Simultaneous determination of piroxicam and norfloxacin in biological fluids by high-performance liquid chromatography with fluorescence detection at zero-order emission mode.

A new highly sensitive high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) in zero-order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero-order mode for emission. The zero-order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001-20 µg/ml and 0.00003-0.035 µg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10-4 and 1.32 × 10-5 µg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10-3 and 4.01 × 10-5 µg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF.

Determination of Norfloxacin Using a Tetraoxocalix[2]arene[2]triazine Covalently Functionalized Multi-walled Carbon Nanotubes Modified Electrode.

This paper presents a sensitive voltametric procedure for the determination of norfloxacin (NF) by a tetraoxocalix[2]arene[2]triazine (TOCT) covalently functionalized multi-walled carbon nanotubes (MWCNTs) modified electrode. The electrochemical sensing of NF was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Through a combination of the excellent selective recognition of TOCT and the outstanding electronic properties of MWCNTs, this electrochemical sensor shows excellent sensitivity and high selectivity for an electrochemical detection of NF. The stripping response is highly linear (R = 0.996) over the NF concentration range of 0.5 - 8.0 µM with the LOD of 0.1 µM. The fabricated sensors were successfully applied for quantitative detection of NF in pharmaceutical formulations and human urine samples. A high anti-interference ability to common interferences and satisfactory results were obtained. This is expected to play a huge potential in the real-time monitoring of NF in clinical applications.

Square-wave adsorptive anodic stripping voltammetric determination of norfloxacin using a glassy carbon electrode modified with carbon black and CdTe quantum dots in a chitosan film.

A glassy carbon electrode was modified with carbon black and CdTe quantum dots in a chitosan film to obtained a sensor for norfloxacin (NOR) in the presence of dopamine, caffeine, and uric acid. The morphological, structural and electrochemical characteristics of the nanostructured material were evaluated using spectrophotometry, X-ray diffraction, transmission electronic microscopy and voltammetry. The high electrochemical activity, fast electron transfer rate and high surface area enhanced the oxidation peak currents and shifted the peak potentials of NOR for more negative values (typically at 0.95 V vs. Ag/AgCl). Electrochemical determination of NOR was carried out using square-wave adsorptive anodic stripping voltammetry (SWAdASV). Response is linear in the 0.2 to 7.4 µmol L-1 NOR concentration range, and the detection limit is as low as 6.6 nmol L-1. The method was successfully applied to the determination of norfloxacin in pharmaceutical formulation, synthetic urine and spiked serum. Graphical abstract Schematic presentation of a voltammetric method using a glassy carbon electrode modified with carbon black and CdTe quantum dots in a chitosan film for the determination of norfloxacin in serum and urine samples.

Determination of norfloxacin in urine and pharmaceutical samples using terbium doped zinc sulphide nanomaterials-sensitized fluorescence method.

Highly crystalline polyethylene glycol (PEG) coated Tb3+ doped ZnS nanoparticles have been synthesized and successfully used for norfloxacin sensing. The crystallographic and morphological analyses of PEG coated Tb3+ doped ZnS nanoparticles were performed by X-ray diffraction and Transmission electron microscopy, respectively. The confirmation of Tb3+ doping in ZnS host matrix was done by emission spectroscopy and energy dispersive X-ray spectroscopy. Further, the interaction of norfloxacin with PEG coated Tb3+ doped ZnS nanomaterials was confirmed by optical analysis: spectrophotometrically and spectrofluorimetrically. Norfloxacin sensing was measured by luminescence intensity which increased with increase in concentration of norfloxacin in range from 2.0 × 10-9-8.0 × 10-7 mol L-1, with its correlation coefficient 0.9991. The detection limit of proposed method was 0.05 × 10-9 mol L-1. The developed luminescence method was successfully applied for the determination of norfloxacin using PEG coated Tb3+ doped ZnS nanoparticles in urine and pharmaceutical samples.

High performance liquid chromatography coupled with resonance Rayleigh scattering for the detection of three fluoroquinolones and mechanism study.

A reliable and versatile high performance liquid chromatography coupled with resonance Rayleigh scattering method was established for the determination of three fluoroquinolones, including levofloxacin, norfloxacin and enrofloxacin in water sample and human urine sample. In pH 4.4-4.6 Britton-Robinson buffer medium, the fluoroquinolones separated by high performance liquid chromatography could react with erythrosine to form 1:1 ion-association complexes, which could make contributions to the great enhancement of RRS. The resonance Rayleigh scattering signal was recorded at λex=λem=330 nm. The resonance Rayleigh scattering spectral characteristics of the drugs and the experimental conditions such as pH, detection wavelength, erythrosine concentration, flow rate, the length of reaction tube were studied. Quantum chemistry calculation, Fourier transform infrared spectroscopy and absorption spectroscopy were used to discuss the reaction mechanism. The recoveries of samples added standard ranged from 97.53% to 102.00%, and the relative standard deviation was below 4.64%. The limit of detection (S/N=3) of 0.05-0.12 µg mL(-1) was reached, and the linear regression coefficients were all above 0.999. The proposed method was proved as a simple, low cost and high sensitivity method.

Computational simulation and preparation of fluorescent magnetic molecularly imprinted silica nanospheres for ciprofloxacin or norfloxacin sensing.

A magnetic molecularly imprinted fluorescent sensor for the sensitive and convenient determination of ciprofloxacin or norfloxacin in human urine was synthesized and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, ultraviolet/visible spectroscopy, and fluorescence spectroscopy. Both cadmium telluride quantum dots and ferroferric oxide nanoparticles are introduced into the polymer for the rapid separation and detection of the target molecules. The synthesized molecularly imprinted polymers were applied to detect ciprofloxacin or its structural analog norfloxacin in human urine with the detection limit 130 ng/mL. A computational study was developed to evaluate the template-monomer geometry and interaction energy in the polymerization mixture to determine the reaction molar ratio of the template and monomer molecules.

Detection of norfloxacin and monitoring its effect on caffeine catabolism in urine samples.

A multi-walled carbon nano tube (MWCNT) modified pyrolytic graphite (MPG) electrode is prepared and applied to detect norfloxacin (NFX) based on its electrochemical reduction. The experimental parameters affecting the NFX determination were optimized in terms of MWCNT amount, pH, reaction time, and square wave frequency. The dynamic range for the NFX analysis ranged between 1.2 and 1000µM with a detection limit of 40.6±3.3nM. The effect of NFX on the catabolism of caffeine has been studied by determining its concentration in the urine samples after the prolonged administration of NFX using the MPG electrode. The results show that the catabolism of caffeine is inhibited by ~65% after five days of NFX administration, consequently the caffeine concentration in the urine sample is increased, which is reflected in terms of ~2.5 times increase in the peak current of caffeine. The determinations of NFX and caffeine were selective and the method was successfully applied in biological fluids and pharmaceutical tablets for the test compound analysis. In future this method can be useful for the selective determination of NFX and studying its effect on caffeine catabolism.

Preparation of an amperometric sensor for norfloxacin based on molecularly imprinted grafting photopolymerization.

A sensitive amperometric sensor for norfloxacin (NF) was introduced. The receptor layer was prepared by molecularly imprinted photopolymerization of acrylamide and trimethylolpropane trimethacrylate on the surface of a gold electrode. The binding mechanism of the molecularly imprinted polymer was explored by ultraviolet (UV) and infrared (IR) spectroscopy. The chemosensor was characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), electrochemical impedance (EI), and scanning electron microscopy (SEM). The electrode prepared by photopolymerization has a better recognition ability to template molecules than that of electropolymerization and NIP. Some parameters affecting sensor response were optimized. Norfloxacin was detected by measurements of an amperometric i-t curve. The linear relationships between current and logarithmic concentration are obtained from 1.0 × 10(-9) to 1.0 × 10(-3) mol L(-1). The detection limit of the sensor was 1.0 × 10(-10) mol L(-1). The proposed method is sensitive, simple, and cheap, and is applied to detect NF in human urine successfully.

Antimutagenic evaluation of vitamins B1, B6 and B12 in vitro and in vivo, with the Ames test.

The aim of this work is to evaluate vitamins B antimutagenic effect against alkylatings methyl-N-nitro-N-nitrosoguanidine (MNNG), ethyl-N-nitro-N'- nitrosoguanidine (ENNG), frameshift mutagens 2-aminoanthracene (2AA) and 2-acetyl-amino-fluorene (2AF) and ROS-generating antibiotics norfloxacin (NOR) and nalidixic acid (NLX), using the in vitro Ames test. In vivo antimutagenesis studies were performed against urinary mutagens induced by NOR (70 mg/kg) or NLX (100 mg/kg) in CD1 mice. Vitamin B1 was antimutagenic against alkylatings MNNG (P<0.05) or ENNG (P<0.001). In fact as per the results observed during the current study, none of the vitamins reduced mutagenesis caused by frameshift mutagens. All of them reduced mutagenesis of NOR or NLX (P<0.001). In vivo studies showed that vitamins B1 and B6 (10 or 100 mg/kg) reduced urinary mutagens from NOR (P<0.001) or NLX (P<0.02) either free or ß-glucoronidase-conjugates. None of the studied samples were toxic for the employed antimutagenic system. Vitamin B12 (4 mg/kg) reduced urinary mutagens of NOR or NLX (P<0.02). Vitamins B inhibited DNA mutations induced by ROS generated by NLX or NOR, both in vitro and in vivo. Vitamin B1is antimutagenic against mutations induced by the alkylating MNNG or ENNG. Based on the observations, employment of vitamins B in vivo can be a promising alternative to reduce genotoxic risk exposure to ROS.

Electrochemical sensor for the sensitive determination of norfloxacin in human urine and pharmaceuticals.

Electrochemical determination of Norfloxacin (NF) has been presented at edge plane (EPPGS) and basal plane pyrolytic graphite sensors (BPPGS) by using square wave voltammetry at physiological pH 7.2. An increased peak current with a shift of peak potential to less positive value was observed at EPPGS as compared to BPPGS. The effect of pH, scan rate and analyte concentration has been examined. The peak current was found to be linear to the concentration of NF in the range 0.5 × 10(-6) to 50.0 × 10(-6)mol L(-1) for EPPGS and the detection limit (3σ/b) was found to be 28.3 × 10(-8)mol L(-1). The method has been successfully used to determine the content of NF in the pharmaceutical preparations. Biological relevance of the developed method has been described by the determination of NF in human urine samples of the patients undergoing treatment with NF. The method is selective and NF can be determined without any interference from common urine metabolites such as uric acid and ascorbic acid.

Chemiluminescence determination of fluoroquinolones using Fenton system in the presence of terbium(iii) ions.

A simple new chemiluminescent, CL, method is described for the determination of fluoroquinolones such as: ciprofloxacin (CF), norfloxacin (NF), and ofloxacin (OF). This method is based on the measurement of terbium(iii) emission. This emission follows an energy transfer to the uncomplexed terbium(iii) ions from the excited products of fluoroquinolone oxidations. Under optimum conditions, calibration graphs were obtained for 2 × 10(-8)-2 × 10(-6) mol L(-1) of NF; 3 × 10(-8)-2 × 10(-6) mol L(-1) of CF and 4 × 10(-7)-5 × 10(-5) mol L(-1) of OF. The detection limits are 7 × 10(-9) mol L(-1) norfloxacin, 1 × 10(-8) mol L(-1) ciprofloxacin and 1.5 × 10(-7) mol L(-1) ofloxacin. The method was successfully applied to the determination of these drugs in pharmaceutical formulations.

A simple chromatographic method for determining norfloxacin and enoxacin in pharmacokinetic study assessing CYP1A2 inhibition.

We developed a simple assay method for the determination of serum and urine norfloxacin and enoxacin using reversed-phase high-performance liquid chromatography and perchloric acid precipitation for sample pre-treatment. Optimized conditions can permit detection of norfloxacin and enoxacin in the same chromatogram, so either compound can be used as an internal standard for another determinant. Supernatants of the precipitated samples were analyzed by the octadecylsilyl silica-gel column under ambient temperature and an ultraviolet wavelength of 272 nm. A mobile phase solvent consisting of 20 mm sodium dihydrogenphosphate (pH 3.0) and acetonitrile (85:15, v/v) was pumped at a flow rate of 1.0 mL/min. The calibration curves for norfloxacin and enoxacin at a concentration of 62.5-1000 ng/mL for serum and 250-4000 ng/mL for urine were linear (r > 0.9997). The recoveries of norfloxacin and enoxacin from serum and urine were >94% with the coefficient of variations (CV) <5%. The CVs for intra- and inter-day assay of norfloxacin and enoxacin were <4.2 and <5.5%, respectively. This method can be applied to the pharmacokinetic study of norfloxacin and enoxacin after repeated administration to assess changes in CYP1A2 activity in healthy subjects.

A novel chemiluminescence reaction system for the determination of norfloxacin with Ag(III) complex.

A novel chemiluminescence (CL) system for the determination of norfloxacin (NFLX) is developed based on the direct CL reaction of [Ag(HIO(6))(2)](5-)-H(2)SO(4)-NFLX system. The possible mechanism of CL emission and enhancing effect was discussed by comparing UV, fluorescence and CL spectra. [Ag(HIO(6))(2)](5-) in the presence of H(2)SO(4) could produce CL emission at 490 nm, this might be caused by the excited state (O(2))(2)*. The enhancing effect of NFLX may be produced through an intermolecular energy transfer from part of (O(2))(2)* to NFLX molecule and complex of Ag(3+) and NFLX. The CL intensity emission intensity was linear in the range 1.34 x 10(-8) to 5.44 x 10(-6) gmL(-1) with correlation coefficient of 0.9982. The detection limit (s/n=3) was 3.10 x 10(-9) gmL(-1). The recovery was in the range of 90.0-104% with the RSD of 1.1-2.8%. The proposed flow injection CL method was applied satisfactorily for the determination of NFLX in capsule, human serum and urine.

[Norfloxacine efficacy in acute cystitis in the region with 10% resistance of E. coli to fluoroquinolones: a comparative randomized study].

We have conducted a clinicomicrobiological study the first stage of which consisted in collection of information on uropathogens resistance to fluoroquinolones in 89 females with uncomplicated urinary infections (UI). Sensitivity to antibacterial drugs was determined by the agar dilution test (CLSI, 2007). At stage two of the study we made a prospective multicenter randomized trial including 108 females aged 18-55 years with acute uncomplicated cystitis. The patients were randomized into two groups: group 1 (n = 55) received norfloxacin (400 mg twice a day for 3 days); group 2 (n = 53) received phosphomycin (a single 3.0 g dose). Clinical and microbiological assessment of efficacy and safety was performed before the treatment, on treatment day 5, 10 and 28. Isolation of E. coli strains in the region highly and moderately resistant to cyprofloxacine was rather high--10 and 1.1%, respectively. Before the treatment E. coli was isolated from the urine in 82.4% cases, E. faecalis--5.9%, S. saprophyticus--3.9%, Staphylococcus spp--2.0%, others--< 1%. No significant differences by bacteriological and clinical efficacy were found between the groups. Eradication of the agent and persistent bacteriological response were seen in 100 and 95.2%, 95.8 and 100% patients of groups 1 and 2, respectively. Complete and partial responses were registered in 68.5 and 76%, 76 and 98% patients of groups 1 and 2, respectively. Thus, treatment with norfloxacin in a dose 400 mg twice a day for 3 days and with phosphomycin in a 3.0 g single dose have high clinical and microbiological efficacy in uncomplicated cystitis. Norfloxacin retains clinical and microbiological efficacy in acute uncomplicated cystitis even in conditions of 10% prevalence of fluoroquinolone-resistant strains. We think that there are reasons for revision of criteria of interpretation of uropathogens sensitivity to fluoroquinolones in the treatment of uncomplicated UI.

Gold nanoparticle-enhanced chemiluminescence detection for CE.

Introducing gold nanoparticles (AuNPs) to the running buffer further improved the sensitivity of luminol-H(2)O(2) chemiluminescence (CL) detection for CE. This has led to the development of sensitive CE-CL assays of biomedically interesting compounds. Epinephrine and norfloxacin were taken as the model analytes. Epinephrine inhibited light emission from the AuNP-catalyzed CL while norfloxacin enhanced it. The CE-CL assays had detection limits of 6.9x10(-9) M for epinephrine and 7.3x10(-9) M for norfloxacin. It was noted that in the absence of AuNPs no CE-CL analytical signal was produced by epinephrine at 4.0x10(-5) M or norfloxacin at 1.5x10(-3) M under similar experimental conditions. Fluorescence spectroscopic measurements showed that although the fluorescence excitation/emission maxima remained the same, the fluorescence lifetime of luminol increased significantly in the presence of AuNPs (tau(2) increased from 8.49+/-0.12 to 9.18+/-0.047 ns in a two-exponential fit), indicating that the excited states of luminol molecules were stabilized through the interaction between luminol molecules and AuNPs. Finally, quantitation of epinephrine and norfloxacin in biological samples such as human urine by using the present AuNP-enhanced CE-CL method was demonstrated.

Water-compatible molecularly imprinted polymer for the selective recognition of fluoroquinolone antibiotics in biological samples.

A novel water-compatible molecularly imprinted polymer (MIP), prepared with enrofloxacin (ENR) as the template, has been optimised for the selective extraction of fluoroquinolone antibiotics in aqueous media. The results of a morphological characterisation and selectivity tests of the polymer material for ENR and related derivatives are reported. High affinity for the piperazine-based fluoroquinolones marbofloxacin, ciprofloxacin, norfloxacin and ofloxacin was observed, whereas no retention was found for nonrelated antibiotics. Various parameters affecting the extraction efficiency of the polymer have been optimised to achieve selective extraction of the antibiotics from real samples and to reduce nonspecific interactions. These findings resulted in a MISPE/HPLC-FLD method allowing direct extraction of the analytes from aqueous samples with a selective wash using just 50% (v/v) organic solvent. The method showed excellent recoveries and precision when buffered urine samples fortified at five concentration levels (25-250 ng mL(-1) each) of marbofloxacin, ciprofloxacin, norfloxacin, enrofloxacin and sarafloxacin were tested (53-88%, RSD 1-10%, n = 3). Moreover, the biological matrix of the aqueous samples did not influence the preconcentration efficiency of the fluoroquinolones on the MIP cartridges; no significant differences were observed between the recovery rates of the antibiotics in buffer and urine samples. The detection limits of the whole process range between 1.9 and 34 ng mL(-1) when 5-mL urine samples are processed. The developed method has been successfully applied to preconcentration of norfloxacin in urine samples of a medicated patient, demonstrating the ability of the novel MIP for selective extraction of fluoroquinolones in urine samples.

Capillary electrophoresis-chemiluminescence determination of norfloxacin and prulifloxacin.

A capillary electrophoresis (CE)-chemiluminescence (CL) method for determining norfloxacin (NFLX) and prulifloxacin (PFLX) was developed based on the enhanced CL intensity of the cerium(IV)-sulfite-fluoroquinolone (FQ) reaction sensitized by terbium(III). The separation was conducted in buffer composed of 20 mM sodium citrate, 4 mM citric acid and 10 mM sodium sulfite at pH 6.1. The CL reagent solution consisted of 2 mM cerium(IV), 4 mM terbium(III) and 1.1 mM hydrochloric acid. NFLX and PFLX were baseline separated within 11 min with detection limits (S/N=3) of 0.057 and 0.084 microg mL(-1), respectively. The maximum intra- and inter-day relative standard deviations (R.S.D.s) of migration time of the analytes were less than 4.0% and 4.2%, respectively. The proposed method was applied to detect NFLX and PFLX in fortified urine sample and the results were comparable to high-performance liquid chromatography (HPLC)-UV method. Moreover, the high selectivity of the CL detection and the high-separation efficiency of CE render the method the potential of quick analyzing fluoroquinolones in real complex matrix.

Determination of levofloxacin and norfloxacin by capillary electrophoresis with electrochemiluminescence detection and applications in human urine.

A novel method for the determination of norfloxacin (NOR) and levofloxacin (LVX) was developed by CE separation and electrochemiluminesence detection (ECL). The methods for capillary conditioning and the effect of solvent type were studied. Parameters affecting the CE and ECL were also investigated. Under the optimum conditions, the two analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 4.8 x 10(-7) mol/L for NOR and 6.4 x 10(-7) mol/L for LVX, respectively. The precisions of intraday and interday are less than 4.2 and 8.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 1.2 x 10(-6) mol/L for NOR and 1.4 x 10(-6) mol/L for LVX, respectively. The applicability of the proposed method was illustrated in the determination of NOR and LVX in human urine samples and the monitoring of pharmacokinetics for NOR. The recoveries of NOR and LVX at different levels in human urine samples were between 84.3 and 92.3%.

Determination of norfloxacin and enrofloxacin by solid-phase microextraction/high-performance liquid chromatography.

A solid-phase microextraction (SPME) method followed by separation with high-performance liquid chromatography and subsequent UV detection was developed for the determination of norfloxacin and enrofloxacin. The simple and sensitive preconcentration technique uses 280 nm wavelength in mobile phase of citrate buffer (0.01 M), pH 3.8, prepared in water (A) and acetonitrile (B), with composition of the mobile phase A:B, 40:60, at a flow rate of 1.0 mL/min. A C18 reversed-phase analytical column (5 microm) was selected as separation medium for the technique. To obtain optimum extraction efficiency, several parameters relating to SPME were investigated. The method was linear over the range of 10-100 ng/mL for norfloxacin and enrofloxacin with a correlation coefficient (R2) value of 0.9972 and 0.9980 for norfloxacin and enrofloxacin, respectively. Using the SPME method, the detection limits (signal-to-noise ratio = 3) are 0.17 and 0.12 ng/mL for norfloxacin and enrofloxacin, respectively.

Comparative bioavailability of norfloxacin tablets based on blood and urine data.

OBJECTIVE: To assess the bioavailability of norfloxacin from urinary excretion relative to plasma concentration. MATERIALS AND METHODS: Twelve healthy volunteers (22-33 years) participated in the study. Each received a previously developed (M), a local (L) and a multinational (Noroxin) tablet (Ref), 400 mg each, according to a random balanced three-way crossover design on 3 different days. Blood samples were collected over a 12-hour period and urine over a 24-hour period. Norfloxacin concentrations were analyzed by a validated HPLC method. RESULTS: An initial estimate of bioequivalence of the three products was obtained using analysis of variance on transformed data and based on confidence interval calculation. Elimination pharmacokinetic parameters (half-life and renal clearance) calculated from plasma concentration and urinary excretion data (mean values, n = 36) were comparable to reported values for norfloxacin. Interproduct differences in elimination parameters (mean values, n = 12) were statistically insignificant (F values, ANOVA). Strong association was found between the mean of plasma concentration and urinary excretion rates for many volunteers (F values, regression analysis). Relative bioavailability values calculated for the local and previously developed products relative to Noroxin were higher than 85% based on area under the curve and urinary excretion. Bioequivalence could not be established among the three tested products based on calculated 90% confidence intervals. CONCLUSION: Urinary excretion of norfloxacin may be a useful noninvasive tool for bioavailability assessment of norfloxacin oral formulations.