes, and the microbial community diversity changed with the addition of 250 μg L−1 of TC. The genes of tetA and tetC, tetM and tetS, tetA and tetM, tetS and tetA showed significant correlation with each other (P < 0.05).

Fate of trace tetracycline with resistant bacteria and resistance genes in an improved AAO wastewater treatment plant

The fate of trace tetracycline, tetracycline resistant bacteria (TRB) and tetracycline resistant genes (TRGs) in an improved anaerobic-anoxic-oxic (AAO) wastewater treatment plant (WWTP) was investigated in this study. Quantitative real-time polymerase chain reaction (qPCR) and conventional heterotrophic plate count method were used to measure eight tet genes (tetA, tetB, tetC, tetE, tetM, tetO, tetS and tetX) and TRB, respectively. The TRB percent of total heterotrophic bacteria (THB) is about 1.31–24.1% in WWTP influent. Tet gene abundance in the WWTP varied greatly among the gene types. The concentrations of TRGs in effluent samples ranged from 7.11×10−9 to 1.53×10−4 copies/copy 16S rRNA gene. TRB and THB, tetM and tetO, tetE and tetX, but not the others, showed a significant correlation with each other (p<0.01). The relationships between ribosomal protection protein genes, enzymatic modification gene and corresponding concentrations of antibiotics were found to be considerably significant (R2=0.898, p<0.01 for ribosomal protection protein genes and R2=0.872, p<0.05 for enzymatic modification gene).

Ultrasensitive Resonance Scattering (RS) Spectral Detection for Trace Tetracycline in Milk Using Aptamer-Coated Nanogold (ACNG) as a Catalyst

This paper reports an ultrasensitive resonance scattering (RS) method to detect tetracycline (TET) in milk based on the competition of aptamers between nanogold and TET, aggregation of naked nanogold, nanocatalytic Fehling reaction, and RS signals of catalytic product Cu₂O cubic. The detection principle was confirmed by the nanoparticle size analyzer (NANOS), scanning electron microscopy (SEM), and X-ray diffraction (XRD). The variations of RS intensity had good linear correlation with TET concentrations, and the limit of detection was calculated as 11.6 nM. The proposed method was successfully applied for analysis of TET in milk, with total recoveries ranging from 105 to 109%.