Developing and validation of impurity and the simultaneous quantity determination methods for tablet forms containing Ciprofloxacin HCl and Ornidazole.

In this study simple, effective, fast and economic reverse-phase HPLC methods have been developed for the purposes of identifying impurities and quantities of the two active ingredients in the tablet dosage products containing Ciprofloxacin HCl and Ornidazole together. These methods have been validated according to the parameters of International Conference on Harmonization (ICH) and Centre for Drug Evaluation and Research (CDER) to prove reliability and applicability of the methods. In the validation studies, all of the parameters defined in ICH as selectivity, precision, accuracy, linearity, LOD, LOQ, recovery, robustness and solution stability were tested. In the impurity method new developed, Inertsil ODS-3 HPLC column, buffer solution with a pH of 3 and acetonitrile mobile phase mixture in isocratic elution were used. Mobile phase flow rate was 1.0 ml/min and detector wavelength were adjusted to be 330 nm. In the quantity determination method, Inertstil C8 HPLC column, a buffer solution having a pH of 3, methanol and acetonitrile mobile phase mixture in gradient elution was employed. In this method also, mobile phase flow rate was preferred to be 1.5 ml/min and detector wavelength as 330 nm.

Solvothermal and Ultrasonic Preparation of Two Unique Cluster-Based Lu and Y Coordination Materials: Metal-Organic Framework-Based Ratiometric Fluorescent Biosensor for an Ornidazole and Ronidazole and Sensing Platform for a Biomarker of Amoeba Liver Abscess.

Through powerful solvothermal and facile ultrasonic synthetic strategies, two unique cluster-based lanthanide Lu and Y nanoporous metal organic frameworks (MOFs) have been successfully prepared, namely, {[Lu2(L)2]·2DMF·H2O}n (Lu-MOF) and [Y(L)(DMF)0.75]n (Y-MOF) (H3L = terphenyl-3,4'',5-tricarboxylic acid). In addition, both the morphologies and nanosizes of Lu-MOF and Y-MOF materials also have been deliberately tuned by adjustable ultrasonic conditions including irradiation time (40, 60, and 80 min) and power (70 w, 100 w). Currently, it is noted that the abuse of antibiotics such as ornidazole and ronidazole leads to great damage to human health, and therefore the development of highly effective and facile detection methods for ornidazole and ronidazole is quite important. Herein, to improve the fluorescent sensing sensitivity of antibiotics, Eu3+ and Tb3+ have been introduced into Lu-MOF (under a solvothermal preparation method) to fabricate a dual-emission hybrid material Eu3+/Tb3+@Lu-MOF through a postsynthesis strategy, which can be successfully applied as a self-calibrated ratiometric fluorescent sensor for ornidazole and ronidazole with high selectivity and sensitivity (the Ksv value for ornidazole is 1.0854 × 106 [M-1], and the Ksv value for ronidazole is 1.0595 × 107 [M-1]) and low detection limit values (2.85 nM for ornidazole and 26.7 nM for ronidazole). On the other hand, amoeba liver abscess (ALA) will easily lead to irregular fever, night sweats, and other tortured symptoms; C-reactive protein autoantibody (CRP Ab) is the important biomarker for the detection of ALA. Given this, Y-MOF (under the solvothermal preparation method) also has been successfully designed to combine FAM-labeled NH-ssDNA to construct the scarcely reported excellent hybrid FAM-labeled NH-ssDNA/Y-MOF sensing platform for the highly effective discrimination of CRP Ab with excellent sensitivity and selectivity in real samples such as human serum solution.

Determination of the sulfate and glucuronide conjugates of levornidazole in human plasma and urine, and levornidazole and its five metabolites in human feces by high performance liquid chromatography-tandem mass spectrometry.

Levornidazole is a novel third-generation nitroimidazoles antibiotic which metabolism and disposition in human are not well known. We have previously developed two methods to quantify levornidazole and its phase I metabolites, Ml (Hydroxylation metabolite), M2 (N-dealkylation metabolite) and M4 (Oxidative dechlorination metabolite), in human plasma and urine. In this study, we developed three novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods and analyzed its phase II metabolites, sulfate conjugate (M6) and glucuronide conjugate (M16), in human plasma and urine, and the parent drug and above-mentioned five metabolites in human feces samples. Analytes and internal standard (IS) in human plasma were extracted by a solid-phase extraction procedure and separated on an ACQUITY UPLC CSH C18 column in gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase. The pretreatment procedures for urine and feces homogenate samples involved a protein precipitation followed by liquid-liquid extraction, and chromatographic separations were performed on the Atlantis T3 columns of different lengths and particle sizes (2.1 × 50 mm, 3 µm and 2.1 × 150 mm, 5 µm), respectively. The mobile phases consisted of formic acid and acetonitrile-methanol solution (v/v, 50:50) in gradient elution. The MS/MS analysis was conducted on TSQ Quantum triple quadrupole mass spectrometer using electrospray ionization with selected reaction monitoring (SRM) in the positive ion mode. The calibration curves for all analytes were linear and the validation ranges were as follows: 0.005-0.500 µg/mL for M6 and 0.005-2.500 µg/mL for M16 in plasma; 0.010-10.000 µg/mL for M6 and M16 in urine; 0.005-1.000 µg/mL for levornidazole, M2, M4 and M16, and 0.010-2.000 µg/mL for M1 and M6 in human feces homogenate. Across these matrices, mean intra- and inter- batch accuracy values were in the ranges of 80.0%-120.0%, and intra- and inter-batch precision values did not exceed 20%. It was fully validated including selectivity, linearity, matrix effect, extraction recovery, stability, dilution integrity, carryover and incurred sample analysis (ISR). These newly developed methods were successfully applied in pharmacokinetics, metabolism and disposition study of levornidazole in 12 healthy Chinese subjects.

Preparation of monoclonal antibody and development of an indirect competitive enzyme-linked immunosorbent assay for ornidazole detection.

A monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for ornidazole (ONZ) detection were developed. ONZ was conjugated with cationic bovine serum albumin as a hapten to generate the artificial immunogens and coating antigens. BALB/c mice were immunized, and mAbs were obtained. The competitive inhibition curve of ic-ELISA was y=0.0438x2-0.2101x+0.2925, with R2=0.9941. The 50% inhibition concentration, the limit of detection, and limit of quality for ONZ were 0.15, 0.01, and 0.05µg/kg, respectively. The cross-reactivity of the mAbs to secnidazole was 0.33%. The recoveries were from 89.18% to 101.63% and the coefficient of variation was less than 7.15% in chicken, chicken liver, and honey samples, all of which had ONZ concentrations of 0.05 and 0.1µg/kg. Results showed that the ic-ELISA based on mAb could be used for the rapid detection for ONZ.

Two-way and three-way approaches to ultra high performance liquid chromatography-photodiode array dataset for the quantitative resolution of a two-component mixture containing ciprofloxacin and ornidazole.

Two-way and three-way calibration models were applied to ultra high performance liquid chromatography with photodiode array data with coeluted peaks in the same wavelength and time regions for the simultaneous quantitation of ciprofloxacin and ornidazole in tablets. The chromatographic data cube (tensor) was obtained by recording chromatographic spectra of the standard and sample solutions containing ciprofloxacin and ornidazole with sulfadiazine as an internal standard as a function of time and wavelength. Parallel factor analysis and trilinear partial least squares were used as three-way calibrations for the decomposition of the tensor, whereas three-way unfolded partial least squares was applied as a two-way calibration to the unfolded dataset obtained from the data array of ultra high performance liquid chromatography with photodiode array detection. The validity and ability of two-way and three-way analysis methods were tested by analyzing validation samples: synthetic mixture, interday and intraday samples, and standard addition samples. Results obtained from two-way and three-way calibrations were compared to those provided by traditional ultra high performance liquid chromatography. The proposed methods, parallel factor analysis, trilinear partial least squares, unfolded partial least squares, and traditional ultra high performance liquid chromatography were successfully applied to the quantitative estimation of the solid dosage form containing ciprofloxacin and ornidazole.

Development of a Single Ion Pair HPLC Method for Analysis of Terbinafine, Ofloxacin, Ornidazole, Clobetasol, and Two Preservatives in a Cream Formulation: Application to In Vitro Drug Release in Topical Simulated Media-Phosphate Buffer Through Rat Skin.

Present work reports an HPLC method with UV detection for quantification of terbinafine, ofloxacin, ornidazole, and clobetasol in a cream formulation along with two preservatives methyl and propyl paraben. The chromatographic separation and quantification was achieved by an octyl bonded column and a gradient elution program involving an ion-pairing reagent, hexanesulfonic acid (0.2%, pH modified to 2.7 using orthophosphoric acid) and acetonitrile. The method was simple and devoid of buffer salts and therefore advantageous for system and column life. The three step gradient program was initiated with 30% (v/v) acetonitrile for the first 5 min and ramped linearly to 60% in the next 7 min. The mobile phase remained constant for the next 11 min and then concluded at 30% (v/v) of acetonitrile. Flow rate throughout was 0.8 mL/min, and all the signals were monitored at 243 nm. The method was applied for assay of a cream formulation and its in vitro permeation studies to determine the penetration profile of the four drugs and two preservatives. A marketed cream formulation was selected for the permeation study, which was carried out using a diffusion cell consisting of topical simulated media, phosphate buffer (pH=6.8) solution containing 1% sodium lauryl sulfate as a receiver medium.

A dispersive liquid-liquid micellar microextraction for the determination of pharmaceutical compounds in wastewaters using ultra-high-performace liquid chromatography with DAD detection.

A dispersive liquid-liquid micellar microextraction (DLLMME) method coupled with ultra-high-performance liquid chromatography (UHPLC) using Diode Array Detector (DAD) detector was developed for the analysis of five pharmaceutical compounds of different nature in wastewaters. A micellar solution of a surfactant, polidocanol, as extraction solvent (100 µL) and chloroform as dispersive solvent (200 µL) were used to extract and preconcentrate the target analytes. Samples were heated above critical temperature and the cloudy solution was centrifuged. After removing the chloroform, the reduced volume of surfactant was then injected in the UHPLC system. In order to obtain high extraction efficiency, the parameters affecting the liquid-phase microextraction, such as time and temperature extraction, ionic strength and surfactant and organic solvent volume, were optimized using an experimental design. Under the optimized conditions, this procedure allows enrichment factors of up to 47-fold. The detection limit of the method ranged from 0.1 to 2.0 µg/L for the different pharmaceuticals. Relative standard deviations were <26% for all compounds. The procedure was applied to samples from final effluent collected from wastewater treatment plants in Las Palmas de Gran Canaria (Spain), and two compounds were measured at 67 and 113 µg/L in one of them.

Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole in human saliva.

The aim of this study was to develop a rapid and sensitive method for the simultaneous quantification of metronidazole (MEZ), tinidazole (TNZ), ornidazole (ONZ) and morinidazole (MNZ) in human saliva. A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection at 318 nm was carried out on a C18 column, using a mixture of potassium dihydrogen phosphate buffer, acetonitrile, and methanol (55:15:30, v/v/v) as a mobile phase with a flow rate of 1.0 ml/min. The saliva samples (100 µl) were firstly deproteinized by precipitation with methanol (400 µl), after which they were centrifuged and the supernatants were directly injected into the HPLC system. This method produced linear responses in the concentration ranges of 25.2-5040.0, 23.9-4790.0, 25.4-5080.0, 25.0-5000.0 ng/ml with detection limits of 6.0, 17.6, 10.0 and 11.3 ng/ml for MEZ, TNZ, ONZ and MNZ (S/N=3), respectively. The methods were validated in terms of intra- and inter-batch precision (within 7.3% and 9.1%, respectively), accuracy, linearity, recovery and stability. The study proved that HPLC is both sensitive and selective for the simultaneous quantification of MEZ, TNZ, ONZ and MNZ in human saliva using a single mobile phase.

[Far-IR and THz absorption spectra studies of metronidazole, tinidazole and ornidazole].

Metronidazole, tinidazole and ornidazole are 5-nitro-imidazole medicines used particularly for anaerobic bacteria and protozoa infections. The present paper reports that terahertz time-domain spectroscopy (THz-TDS) and Fourier transform infrared spectroscopy (Far-FTIR) were used to measure the fingerprint spectra of metronidazole, tinidazole and ornidazole in the frequency range of 0.9 - 19.5 THz under the room temperature. In addition, THz-TDS was also used to measure the absorption spectra of pure tinidazole and tinidazole tablets from different manufactures between 0.2 and 2.2 THz. In parallel with the experimental study, the cross correlation analysis was applied to compare the array of correlation coefficients between pure tinidazole and different tinidazole tablets. Results show that the method is rapid, simple and accurate to identify their effective chemical compositions and stability when the FTIR and THz spectra data combine with the array of correlation coefficient. Our research provides a visual approach to the standardization and modernization of the quality control in the production and sale of such drugs.

Extractional spectrophotometric analysis of metronidazole, tinidazole, ornidazole and secnidazole bases through acid-dye complexation using bromothymol blue dye.

An easy, precise and valid extractional-spectrophotometric technique is described for the assessment of metronidazole (MNZ), tinidazole (TNZ), ornidazole (ONZ) and secnidazole (SNZ) in pure state and in their pharmaceutical formulations. The technique includes first the reduction of above cited drugs using HCl and zinc powder, then the formation of intense yellow colored ion-association complex species (1:3 drug/dye) using bromothymol blue (BTB) in a buffered aqueous acidic medium at pH 3-3.50. The colored products are extracted into dichloromethane and quantitatively determined at 416-420 nm. The experimental operating factors influencing the ion-pairs development were studied and optimized to obtain the maximum color intensity. The Beer plots are obeyed in the concentration ranges 2.50-22.50, 2.50-30, 7.50-35 and 5-30 µgml-1 for MNZ, TNZ, ONZ and SNZ, respectively, with correlation coefficients not less than 0.9995. The proposed technique is recommended for the routine quality control analysis of the investigated drugs in commercial tablets with no observed interference from common pharmaceutical adjuvants. Results of such analysis were statistically validated and through recovery studies, showing excellent agreement with those achieved by the reported techniques.

[Determination of nitroimidazoles in oral hygiene by ultra-performance liquid chromatography-tandem mass spectrometry].

A method for the simultaneous determination of metronidazole, tinidazole, ornidazole, ronidazole and dimetridazole in oral hygiene by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed. The sample was diluted with 0.1% formic acid/acetonitrile (95:5, v/v), then centrifuged and filtered with a membrane. The separation was carried out on a Cloversil C18 column (100 mm x 2.1 mm, 3.5 microm) with the gradient elution of 0.1% formic acid and acetonitrile as the mobile phases. The analytes were determined by UPLC-MS/MS and quantified by external standard method. The calibration curves showed good linearity in the range of 1.0-60.0 microg/L with r > or = 0.9992. The recoveries were 91.5%-108% at the three spiked levels of 10.0, 20.0 and 100 mg/kg, and the relative standard deviations were 1.14%-5.22%. This method is easy, sensitive and suitable for the determination of nitroimidazoles in oral hygiene.

[Correlation of ornidazole concentration in saliva and serum of healthy volunteers].

OBJECTIVE: To investigate the distribution of ornidazole in the salivary and serum of healthy adults and explore the feasibility of monitoring serum drug concentration with salivary. METHODS: Six volunteers received a single dose of 0.6 g ornidazole via intravenous infusion. The concentrations of ornidazole in the saliva and serum were assayed by high-performance liquid chomatography, and the correlation of the drug concentrations in saliva to that in serum was analyzed. RESULTS: The concentration of ornidazole in the saliva was strongly associated with that in the serum (r = 0.825-0.969), and the ratio of saliva-to-serum concentration (S/P) of ornidazole was 0.99 ± 0.13. CONCLUSION: Detection of saliva ornidazole concentration is feasible for monitoring the therapeutic concentration of ornidazole.

A molecularly imprinted monolith for the fast chiral separation of antiparasitic drugs by pressurized CEC.

Molecularly imprinted polymer (MIP) monoliths with (S)-ornidazole ((S)-ONZ) as the template molecule have been designed and prepared by the simple thermal polymerization of methacrylic acid, 4-vinylpyridine, and ethylene dimethacrylate in the presence of a binary porogenic mixture of toluene and dodecanol. The influences of polymerization mixture composition on the chiral recognition of ONZ have been evaluated, and the imprint effect in the optimized MIP monolith has been clearly demonstrated. The new monolithic stationary phase with optimized porous property and good selectivity was used for the chiral separation of ONZ by pressurized CEC. The pressurized CEC conditions were also optimized to obtain the good chiral separation. The enantiomers were rapidly separated within 9 min on the MIP-based chiral stationary phase, whereas the chiral separation was not obtained on the nonimprinted polymer. Additionally, the proposed method has been successfully applied to the chiral separation of ONZ in tablet samples by injection of the crude sample. The cross-selectivity for similar antiparasitic drug was investigated. The results indicated that the chiral separation of secnidazole could also be obtained on the optimized MIP monolith within 14 min.

HPTLC determination of cefuroxime axetil and ornidazole in combined tablet dosage form.

A new simple high-performance thin layer chromatographic method for determination of cefuroxime axetil and ornidazole in combined tablet dosage form is developed and validated. Cefuroxime axetil is second-generation cephalosporin used to treat or prevent infections that are proven or strongly suspected to be caused by bacteria. Ornidazole is used to cure protozoan infections. The separation is carried out on Merck precoated silica gel aluminium plate 60 F(254) using toluene-n-butanol-triethylamine (8.5:2:0.5, v/v/v) as mobile phase. Quantitative determination of drugs is carried out by densitometric scanning of plates at 285 nm. The retention factor for ornidazole and cefuroxime axetil is found to be 0.51 +/- 0.007 and 0.67 +/- 0.009, respectively. The method is validated with respect to linearity, accuracy, precision, and robustness. Response found to be linear in the concentration range of 100-500 ng/band for both cefuroxime axetil and ornidazole. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. The % assay is found to be 102.36 +/- 0.775 and 101.00 +/- 1.192 for cefuroxime axetil and ornidazole, respectively.

Simultaneous determination of ofloxacin and ornidazole in pharmaceutical preparations by capillary zone electrophoresis.

A simple, rapid and validated capillary electrophoretic method has been developed for the separation and determination of ofloxacin and ornidazole in pharmaceutical formulations with detection at 230 nm. Optimal conditions for the quantitative separations were investigated. Analysis times shorter than 4 min were obtained using a background electrolyte solution consisting of 25 mmol/L phosphoric acid adjusted with 1 M Tris buffer to pH 8.5, with hydrodynamic injection of 5 s and 20 kV separation voltage. The validation criteria for accuracy, precision, linearity and limits of detection and quantitation were examined and discussed. An excellent linearity was obtained in concentration range 25-250 microg/mL. The detection limits for ofloxacin and ornidazole were 1.03 +/- 0.11 and 1.80 +/- 0.06 microg/mL, respectively. The proposed method has been applied for the analysis of ofloxacin and ornidazole both individually and in a combined dosage tablet formulation. The proposed validated method showed recoveries between 96.16 and 105.23% of the nominal contents.

Development and validation of column high-performance liquid chromatographic and derivative spectrophotometric methods for determination of levofloxacin and ornidazole in combined dosage forms.

The manuscript describes validated reversed-phase column high-performance liquid chromatographic (RP-HPLC) and first-derivative UV spectrophotometric methods for the estimation of levofloxacin (LFX) and ornidazole (ORNI) in combined dosage forms. The RP-HPLC separation was achieved on a Phenomenex C18 column (250 mm x 4.6 mm id, 5 microm) using KH2PO4 buffer (pH 6.8)-methanol-acetonitrile (70 + 15 + 15, v/v/v) mobile phase at a flow rate of 1.5 mL/min and ambient temperature (25 +/- 2 degrees C). Quantification was achieved with photodiode array detection at 295 nm over the concentration range of 1-10 microg/mL for both LFX and ORNI, with mean recovery of 101.7 +/- 0.23 and 99.23 +/- 1.57%, respectively, by the RP-HPLC method. The derivative spectrophotometric method was based on the determination of both the drugs at their respective zero crossing point (ZCP). The first-order derivative spectra were obtained at N = 1 (scaling factor), Deltalambda = 2.0 nm (wavelength interval), and the determinations were made at 310 nm (ZCP of ORNI) for LFX and 295 nm (ZCP of LFX) for ORNI over the concentration range of 2-40 microg/mL for both LFX and ORNI. Mean recovery was 99.46 +/- 0.96 and 100.9 +/- 0.72%, respectively, by the first-derivative UV spectrophotometric method. Standard and sample solutions were prepared with methanol as the solvent in both of the methods. These methods were found to be simple, accurate, precise, and sensitive and were applicable for the simultaneous determination of LFX and ORNI in combined dosage forms.

Simultaneous determination of cefuroxime axetil and ornidazole in tablet dosage form using reversed-phase high performance liquid chromatography.

A simple, accurate and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of cefuroxime axetil and ornidazole in combined tablet dosage form has been developed. The method was performed with a HiQ-SiL C18 column (250 mm x 4.6 mm) and photodiode array (PDA) detector, using 0.01 mol/L potassium dihydrogen orthophosphate-methanol (56 : 44, v/v) as the mobile phase and tinidazole as the internal standard. Beer's law obeys in the concentration ranges of 5 - 25 microg/mL and 10 - 50 microg/mL for cefuroxime axetil and ornidazole, respectively. The method has been successfully validated statistically and applied for the analysis of the drugs in pharmaceutical formulation.

Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form.

A simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method is described for the simultaneous determination of levofloxacin hemihydrate and ornidazole in tablet dosage form. The method is based on the HPTLC separation of the two drugs followed by densitometric measurements of their spots at 298 nm. The separation is carried out on Merck TLC aluminium sheets of silica gel 60 F254 using n-butanol-methanol-ammonia (5:1:1.5, v/v/v) as mobile phase. The linearity is found to be in the range of 50-250 and 100-500 ng/spot for levofloxacin hemihydrate and ornidazole, respectively. The method is successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients are found. The suitability of this HPTLC method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines.

Determination of gatifloxacin and ornidazole in tablet dosage forms by high-performance thin-layer chromatography.

A simple and sensitive high-performance thin-layer chromatography (HPTLC) method has been developed for the quantitative estimation of gatifloxacin and ornidazole in its combined dosage forms. Gatifloxacin and ornidazole were chromatographed on silica Gel 60 F(254) TLC plate using n-butanol:methanol:ammonia (6 M) (8:1:1.5 v/v) as the mobile phase and scanned at 302 nm using a Camag TLC Scanner 3. The R(f) value of gatifloxacin and ornidazole was found to be 0.21 +/- 0.02 and 0.76 +/- 0.04, respectively. The linearity of gatifloxacin and ornidazole were in the range of 100 - 500 ng/spot and 250 - 1250 ng/spot, respectively. The limit of detection was found to be 40 ng/spot for gatifloxacin and 100 ng/spot for ornidazole. The proposed method was applied for the determination of gatifloxacin and ornidazole in combined dosage forms.