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.     

Direct determination of four fluoroquinolones,enoxacin, norfloxacin,ofloxacin, and ciprofloxacin,in pharmaceuticals and blood serum by HPLC

A rapid, accurate and sensitive method has been developed for the quantitative determination of four fluoroquinolone antimicrobial agents, enoxacin, norfloxacin, ofloxacin and ciprofloxacin, with high in-vitro activity against a wide range of Gram-negative and Gram-positive organisms.A Kromasil 100 C(8) 250 mm x 4 mm, 5 microm analytical column was used with an eluting system consisting of a mixture of CH(3)CN-CH(3)OH-citric acid 0.4 mol L(-1) (7:15:78 %, v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in limits of detection: 0.02 ng per 20 microL injection for enoxacin and 0.01 ng for ofloxacin, norfloxacin and ciprofloxacin. Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng microL(-1). A rectilinear relationship was observed up to 2 ng microL(-1) for enoxacin, 12 ng microL(-1) for ofloxacin, 3 ng microL(-1) for norfloxacin, and 5 ng microL(-1) for ciprofloxacin. Separation was achieved within 10 min. The statistical evaluation of the method was examined by performing intra-day (n=8) and inter-day precision assays (n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97+/-6% over the range 0.1-0.5 ng microL(-1).     

Formation of secondary triplet species after excitation of fluoroquinolones in the presence of relatively strong bases

Laser flash photolysis of 7-(piperazin-1-yl) fluoroquinolones leads to the formation of a triplet excited state (3A*) at the end of the pulse (lambdamax 520, 610, and 620 nm for enoxacin, ciprofloxacin, and norfloxacin, respectively). Phosphate and bicarbonate buffers react with 3A* to form a secondary triplet (3B*, reaction rates (0.8-9.9) x 108 M-1 s-1), whose T-T absorption is red-shifted (lambdamax 670 nm for enoxacin, 700 nm for ciprofloxacin and norfloxacin). The formation of a secondary triplet is not a common process and disagrees with previous work suggesting that electron transfer occurs between phosphate buffer and the primary triplet excited state with the formation of the anion radical of the fluoroquinolone (FQ.-). We have shown that the FQ.- transient absorption spectrum is quite distinct from that of 3B*. The photophysical characteristics of 3B* have been determined by energy transfer to naproxen, and it has been found that its energy is lower than that of 3A*.     

Spectrofluorimetric determination of moxifloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine the antibiotic moxifloxacin is proposed and was applied to pharmaceuticals, human urine and serum. The fluorimetric method allows the determination of 30-300 ng mL-1 moxifloxacin in aqueous solution containing phosphoric acid-phosphate buffer (pH 8.3) with lambda exc = 287 nm and lambda em = 465 nm. Detection and quantification limits were 10 and 30 ng mL-1, respectively, with a relative standard deviation (n = 10) of 2%. This method was applied to the determination of moxifloxacin in three Spanish commercial pharmaceutical formulations. Another variant of the method in micellar medium allows the direct measurement of moxifloxacin in human serum and urine by standard additions. The enhanced fluorescence of moxifloxacin in 8 mM sodium dodecyl sulfate (SDS) solution at pH 4.0 (acetic acid-acetate buffer) for lambda exc = 294 nm and lambda em = 503 nm shows the same linear range as the aqueous method with a 25% lower slope (with detection and quantification limits of 15 and 60 ng mL-1, respectively, and a relative standard deviation of 1.3%), but permits the background fluorescence for urine and serum blanks to be minimized. Hence, sufficient sensitivity is reached to determine therapeutic concentrations of the drug in urine (average recovery 102 +/- 2%) and serum (average recovery 105 +/- 2%) samples.     

硫胺素-亚硝酸根体系的荧光性质研究及应用

研究了在碱性溶液中硫胺素（VB1）与亚硝酸根的荧光反应,建立了荧光光度法测定硫胺素的新方法。方法线性范围为1．28－324ng/mL,检出限0．52ng/mL,对15个浓度为100ng/mL的硫胺素溶液进行测定,相对标准偏差为1．4％。应用于药物中硫胺素的测定,回收率在96．5％－101．8％之间。     

Spectrophotometric and spectrofluorimetric methods for the determination of pregabalin in bulk and pharmaceutical preparation

Two new, sensitive and selective spectrofluorimetric and spectrophotometric methods have been developed for the determination of the gamma-amino-n-butyric acid derivative pregabalin (PGB) in bulk drug and capsule. Pregabalin, as a primary amine compound, reacts with 7-chloro-4-nitrobenzofurazon (NBD-Cl) which is a highly sensitive fluorogenic and chromogenic reagent used in many investigations. According to this fact, spectrophotometric and spectrofluorimetric methods for the determination of pregabalin in capsules were developed for the first time. The relation between the absorbance at 460 nm and the concentration is rectilinear over the range 0.5-7.0 microg mL(-1). The reaction product was also measured spectrofluorimetrically at 558 nm after excitation at 460 nm. The fluorescence intensity was directly proportional to the concentration over the range 40-400 ng mL(-1). The method was applied successfully to the determination of this drug in pharmaceutical dosage form. The mean recovery for the commercial capsules was 99.93% and 99.96% for spectrophotometric and spectrofluorimetric study, respectively. The suggested procedures could be used for the determination of PGB in pure and capsules being sensitive, simple and selective.     

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. Copyright © 2010 John Wiley & Sons, Ltd.     

Flow-Injection Chemiluminescence Determination of Fluoroquinolones

ABSTRACT

A new flow-injection CL method was developed for the determination of fluoroquinolones including ofloxacin, norfloxacin, ciprofloxacin and lomefloxacin in pharmaceutical preparations, based on the chemiluminescence reaction of sulphite with cerium(IV) sensitized by these compounds. The linear ranges are 0.04 to 4.0 μg ml^?1 for ofloxacin and 0.4 to 40.0 μg ml^?1 for norfloxacin, ciprofloxacin and lomefloxacin, respectively. The detection limits are 0.016 μg ml^?1 for ofloxacin and 0.16 μg ml ^?1 for norfloxacin, ciprofloxacin and lomefloxacin, respectively. The relative standard deviations (RSD) are 2.1 to 2.6% (n=10) for these fluoroquinolones. The analytical procedure has been applied to the determination of the fluoroquinolones in pharmaceutical commercial formulations. The results are in agreement with those obtained by the official methods.     

Study of fluorescence characteristics of the charge-transfer reaction of quinolone agents with bromanil

A spectrofluorimetric method was discussed for the determination of three antibacterial quinolone derivatives, ofloxacin (OFL), norfloxacin (NOR) and ciprofloxacin (CIP) through charge-transfer complexation (CTC) with 2,3,5,6-tetrabromo-1,4-benzoquinone (bromanil, TBBQ). The method was based on the reaction of these drugs as n-electron donors with the pi-acceptor TBBQ. TBBQ was found to react with these drugs to produce a kind of yellow complexes and the fluorescence intensities of the complexes were enhanced by 29-36 times more than those of the corresponding monomers. UV-vis, (1)H NMR and XPS techniques were used to study the complexes formed. The various experimental parameters affecting the fluorescence intensity were studied and optimized. Under optimal reaction conditions, the rectilinear calibration graphs were obtained in the concentration range of 0.021-2.42 microg mL(-1), 0.017-2.63 microg mL(-1) and 0.019-2.14 microg mL(-1) for OFL, NOR and CIP, respectively. The methods developed were applied successfully to the determination of the subject drugs in their pharmaceutical dosage forms with good precision and accuracy compared to official and reported methods as revealed by t- and F-tests.     

A flow injection chemiluminescence method for the determination of fluoroquinolone derivative using the reaction of luminol and hydrogen peroxide catalyzed by gold nanoparticles

Based on the enhancement of chemiluminescence (CL) of luminol-hydrogen peroxide-gold nanoparticles system by fluoroquinolones (FQs), a novel and rapid CL method is reported for the determination of FQs derivatives. Under the optimum conditions, the CL intensity is proportional to the concentration of FQs derivative in solution. The corresponding linear regression equations are established over the range of 0.08-1.28 μg/mL for norfloxacin, 0.013-1.32 μg/mL for ciprofloxacin, 0.014-1.4 μg/mL for lomefloxacin, 0.029-1.46 μg/mL for fleroxacin, 0.02-1.0 μg/mL for ofloxacin and 0.01-1.44 μg/mL for levofloxacin, respectively. The limits of detection (S/N = 3) are 3.2, 9.5, 7.0, 9.0, 8.0, and 8.0 ng/mL with the relative standard deviation (n = 11) 4.3, 1.5, 1.9, 1.3, 1.6 and 2.1% for norfloxacin, ciprofloxacin, lomefloxacin, fleroxacin, ofloxacin and levofloxacin, respectively. This proposed method has been applied to detect FQs derivatives in human urine successfully.     

Sequential injection technique employed for stoichiometric studies, optimization and quantitative determination of some fluoroquinolone antibiotics complexed with iron(III) in sulfuric acid media

A sequential injection spectrophotometric method for stoichiometric studies, optimization and quantitative determination of ciprofloxacin and norfloxacin was developed. The work is based on the complexation reaction of ciprofloxacin and norfloxacin with iron(III) in sulfuric acid media and a spectrophotometric measurement of absorbances of the corresponding complexes at 447 and 430 nm respectively. The stoichiometries and formation constants were determined. A 1:2 iron(III) to drug mole ratio was found to give the most predominant complexes for both drugs with 5.00 x 10(-3) M H(2)SO(4) and at 0.20 M ionic strength utilizing Job's method and the molar ratio method. A numerical method was utilized for the calculation of the formation constants, the logarithms of which were found to be 7.756 +/- 0.121 and 7.839 +/- 0.056, for ciprofloxacin and norfloxacin respectively. A factorial design together with the all-model-search method was utilized for the optimization of the concentration and aspiration volume of iron(III) as these were the variables which most affected peak absorbance. Working dynamic ranges of 50-500 ppm and 50-400 ppm were obtained for ciprofloxacin and norfloxacin respectively. The method was found to be suitable for the determination of these compounds in pharmaceutical preparations.     

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.     

Spectrofluorimetric Determination of Methyl Paraben in Pharmaceutical Preparations by Means of its Dansyl Chloride Derivative

 A spectrofluorimetric method for the determination of methyl paraben based on derivatization with the labelling reagent dansyl chloride (DNS-Cl), is presented. The effect of the reaction variables (pH, DNS-Cl concentration, temperature, reaction time) and instrumental parameters, has been examined. A linear calibration graph in the ng/ml range has been established. The limit of detection is 18 ng/ml with relative standard deviation less than 3%. The proposed method has been satisfactorily applied to determination of the paraben in two pharmaceutical preparations. Received May 25, 1999. Revision October 20, 1999.     

Simple and rapid determination of the drug naproxen in pharmaceutical preparations by heavy atom-induced room temperature phosphorescence

A simple, selective and sensitive heavy atom-induced room temperature phosphorimetric method (HAI-RTP) is described for the determination of naproxen (NAP) in pharmaceutical preparations. The phosphorescence signals are a consequence of intermolecular protection when analytes are, exclusively, in presence of a heavy atom salt and sodium sulfite as an oxygen scavenger to minimize RTP quenching. These variables selection constitute the basis of a HAI-RTP method for the determination of naproxen (detection limit 17.6 ng ml(-1); 1.71% relative standard deviation at 250 ng ml(-1)). The method has been applied satisfactorily to the analysis of pharmaceutical preparations.     

Determination of fluoroquinolones in edible animal tissue samples by high performance liquid chromatography after solid phase extraction

In the present work, a rapid, accurate, and sensitive method has been developed for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in edible animal tissues (muscle tissue, liver, kidney, and eggs). The separation was accomplished on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature within 15 min. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v). UV detection at 275 nm yielded the following limits of detection: 100 pg per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 20 pg for ofloxacin, and 200 pg for enrofloxacin. Peaks in real samples were identified by means of a photodiode array detector. The method was validated in terms of intra-day (n = 8) and inter-day (n = 8) precision and accuracy. Tissue samples were purified from endogenous interference by solid-phase extraction using Oasis HLB cartridges. The solid-phase extraction protocol was optimized in terms of retention and elution. Recovery rates at fortification levels of 40, 60, and 80 ng/g ranged from 82.5% to 111.1%. The applicability of the method was examined using real samples from a chicken treated orally with the five studied fluoroquinolones.     

High-performance thin-layer chromatography with densitometry for the determination of ciprofloxacin and impurities in drugs

A thin-layer chromatographic (TLC)-densitometric method has been developed for identification and quantification of ciprofloxacin (Rf = 0.61) and an ethylenediamine compound (Rf = 0.42), a desfluoro compound (Rf = 0.48), by-compound A (Rf = 0.53), and fluoroquinolonic acid (Rf = 0.68) as ciprofloxacin degradation products in pharmaceutical preparations. By using chloroform-methanol-25% ammonia (43 + 43 + 14, v/v/v) as the mobile phase and silica gel 60 F254 high-performance TLC plates as the stationary phase, it was possible to separate individual constituents that, when subjected to ultraviolet (UV) densitometric analysis at 330 nm for fluoroquinolonic acid and 277 nm for the other compounds, gave well developed peaks allowing easy qualitative and quantitative analyses. DMSO-methanol (1 + 1) was used to extract drug constituents. The method showed high sensitivity (limit of detection 10 to 44 ng), a wide linearity range (3 to 20 microg/mL), and good precision (2.32 to 6.46% relative standard deviation) and accuracy (percentage recoveries 98.62 to 101.52%) for individual constituents.     

Highly sensitive spectrofluorimetric determination of ephedrine hydrochloride in pharmaceutical preparations

A sensitive and specific spectrofluorimetry method was developed and validated for the quantification of ephedrine (EP) in pharmaceutical preparations. The method is based on the fluorescent enhancing reaction of EP with 7-chloro-4-nitrobenzofurazan (NBD-C1; derivatization reagent), in borate buffer of pH 9 to yield a yellow, fluorescent product. Under these experimental conditions, the derivatized product of EP had excitation and emission wavelength maxima at 458 and 516 nm, respectively. The linear range of this method was 20-2500 ng/mL. The detection limit was 7.3 ng/mL EP. Intra- and interday precisions of the assay at 3 concentrations within this range were 0.037-1.77%. The low relative standard deviation values indicate good precision, and high recovery values indicate excellent accuracy of the method. The proposed method was applied to the determination of the examined drugs in pharmaceutical formulations, and the results indicate that the method is equally as accurate, precise, and reproducible as the official method.     

铽增敏高效液相色谱柱后衍生荧光检测法同时检测鸡肉中的三种氟喹诺酮类药物残留

基于氟喹诺酮类药物与铽离子形成配合物后的荧光增强作用,建立了同时检测鸡肉中氟喹诺酮类(FQs)药物环丙沙星、诺氟沙星和恩诺沙星残留的Tb3+增敏高效液相色谱（HPLC）柱后衍生荧光检测方法。优化的实验条件如下：流动相为0.05 mol/L 醋酸/醋酸钠缓冲液(pH 6.0)-乙腈(体积比为89∶11),色谱柱为Hypersil BDS-C18,柱温40 ℃,流速0.8 mL/min;Tb3+浓度为8×10-5 mol/L;衍生反应温度40 ℃,衍生泵流速0.5 mL/min;荧光检测激发波长271 nm,发射波长545 nm。实验结果表明,将上述3种药物以1.0,10.0,50.0,100.0 ng/g水平添加到鸡肉后的回收率范围为66.3%~88.0%,相对标准偏差（RSD）均小于15.0%。定量分析的线性范围为0.1~500 ng/mL,方法的日内和日间RSD均小于13.0%;最低检出限分别为0.05(环丙沙星)、0.05(诺氟沙星)和0.08(恩诺沙星)ng/g,比前人报道的非衍生高效液相色谱荧光检测法检测FQs药物的灵敏度有极大的提高。该项研究为FQs药物多残留检测提供了灵敏度更高的新方法。     

Determination of quinolones in animal tissues and eggs by high-performance liquid chromatography with photodiode-array detection

A rapid, specific reversed-phase HPLC method is described, with solid-phase extraction, for assaying five quinolones (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin and marbofloxacin) with confirmative diode-array detection in samples of bovine kidney, muscle and eggs. The least efficient extraction was marbofloxacin from kidney tissue (64%). The lower detection limit for each quinolone was: enrofloxacin and ciprofloxacin, 1 ng; norfloxacin and difloxacin, 2 ng; marbofloxacin, 4 ng injected. The intra-day relative standard deviations were lower than 7.9% and lower than 8.6% for inter-day assays. These results indicate that the developed method had an acceptable precision.     

Determination of the antibacterial drug sulfamethoxazole in pharmaceutical preparations containing trimethoprim by spectrofluorimetry after derivatization with fluorescamine

An analytical method was developed for the determination of sulfamethoxazole (SMZ) in the presence of trimethoprim (TMP) by normal fluorescence. When both analytes are present a selective derivatization with fluorescamine of SMZ gives an intense fluorescent derivative with no interference from the other compound. The reaction was optimized to obtain the best analytical performance. The detection limit and the lower limit of quantitation of SMZ in the reaction medium was 5.2 ng/mL. The intra-day precision (relative standard deviation) was 1.51% for a 100 ng/mL SMZ standard solution and the inter-day precision over 7 days for a 100 ng/mL solution in the presence of 20 ng/mL TMP solution was 2.5%. The method has been applied to three pharmaceutical preparations containing both compounds, without any separation steps required. Received: 28 December 1998 / Revised: 28 April 1999 / Accepted: 30 April 1999