High-Performance Liquid Chromatography of Derivatives Involved in the Terminal Steps of Tetracycline Biosynthesis in Streptomyces Aureofaciens

Abstract

A simple and reproducible method was developed for the analysis of tetracycline derivatives involved in the last two steps of tetracycline biosynthetic pathway in Streptomyces aureofaciens. The method is based on a gradient liquid chromatographic separation of the compounds using a microbore octadecyl silica column. Beside separation of a mixture of standards, the method was used for separation, detection and quantitation of dehydrotetracycline and tetracycline prepared enzymatically in vitro using anhydrotetracycline oxygenase and tetracycline dehydrogenase, respectively, isolated from S. aureofaciens. The method permits a simple and accurate characterization of kinetics of corresponding enzymatic activities.     

Spectrophotometric Determination of Tetracyclines in Pure Form and in Pharmaceutical Preparations,by a Molybdenum Blue Method

Abstract

A new and sensitive spectrophotometric method has been developed for the determination of tetracyclines either in a pure form or in Pharmaceuticals, by a molybdenum blue method. The procedure is based on the observation that, in sulphuric acid medium, tetracyclines reduce ammonium molybdate to molybdenum blue, the absorbance of which is proportional to the amount of antibiotic present. The variables affecting development of the color have been investigated and the conditions optimized. Beer's law is obeyed for up to 20 μg/ml of tetracycline HCl and oxytetracycline HCl, 28 μg/ml of demeclocycline HCl, 18 μg/ml of chlortetracycline HCl, 32 μg/ml of doxycycline HCl and 40 μ/ml of rolitetracycline. Molar absorptivities (1 mol^?1 cm^?1) and Sandell's sensitivities (μ cm^?2 per 0.001 absorbance unit) are, respectively: tetracycline HCl 4.9×10⁴ and 0.0098, oxytetracycline HCl 5.4×10⁴ and 0.0092, demeclocycline HCl 1.6×10⁴ and 0.0313, chlortetracycline HCl 5.5×10⁴ and 0.0094, doxycycline HCl 3.4×10⁴ and 0.0141, rolitetracycline 2.7×10⁴ and 0.0195.     

4-Aminoantipyrine as an Analytical Reagent for the Colorimetric Determination of Tetracycline and Oxytetracycline

Abstract

A spectrophotometric method is proposed for the determination of tetracycline and oxytetracycline and their dosage forms. The suggested method depends on the reaction with 4-aminoantipyrine in the presence of an alkaline oxidising agent. A red antipyrine dye is produced. The reaction ratio has been determined. Variables such as pH, temperature, reagent concentration, stability of the colour produced have been evaluated to permit selection of the most advantageous technique. Beer's law was obeyed over the concentration range 0.04 - 0.12 mg/ml and 0.04 - 0.16 mg/ml for tetracycline and oxytetracycline respectively.     

Microbiological Assay of Tetracycline with a Potentiometric CO2 Gas Sensor

Abstract

A novel potentiometric method for the determination of the antibiotic tetracycline in pharmaceutical preparations is reported. The method is based on the inhibition of respiration of a suspension of Escherichia cole, which is measured with a potentiometric carbon dioxide sensor. The dose-response curve of carbon dioxide produced versus the logarithm of the tetracycline hydro-chloride concentration is linear from 33 to 167 μg/ml. Good agreement with the label claim was obtained in the assay of a pharmaceutical preparation of tetracycline hydrochloride.     

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     

Highly-sensitive ion selective electrode based on molecularly imprinted polymer particles for determination of tetracycline in aqueous samples

In this work, a highly-sensitive polymeric membrane ion selective electrode for determination of tetracycline was constructed by using molecularly imprinted polymer (MIP) particles as quasi-ionophore. The water-compatible MIP particles targeting tetracycline were synthesized with tetracycline as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2′-azobisisobutyronitrile as an initiator and lanthanum ion as a mediator. Benefited from the distinctive performance of the quasi-ionophore and the optimized composition of the membrane and the inner filling solution, the lower detection limit of the electrode was decreased to about 1 × 10⁻⁸ mol/l. It exhibited a good electrode slope 59.8 mV/decade near the theoretical Nernstian one, with a wide linear working range from 2.0 × 10⁻⁸ to 1.0 × 10⁻³ mol/l. Due to the specific recognition of tetracycline by the MIP particles, the selectivity coefficients for routine interferences were less than 10⁻⁴. The fabricated electrode should be used in pH 2–4, response time of which was less than 200 s when the concentration of tetracycline was higher than 1.0 × 10⁻⁶ mol/l and no more than 30 min at the concentration of 1.0 × 10⁻⁸ mol/l. Finally, the proposed highly-sensitive ion selective electrode has been successfully applied to the determination of tetracycline in aqueous samples.     

Development and validation of an HPLC method for tetracycline‐related USP monographs

A novel reversed‐phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4‐epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4‐epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H₃PO₄ as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80–120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50–150% of the acceptance criteria specified in the individual USP monographs for 4‐epianhydrotetracycline hydrochloride. The limit of quantification for 4‐epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.     

Monitoring Pharmaceuticals Photo-Fenton Degradation Process by Using Solid Phase Extraction and Liquid Chromatography

Abstract

The recovery of the pharmaceuticals bezafibrate and tetracycline from water was evaluated, using Solid Phase Extraction (SPE) with the aim of applying this technique to interrupt the pharmaceuticals' photodegradation by photo-Fenton process for further analysis. Sep-Pack C-18, Strata X, and Oasis HLB cartridges were evaluated. Oasis HLB showed the most satisfactory recovery and repeatability results: 98% (CV = 1%) for bezafibrate (20.0 mg L^?1) and 76% (CV = 1%) for tetracycline (25.0 mg L^?1). There was not a significant decrease in recovery at lower concentrations of the pharmaceuticals, and neither when present in Sewage Treatment Plant (STP) effluent matrix.     

Photocatalytic removal of tetracycline by a Z-scheme heterojunction of bismuth oxyiodide/exfoliated g-C3N4: performance,mechanism, and degradation pathway

Antibiotics, once being released into the environment, become recalcitrant organic pollutants, which pose a potential risk to ecological balance and human health. In this study, a Z-scheme heterojunction of bismuth oxyiodide (BiOI)/exfoliated g-C₃N₄ (BiOI/ECN hereafter) was synthesized by the combination of thermal exfoliation of g-C₃N₄ and chemical precipitation of BiOI for efficient photocatalytic degradation of tetracycline in aqueous solutions under visible light irradiation. The optimized BiOI/ECN delivered an outstanding degradation rate at circa 0.0705 min^?1, which was 10 times higher than that of the bulk g-C₃N₄. The photocatalytic degradation efficiency of tetracycline remained almost unchanged in a pH range of 3–11, and the BiOI/ECN displayed an excellent photostability upon recycled usage. The photocatalytic mechanism of tetracycline was ascribed to the main reactive oxidation species of photogenerated holes and superoxide radicals. In addition, the possible degradation pathways of tetracycline were investigated by HPLC-MS to identify intermediates. The toxicity of photocatalytic-generated intermediates of tetracycline was found significantly alleviated according to the calculation of quantitative structure–activity relationship prediction. This work not only provides an attractive photocatalyst for the removal of tetracycline but also opens a new avenue for rational design of Z-scheme heterojunction composites for tetracycline degradation.     

Feasibility of Fluorescence Detection of Tetracycline in Media Mixtures Employing A Fiber Optic Probe

Abstract

This work assesses the feasibility of fluorescence detection of tetracycline in very optically dense mixtures of highly fluorescent media ingredients used in tetracycline production by fermentation. The fluorescence measurements are accomplished with a fiber optic probe. Seven different mixtures were examined in this study. Each one contained a nonfluorescent base of nutrients and salts along with one of the following media ingredients at 5 g/100 mL: cottonseed flour, corn gluten meal, soybean flour, distiller 's grains and solubles, corn steep liquor, brewer's yeast, and molasses. The concentration of tetracycline was varied in each mixture and fluorescence measurements were made at every concentration step. Excitation light of 390 nm was used to probe the samples, and emission spectra were obtained over the wavelength range from 400 to 600 nm. In most of the samples studied, the fluorescence intensity in the wavelength range corresponding to background media fluorescence (420–480 nm) decreased as the tetracycline concentration increased. The decreases in the short wavelength range might be explained by the absorption by tetracycline of 390 nm excitation light (in competition with absorption by the media) and/or by absorption of background media fluorescence by tetracycline. Frequently, the maximum emission of the mixtures shifted to longer wavelengths. The maximum approached that of tetracycline (approximately 520 nm). Plots of integrated fluorescence intensity, in the emission wavelength regions of 420-480 nm and 500-560 nm, versus tetracycline hydrochloride concentration reflect these shifts. We have found that the changes in fluorescence intensity in these two wavelength regions during tetracycline addition depend on the identity of the media component in the mixture. For corn meal, soybean, brewer's, and molasses media, the fluorescence in the short emission wavelength range decreases while that in the long region increases. In the case of distiller's and corn steep media, the fluorescence changes very little during tetracycline addition. Finally, in cottonseed medium, the fluorescence increases in both wavelength ranges. The data show that fluorescence can be used to detect tetracycline, at least qualitatively, in the presence of the highly fluorescent media ingredients.     

Synthesen von α,β-ungesättigten 2-(2-Oxo-benzazolin-3-yl)-3-hydroxy-dithio-carbonsäure-alkylestern und entsprechend substituierten Keten-S,S-acetalen sowie deren Kristall- und Molekülstruktur

Summary The new alkyl 2-(2-oxo-benzazoline-3-yl)-3-hydroxy dithiocrotonates and dithiocinnamates4 and the corresponding ketene dithioacetals2 and5 are obtained by dithiocarboxylation of the 3-acceptormethyl substituted benzazoline-2-ones1 or3. Alkylation at room temperature gives compounds4 whereas at higher alkylation temperature2 or5 are formed. The results of X-ray analyses performed for methyl 3-hydroxy-p-chloro-dithiocinnamate4d and of N-[1-(4-chloro-benzoyl)-2,2-bis(methylthio)-vinyl]-benzothiazoline-2-one5e are discussed.

     

New Colorimetric Method for Determination of Tetracycline and Oxytetracycline Via Charge Transfer Complex Formation

Abstract

A colorimetric method for the determination of tetracycline and oxytetracycline is developed. It depends on nitration and reaction of the nitroderivatives with acetone in the presence of potassium hydroxide. A highly colored complex is produced. Variables such as temperature) time of heating, volume of nitrating mixture, acetone and potassium hydroxide have been evaluated to permit the selection of the most advantageous technique. Beer's Law is obeyed over the concentration range from 0.004 – 0.04 mg ml for tetracycline and oxytetracycline.     

N-COORDINATED COPPER(II) COMPLEXES WITH 2-S-METHYL-5,5-DIMETHYLIMIDAZOLIN-4-ONE (L). CRYSTAL STRUCTURE OF [CuL4H2O][CuCl4]

Abstract

The reaction between CuX₂ (X = Cl, Br, NO₃, ClO₄) and 2-S-methyl-5,5-dimethylimidazolin-4-one (L) yields complexes having stoichiometry CuL₂Cl₂ · 1/2H₂O (1), CuL₂Br₂ (2), CuL₄X₂ · 2H₂O [X = NO₃ (3), ClO₄ (4)]. The X-ray crystal structure solved for 1 showed that it contains discrete [CuL₄ · H₂O]²⁺ and [CuCl₄]^2- ions, separated by normal van der Waals contacts. The [CuL₄H₂O]²⁺ cation has crystallographic C ₄ symmetry, with the copper atom and the oxygen of the water molecule lying on a four-fold axis of the space group P4/n and the metal atom in a square-pyramidal coordination geometry, obtained by a square basal plane defined by four N atoms and the apex occupied by the water molecule. The [CuCl₄]^2- anion shows a very flattened tetrahedral geometry. FT-IR and FT-Raman spectra of 1–4 show that 2,3 and 4 also are N(3)-coordinated to copper; nitrate and perchlorate do not coordinate. The v(CuO) vibrations are present at about 380 cm^?1 for 3 and 4, while for 2 v(CuBr) are at 224 and 215 cm^?1. Esr data for 1–4 recorded on solid samples highlight a d_x ² - y² ground state for the cation of 1, 3, and 4 which is typical for tetragonally elongated monomeric copper(II) complexes. Compound 2 exhibits two couples of g values characteristic of two independent copper centres both with compressed tetrahedral geometry. The deconvoluted reflectance spectra of 1–4 substantially confirm the presence of CuN₄ and CuBr₄ chromophores in 2 and a trans-CuN₄O₂ chromophore in 3 and 4. For the latter complexes an orbital sequence d_{x² - y²} > d_xy > d_z² > d_xy, d_yz is also suggested on the basis of the similarity between L and imidazole type ligands.     

Isatin Imines in the Reaction with Iminophosphine: X-Ray Structure of Phospholane Derivatives

3-Imino-1-methyl-indolin-2-one (2a) reacts with phosphorus hexamethyltriamide 1(1:1 mol) in dry toluene at room temperature to give the dimeric spirophospholane 4a. On the other hand, the reaction of 3-(4-arylimino)-1-methylindoline-2-ones 2b–2c with trisdimethylaminophosphine 1 in boiling toluene gave phosphoramides 4b and 4c. Furthermore, 1-benzyl-3-(4-arylimino)indolin-2-ones 3a–3c react with aminophosphine 1 in excess as solvent at 110 °C for 6–8 h to give phosphonamides 5a–5c. Possible reaction mechanism are considered and the structural assignments are based on spectroscopic data and x-ray analysis.     

Nebenprodukt bei der Herstellung von 4-Hydroxy-3-phenylcumarin

The synthesis of 4-hydroxy-3-phenylcoumarin (3a) from phenol (1) and diethylphenylmalonate (2) yields the isocoumarin derivative4 as a by-product (1–2%). If3a is heated with2 or diphenylcarbonate (6) at 300°C the coumarino-isocoumarin4 is formed in 83% and 85% yield, respectively.     

Investigations on the amalgamation of gold nanorods by iodine and the detection of tetracycline

The morphological transformation process of gold nanorods (Au-NRs) resulting from the reaction between tetracycline and iodine was monitored by the plasmon resonance absorption (PRA) spectra and the scanning electron microscope (SEM) images. It was found that iodine could fuse Au-NRs into sphericity with the lower aspect ratio and blue shift of the longitudinal PRA band. It was found, however, that the presence of tetracycline, since it can react with I₂, decreases the effective concentration of I₂ and its fusion effect on Au-NRs. As a result, the longitudinal PRA of Au-NRs shifts to longer wavelength linearly with increasing the concentration of tetracycline. With that, tetracycline can be detected in the range of 5.0×10^∮5- 5.0×10⁻⁴ mol·L⁻¹, with a limit of determination (LOD) of 2.4×10⁻⁶ mol·L⁻¹ (3σ). Most foreign substances in the samples did not interfere in the detection, and tetracycline in the synthetic samples could be detected with the recovery in the range of 92.8%–107.2%, and RSD lower than 4.3%. The concentration of tetracycline in milk detected with standard addition method was so low that it accorded with the safety regulation. Supported by the National Natural Science Foundation of China (Grant Nos. 30570465 and 20425517)     

Efficient Synthesis of Novel 3-[5-(1H-Benzimidazol-2-Ylmethanesulfonyl)-4-Phenyl-4H-(1,2,4)Triazol-3-Yl]-1H-(1,8)Naphthyridin-4-One Derivatives

Abstract

of 1,4-dihydro-4-oxo-1,8-naphthyridine-3-carbohydrazide (4) with substituted phenyl isothiocyanates (5) in ethanol under reflux for 30 min gave thiosemicarbazide derivatives 6, which on cyclization in 2N NaOH under refluxing conditions for 1 h resulted in 3-(5-mercapto- 4-phenyl-4H-1,2,4-triazol-3-yl)-1,8-naphthyridin-4(1H)-one (7). Alternatively, 7 could also be prepared from following sequence of reactions, i.e., 4 → 8 → 7. In another sequence of reactions, condensation of 7 with chloroacetic acid in dimethylformamide (DMF) and K₂CO₃ as a mild base at 120 °C for 2 h resulted in 2-((5-(1,4-dihydro-4-oxo-1,8-naphthyridin-3-yl)-4-phenyl-4H-1,2,4-triazol-3-yl)sulfanyl)acetic acid (10). The latter, on reaction with substituted o-phenylenediamine (11) in 6N HCl for 4 h yielded 3-(5-((1H-benzo[d]imidazol-2-yl)methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,8-naphthyridin-4(1H)-one (12). Alternatively, 12 could also be prepared by reacting 7 with 13 in DMF and K₂CO₃ as a mild base at 120 °C for 2 h, followed by oxidation with H₂O₂ resulting in the corresponding sulfonyl derivatives 14.     

Pentacoordinate spirosilicate anion, bis(2,2′-biphenyldiyl)methylsilicate, synthesized by the lithium cleavage of dimethoxyethane

The reaction of spirobisilafluorene (1) with lithium in dimethoxyethane produces lithium 1-methyl-spirobisilafluorenide (2), a stable pentacoordinate silicon compound with five carbon ligands, and lithium 2-methoxyethoxide, which was identified by trapping with Ph₃SiCl to give Ph₃Si-OCH2CH2OCH3 (4). The X-ray crystal structure of 2 shows that the geometry at silicon is an idealized trigonal bipyramid, slightly distorted toward a square pyramid. Methanolysis of 2 cleaves a Si-aryl bond producing a methyl biphenylsilafluorene, 3. Crystal structures are reported for 3 and 4.     

New Azothiacalix[4]arenes Containing Biheterocyclic Subunits: Extraction and Complexation Properties

New thiacalix[4]arenes 2a, 3a, 4 and 5 functionalized with biheterocyclic and azophenyl groups at the lower rim and the upper rim of the macrocycle, respectively, were synthesized and their extraction properties towards Cu²⁺, Ag⁺, Ni²⁺ and Pb²⁺ studied. The complexation properties of the bithiazolyl receptors 2a, 4 and the bipyridyl receptors 3a, 5 were investigated by fluorescence and UV–visible titrations, respectively. The stoichiometries of the complexes were determined. A stoichiometry of 1:1 was found for the Cu-2a, Ag-3a complexes and 2:1 for the Cu₂-4, Ag₂-5 complexes as a function of the number of grafted bithiazolyl groups. The association constants for the 1:1 complexes were calculated using the Benesi–Hildebrand plot and by linear regression analysis.     

Synthesis and Reactions of Polynuclear Heterocycles: Azolothienopyrimidines and Thienothiazolopyrimidines

We prepared a thieno[2,3-d]pyrimidine compound fused with a thiazolo ring to produce biologicaly active compounds. In a one-step reaction, 2-arylmethylene derivative (3) was prepared via the reaction of a ternary mixture of 2-thioxo-1,2,3,4-tetrahydrocyclohepteno[4,5]thieno[2,3-d]pyrimi-dine-4-one (2), cloroacetic acid, and a proper aldehyde. The reaction of 2 with 3-chloropent-2,4-dione in ethanolic potassium hydroxide yielded the S-acetylacetone derivative 4e. The latter compound reacted with hydrazine hydrate and phenyl hydrazine to give 2-pyrazolthio derivatives 8a,b, respectively. Compound 4e also underwent cyclization on boiling with acetic anhydride/pyridine solution to form 2-acetyl-3-methyl thiazolo[3,2-a]cyclohepteno[4,5]thieno[2,3-d] pyrimidine-5-one (9). To support the structure 9, it gave a characteristic reaction for the 2-acetyl group. The 2-methylthio derivatives 4a underwent further alkylation at N₃ to give 6a,b. The purpose of the synthesis of thienopyrimidine derivatives is due to high biological activities. The 4-oxo-thienopyrimidine derivatives acted as inhibitors of adenosine kinase, platelet aggregation, antilukemia, and anticancer activities.