Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.     

NMR and IR characterization of the aluminium complexes of norfloxacin and ciprofloxacin fluoroquinolones

A set of new aluminium complexes of norfloxacin (NOR) and ciprofloxacin (CIP) that show an improvement in their pharmaceutical properties were studied using solution and solid-state nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. The complexes synthesized with two different methods were compared. One of these methods will allow formulation of the compounds at production scale. High-resolution (13)C spectra were obtained with the cross-polarization and magic angle spinning (CP-MAS) experiment. These spectra were assigned by comparing them with the solution data of the pure drug and by using a quaternary carbon edition technique. The carbon relaxation times in the rotating frame, T(1rhoC), were measured for all the complexes. A two-exponential decay evidences that the complexes are nonhomogeneous. The short T(1rhoC) values are in the range 320-1100 micros and the long values in the range 1.8-7 ms. (27)Al MAS NMR spectra revealed an octahedral coordination between the aluminium ion and oxygens of the pure drug, supporting a 3:1 ligand:metal stoichiometry in both CIP and NOR complexes. The stretching and deformation modes of carboxylic acid and carboxylate and keto groups were analyzed by IR. This technique shows that the same modes are present in the aluminium complexes obtained by the two methods and that the coordination of the fluoroquinolone to aluminium occurs through the 4-keto and 3-carboxylic groups.     

Supercritical fluid extraction of the fluoroquinolones norfloxacin and ofloxacin from orally treated-chicken breast muscles

Supercritical fluid extraction (SFE) of the fluoroquinolones norfloxacin and ofloxacin from chicken breast muscles was examined. A liquid chromatography with fluorescence detection was used for the determination of the fluoroquinolones. Extraction conditions of the SFE were optimized by determining the extraction parameters to achieve a sufficiently high recovery of each fluoroquinolone in fortified-muscle samples. Recovery values for the extraction of the fluoroquinolones using the SFE ranged from 70 to 87%. Chickens were treated orally with each fluoroquinolone and their muscles were extracted at set time intervals for time-course determination of the fluoroquinolones in chickens. The SFE combined with liquid chromatographic analysis showed that the concentrations of the fluoroquinolones decreased gradually with time in the chicken muscles after oral treatment, giving a concentration less than 5 ng/ml in 120 h. No further sample cleanup procedures were required after the SFE. These results suggest that SFE method is an extraction method for the determination of norfloxacin and ofloxacin in chicken muscle.     

Simultaneous determination of trimethoprim,enrofloxacin, and ciprofloxacin in blood serum of poultry

A method is proposed for the simultaneous determination of trimethoprim, enrofloxacin, and ciprofloxacin in blood serum of poultry using HPLC. Samples were prepared using protein precipitation. The chromatographic separation of substances was attained on a C18 column using a mixture of a 50 mM acetate buffer solution (pH 3.0) with acetonitrile (86: 14) as a mobile phase. Detection was performed at 278 nm. Linearity was observed in the concentration range 50–5000 ng/mL for trimethoprim and 10–5000 ng/mL for ciprofloxacin and enrofloxacin. Precision was ≤2.1% for enrofloxacin, ≤1.6% for ciprofloxacin, and ≤14% for trimethoprim. Accuracy was ≤10.2% for enrofloxacin, ≤9.9% for ciprofloxacin, and ≤11.9% for trimethoprim. The method was applied to pharmacokinetic studies of complex antibacterial drugs containing trimethoprim and enrofloxacin as active substances.     

Differential pulse polarographic determination of ofloxacin in pharmaceuticals and biological fluids

A sensitive method is described for the determination of ofloxacin in its pure form, dosage forms and biological fluids. The proposed method depends upon the polarographic activity of ofloxacin in Britton Robinson buffers, whereby a well-defined cathodic wave is produced over the pH range 4.1-10.3. The wave was characterized as being irreversible, diffusion-controlled with limited adsorption properties. The current-concentration relationship was found to be rectilinear over the range 5x10(-5)-5x10(-4) M and 1x10(-5)-5x10(-4) M using the DC(t) and DPP modes respectively, with a minimum detectability (S/N=3) of 3x10(-7) M. The proposed method was successfully applied to the determination of ofloxacin in tablets and biological fluids. The results obtained were found to be in agreement with those obtained by a reference method.     

Direct determination of antibacterial norfloxacin in urine by isopotential fluorimetry

A novel and simple method is presented for the determination of norfloxacin in human urine by matrix isopotential synchronous fluorescence spectrometry (MISF). This method is useful for the determination of compounds in samples with unknown background fluorescence, such as norfloxacin in urine, without the need for tedious sample pre-treatment. The method was performed in ethanol-water medium (10% v/v), at an apparent pH of 4.8 provided by adding sodium acetate-acetic acid buffer solution. In the determination of norfloxacin in urine the fluorescent intensity varied linearly with its concentration from 20 to 200 ng/mL. An exhaustive statistical analysis has been developed to all calibration graphs, this treatment includes robust regression such as least median of squares, which also detects outliers and leverage points. The overall least-squares regression has been applied to find the more exact straight line that fits the experimental data. The error propagation has been considered to calculate the detection limit and the repeatability of the method. The method shows very low detection limits with acceptable recoveries and precisions. The applicability of the proposed method was illustrated in the determination of norfloxacin in human urine sample without sample pre-treatment.     

Separation of seven fluoroquinolones by microemulsion electrokinetic chromatography and application to ciprofloxacin, lomefloxacin determination in urine

A simple, reliable microemulsion electrokinetic chromatography (MEEKC) method is developed for the simultaneous separation of seven fluoroquinolones (FQs). The best separation is achieved in a carrier electrolyte containing 1% (v/v) heptane, 100 mmol/L sodium dodecyl sulfate (SDS), 10% (v/v) 1-butanol, and 8 mmol/L phosphate-sodium tetraborate buffer at pH 7.30. The proposed method was directly applied to the determination of ciprofloxacin (CPF) and lomefloxacin (LMF) in urine samples of subjects administered either with CPF or LMF.     

Determination of ofloxacin, norfloxacin, and ciprofloxacin in sewage by selective solid-phase extraction, liquid chromatography with fluorescence detection, and liquid chromatography--tandem mass spectrometry

A solid-phase extraction (SPE) and liquid chromatographic (LC) method was developed for the determination of selected fluoroquinolone (FQ) drugs including ofloxacin, norfloxacin, and ciprofloxacin in municipal wastewater samples. Extraction of the FQs was carried out with a weak cation exchanger SPE cartridge, the Oasis WCX. The cartridge was washed with water and methanol as a cleanup before the FQs were eluted by a mixture of methanol, acetonitrile, and formic acid. Separation of the FQs was achieved by using a Zorbax SB-C8 column under isocratic condition at a flow rate of 0.2mL/min. Recoveries of the FQs in spiked final effluent samples were between 87 and 94% with a relative standard deviation of less than 6%. Several techniques have been evaluated for the detection of FQs in sewage extracts; they included fluorescence detection and electrospray ionization (ESI) mass spectrometry using either mass-selective detection or tandem mass spectrometry (MS/MS). When they were applied to sewage influent and effluent samples, the LC-MS/MS technique operating in the multiple reaction monitoring (MRM) mode proved to be best suited for the determination of FQs in sewage samples as it provided the highest sensitivity (limit of quantification 5ng/L) and selectivity. The observation of signal suppression (matrix effect) for some FQs in ESI LC-MS and LC-MS/MS is discussed and a solution is proposed. The three FQs were detected in all the sewage samples tested in this work, with median concentrations between 34 and 251ng/L.     

Simultaneous determination of dexamethasone and betamethasone in pharmaceuticals by reversed-phase HPLC

Chromatographia - An HPLC method for the simultaneous determination of the glucocorticoids betamethasone and dexamethasone is described. The method based on the separation of these compounds using...     

Column high-performance liquid chromatographic determination of norfloxacin and its main impurities in pharmaceuticals

A gradient reversed-phase column high-performance liquid chromatographic method was developed for the detection and quantification of norfloxacin and its major impurities in norfloxacin-containing pharmaceuticals. Chromatographic separations were performed under the following experimental conditions: column, Zorbax SB RP-18 (5 microm, 250 x 4.6 mm); injection volume, 20 microL; mobile phase, 0.05 M NaH2PO4 (pH 2.5)-acetonitrile (87 + 13) for 16 min and (58 + 42) for 9 min (stepwise gradient); and flow rate, 1.3 mL/min. All analyses were performed at 25 degrees C, and the eluate was monitored at 275 nm using a diode array detector. Linearity (correlation coefficient = 0.999), recovery (99.3-101.8%), relative standard deviation (0.2-0.7%), and quantitation limit (0.12-0.47 microg/mL) were evaluated and found to be satisfactory. The method is simple, rapid, and convenient for purity control of norfloxacin in both raw materials and dosage forms.     

Direct determination of arsenic in blood serum by electrothermal atomic absorption spectrometry

A method for the direct determination of arsenic in human blood serum is described. To suppress loss of arsenic by volatilization anal to remove chemical interferences in graphite-furnace atomic absorption spectrometry, the formation of involatile compounds with graphite, or with a matrix modifier is tested. With aqueous solutions, two sorts of interactions between graphite and arsenic are shown. But, in presence of serum, these interactions do not occur, Among 18 matrix modifiers tested, nickel gives the best sensitivity when used at high concentrations in the presence of Triton X-100. The proposed method allows direct arsenic determination, based on calibration with aqueous solutions. The method is applied to the serum of 20 normal subjects. The limit of detection is 0.4 μg l^?1 arsenic.     

Electronic structure and specroscopic properties of norfloxacin,enoxacin, and nalidixic acid

The X-ray photoelectron spectra (XPS) of the core electrons of the carbon, oxygen, nitrogen, and fluorine atoms of antibiotics from the class of quinolones (nalidixic acid, norfloxacin, and enoxacin), having different antibacterial activities, were measured and interpreted. The integrated intensities of the π-π* and n-π* transition bands in the absorption spectra of the compounds were analyzed. The maxima of the luminescence spectral bands are given along with the lifetimes of the excited states and quantum yields of the protolytic forms of the compounds in aqueous media. The XPS data were compared with the Mulliken charges on heteroatoms obtained by quantum-chemical calculations.     

铁络合光度法测定氧氟沙星和诺氟沙星含量

研究了氧氟沙星、诺氟沙星与Fe(III)在室温下的络合反应.在酸性介质中,氧氟沙星、诺氟沙星与Fe(III)形成的黄色配合物最大吸收波长为436nm;氧氟沙星和诺氟沙星的浓度分别在2.24~192mg/L和8.0~248mg/L范围内与体系的吸光度呈良好的线性关系,其线性回归方程分别为:A=0.009 06+0.005 7 c(c,mg/L;r=0.999 3;氧氟沙星)和A=0.001 23+0.003 04 c(c,mg/L;r=0.999 8;诺氟沙星),方法的检出限分别为0.41和0.76mg/L.对浓度均为64.0mg/L的氧氟沙星和诺氟沙星平行测定11次,其相对标准偏差分别为0.6%和0.8%.应用拟定的方法对氧氟沙星和诺氟沙星药物制剂中主成分的含量进行了测定,回收率在99.7%~104.7%范围内.     

Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization

Capillary electrophoresis (CE) with UV photo-diode array detection technique was utilized to adopt new method for the analysis of norfloxacin and tinidazole in pharmaceuticals. Many CE aspects including separation, rapidity, sensitivity, ruggedness as well as the repeatability of qualitative and quantitative analyses were considered simultaneously for the purpose of optimization. Experimental design approach including factorial design and response surface methods were applied to optimize electrolyte concentration and the pH while injection time, voltage and column temperature were optimized using the univariate method. Successful results were obtained using 32.5 mmol l^−l phosphate electrolyte at pH 2.5, injection time 8.0 s, voltage 25 kV and column temperature 25 °C with detection at wavelength 301 nm. The analytical characteristics including recovery, intermediate precision, linear dynamic ranges, linearity and selectivity as well as limits of detection and quantification were demonstrated and the applicability to pharmaceuticals was studied. The newly provided method enjoys the advantages of CE over HPLC with respect to rapidity, ruggedness, simplicity in reagents and sample preparation as well as saving in reagents and samples.     

Concurrent determination of four fluoroquinolones in catfish,shrimp, and salmon by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method with fluorescence detection was developed for concurrent determination of 4 fluoroquinolones: ciprofloxacin (CIPRO), enrofloxacin (ENRO), sarafloxacin (SARA), and difloxacin (DIFLX) in catfish, shrimp, and salmon. The procedure consists of extraction from fish tissue with acidified ethanol, isolation and retention on a cation exchange solid-phase extraction column, elution with basic methanol, and LC analysis with fluorescence detection. LC is performed by isocratic elution with acetonitrile-2% acetic acid (16 + 84) mobile phase, and a PLRP-S polymer column with fluorescence detection, excitation 278 nm and emission 450 nm. A target level of 20 ppb for each of the 4 fluoroquinolones has been established for this method. Fortified and incurred fish sample results are based on a 5-point standard curve calculation (10-160 ppb). Overall percent recoveries (% relative standard deviation) from fortified catfish were 78 (10), 80 (11), 70 (9.4), and 78 (10); from fortified shrimp, 69 (5.9), 85 (4.9), 79 (5.9), and 90 (4.5); and from fortified salmon, 56 (15), 93 (5.6), 61 (11), and 87 (5.0) for CIPRO, ENRO, SARA, and DIFLX, respectively. Data from the analysis of fluoroquinolone-incurred catfish, shrimp, and salmon are presented.     

HPLC determination of ciprofloxacin, cloxacillin, and ibuprofen drugs in human urine samples

This paper reports, for the first time, a liquid chromatographic method for the simultaneous determination of three frequently co-administered active principles, two antibiotics, ciprofloxacin (CIPRO) and cloxacillin (CLOXA) belonging to the fluoroquinolones and beta-lactam families, respectively, and ibuprofen (IBU), a non-steroidal anti-inflammatory drug. The chromatographic separation was performed on a C-18 analytical column, using isocratic elution with methanol-acetonitrile-pH 3 formate buffer (CT = 0.1 M) (15:12:73, v/v/v) for 3 min and, after that, a linear gradient with methanol-acetonitrile (88:12, v/v) for 8 min. Several absorption spectra were obtained for each peak using a DAD detector. Chromatograms at the maximum absorption wavelength for each analyte, 220 nm for both IBU and CLOXA, and 280 nm for CIPRO were selected as the most suitable. The proposed chromatographic method requires about 15 min per sample. The presence of a urine background was tested and no interference was found. The method was satisfactorily applied to the determination of CIPRO, CLOXA, and IBU, in fortified urine, and in urine samples from a patient undergoing treatment with these three active principles, among others. Limits of quantification in urine were 1.00, 1.70, and 2.87 microg/mL for CIPRO, CLOXA, and IBU, respectively.     

Determination of norfloxacin in pharmaceuticals,human serum,and urine using a luminol—dissolved oxygen chemiluminescence system

A sensitive chemiluminescence method, based on the enhancive effect of norfloxacin on the reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of norfloxacin. The increment of the chemiluminiscence intensity was proportional to the concentration of norfloxacin, giving a calibration graph linear over the concentration from 0.4 ng mL⁻¹ to 400.0 ng mL⁻¹ (r ² = 0.9988) with the detection limit of 0.1 ng mL⁻¹ (3 × σ _noise). At the flow rate of 2.0 mL min⁻¹, a complete determination of norfloxacin, including sampling and washing, could be accomplished in 30 s with the relative standard deviation lower than 3.0 %. The proposed method was applied successfully to determine norfloxacin in pharmaceuticals, human urine, and serum. Possible mechanism of the reaction was also discussed.     

Separation of fifteen quinolones by high performance liquid chromatography: application to pharmaceuticals and ofloxacin determination in urine

A simple chromatographic method is described for assaying 15 quinolones and fluoroquinolones (pipemidic acid, marbofloxacin, enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, nalidixic acid, flumequine and piromidic acid), in urine and pharmaceutical samples. The determination was achieved by LC using an RP C18 analytical column. A mobile phase composed of mixtures of methanol-ACN-10 mM citrate buffer at pH 3.5 and 10 mM citrate buffer at pH 4.5, delivered under an optimum gradient program, at a flow rate of 1.5 mL/min, allows to accomplish the chromatographic separation in 26 min. For detection, diode-array UV-Vis at 280 nm and fluorescence detection set at excitation wavelength/emission wavelength: 280/450, 280/ 495, 280/405 and 320/360 nm were used. Detection and quantification limits were between 0.3-18 and 0.8-61 ng/mL, respectively. The method was validated in terms of interday (n = 6) and intraday (n = 6) precision and accuracy. The procedure was successfully applied to the analysis of human and veterinary pharmaceuticals. Also, ofloxacin was determined in human urine samples belonging to a patient undergoing treatment with this active principle, among others.     

Development of a validated HPLC method for the determination of four penicillin antibiotics in pharmaceuticals and human biological fluids

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.