Chemiluminescence Determination of Tetracyclines via Aluminum Sensitized Fluorescence

Tetracycline, oxytetracycline, doxycycline, and chlortetracycline have been determined by chemiexcitation of the corresponding Al(III) highly fluorescent complex from the permanganate or cerium(IV)-sulphite chemiluminogenic reactions. Limits of detection and ranges of linearity are equal to 0.024, 0.015, 0.014, and 0.050 µg mL^?1 and 0.067–3.20, 0.042–1.70, 0.042–3.00, and 0.103–2.80 µg mL^?1 for tetracycline, oxytetracycline, doxycycline, and chlortetracycline, respectively. Average recovery of tetracyclines from solutions of commercial formulations was equal to 99.8% and the procedure was successfully applied to the determination of tetracyclines in commercial products with mean relative error equal to 3.4% (range 1.4–5.0%).     

Confirmatory development and validation of HPLC-DADmethod for the determination of tetracyclines in gilthead seabream (Sparus aurata) muscle tissue

A confirmatory high-pressure liquid chromatographic method for the determination of nine tetracyclines in Sparus aurata (gilthead seabream) muscle tissue is developed and presented herein. Tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, chlortetracycline, 4-epi-chlortetracycline, doxycycline, methacycline and demeclocycline were separated on a Kromasil, C18 (250 mm × 4 mm, 5 μm) analytical column by gradient elution with a mobile phase consisting of 0.001 M ethylenediaminetetraacetic acid/sodium salt and acetonitrile at 25°C. Diode array detection with monitoring at 280 nm (for the determination of chlortetracycline, 4-epi-chlortetracycline, methacycline and demeclocycline) and 355 nm (for tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline and demeclocycline) was applied for peak identification and quantification of analytes. Examined antibiotics were isolated from gilthead seabream tissue by leaching using a citrate buffer (pH 4.0) and purified by solid phase extraction using Oasis HLB(200 mg/6 mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC.     

Determination of tetracycline residues in honey by CZE with ultraviolet absorbance detection

We have developed a sensitive CE method to determine eight tetracyclines (TCs) (chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, TC, and rolitetracycline (RTC)) in honey samples. The running buffer was 150 mM sodium borate (pH 9.8) and 2.5% 2-propanol with 15 s hydrodynamic injection at 25 kV. We have also developed an SPE procedure with a C18 cartridge as a clean-up step. Analytes were detected at 360 nm in less than 16 min. LODs ranged in honey from 23.9 microg/kg for TC to 49.3 microg/kg for RTC. Seven samples of Spanish honey of different floral origins were examined. None of them showed contamination with these antibiotics using the proposed method.     

Trace measurements of tetracyclines using adsorptive stripping voltammetry

The surface-active properties of tetracycline antibiotics are exploited for developing a sensitive adsorptive stripping method for trace measurements of these compounds. Controlled interfacial accumulation at the h.m.d.e. permits convenient quantitation at the submicromolar and nanomolar concentration levels. With 210 s accumulation, the method provides 28, 27, 26 and 23 signal enhancements for tetracycline hydrochloride, oxytetracycline, chlortetracycline and doxycycline, respectively. The adsorptive stripping response is evaluated with respect to accumulation time and potential, stripping mode, concentration dependence, electrolyte and pH, and other variables. Detection limits are 6 × 10⁻¹⁰ M for doxycycline, 1 × 10⁻⁹ M for oxytetracycline and chlortetracycline, and 2 × 10⁻⁹ M for tetracycline hydrochloride with 300 s accumulation. The reproducibility of the determination (at the 1 × 10⁻⁷ M level) expressed in terms of relative standard deviation, ranges from 0.8 to 2.0%.     

Reversed-phase high-performance liquid chromatography coupled to ultraviolet and electrospray time-of-flight mass spectrometry on-line detection for the separation of eight tetracyclines in honey samples

Eight antibiotics, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline and rolitetracycline, were separated and quantified in Spanish honey extracts of different floral origin using a commercial RP-C18 HPLC column and two different on-line detectors (diode array and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) systems). Operating a linear gradient at a flow of 2 ml min(-1) the HPLC separation of the eight antibiotics was obtained within 10 min with good peak symmetry and an acceptable resolution (2.1) for the critical band pair rolitetracycline and oxytetracycline. Values of the numbers of theoretical plates (N) were comprised between 2328 and 19448 while the limits of detection in honey were within 0.02-1.03 microg kg(-1) in the case of UV detection and 0.05-0.76 microg kg(-1) for ESI-TOF-MS detection (operating in negative mode). A recovery study was carried out by preparing some quality control samples at four levels of concentration (10, 25, 50 and 100 microg kg(-1)) and percentages between 72% and 98% were attained.     

Determination of Tetracyclines in Honey Using Liquid Chromatography with Ultraviolet Absorbance Detection and Residue Confirmation by Mass Spectrometry

A determination method has been optimized and validated for the simultaneous analysis of tetracycline （TC）, oxytetracycline （OTC）, chlortetracycline （CTC） and doxycycline （DC） in honey. Tetracyclines （TCs） were removed from honey samples by chelation with metal ions bound to small Chelating Sepharose Fast Flow columns and eluted with Na2EDTA-Mcllvaine pH 4.0 buffers. Extracts were further cleaned up by Oasis HLB solid-phase extraction （SPE）, while other solid-phase extraction cartridges were compared. Chromatographic separation was achieved using a polar end-capped C 18 column with an isocratic mobile phase consisting of oxalic acid, acetonitrile and methanol. LC with ultraviolet absorbance at 355 nm resulted in the quantitation of all four tetracycline residues from honey samples fortified at 15, 50, and 100 ng/g, with liner ranges for tetracyclines of 0.05 to 2 μg/mL. Mean recoveries for tetracyclines were greater than 50% with R.S.D. values less than 10% （n= 18）. Detection limits of 5, 5, 10, 10 ng/g for oxytetracycline, tetracycline, chlortetracycline and doxycycline, respectively and quantitation limits of 15 ng/g for all the four tetracyclines were determined. Direct confirmation of the four residues in honey （2-50 ng/g） was realized by liquid chromatography-tandem mass spectrometry （LC/MS/MS）. The linear ranges of tetracyclines determined by LC/MS/MS were between 5 to 300 ng/mL, with the linear correlation coefficient r〉 0.995. The limits of detection of 1 to 2 ng/g were obtained for the analysis of the TCs in honey.     

Flow Injection Chemiluminescence Determination Of Tetracyclines

Abstract

A fast and simple flow injection chemiluminescence method for the determination of trace amounts of tetracyclines is proposed based on the chemiluminescence of the KMnO₄ - TCs redox reaction in acidic medium in the presence of a sensitizer, OP. The response to the concentration of tetracyclines is linear in the range of 1.00 to 1000 μg mL^?1 with a relative standard deviation < 2.3 % for determination of 50.0 μg mL^?1 of each antibiotic (n = 11). The detection limits for tetracycline, oxytetracycline and chlortetracycline are 0.40, 0.52 and 0.60 μg mL^?1, respectively. The recommended method is suitable for automatic and continuous analysis and has been successfully tested for determination of tetracyclines in commercial formulations. The chemiluminescence intensity was found to be remarkably enhanced when in the presence of OP micellar system; possible reasons for this observed micellar induced enhanced chemiluminescence is given.     

Complexometric-spectrophotometric assay of tetracyclines in drug formulations

An accurate, rapid and very simple spectrophotometric method for the assay of tetracyclines (tetracycline.HCl, chlorotetracycline.HCl, demeclocycline, oxytetracycline.HCl and doxycycline) has been developed. The method is based on the complexation of iron(III) with tetracyclines in 0.001M sulphuric acid. It has been successfully applied to the assay of tetracyclines in drug formulations, and the interferences of excipients have been examined. The results have been statistically compared with those obtained by two standard methods and found to be very satisfactory.     

Spectrophotometric determination of tetracyclines and cephalosporins with ammonium vanadate

A spectrophotometric method for the determination of some tetracyclines as well as some cephalosporins is described. The drug is boiled with ammonium vanadate solution in sulphuric acid medium for 10 min and the absorbance of the colour developed is measured at 750 nm. The proposed method can be successfully applied to the determination of tetracycline hydrochloride, oxytetracycline hydrochloride, doxycycline hyclate, demeclocycline hydrochloride, chlortetracycline hydrochloride, cephalothin sodium, cephaloridine and cephapirin sodium. These drugs can be determined either in pure form or in pharmaceutical preparations.     

规模化养殖场废水中抗生素种类及残留特征研究

2010年调查了海南省6家规模化养猪场抗生素使用品种及来源,并采集养殖废水样品,用高效液相色谱紫外检测器检测了样品中4种四环素(四环素、土霉素、金霉素和强力霉素)和8种磺胺类药物(磺胺嘧啶、磺胺甲噻唑、磺胺噻唑、磺胺二甲基嘧啶、磺胺氯哒嗪、磺胺邻二甲氧嘧啶、磺胺间二甲氧嘧啶和磺胺喹恶啉)的残留浓度水平。结果显示,6家养殖场废水中土霉素、四环素和磺胺嘧啶的检出率及检出浓度较高,其中土霉素检出率为100%,最高浓度为71.75μg/L;四环素检出率为63%,最高检出浓度为24.83μg/L;磺胺嘧啶检出率为83%,最高检出浓度为17.69μg/L。4种四环素类检出总量变化范围为18.25～99.64μg/L,8种磺胺类检出总量变化范围为3.45～24.49μg/L;从养殖规模上看,小规模养殖场抗生素类检出品种较多,检出浓度也较高。     

Identification of tetracycline antibiotics by electrospray ionization in a quadrupole ion trap

Tetracycline antibiotics, tetracycline, chlortetracycline, demeclocycline, doxycycline, minocycline, methacycline, oxytetracycline, and anhydrotetracycline, are examined by electrospray ionization in a quadrupole ion trap. Studies were undertaken to evaluate the use of metal complexation as an alternative to conventional proton attachment. A variety of metal cationization processes, including attachment of Na⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, and Cu²⁺ were probed. Infrared multiphoton photodissociation and collisionally activated dissociation (CAD) were compared for generation of diagnostic fragmentation patterns of protonated and metal cationized tetracyclines. The photodissociation spectra provide a more informative signature, including more low mass ions that are not observed upon CAD. The metal complexes dissociate by pathways that are similar to those observed for the protonated molecules.     

Tetracycline Potentiometric Sensor Based on Cyclodextrin for Pharmaceuticals and Waste Water Analysis

In this work, the construction and performance evaluation of PVC membranes ion‐selective electrodes for the determination of tetracycline type antibiotics is described. Electrodes with the best responses were based on plastic membranes containing 31% (w/w) PVC, 68% (w/w) of dibutylphthalate as plasticizer and 1% (w/w) of β‐cyclodextrin as ionophore. The electrodes were responsive over 6 months to tetracycline, oxytetracycline, doxycycline and chlortetracycline in glycine buffer solution (pH 2), in the dynamic range 2×10^?5–10^?2 mol L^?1 with a constant slope of about 55 mV/dec To enable large scale analysis reducing wastes as associated costs tubular shape electrodes were coupled to a sequential‐injection analysis system and its performance evaluated using pharmaceutical samples and waste waters from a treatment facility. The sample throughput of 51 samples h^?1 was enabled by the system as well as results that favorably agree with those provided by chromatographic analysis.     

Temperature-Induced Ionic Liquids Dispersive Liquid�CLiquid Microextraction of Tetracycline Antibiotics in Environmental Water Samples Assisted by Complexation

In this study, dispersive liquid?Cliquid microextraction (DLLME) combined with ultra high pressure liquid chromatography (UHPLC)?Ctunable ultraviolet detection (TUV), was developed for pre-concentration and determination of trace levels of tetracyclines, including 4-epitetracycline, 4-epichlortetracycline, doxycycline, chlortetracycline oxytetracycline, tetracycline, 4-epianhydrotetracycline and anhydrotetracycline, in aqueous samples. La (III) was used as the chelating agent to form a hydrophobic complex compound with tetracyclines, followed by extraction with ionic liquids. Some important parameters that may affect extraction efficiencies were examined and optimized. Under the optimum conditions, linearity of the method was observed in the range of 0.1?C200 ??g L^?1, with correlation coefficients (r ²) >0.992. The limits of detection and quantification were 0.031?C0.079 and 0.10?C0.26 ??g L^?1, respectively. The spiked recoveries of eight target compounds in river water, fishpond water and hog leachate were achieved in the range of 62.6?C96.3, 58.9?C94.5, 55.1?C86.1%, respectively.     

Determination of Tetracyclines by Solid-Phase Extraction with a Molecularly Imprinted Polymer and High-Performance Liquid Chromatography

Tetracyclines are widely used antibiotics classified as emerging pollutants and may lead to an increase in bacterial resistance in the environment. In order to determine these compounds at low concentrations, a water-compatible molecularly imprinted polymer was developed for solid-phase extraction followed by high-performance liquid chromatography analysis. The monomers 2-hydroxyethyl methacrylate and glycerol dimethacrylate were added 1 h after the start of the synthesis to provide hydroxyl groups on the polymer surface. This hydrophilic layer established hydrogen bonds with water, minimizing interferences of this solvent in the analyte-polymer complex, increasing analyte adsorption. The polymer was then used for solid-phase extraction to preconcentrate the tetracyclines. The method provided low limits of quantification (5 µg L^?1), good linearity, precision, and accuracy for tetracyclines, with preconcentration factors of 14, 19, 29, and 41 for oxytetracycline, tetracycline, chlortetracycline, and doxycycline, respectively.     

Magnetic solid phase extraction based on phenyl silica adsorbent for the determination of tetracyclines in milk samples by capillary electrophoresis

A magnetic solid phase extraction method coupled to capillary electrophoresis is proposed for the determination of tetracycline, oxytetracycline, chlortetracycline and doxycycline in milk samples. Five different magnetic phenyl silica adsorbents covered with magnetite were synthesized by varying the molar ratio of phenyltrimethylsilane and tetramethylorthosilicate; these adsorbents were evaluated in terms of their pH and degree of hydrophobicity for tetracycline retention. The optimal, selected combination of conditions was a pH of 10.0 and a magnetic sorbent ratio of 4:1; under these conditions, the retention capacity ranged from 99.7% to 101.2% for the four tetracyclines analyzed. The elution conditions and initial sample volume of the proposed extraction method were also optimized, and the best results were obtained with 1×10(-3) M acetic acid in methanol as eluent and a 200 ml of sample volume. Under optimal conditions, average recoveries ranged from 94.2% to 99.8% and the limits of detection ranged from 2 to 9 μg l(-1) for the four tetracyclines. After the proposed method was optimized and validated, 25 milk samples of different brands were analyzed, oxytetracycline residues were detected in five samples, in concentrations ranging from 98 to 213 μg l(-1). Subsequent analysis of positive samples by SPE-CE and magnetic solid phase extraction-HPLC revealed than no significant differences were found from results obtained by the proposed methodology. Thus, the developed magnetic extraction is a robust pre-concentration technique that can be coupled to other analytical methods for the quantitative determination of tetracyclines.     

Simultaneous Determination of Oxytetra- cycline, Doxycycline, Tetracycline and Chlortetracycline in Tetracycline Antibiotics by High-Performance Liquid Chromatog- raphy with Fluorescence Detection

A simple, rapid and sensitive high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of oxytetracycline, doxycycline, tetracycline and chlortetracycline was developed, and successfully applied to the analysis of commercial tetracycline antibiotics. The separation was performed on a reverse-phase C₁₈ column with a gradient elution composed of methanol and sodium acetate buffer (containing disodium ethylenediaminetetraacetate and calcium chloride, pH 8.10) as the mobile phase, and fluorescence detection at 532 nm (excitation at 380 nm). The detection limits for oxytetracycline, doxycycline, tetracycline and chlortetracycline were 0.1, 0.5, 0.3 and 0.4 g L^–1, respectively. Data with respect to precision and accuracy were reported and discussed.     

Phosphorimetric Detection in HPLC VIA Trivalent Lanthanides: High Sensitivity Time-Resolved Luminescence Detection of Tetracyclines Using Eu3+ in a Micellar Post Column Reagent

Abstract

The detection of tetracyclines in HPLC by sensitized Europium phosphorescence has been reinvestigated using a dedicated LC detector with time-resolved luminescence capability. A micellar Eu³⁺ post column reagent was developed which contained 1% Triton X-100 and 200uM tri-n-octyl phosphine oxide buffered at pH 10.5. This reagent provided optimized time-resolved detection and was found to be compatible with a several mobile phases used in reversed phase chromatography of tetracyclines. The sensitivity and quantitative linearity of the technique was determined using standard fluorometric and time-resolved modes of detection with both grating and filter emission monochromation. Peak areas were linear with concentration from 2 ug on column to the limit of detection. Subnanogram limits of detection were obtained for both tetracycline and oxytetracycline. The relative sensitivity of the technique for compounds tested was oxytetracycline=tetracycline > doxycycline > chlortetracycline > minocycline. The high selectivity of this method promises to be useful in     

高效液相色谱法检测畜禽肉中四环素类药物残留的条件优化

对畜禽肉中四环素类残留的检测方法进行改进,选用Na2EDTA-McIlvaine缓冲溶液作为提取液进行超声提取,经C18固相萃取小柱净化,脱溶剂后用流动相三氟乙酸-乙腈溶解,使用高效液相色谱-PDA检测器分段采集355 nm及270 nm波长下的色谱信息,对前处理方法及色谱条件进行优化。四环素、土霉素在0.01～1μg/g范围内,金霉素、多西环素在0.02～2μg/g范围内回收率为60%～85%,相对标准偏差小于5%,可以满足国家残留限量0.1～0.6μg/g的检测要求。     

Spectrophotometric determination of molybdenum with phenylfluorone

Spectrophotometric studies on the reaction between molybdenum as molybdate (MoO₄^2?) and phenylfluorone are presented. The reaction conditions are optimized to develop an intense color (molar absorptivity is 3.8 × 10³) selective and sensitive for the Spectrophotometric determination of molybdenum. The absorbance is measured at 560 nm, at a pH of 1.5–3. The colored complex is stable for up to 24 hr, Beer's law is obeyed, over the concentration range of 1 to 4 μg/25 ml. The relative standard deviation is 2% and the sensitivity of the method is 1.60 × 10^?4 mg/ml.     

CUTANEOUS PHOTOTOXICITY OF TETRACYCLINE ANTIBIOTICS: GENERATION OF FREE RADICALS and SINGLET OXYGEN DURING PHOTOLYSIS AS MEASURED BY SPIN-TRAPPING and THE PHOSPHORESCENCE OF SINGLET MOLECULAR OXYGEN

Chlortetracycline (CTC) generated an aryl radical in aqueous buffer (pH 7.4) during near UV irradiation, as evidenced by the formation of 2-methyl-2-nitrosopropane spin adducts. The radical was produced via dechlorination, a photoprocess not previously reported for tetracyclines. Demeclocyc-line (DEM), another chlorinated tetracycline, did not produce detectable aryl radicals. Relative ¹O₂ yields obtained by direct luminescence measurements at 1268 nm for five tetracyclines in alkaline ethanol (demeclocycline · tetracycline · chlortetracycline · doxycycline · minocycline) showed that DEM produced approximately three times as much singlet oxygen as CTC. This constitutes direct evidence that tetracyclines sensitize both Type I and Type II photoreactions.