Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities.

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 µm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.

The effect of calcium hydroxide on the steroid component of Ledermix and Odontopaste.

AIM: To investigate the chemical interaction of calcium hydroxide with the corticosteroid triamcinolone acetonide in Ledermix Paste and in Odontopaste, a new steroid/antibiotic paste. METHODOLOGY: Validated methods were developed to analyse the interaction of calcium hydroxide in two forms, Pulpdent Paste and calcium hydroxide powder, with triamcinolone acetonide within Odontopaste and Ledermix Paste. High-performance liquid chromatography (HPLC) was used to analyse the mixed samples of the pastes and calcium hydroxide. The concentration of triamcinolone acetonide within the pastes was determined over 0, 2, 6, 24 and 72-h time-points. All tests with the HPLC involved the testing of the standard with triplicate injections alongside the samples. All samples were tested in duplicate with each injected twice; therefore, four tests were performed for each investigation. Linearity, precision and specificity of the testing procedures and apparatus were validated. Descriptive statistics are provided. RESULTS: In both pastes, there was a marked rapid destruction of the triamcinolone acetonide steroid upon mixing with calcium hydroxide. Odontopaste suffered a lower rate of destruction of the triamcinolone acetonide component than Ledermix Paste, but both pastes showed very similar degrees of steroid destruction after 72 h. When using calcium hydroxide powder with Ledermix Paste, the triamcinolone was destroyed entirely and immediately. CONCLUSION: The addition of calcium hydroxide to Odontopaste or Ledermix Paste results in the rapid destruction of the steroid.

A rapid method based on hot water extraction and liquid chromatography-tandem mass spectrometry for analyzing tetracycline antibiotic residues in cheese.

A rapid, specific, and sensitive procedure for determining residues of 4 widely used tetracycline antibiotics and 3 of their 4-epimers in cheese is presented. The method is based on the matrix solid-phase dispersion (MSPD) technique followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). After dispersing samples of mozzarella, asiago, parmigiano, gruyere, emmenthal, and camembert on sand, target compounds were eluted from the MSPD column by passing through it 6 mL water heated at 70 degrees C. After acidification and filtration, 200 microL of the aqueous extract was directly injected into the LC column. For analyte identification and quantification, MS data acquisition was performed in the multireaction monitoring mode, selecting 2 precursor ion-to-product ion transitions for each target compound. Hot water appeared to be an efficient extractant, because absolute recoveries were no lower than 78%. Using demeclocycline as a surrogate analyte, recoveries of analyte added to the 6 types of cheeses at the 30 ng/g level were 96-117%, with relative standard deviation (RSD) not higher than 9%. Statistical analysis of the mean recovery data showed that the extraction efficiency was not dependent on the type of cheese analyzed. This result indicates that this method could be applied to other cheese types not considered here. At the lowest concentration considered, i.e., 10 ng/g, the accuracy of the method ranged between 90 and 107%, with RSDs not larger than 12%. Based on a signal-to-noise ratio of 10, limits of quantitation were estimated to be 1-2 ng/g.

Improvements in europium sensitized fluorimetric determination of demeclocycline and methacycline.

Demeclocycline (DM) and methacycline (MT) have been determined by europium-sensitized fluorescence, using EDTA as co-ligand and cetyltrimethylammonium chloride as surfactant. The methods have been developed in slightly alkaline solutions, with the formation of a new chelate where the lanthanide ion is bound to the beta-diketone group. Calibration graphs between 0.01 and 0.1 microg mL(-1) have been obtained for DM and MT determination. Both methods have been applied to the determination of these tetracyclines in serum samples with satisfactory recovery results.

Optimized methods for capillary electrophoresis of tetracyclines.

Optimized methods for the analysis of some tetracyclines by capillary electrophoresis are described. Different buffer systems were employed for the separation of tetracycline, oxytetracycline and demeclocycline from their respective major impurities, including the 2-acetyl-2-decarboxamido derivatives. The influence of buffer pH and buffer concentration was systematically investigated. Non-ionic surfactant Triton X-100 and methyl-beta-cyclodextrin were used to obtain improved selectivity in the case of oxytetracycline and demeclocycline. The results are compared with those of previously established liquid chromatography methods. Good correlations were obtained.

Quantitative analysis of demeclocycline by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method suitable for the quality control of demeclocycline is described. The stationary phase is a poly(styrene-divinylbenzene) copolymer, kept at 60 degrees C. The mobile phase comprises 2-methyl-2-propanol-0.2 M potassium phosphate buffer (pH 9.0)-0.02 M tetrabutylammonium hydrogen sulphate (pH 9.0)-0.01 M sodium edetate (pH 9.0)-water (8:10:15:10:57, m/v/v/v/v). The flow rate is 1 ml min-1 and detection is performed at 254 nm. Official standards are compared and results for the analysis of a number of commercial bulk samples and preparations are presented. 4-Epidemeclocycline and demethyltetracycline are the main impurities. 4-Epidemethyltetracycline and 2-acetyl-2-decarboxamido-demeclocycline can also be present.

Evaluation of amperometric detection for the liquid-chromatographic determination of tetracycline antibiotics and their common contaminants in pharmaceutical formulations.

A sensitive high-performance liquid chromatographic technique with amperometric detection has been developed for the determination of seven commercially used tetracyclines in bulk powders and pharmaceutical preparations. The technique is based on the oxidation of these compounds and their contaminants at the glassy carbon electrode. The extraction procedures are simple and the HPLC conditions separate the tetracyclines from their major degradation products. The chromatography was performed using a commercially available octadecylsilane column, with a mobile phase: KH2 PO4 (pH = 2.5; 0.05 M) - acetonitrile (84:16, v/v) and detection at 1.2 V. The technique permits the simultaneous determination of trace amounts of chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline and tetracycline as well as the separation of their common impurities (epi, anhydro and epianhydro contaminants) with detection limits of 0.1-1.0 ng microl(-1) and recoveries of 99.1-100.4%. No interference was observed from the commonly present excipients in pharmaceutical formulations.

Complexation of tetracyclines with metal ions in relation to photosensitization.

It has been shown by spectroscopy and by a Photosensitive Index method, involving measurements of interfacial tension, that Cu2+ and Ni2+ form 2:1 complexes with oxytetracycline and 1:1 complexes with demethylchlortetracycline. Fe2+, Zn2+, Mg2+ and Ca2+ form weaker 1:1 complexes with both drugs. The possibility is discussed of using metal ions to reduce photosensitization by tetracyclines in-vivo.

[Reversed phase high performance liquid chromatography of tetracyclines].

The present paper describes a quantitative determination method of tetracyclines by reversed phase high performance liquid chromatography. The method used a Waters 10 C18 4.6 nm ID x 250 nm reversed phase column, with a mobile phase composed of 14% acetonitrile-3% dimethylformamide-83% water containing 0.02 mol/L citric acid (pH-2.5), the detector wavelength was set at 350 nm, and the flow rate was maintained at 1.0 ml/min. Demethylchlortetracycline, epi-demethylchlortetracycline, demethyltetracycline, epi-demethyltetracycline, tetracycline, epi-tetracycline, chlortetracycline and epi-chlortetracycline can be separated highly efficiently. The tetracyclines and their anhydro- products can be separated on the same column with 15% acetonitrile- 15% dimethylformamide- 70% 0.02 mol/L citric acid as the mobile phase, and detected at UV 270 nm.

Analysis of tetracyclines in honey by high-performance liquid chromatography/tandem mass spectrometry.

A confirmatory method coupling liquid chromatography to tandem mass spectrometry (LC/MS/MS) is described for the determination of tetracycline, oxytetracycline, doxycycline and chlortetracycline in honey. Demeclocycline, another tetracycline molecule not reported for its usage in honey, was used as internal standard to quantify the four analytes. The sample preparation entails a clean-up on an Oasis HLB solid-phase extraction cartridge and analyses were realised by LC/MS/MS in selected reaction monitoring mode. The stability of tetracyclines was checked under various storage conditions at -20, +4 and +20 degrees C (both under dark and light exposures). Indeed, tetracyclines are not stable molecules and the epimerisation phenomenon was evaluated in this work. Appropriate correction factors of the MS/MS responses of each epimer were studied for each of the four tetracyclines to accurately quantify them. Moreover, the matrix effects encountered during the LC/MS/MS analyses were also studied in spiked experiments from blank honey samples of various geographical origins and different flower types.

The release and diffusion through human coronal dentine in vitro of triamcinolone and demeclocycline from Ledermix paste.

Crowns from freshly extracted human third molar teeth were used to quantify the release and diffusion of corticosteroid and antibiotic tracer molecules from Ledermix paste used as an indirect pulp-capping agent. These molecules readily diffused through dentine and reached a peak rate of diffusion at 2 h. The rate then decreased exponentially with time. The concentrations of the drugs in the dentine were calculated; this showed that a gradient existed from the cavity floor to the pulp space. The data obtained appeared to have clinical relevance and helped explain the therapeutic benefits of this medicament when used as an indirect pulp-capping agent.

On-line metal chelate affinity chromatography clean-up for the high-performance liquid chromatographic determination of tetracycline antibiotics in animal tissues.

An on-line high-performance liquid chromatographic (HPLC) method for the determination of tetracycline, oxytetracycline, chlortetracycline and demeclocycline using metal chelate affinity chromatography-reversed-phase HPLC has been developed. The drugs were extracted with succinate buffer and the extract diluted with EDTA-pentanesulphonate buffer. Diluted extract was then absorbed onto a C8 or XAD-2 solid-phase extraction (SPE) cartridge and eluted with methanol. The eluate was then injected onto a TSKgel chelate column which had been preloaded with copper(II). The tetracyclines were eluted from this column onto the analytical column (Polymer Labs. PLRP-S) with an EDTA-containing buffer. Elution of the analytical column was via a methanol-acetonitrile gradient and detection was by UV at 350 nm. Average recoveries at the 10, 20, 50 and 300 micrograms kg-1 levels were 50-80%. The limit of detection (LOD) was 10 micrograms kg-1 for oxytetracycline and tetracycline and 20 micrograms kg-1 for chlortetracycline and demeclocycline. The method was validated for sheep liver and cattle kidney.