Tetracycline Residues In Milk Research Articles

ELISA based Monitoring and Quantification of Tetracycline Residues in Fresh and Powdered Cow Milk Commercialized in Constantine Region (Northeast Algeria)

Background: This study aimed to identify tetracycline residues in milk consumed in the region of Constantine (northeast Algeria) using ELISA based tetracyclin kit. Methods: A total of 180 samples were analyzed (fresh cow milk and imported powdered milk). To compare ELISA and HPLC detection values, 22 fresh milk ELISA positive samples were confirmed by HPLC analysis. Result: 92.5% of fresh milk samples contained tetracycline residues at values between 5 and 74 µg/L and 33.3% of the samples showed concentrations between 49 and 74 µg/L, that exceed the MRLs recommended by the FDA. No significant differences (p greater than 0.01) were found between the values obtained by the two methods.

Antibiotic-Fe3O4 nanoparticles with highly efficient catalytic activity for enhanced chemiluminescence detection of tetracyclines residues in foods

Tetracyclines (TCs) are the most commonly antimicrobial agents that used in livestock production worldwide. It is important to supervise tetracyclines residues in food for environmental monitoring and food safety. In this study, a novel, label-free chemiluminescence (CL) assay without antibody was established. Fe3O4 NPs could facilitate the CL interaction between luminol and H2O2. Interestingly, TCs could enhance the catalytic ability of Fe3O4 NPs and result in a further amplification of the CL intensity. The CL intensity varied linearly with the concentration of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and ranging from 10–2400, 10–2800, and 5–2100 nmol/L, respectively; The limits of detection were 4 nmol/L for TC, 6 nmol/L for OTC, and 2 nmol/L for CTC. This CL strategy was applied successfully in testing three TCs residues in milk, eggs and honey samples with more sensitive results, which provided an alternative strategy for monitoring the correct use of TCs.

On-line temperature-responsive restricted access material of sample preparation technique coupling with liquid chromatography for detection of tetracycline residues in milk

Effort of sample preparation has been working towards the automation and improvement of accuracy. Development of on-line sample preparation coupled with liquid chromatography (LC) is a promising way. In this study, an on-line extraction-LC automated analysis was proposed based on a temperature-sensitive restricted access material (RAM) for the determination of tetracycline residues (TCs) in milk. The RAM was synthesized using silica as substrate by the two-step surface initiated-atom transfer polymerization (SI-ATRP), styrene (St), sodium 4-vinylbenzenesulfonate (Nass) and a lower critical solution temperature (LCST)-responsive n-vinylcaprolactam (VCl) were conjugated to fabricate multiple interaction-adsorption sites of P(St-co-Nass-co-VCl) on silica interior, while an upper critical solution temperature (UCST)-responsive sulfobetaine methacrylate (SBMA) was then block polymerized to form zwitterionic exclusion sites on exterior. Thereafter, the RAM was packed as extraction column to be fixed into LC ahead of C18 analytical column, establishing automatic analysis by rotation of switching valve. In conjunction with LCST and UCST dual-responsive properties to adjust the hydrophobic-hydrophilic transition of the grafted P(St-co-Nass-co-VCl)-b-PSBMA chains, the RAM column acquires the best extraction of TCs and meanwhile the furthest removal of proteins at about 30 ℃ of column temperature. And using the proposed automatic analysis for monitoring of TCs in milk, a wide linearity (20–900 ng mL−1), low limit of detection (1.0 ng mL−1) and limit of quantification (2.5 ng mL−1) as well as good inter-day (<3.9 % RSD) and intra-day precision (<4.8 % RSD) with satisfactory recoveries (97.2–101.9 %) show the superior efficiency. It is believed that the introduction of temperature-sensitive RAM to construct on-line extraction is a powerful tool for detecting trace substances in scientific analyses.

Green hairy basil seed mucilage biosorbent for dispersive solid phase extraction enrichment of tetracyclines in bovine milk samples followed by HPLC analysis

Unmodified hairy basil seed mucilage (Ocimum basilicum L.), with attractive features as structural functionality and adsorption capacity, was employed as a green biosorbent for dispersive solid phase extraction and enrichment of oxytetracycline, tetracycline, and doxycycline before quantitation by HPLC-UV for the first time. Hairy basil crushed seed increased the contacting surface area and was completely dispersed in the sample solution to extract tetracyclines under acidic condition with the assistance of ultrasonic waves. The analytes in the extraction phase were separated on a C18 column under isocratic condition with a mobile phase consisted of acetonitrile and trifluoroacetic acid. Influence of chemical and physical variables on the extraction efficiency of the developed method was investigated and optimized systematically. Under the optimal condition of all experimental parameters, good linear ranges were obtained at 15.0–500 μg L−1 for tetracyclines with determination coefficients more than 0.9994. Limits of detection (LODs) and limits of quantitation (LOQs) ranged 5.0–7.0 and 15.0 μg L−1, respectively. Relative standard deviations (RSDs) of the proposed method at 100 and 300 μg L−1 for TCs were less than 13 % and 10 %, respectively with percentage TC recoveries from spiked standard ranging 83.1–109.9 %. This simple, reliable, cost-effective, and environmentally friendly method was successfully applied for the analysis of tetracycline residues in milk. The greenness of the proposed method was assessed using the Analytical Eco-Scale and AGREE protocol.

Towards direct detection of tetracycline residues in milk with a gold nanostructured electrode.

Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl3. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class.

Copper doped zinc sulfide quantum dots as ratiometric fluorescent probes for rapid and specific detection of tetracycline residues in milk

In this work, copper-doped zinc sulfide quantum dots (ZnS:Cu QDs) were developed as ratiometric fluorescent probes to detect tetracycline (TC) in milk. With the increasing of TC concentration, the fluorescence at 578 nm of ZnS:Cu QDs was quenched, while a new fluorescence at 520 nm was significantly enhanced due to the formation of a green-emitting complex of Zn2+ and TC on the surface of probes. The fluorescence intensity ratio (I520/I578) have a good linear relationship (R2 = 0.98) with the concentration of TC in the range of 0.2–2.0 μM. The detection limit was calculated to 149 nM, which is lower than the allowable level of TC (about 180 μM) in milk set by the US Food and Drug Administration (FDA). Finally, the fluorescent test papers have been also prepared using ZnS:Cu QDs as ink by painting on a piece of filter paper. With the addition of TC, the orange test paper produced green spots that became more and more obvious with a dose-discerning ability as low as 2 μM. This work demonstrated a great potential of ratiometric fluorescent probe for the easy and visual detection of TC in complicated real samples.

Mixed-mode fabric phase sorptive extraction of multiple tetracycline residues from milk samples prior to high performance liquid chromatography-ultraviolet analysis

Herein, a new extraction mode of classical fabric phase sorptive extraction (FPSE), namely mixed-mode strong cation exchanging FPSE is presented and evaluated for the first time for the determination of tetracycline residues in milk, after protein precipitation. FPSE is a microextraction technique characterized by high chemically stable sorbent, higher analyte retention capacity and faster analyte diffusion capability, where the target analytes interact with the extraction sorbent during the pre-treatment process via a plethora of intermolecular/interionic interactions exerted by different functional moieties implanted on the sponge-like porous sol–gel sorbent network. Subsequently, the selectively retained analytes are eluted using a small volume of solvent system. A novel extraction membrane containing sol–gel C18/propyl sulfonic acid, a mixed mode sorbent consisting of neutral, C18 and strong cation exchanger, propyl sulfonic acid (C3H6SO3−) coated on 100% cotton cellulose fabric substrate was designed, synthesized, characterized and evaluated for the selective isolation of four tetracycline antibiotic residues from milk samples.For quantitative analysis, high performance liquid chromatography coupled with a UV detector (at 350 nm) was used. The method was validated according to the 2002/657/EC decision with regards to linearity, selectivity, stability, limits of detection and quantitation (LOD/LOQ), decision limit, detection capability, trueness, precision and ruggedness, according to the Youden’s test. Recoveries ranged between 88.9 and 122.4%, while the LOD and LOQ were obtained at 15 μg kg−1 and 50 μg kg−1, respectively. Finally, the composite analytical method was applied to monitor the presence of residual tetracyclines in milk samples purchased from a local Greek market.

Ratiometric visual detection of tetracycline residues in milk by framework-enhanced fluorescence of gold and copper nanoclusters.

We reported a novel dual-emission fluorescence probe fabricated by encapsulating both gold (AuNCs) and copper nanoclusters (CuNCs) into zeolitic imidazolate framework-8 (ZIF-8). Obtained composite (AuCuNCs@MOF) was utilized for ratiometric determination of Tetracycline (Tcy) antibiotic. Under excitation at 400nm, the AuCuNCs@MOF composite displayed two emission peaks at 520 and 650nm which were originated from AuNCs and CuNCs, respectively. Upon the addition of Tcy, the red emission intensity of the nanoparticles at 615nm was significantly decreased, while the green emission at 520nm stayed almost constant which resulted in a clear fluorescence color change from red to green under a UV lamp. The logarithm of the fluorescence ratio against the concentration of Tcy exhibited a satisfactory linear relationship from 20 to 650nM with a detection limit (LOD) of 4.8nM. Current probe was applied for Tcy quantification in milk samples with superior results.

Effect of chloramphenicol on a bioassay response for the detection of tetracycline residues in milk

The tetracyclines are commonly used in veterinary medicine, and yet the residues are not always detected by microbiological inhibitor tests using Geobacillus stearothermophilus subsp. calidolactis C-953 at their Maximum Residue Limit levels. In order to improve the sensitivity of these methods, a bioassay was evaluated to study the effect produced by the incorporation of different chloramphenicol (CAP) concentrations in the culture medium. The specificity and detection limits of six tetracyclines in milk were determined. As the levels of CAP increased, a decrease in specificity, from 97.9% (for 0, 50, 200 and 400 μg/kg of CAP) to 88.0% (for 600 μg/kg of CAP) was observed. The logistic regression model indicates a significant effect of the CAP concentration. However, the tetracycline-CAP interaction was not significant, and thus, a synergetic effect can not be considered between the two antimicrobials, with only a simple sum of their antimicrobial effects. When the CAP concentration is increased from 0 to 400 μg/kg, the detection limits of chlortetracycline (590 μg/kg-316 μg/kg), doxycycline (115 μg/kg-62 μg/kg), meclocycline (105 μg/kg-52 μg/kg), oxytetracycline (446 μg/kg-273 μg/kg), rolitetracycline (191 μg/kg-134 μg/kg) and tetracycline (302 μg/kg-158 μg/kg) are observed to decrease.

Development of a High Pressure Liquid Chromatography with Diode Array Detection Method for the Determination of Four Tetracycline Residues in Milk by Using QuEChERS Dispersive Extraction

QuEChERS (quick, easy, cheap, effective, rugged and safe) dispersive extraction was applied for the extraction of tetracyclines (oxytetracycline, tetracycline, chlorotetracycline and doxycycline) from milk. Target analytes were determined by an accurate and sensitive chromatographic analytical method, which was validated in terms of 2002/6572/EC decision. The analytes were separated on an Orbit 100C4 (5 μm, 250 × 4.0 mm) analytical column under a gradient mobile phase composed of a mixture of 0.01 M oxalic acid, 10−4 M Na2EDTA and acetonitrile. For the extraction of isolated compounds from sorbent, a methanol and 0.01 M oxalic acid mixture (1:1 v/v) was used, leading to relative recovery rates from 83.07% to 106.3% at concentration levels in the range 100–200 μg/kg. The within-laboratory reproducibility, expressed as a relative standard deviation, was &lt;15.5%. Decision limits ranged between 100.3 μg/kg and 105.6 μg/kg and the detection capability varied between 100.6 μg/kg and 109.7 μg/kg. Ruggedness was evaluated by following the Youden approach, in terms of milk mass, sorbent mass, centrifugation time, vortex time, type and volume of organic solvents and evaporation temperature.

High-throughput amperometric determination of tetracycline residues in milk and quality control of pharmaceutical formulations: flow-injection versus batch-injection analysis

This paper demonstrates a potential use of the reduced graphene-oxide modified electrode associated with FIA and BIA systems for rapid, simple and sensitive determination of tetracycline in milk and pharmaceutical formulations samples.

Solid-Phase Extraction and Large-Volume Sample Stacking-Capillary Electrophoresis for Determination of Tetracycline Residues in Milk.

Solid-phase extraction in combination with large-volume sample stacking-capillary electrophoresis (SPE-LVSS-CE) was applied to measure chlortetracycline, doxycycline, oxytetracycline, and tetracycline in milk samples. Under optimal conditions, the proposed method had a linear range of 29 to 200 µg·L−1, with limits of detection ranging from 18.6 to 23.8 µg·L−1 with inter- and intraday repeatabilities < 10% (as a relative standard deviation) in all cases. The enrichment factors obtained were from 50.33 to 70.85 for all the TCs compared with a conventional capillary zone electrophoresis (CZE). This method is adequate to analyze tetracyclines below the most restrictive established maximum residue limits. The proposed method was employed in the analysis of 15 milk samples from different brands. Two of the tested samples were positive for the presence of oxytetracycline with concentrations of 95 and 126 µg·L−1. SPE-LVSS-CE is a robust, easy, and efficient strategy for online preconcentration of tetracycline residues in complex matrices.

Latex bead and colloidal gold applied in a multiplex immunochromatographic assay for high-throughput detection of three classes of antibiotic residues in milk

Two rapid and sensitive high-throughput immunochromatographic assays were successfully developed for simultaneous determination of 12 sulfonamide, 18 quinolone and six tetracycline residues in milk. Under optimized conditions, residues from these three classes of antibiotics can be qualitatively and quantitatively determined on a single strip within 10 min. The detection limits were much lower than the maximum residue limits established by the European Union. Comparative evaluations of sensitivity, antibody consumption, coefficient of variation and user experience between these two immunochromatographic assays showed that latex beads have advantages over colloidal gold when employed as a label in the assay. Therefore, the latex bead-based high-throughput immunochromatographic assay has the best potential for on-site testing of a large number of samples as part of food safety applications.

An electronic tongue based on conducting electrospun nanofibers for detecting tetracycline in milk samples

Impedimetric e-tongue based on conducting electrospun nanofibers provides a rapid and sensitive means for the detection of tetracycline residues in milk.

An iPhone-based digital image colorimeter for detecting tetracycline in milk

An iPhone-based digital image colorimeter (DIC) was fabricated as a portable tool for monitoring tetracycline (TC) in bovine milk. An application named ColorConc was developed for the iPhone that utilizes an image matching algorithm to determine the TC concentration in a solution. The color values; red (R), green (G), blue (B), hue (H), saturation (S), brightness (V), and gray (Gr) were measured from each pictures of the TC standard solutions. TC solution extracted from milk samples using solid phase extraction (SPE) was captured and the concentration was predicted by comparing color values with those collected in a database. The amount of TC could be determined in the concentration range of 0.5–10μgmL−1. The proposed DIC-iPhone is able to provide a limit of detection (LOD) of 0.5μgmL−1 and limit of quantitation (LOQ) of 1.5μgmL−1. The enrichment factor was 70 and color of the extracted milk sample was a strong yellow solution after SPE. Therefore, the SPE-DIC-iPhone could be used for the assay of TC residues in milk at the concentration lower than LOD and LOQ of the proposed technique.

A trace analysis of oxytetracycline and tetracycline residues in pasteurized milk supplied in Tehran: a one-year study (April 2011-March 2012)

BACKGROUND: Tetracyclines (TCs) are broad-spectrumantibiotics that are widely used in veterinary medicine. Thepresence of TCs residues in milk is a public health concern allover the world. OBJECTIVES:This study aimed to determine TCsresiduals in pasteurized milk marketed by some dairy companiesin Tehran from April 2011 to March 2012. METHODS: 432pasteurized milk samples were purchased from supermarketssupplying the milk products of 12 major dairy companies inTehran (3 samples from each company every month), and theywere stored at -20 0C until analysis. Oxytetracycline (OTC) andTetracycline (TC) residues in each sample were extracted by aliquid - liquid phase procedure and quantitated using a highperformance liquid chromatographic (HPLC) method. Chromatographicconditions included a mobile phase as oxalic acidbuffer- acetonitril (80: 20) with a flow rate of 1mL/min and UVdetectionat 355 nm. RESULTS: TCs residuals in most milksamples were lower than 100 ppb, maximum residue level(MRL); however, in seven samples (1.62%) the total residues ofOTC and TC were more than MRL. In the latter milk samples, themedian total TCs residue was 625 ppb, ranging between 274 and1270 ppb. CONCLUSIONS: Because of the presence of TCresidues above the MRL level in a limited number of milksamples, it is concluded that more studies and supervision ofhealth authorities are needed in this field.

Ultrasound-assisted dispersive extraction for the high pressure liquid chromatographic determination of tetracyclines residues in milk with diode array detection

Ultrasound assisted matrix solid phase dispersive extraction was applied for the selective isolation and clean-up of tetracyclines (oxytetracycline, tetracycline, epi-chlorotetracycline, chlorotetracycline and doxycycline) from milk. Target analytes were determined by an accurate and sensitive chromatographic analytical method, which was validated to meet the European Legislation criteria. The separation was performed on a LiChroCART-LiChrospher® 100 RP-18 (5μm, 250×4mm) analytical column, operated at ambient temperature, followed by diode array detection.Validation included investigation of linearity, selectivity, stability, limits of detection and quantitation, decision limit, detection capability, trueness, precision and ruggedness according to the Youden’s approach. Limits of quantitation of examined tetracyclines were from 14.5 to 56.6μg/kg significantly lower than respective Maximum Residue Limits, whereas recoveries ranged from 82.0% to 108%. The applicability of the method was evaluated using milk samples purchased from local market. Accuracy of the method was additionally proved by analysis of bovine milk certified reference material (BCR®-492).

High Throughput Detection of Tetracycline Residues in Milk Using Graphene or Graphene Oxide as MALDI-TOF MS Matrix

In this work, a new pre-analysis method for tetracyclines (TCs) detection from the milk samples was established. As a good accomplishment for the existing accurate quantification strategies for TCs detection, the new pre-analysis method was demonstrated to be simple, sensitive, fast, cost effective, and high throughput, which would do a great favor to the routine quality pre-analysis of TCs from milk samples. Graphene or graphene oxide was utilized, for the first time, as a duel-platform to enrich and detect the TCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). All together, four TCs were chosen as models: tetracycline, oxytetracycline, demeclocycline, and chlortetracycline. Due to the excellent electronic, thermal, and mechanical properties, graphene and graphene oxide were successfully applied as matrices for MALDI-TOF MS with free background inference in low mass range. Meanwhile, graphene or graphene oxide has a large surface area and strong interaction force with the analytes. By taking the advantage of these features, TCs were effectively enriched with the limit of detection (LOD) as low as 2 nM.

Synthesis and application of molecularly imprinted poly(methacrylic acid)–silica hybrid composite material for selective solid-phase extraction and high-performance liquid chromatography determination of oxytetracycline residues in milk

A novel molecularly imprinted organic–inorganic hybrid composite material (MIP-HCM) was developed based on molecular imprinting technique in combination with hybrid composite synthesis and sol–gel technology for selective solid-phase extraction (SPE) of tetracyclines residues in milk. The MIP-HCM was prepared using oxytetracycline as the template, methacrylic acid as organic functional monomer, tetraethoxysilane as inorganic precursor and methacryloxypropyltrimethoxysilane as the coupling agent. Synthesis conditions are optimized by changing some factors to obtain sorbent with the controllable adsorption capacity, selectivity, hardness and toughness. Binding study demonstrated that the imprinted hybrid composites showed excellent affinity and high selectivity to oxytetracycline. An enrichment factor of 18.8 along with a good sample clean-up was obtained under the optimized SPE conditions. The average recoveries of three tetracyclines antibiotics spiked milk at 0.1, 0.2 and 0.5mgkg−1 were in the range of 80.9–104.3% with the precision of 1.5–5.0%. The limits of detection and quantitation of the proposed method were in a range of 4.8–12.7μgkg−1 and 16.0–42.3μgkg−1, respectively.

Optimization of bioassay for tetracycline detection in milk by means of chemometric techniques

In this study, a microbiological method of dichotomous response using Bacillus cereus was designed and optimized to detect tetracyclines (TCs) at concentrations near to the maximum residue limits (MRLs). In a first stage, the response time of bioassay was reduced to 5 h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (2(6-3)) was analysed using logistic regression model. This design indicates significant effects of log S and chloramphenicol (CAP) on the detection limit (DL) of TC. Then, the response surfaces (RS) of the TCs DTs as a function of log S and CAP were plotted using a Dohlert design and the logistic regression model. These RS show a linear decrease with the raise of CAP and a quadratic effect of log S. Finally, the DTs of TC (109 μg l(-1)) and oxytetracycline (100 μg l(-1)) were adjusted to their MRLs through the desirability function. By successive application of experimental design techniques could be optimized a bioassay for the detection of TC residues in milk. The best conditions have been achieved when the assay was made with log S = 5·12 and CAP = 470 μg l(-1). Experimental design techniques together with the logistic regression model and the desirability function represent an adequate tool for the optimization of a bioassay with binary response.