High enrichment and sensitive measurement of oxytetracycline in tea drinks by thermosensitive magnetic molecular imprinting based magnetic solid phase extraction coupled with boron doped carbon dots.

As a typical kind of new pollutants, there are still some challenges in the rapid detection of antibiotics. In this work, a sensitive fluorescent probe based on boron-doped carbon dots (B-CDs) in combination with thermo-responsive magnetic molecularly imprinted polymers (T-MMIPs) was constructed for the detection of oxytetracycline (OTC) in tea drinks. T-MMIPs were designed, fabricated and employed to enrich OTC at trace level from tea drinks, and B-CDs were utilized as the fluorescent probe to detect the concentration of OTC. The proposed method exhibited good linear relationship with OTC concentration from 0.2 to 60 µg L-1 and the limit of detection was 0.1 µg L-1. The established method has been successfully validated with tea beverages. Present work was the first attempt application of T-MMIPs in combination with CDs in detection of OTC, and demonstrated that the proposed method endowed the detection of OTC with high selectivity, sensitivity, reliability and wide application prospect, meanwhile offered a new strategy for the method establishment of rapid and sensitive detection of trace antibiotics in food and other matrices.

Qualitative immunoassay for the determination of tetracycline antibiotic residues in milk samples followed by a quantitative improved HPLC-DAD method.

Environmental contaminant is one of several problems harming people and wildlife. An example of current emerging contaminants are antibiotics residues that can present in water and food. Although antibiotics are intended to treat or prevent human and animal infections, antibiotics have also been used as animal food supplements for their ability to promote growth and feed efficiency. This overuse of antibacterial has resulted in the accumulation of antibiotics residues in food products which are eventually consumed by human. The continuous unnecessary exposure of human to antibiotics through the direct animals meet or milk, or indirectly through plants or soil can increase the chance of the emergence of multi drug resistance bacteria and consequently adversely affecting human health. New regulations have been imposed regarding antibiotics utilization. Due to the scarce of data regarding antibiotics residue conditions in different types of food intended for human consumption in Saudi Arabia, this study proposed an optimized chromatographic method (HPLC-DAD) followed by an immunoassay approach for specifically detecting tetracyclines antibiotics in animal milk samples. The method was carried out using an RP-C18 column with a mobile phase consisting of 0.01 M KH2PO4: acetonitrile:methanol (70:20:10, v/v/v) adjusted to pH 4. Improvements were observed in the method in terms of resolution and sensitivity. The protein precipitation method used for extraction demonstrated high percent recoveries of 85-101%. The method was validated according to the guidelines of the International Conference for Harmonization (ICH). It is evidently clear from these findings that the presence of tetracycline and oxytetracycline antibiotics residues in milk products from the Saudi market are below the maximum residual limits (MRLs).

High-capacity removal of oxytetracycline hydrochloride from wastewater via Mikania micrantha Kunth-derived biochar modified by Zn/Fe-layered double hydroxide.

Antibiotic contamination in water has been an increasing global concern, and how to effectively remove antibiotics (e.g., oxytetracycline [OTC] hydrochloride) from wastewater becomes imperative. In this study, the biochar derived from an invasive plant (Mikania micrantha Kunth) was synthesized with Zn/Fe- layered double hydroxide (LDH) by co-precipitation method (ZnFe-LDH/MBC) to remove OTC from water. ZnFe-LDH/MBC posed the highest OTC removal performance of 426.61 mg/g. ZnFe-LDH/MBC exhibited stability and efficiency in OTC adsorption at different pH levels and under interfering conditions with co-existing ions, as well as outstanding regeneration capabilities during adsorption-desorption cycles. Furthermore, the removal of OTC by ZnFe-LDH/MBC was mediated by several processes including pore filling, hydrogen bonding force, electrostatic interaction, π-π interaction, as well as complexation. Consequently, ZnFe-LDH/MBC has excellent potential for the purification of OTC pollutants that is low-cost, efficient, and environmentally friendly.

A study of the effect of reactive oxygen species induced by violet and blue light from oxytetracycline on the deactivation of Escherichia coli.

Oxytetracycline (OTC), a tetracycline antibiotic, is a broad-spectrum antibacterial agent. In this investigation, liquid chromatography-mass spectrometry (LC-MS) is utilized to determine the effects of blue light (λ = 448 nm) illumination (BLIA) and violet light (λ = 403 nm) illumination (VLIA) on conformational changes in OTC at pH 7.8. The photochemical effect of OTC that is exposed to BLIA and VLIA on the deactivation of Escherichia coli (E. coli) is studied. The deactivation of E. coli has an insignificant effect on treatment with OTC alone. OTC is relatively unstable under BLIA and VLIA illumination in an alkaline solution, and OTC has been shown to inactivate E. coli by generating reactive oxygen species (ROS). Less anionic superoxide radicals (O2•-) are generated from OTC that is treated with BLIA than that from VLIA treatment, so OTC is more efficient in inactivating E. coli under VLIA. Inactivation of reduction rates of 0.51 and 3.65 logs in E. coli are achieved using 0.1 mM OTC under BLIA for 120 min and VLIA for 30 min, respectively, under the same illumination intensity (20 W/m2). Two photolytic products of OTC (PPOs) are produced when OTC is exposed to BLIA and VLIA, with molecular ions at m/z 447 and 431, molecular formulae C21H22N2O9 and C21H22N2O8, and masses of 446.44 and 430.44 g/mol, respectively. The results show that when exposed to VLIA, OTC exhibits enhanced inactivation of E. coli, suggesting that the photochemical treatment of OTC is a potential supplement in a hygienic process.

Oxytetracycline and paracetamol biodegradation performance in the same enriched feed medium with aerobic nitrification/anaerobic denitrification SBR.

Pharmaceuticals such as oxytetracycline and paracetamol are extensive chemicals in the aquatic systems. In this study, the removal performance of oxytetracycline and paracetamol was investigated in the same enriched feed water medium by sequencing batch aerobic/anaerobic reactor system. In this context, oxytetracycline and paracetamol in the aerobic phase were removed by a maximum of 66 and 99.8% respectively. At the same time, nitrification and denitrification removals were obtained as 95% and 98%, respectively. On the other hand, oxytetracycline and equivalent O2 flux of oxytetracycline maximum were calculated as 1.18 and 2.14 mg/L.d and the maximum removal volumetric flux of paracetamol and its O2 equivalent flux were determined approximately as 136 and 303 mg/L.d, simultaneously. In addition, oxytetracycline and paracetamol were given to the system in an amount of maximum 1 and 500 mg/L, respectively. Paracetamol has not significantly affected nitrification and denitrification up to 120 mg/L, but 500 mg/L paracetamol has completely finished denitrification in this system. On the other hand, the water environment of sequencing batc reactor has turned into a pitch dark state at 500 mg/L paracetamol feeding. As a result, aerobic bacteria preferred paracetamol rather than oxytetracycline. In other words, aerobic bacteria preferred paracetamol/oxytetracycline as the second electron acceptor after O2.

A fluorescent sensing strategy for ultrasensitive detection of oxytetracycline in milk based on aptamer-magnetic bead conjugate, complementary strand of aptamer and PicoGreen.

Misuse of antibiotics in animal husbandry and presence of their residues in animal foods is a serious crisis worldwide and thus, monitoring the level of them in food samples is vital for human health. Herein, a fluorescent aptasensor was developed for highly sensitive quantification of oxytetracycline (OTC) in food samples. This method is based on OTC aptamer conjugated to magnetic beads, functioned as recognition element, complementary strand of OTC aptamer, and PicoGreen (PG) as a sensitive double-stranded DNA (dsDNA) fluorescent dye. Formation of OTC aptamer-magnetic bead conjugate provides the opportunity of sample condensation and separation technology. Additionally, the presence of complementary strand leads to significant fluorescence signal alteration of aptasensor in the presence or absence of target and a noteworthy improvement of the aptasensor sensitivity. In the absence of target, complementary strand could bind to aptamer and form dsDNA on the surface of magnetic bead. As a consequence, adding PG to the sample leads to observation of high fluorescence signal from sample. In contrast, once OTC is added to the sample, it binds to OTC aptamer-magnetic bead complex and prevents hybridization of OTC aptamer and its complementary strand. Hence, after addition of PG to the sample, a weak fluorescence intensity is measured. Under optimized conditions, the linear ranges for OTC detection were 0.2-2 nM and 2-800 nM. The detection limit was calculated to be as low as 0.15 nM for the fabricated aptasensor. Besides the great sensitivity, proposed method demonstrated superior specificity towards OTC once it was used against several antibiotics. More significantly, the recovery rates of OTC in milk ranged from 96.46% to 101.5%, implying the great feasibility of designed sensor as well as its potential to be employed for analysis of OTC in real samples.

Aptamer-based thin film gold electrode modified with gold nanoparticles and carboxylated multi-walled carbon nanotubes for detecting oxytetracycline in chicken samples.

In this work, a disposable and portable aptasensor for the fast and sensitive detection of oxytetracycline (OTC) using gold nanoparticles (AuNPs)/carboxylated multi-walled carbon nanotubes (cMWCNTs)@thionine connecting complementary strand of aptamer (cDNA) as signal tags was constructed. The substrate electrode of the aptasensor was thin film gold electrode (TFGE), which have the advantages of portable and uniform performance. In the presence of OTC, OTC competed with cDNA to combine with aptamer. The bioconjugate (AuNPs/cMWCNTs/cDNA@thionine) was released from the TFGE. Thus, the electrochemical signal declined. Under optimized conditions, the aptasensor exhibited good stability, high selectivity and high sensitivity. Furthermore, the developed electrochemical aptamer-based TFGE had a wide dynamic range of 1 × 10-13-1 × 10-5 g mL-1 for target OTC with a low detection limit of 3.1 × 10-14 g mL-1 and was successfully used for the determination of OTC in chicken sample.

The acute effects of erythromycin and oxytetracycline on enhanced biological phosphorus removal system: shift in bacterial community structure.

Since extensive application, an increasing amount of antibiotics has been released into wastewater treatment plants. In this study, the enhanced biological phosphorus removal (EBPR) system was fed with synthetic wastewater containing erythromycin (ERY) and oxytetracycline (OTC) for 7 days to evaluate the variations of solution ortho-P (SOP), volatile fatty acid (VFA), poly-bhydroxyalkanoates (PHAs), specific oxygen uptake rater (SOUR), and microbial community in the EBPR system. The obtained results showed that the P-removal efficiency decreased to 0.0%, and at the end of the experiment, only less than 20% of the VFA could be consumed. Besides, the variable processes of P and PHAs were destroyed. Moreover, to better grasp the inhibitory mechanism of antibiotics, microbial community compositions of activated sludge sampled in all reactors were investigated by high-throughput sequencing techniques. Results of comparative and evolutionary analysis revealed that high concentrations (5 and 10 mg/L) of ERY and OTC could seriously shift microbial communities, while combined antibiotics could induce more. Additionally, Accumulibacter and Competibacter were two primary microorganisms at the genus level in the EBPR system. Accumulibacter decreased seriously for exposure to antibiotics, while Competibacter increased in all experimental reactors especially in combined antibiotics reactor.

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration.

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying ≥ 2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL-1 to 10ngmL-1, with the detection limits of 0.7pgmL-1 and 0.9pgmL-1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.

Ultrasensitive SERS aptasensor for the detection of oxytetracycline based on a gold-enhanced nano-assembly.

This paper investigated a new detection method of oxytetracycline (OTC) in aquatic products with ultrasensitive detection limit. The method was constructed on the basis of raman hot spot between gold nanoparticles (AuNPs) (13nm and 80nm diameter respectively) linked by an DNA sequence. The DNA sequence combined with the OTC aptamer including its complementary sequence as well as a stem-loop structure. The raman signal molecule (4-MBA) was modified at the surface of 13nm AuNPs. After the exposure of OTC, the aptamer sequence was preferentially combined with OTC and partially dehybridized with its complementary sequence which led the 13nm AuNPs to get more closer to the 80nm AuNPs. The raman intensity was thus increased for the more enhanced hot spot generated. Under the optimal experimental conditions, the SERS signal was positively related to the OTC concentration with a wide working range of 4.60×10-2-4.60×102fg/mL and the limit of detection (LOD) was as low as 4.35×10-3fg/mL. The recovery rates of fishmeal ranged from 91.29-110.98%. The specificity of this method was further examined, and the results showed that the AuNPs based aptasensor was highly selective. This developed ultrasensitive aptamer-based SERS detection platform suggested that it may be a promising strategy for a variety of sensing applications.

Antibacterial activity of oxytetracycline photoproducts in marine aquaculture's water.

Oxytetracycline (OTC) is one of the most used antibiotics in aquaculture. The main concern related to its use is the bacterial resistance, when ineffective treatments are applied for its removal or inactivation. OTC photo-degradation has been suggested as an efficient complementary process to conventional methods used in intensive fish production (e.g.: ozonation). Despite this, and knowing that the complete mineralization of OTC is difficult, few studies have examined the antibacterial activity of OTC photoproducts. Thus, the main aim of this work is to assess whether the OTC photoproducts retain the antibacterial activity of its parent compound (OTC) after its irradiation, using simulated sunlight. For that, three Gram-negative bacteria (Escherichia coli, Vibrio sp. and Aeromonas sp.) and different synthetic and natural aqueous matrices (phosphate buffered solutions at different salinities, 0 and 21‰, and three different samples from marine aquaculture industries) were tested. The microbiological assays were made using the well-diffusion method before and after OTC has been exposed to sunlight. The results revealed a clear effect of simulated sunlight, resulting on the decrease or elimination of the antibacterial activity for all strains and in all aqueous matrices due to OTC photo-degradation. For E. coli, it was also observed that the antibacterial activity of OTC is lower in the presence of sea-salts, as demonstrated by comparison of halos in aqueous matrices containing or not sea-salts.

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline.

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF4:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I530 to I477 nm (I530/I477) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml.

Residues of oxytetracycline in cultured rainbow trout.

Nowadays, antibiotics are widely used in aquatic animals to control and treatment of infections or as food supplement for growth increase and animal output. With increasing use of veterinary drugs in food production, there is global consideration about the consumption of antimicrobial residues in aquatic foods and their effects on human health. This study was aimed to evaluate the Oxytetracycline (OTC) residues in Rainbow trout meat in Shahre-kord (Iran) markets before and after frying. After randomized collection of 50 samples of fish in Shahre-kord markets in a six months period were examined. The prepared samples were examined for OTC residues using HPLC analytical method before and after frying. Results showed that 3 (6%) of the samples before frying and 12 (24%) after frying were having lower than Maximum residual limits (MRLs) in Codex alimentarius. However, mean OTC residues before and after frying samples were above MRLs. The mean amounts of OTC were 2260 +/- 1090 and 1110 +/- 930 ng g(-1) before and after frying, respectively. These findings show that the frying of fish reduces OTC residual. Nevertheless, the usage of OTC should be reduced to an acceptable level in fishery industry.

The operation of two EGSB reactors under the application of different loads of oxytetracycline and florfenicol.

This study evaluated the effect of the antibiotics oxytetracycline (OTC) and florfenicol (FLO) on the operation of two EGSB (expanded granular sludge bed) reactors. The experiment was conducted for 210 d in reactor R1 and 245 d in reactor R2. The reactors were inoculated with granular sludge from a upflow anaerobic sludge blanket (UASB) reactor on a local dairy farm. The sludge had an average pellet size of 2.35 mm, good sedimentability and a high percentage of organic material. The antibiotic tolerance and the inhibitory action on the bacterial population were different for each antibiotic studied. The results showed a more severe inhibitory effect on microorganisms that were in contact with increases in loads of FLO than those that were in contact with increasing loads of OTC, a condition reflected in the chemical oxygen demand (COD) removal efficiency.

A molecularly imprinted photonic polymer sensor with high selectivity for tetracyclines analysis in food.

A molecularly imprinted photonic polymer (MIPP) sensor for respective detection of tetracycline, oxytetracycline and chlortetracycline is developed based on the combination of a colloidal crystal templating method and a molecular imprinting technique. Colloidal crystal templates are prepared from monodisperse polystyrene colloids. The molecularly imprinted polymer, which is embodied in the colloidal crystal templates, is synthesized with acrylic acid and acrylamide as monomers, N,N'-methylene bisacrylamide as a cross-linker and tetracyclines (TCs) as imprinting template molecules. After removal of the colloidal crystal template and the molecularly imprinted template, the resulted MIPP consists of a three-dimensional, highly ordered and interconnected macroporous array with a thin hydrogel wall, where nanocavities complementary to analytes in shape and binding sites are distributed. The response of MIPP to TCs stimulants in aqueous solution is detected through a readable Bragg diffraction red-shift, which is due to the lattice change of MIPP structures responding to their rebinding to the target TCs molecules. A linear relationship was found between the Δλ and the concentration of TCs in the range from 0.04 µM to 0.24 µM. With this sensory system, direct and selective detection of TCs has been achieved without using label techniques and expensive instruments. The developed method has been applied successfully to detect tetracycline in milk and honey samples.

Ultrasonic or accelerated solvent extraction followed by U-HPLC-high mass accuracy MS for screening of pharmaceuticals and fungicides in soil and plant samples.

Different veterinary pharmaceuticals are used in agricultural livestock becoming a source of environment contamination. Furthermore, no regulation exists for the concentration limits of pharmaceuticals in soil or water. Monitoring programs for environment contamination with pharmaceuticals are needed, requiring new sensitive and selective screening methods. The present study focuses on developing a method for the simultaneous scanning of forty-two compounds (pharmaceuticals, azole biocides and fungicides) in soil and plant material samples. For extraction purposes the use of ultrasonic assisted and accelerated solvent extraction (ASE) were compared. The extract was purified and concentrated by applying a solid phase extraction step followed by ultra-high-performance-chromatographic separation and accurate-mass spectrometric detection using Exacte Orbitrap technology (FWHM 50,000). The effects of the different extraction solvents and conditions on the extraction efficiency were tested. Although both extraction approaches are applicable the optimal extraction efficiency was obtained by applying accelerated solvent extraction using solvent mixtures containing acetone for soil and methanol for plant samples. An ASE process has been validated for the determination of selected pharmaceuticals and fungicides in soil and in plant material. The recoveries from soil samples were >70% for more than 68% of the compounds. The levels of detection were ≤10 µg kg(-1) for 93% of the compounds tested. The recoveries from plant material were >70% for 64% of the compounds tested. The levels of detection were ≤10 µg kg(-1) for 66% of the compounds. The developed method was used to screen soil and plant material collected throughout the Netherlands and oxytetracycline residues were detected.

Determination of oxytetracycline/oxytetracycline hydrochloride in animal feed, fish feed, and veterinary medicinal products by liquid chromatography with fluorescence detection: collaborative study.

A method for determining oxytetracycline (OTC) in animal feed, fish feed, and veterinary medicinal products at medicated use and contamination levels was collaboratively studied. The method is applicable to the analysis of animal feeds and mineral premixes containing levels > or =2 mg/kg, and fish feed containing levels > or =10 mg/kg. Oxytetracycline hydrochloride (OTC.HCI) is extracted from ground feed material in acid-methanol solution using mechanical agitation. After centrifugation for 5 min at 1230 x g, an aliquot of the extract is diluted with water andlor acid-methanol so that the concentration of OTC.HCI is approximately the same as that in the working standard, and the solutions contain at least 50% water. Injectable veterinary medicinal materials (also called animal remedy materials) are diluted with water andlor extractant to reach the target concentration. The extracts are filtered and analyzed by reversed-phase liquid chromatography with fluorescence detection with excitation at 390 nm and emission at 512 nm. Twenty-eight test samples of medicated feeds, supplements, and drug premixes, including 4 test samples for trace-level analysis, were sent to 17 collaborators in Canada, The Netherlands, and the United States. Results were received from 11 laboratories. The RSDr values (within-laboratory repeatability) ranged from 1.26 to 9.21%; RSDR values (among-laboratory reproducibility) ranged from 2.14 to 12.9%, and HorRat values ranged from 0.54 to 3.02. It is recommended that this method be adopted AOAC Official First Action.

Effects of European foulbrood treatment regime on oxytetracycline levels in honey extracted from treated honeybee (Apis mellifera) colonies and toxicity to brood.

This study aimed to assess oxytetracycline (OTC) residue levels in honey up to 12 weeks after treatment of honeybee colonies with two methods of application (in liquid sucrose and in powdered icing sugar). Significantly greater mortality was seen in the all stages of brood development for the treated colonies when compared with the controls. Samples of honey were extracted up to 12 weeks after treatment and analysed by HPLC following metal chelation with a limit of quantitation of 0.05 mg/kg. These data showed that the current method of application of Terramycin in liquid form results in very high residue levels in honey with residues of 3.7 mg/kg eight weeks after application. Further work is required to determine whether the levels can be further reduced by changes in the method of application whilst ensuring efficacy and minimizing the effects on brood.

Quantitative analysis of oxytetracycline and its impurities by LC-MS-MS.

A liquid chromatographic-tandem mass spectrometric method using an Xterra MS C(18) chromatographic column ( 100 mm x 2.1 mm i.d., 3.5microm) that allows complete separation of oxytetracycline (OTC) and the impurities: 4-epi-oxytetracycline (EOTC), tetracycline (TC), 4-epi-tetracycline (ETC), 2-acetyl-2-decarboxamido-oxytetracycline (ADOTC), alpha-apo-oxytetracycline (alpha-AOTC) and beta-apo-oxytetracycline (beta-AOTC) was developed. Gradient elution was used and calibration curves were obtained using the scan mode selected reaction monitoring (SRM). Acceptable correlations were obtained for OTC, TC, EOTC and ADOTC whereas the correlations of alpha-AOTC and beta-AOTC were less accurate resulting in higher limits of quantification (LOQ) and limits of detection (LOD) relative to the other compounds. The intraday and interday accuracy varied for all the compounds from 90 to 112% and the intraday and interday precision were lower than 7.1%. The method was applied for analysis of commercial available ointments containing OTC resulting in an acceptable quantification of OTC and the impurities in the drug preparations. The advantage of this method compared to the other separation methods is an empty separation window right after the large peak corresponding to OTC in the chromatogram, which facilitates an accurate determination of ADOTC and the other impurities.