Detection and quantification of acidic drug residues in South African surface water using gas chromatography-mass spectrometry.

A method was optimized for derivatization, separation, detection and quantification of salicylic acid, acetylsalicylic acid, nalidixic acid, ibuprofen, phenacetin, naproxen, ketoprofen, meclofenamic acid and diclofenac in surface water using gas chromatography-mass spectrometry. For most of the acidic drugs, recovery was in the range 60-110% and the percent standard deviation was below 15% for the entire method, with limits of detection ranging from 0.041 to 1.614 µg L-1. The developed method was applied in the analysis of acidic drugs in Umgeni River system, KwaZulu-Natal South Africa. All of the selected acidic drugs were detected and quantified, their concentration in Umgeni River system ranged from 0.0200 to 68.14 µg L-1.

A flow injection chemiluminescence method for determination of nalidixic acid based on KMnO4-morin sensitized with CdS quantum dots.

A simple and sensitive flow injection chemiluminescence (CL) method was developed for determination of nalidixic acid by application of CdS quantum dots (QDs) in KMnO4-morin CL system in acidic medium. Optical and structural features of L-cysteine capped CdS quantum dots which were synthesized via hydrothermal approach were investigated using X-ray diffraction (XRD), scanning electron microscopy (SEM), photoluminescence (PL), and ultraviolet-visible (UV-Vis) spectroscopy. Moreover, the potential mechanism of the proposed CL method was described using the results of the kinetic curves of CL systems, the spectra of CL, PL and UV-Vis analyses. The CL intensity of the KMnO4-morin-CdS QDs system was considerably increased in the presence of nalidixic acid. Under the optimum condition, the enhanced CL intensity was linearly proportional to the concentration of nalidixic acid in the range of 0.0013 to 21.0 mg L(-1), with a detection limit of (3σ) 0.003 mg L(-1). Also, the proposed CL method was utilized for determination of nalidixic acid in environmental water samples, and commercial pharmaceutical formulation to approve its applicability. Furthermore, corona discharge ionization ion mobility spectrometry (CD-IMS) method was utilized for determination of nalidixic acid and the results of real sample analysis by two proposed methods were compared. Comparison the analytical features of these methods represented that the proposed CL method is preferable to CD-IMS method for determination of nalidixic acid due to its high sensitivity and precision.

Occurrence of fluoroquinolones and fluoroquinolone-resistance genes in the aquatic environment.

Fluoroquinolones (FQs) have been detected in aquatic environments in several countries. Long-term exposure to low levels of antimicrobial agents provides selective pressure, which might alter the sensitivity of bacteria to antimicrobial agents in the environment. Here, we examined FQ levels and the resistance of Escherichia coli (E. coli) to FQs by phenotyping and genotyping. In the aquatic environment in Osaka, Japan, ciprofloxacin, enoxacin, enfloxacin, lomefloxacin, norfloxacin, and ofloxacin were detected in concentrations ranging from 0.1 to 570 ng L(-1). FQ-resistant E. coli were also found. Although no obvious correlation was detected between the concentration of FQs and the presence of FQ-resistant E. coli, FQ-resistant E. coli were detected in samples along with FQs, particularly ciprofloxacin and ofloxacin. Most FQ-resistant E. coli carried mutations in gyrA, parC, and parE in quinolone resistance-determining regions. No mutations in gyrB were detected in any isolates. Amino acid changes in these isolates were quite similar to those in clinical isolates. Six strains carried the plasmid-mediated quinolone resistance determinant qnrS1 and expressed low susceptibility to ciprofloxacin and nalidixic acid: the minimum inhibitory concentrations ranged from 0.25 µg mL(-1) for ciprofloxacin, and from 8 to 16 µg mL(-1) for nalidixic acid. This finding confirmed that plasmids containing qnr genes themselves did not confer full resistance to quinolones. Because plasmids are responsible for much of the horizontal gene transfer, these genes may transfer and spread in the environment. To our knowledge, this is the first report of plasmid-mediated quinolone resistance determinant qnrS1 in the aquatic environment, and this investigation provides baseline data on antimicrobial resistance profiles in the Osaka area.

Ciprofloxacin susceptibility reduction of salmonella strains isolated from outbreaks

The antimicrobial susceptibility of 212 Salmonella strains isolated from patients and foods was evaluated and 45 percent were found to be resistant to nalidixic acid. Nalidixic acid resistant strains showed a higher minimal inhibitory concentration for ciprofloxacin than sensitive strains. During the study an increase of strains with reduced susceptibility to ciprofloxacin was also observed.

Supported imidazolium ionic liquid phases: a new material for solid-phase extraction.

This study reports a material that is based on the concept of ionic liquid analogue: a slightly crosslinked polymer-supported imidazolium trifluoroacetate salt (IL-CF(3)COO(-)) that favorably combines the properties of ionic liquids (ILs) and the advantages of a solid support. The ionic liquid-supported material was evaluated for the first time as a solid-phase extraction (SPE) sorbent for selectively and quantitatively extracting pharmaceuticals from aqueous samples. The novel IL-CF(3)COO(-) was evaluated under reversed-phase (RP), weak anion exchange (WAX), strong anion exchange (SAX) and strong cation exchange (SCX) SPE procedures, and we found that SAX conditions are the most suitable for investigating the behaviour of the IL-CF(3)COO(-) material. Under SAX conditions, the IL-CF(3)COO(-) material was capable of selectively and quantitatively extracting a group of acidic compounds from aqueous samples, while washing basic analytes that were also present in the sample. The SPE method using IL-CF(3)COO(-) material was used to analyse 1000 ml of different aqueous samples (ultrapure, tap and river) with complete recovery of the acidic compounds studied. Moreover, the method provided clean chromatogram and high recoveries when percolating complex real samples, such as 1000 ml of river water and 250 ml of effluent wastewater from a sewage treatment plant spiked at low levels with the analytes studied.

Evaluation of the extent of protein binding by means of NMR diffusion and relaxation experiments, and automated continuous ultrafiltration.

The extent of protein binding is one of the key parameters in both pharmacodynamics and pharmacokinetics. Since a high protein binding prevents the drug to reach the target enzyme or receptor in the organism and the metabolism and excretion of drug, a low protein binding is preferable. Thus, the protein binding has to be considered in the process of drug development. In order to keep the extent of protein binding low it is necessary to know the mechanism of protein-drug interaction. Because NMR methods are able to provide such structural information the purpose of this paper is to compare relaxation and diffusion NMR experiments with a classical protein binding determination method, i.e. ultrafiltration. All of them gave comparable results for the gyrase inhibitors nalidixic acid, ofloxacin, and gatifloxacin with regard to the extent of protein binding. The relaxation measurements revealed the quinolone moiety to be the main interaction partner with the protein whereas the piperazine moieties are not involved in the binding process.

Adsorption of the quinolone antibiotic nalidixic acid onto anion-exchange and neutral polymers.

Pharmaceutical products are being found in surface and ground waters around the world. While the consequences to human health are unknown, it is suspected that these contaminants adversely alter aquatic ecosystems. This study presents adsorption results for one pharmaceutical product, nalidixic acid (NA), on neutral and anion-exchange polymers at three pH values. The adsorption of NA below and above its pKa of approximately 6 on polymers with different matrices, forms, and degrees of polarity were evaluated. By comparing the Freundlich constants KF and n, the results show that the neutral form of NA adsorbs to a greater extent on neutral polymers, and that the anionic form of NA adsorbs more to anion-exchange polymers. Also, results of the adsorption experiments suggest that aromatic ring interactions between NA and the surface of both neutral and anion-exchange polymers are important in the adsorption process. These results have important implications for the treatment of pharmaceutical-contaminated water, as many pharmaceutical contaminants are ionizable and have aromatic rings in their structure. This study illustrates the importance of pH and sorbate and sorbent structure in considering polymer sorption for treatment of pharmaceutical-contaminated aqueous systems.

Nalidixic acid screening test in detection of decreased fluoroquinolone susceptibility in Salmonella typhi isolated from blood.

OBJECTIVE: To determine the validity of nalidixic acid screening test in the detection of high MICs of fluoroquinolone against Salmonella(S.) typhi isolated from blood and correlate zone diameters of ofloxacin with that of MIC value for nalidixic acid sensitive and resistant strains. DESIGN: Cross-sectional analytical study. PLACE AND DURATION OF STUDY: Clinical Microbiology Laboratory of the Aga Khan Hospital, Karachi from January 2002 to December 2003. PATIENTS AND METHODS: Two hundred S. typhi isolates from blood were included for nalidixic acid screening and ofloxacin susceptibility. Antibiotic susceptibilities for both the antibiotics were obtained by disc diffusion method whereas MICs were determined by standard agar dilution method as recommended by NCCLS guidelines. Sensitivity, specificity and correlation between both antimicrobial susceptibility methods were calculated and results expressed as scattergrams. RESULTS: The results broadly classify S. typhi isolates into nalidixic acid resistant strains with no zone of inhibition around 30 mug nalidixic acid disc and nalidixic acid sensitive strains with mean zone of inhibition of 24.9 mm. All S. typhi isolates with ofloxacin MIC of capital ZHE, Cyrillic 0.125 microg/ml were found to be nalidixic acid resistant (MIC capital ZHE, Cyrillic32 microg/ml) whereas the isolates with ofloxacin MIC 0.06 microg/ml were nalidixic acid sensitive (MIC 8 microg/ml). Screening for nalidixic acid resistance was found to be 100% sensitive and 97% specific in identifying S. typhi strains with reduced susceptibility to fluoroquinolone (MIC capital ZHE, Cyrillic 0.125 microg/ml). CONCLUSION: Nalidixic acid resistance as a screening method is proved to be significant in identifying S. typhi isolates with reduced susceptibility to fluoroquinolones. It is also suggested that inhibition zone of 25 mm around 5 microg ofloxacin disc is appropriate as a selection criterion to detect S. typhi isolates with reduced susceptibility to fluoroquinolones.

Multiresidue method for simultaneous determination of ten quinolone antibacterial residues in multimatrix/multispecies animal tissues by liquid chromatography with fluorescence detection: single laboratory validation study.

Quinolone antibacterials are veterinary drugs authorized for use in food animal production. The analysis of residual amounts of drugs in food from animal origin is important for quality control of products for consumers. For this purpose, Maximum Residue Limits (MRLs) have been set up by a European Union Council Regulation on Veterinary Drug Residues (No. 90/2377/EEC and subsequent), and 8 quinolones received MRLs at concentration levels depending on both the matrix and the animal species of interest. A method was developed for screening and confirming 10 quinolone residues (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid, sarafloxacin) in a wide variety of matrixes of different animal species. It involves extraction of the residues from the biological tissues/fluids by acidic aqueous solution, centrifugation and filtration prior to injection on a C18 narrow-bore column, and detection through a 3-step-mode fluorescence detector. The method was validated during a 2-week study for a set of 8 species-matrixes (i.e., bovine raw milk, bovine muscle, porcine muscle, porcine kidney, porcine liver, fish flesh and skin, poultry muscle, whole egg). Residues were quantified down to 15 microg/kg with limits of detection and quantitation ranging from 4 to 11 and 13 to 36 microg/kg, respectively, which are sufficient compared to the wide range of MRLs set for these substances (from 30 microg/kg for danofloxacin in milk to 1900 microg/kg for difloxacin in poultry liver). The limit of performance of the method in terms of CCalpha and CCbeta, the critical concentrations stated in the Decision No. 2002/657/EC and the ISO Standard No. 11843, has been calculated for the authorized (MRL) substances but only estimated in the case of the nonauthorized (non-MRL) substances.

The simultaneous separation and determination of five quinolone antibiotics using isocratic reversed-phase HPLC: application to stability studies on an ofloxacin tablet formulation.

A rapid and reliable HPLC method was developed for the simultaneously separation and quantitation of five quinolones antibiotics; nalidixic acid, norfloxacin, ofloxacin, ciprofloxacin and lomefloxacin. All five tablet formulations of individual quinolone antibiotics were routinely assayed without interference. The calibration curves were linear (r2> or =0.999) over the concentration range of 1.20-4.8 mg/100 ml. Selectivity, precision, sensitivity and accuracy were established and the method is stability indicating with respect to ofloxacin. The limit of detection and quantitation for ofloxacin was 18 and 36 microg/100 ml, respectively. The separation was performed on a Phenomenex ODS C18 column using an isocratic, ion-pairing mobile phase consisting of 35% (v/v) aqueous acetonitrile together with tetrabutylammonium acetate, sodium dodecyl sulphate and citric acid (pH* 3.4). All analyses were conducted at ambient temperature and was monitored using a Diode Array UV/VIS detector set at wavelengths 235, 254, 275 and 300 nm.

Micellar electrokinetic chromatography for determination of drug partition in phospholipids.

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely L-alpha-dilauroyl phosphatidylcholine (DLPC) or L-alpha-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.

Biological laser printing of genetically modified Escherichia coli for biosensor applications.

One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed a technique, biological laser printing or BioLP, which satisfies these requirements and has advantages over current technologies. BioLP is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates. The BioLP technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.

Determination of nalidixic acid by fluorometry with sodium borohydride and hydrogen peroxide.

A simple and sensitive fluorometric method for the determination of nalidixic acid was established by using 0.75M sodium borohydride and 7.5% hydrogen peroxide solution as fluorogenic reagents. Analyte concentrations of 0.0232-11.6 pg/mL could be determined with high precision and accuracy by the method. A relative standard deviation of 1.75% was obtained for a nalidixic acid concentration of 0.232 pg/mL. The method was satisfactorily applied to the determination of nalidixic acid in human serum, fish muscle, and chicken muscle, and the calibration curves were linear from 0.23 to 58.00 pg/mL, from 9.28 to 32.48 mg/kg, and from 4.64 to 23.20 mg/kg, respectively. The specificity of the reaction is also discussed.

Application of derivative UV spectrophotometry for the determination of enoxacin and nalidixic acid in tablets.

First-, second-, third- and forth-order derivative spectrophotometric methods, using "peak-zero" (P-O) and "peak-peak" (P-P) techniques of measurement have been developed for the determination of enoxacin and nalidixic acid in tablets. The calibration curves were linear in the concentration range of 2.0-12.0 micrograms ml-1 for the analysed quinolones. The procedure was simple, rapid and the results were reliable.

Single-use phosphorimetric sensor for the determination of nalidixic acid in human urine and milk.

A single-use phosphorimetric sensor to determine the germicide nalidixic acid is proposed. The sensing action is based on the absorption of the analyte into the sensing zone and the subsequent measurement of the phosphorescence intensity emitted by the analyte fixed in the sensor. This plane drop sensor is made up of a 3 x 1.6 cm sheet of the polyester Mylar as solid support, and a circular film 5 mm in diameter and 20 microns in thickness, formed by poly(vinyl chloride) and tributyl phosphate as the plasticizer, adhered to its surface. The sensor is introduced for 2 h into the sample solution, after which it is dried and the phosphorescence intensity is measured directly at lambda ex = 332 nm, lambda em = 412 nm, with a delay time of 0.15 ms and a gate time of 10 ms, under a dry nitrogen stream. The characteristic parameters of the construction of the sensing zone and of the processes of fixing the analyte along with the emission of phosphorescence were studied. The applicable concentration range was from 60 to 1500 ng ml-1, with a detection limit of 20 ng ml-1 and a precision of 2% expressed as relative standard deviation. The method was applied to the determination of nalidixic acid in milk and human urine with recoveries ranging between 96.0 and 103.7%. The calibration process was carried out by applying a mathematical method of finite elements that expresses the analytical signal as a function of the analyte concentration and equilibration time between the sensor and the sample solution.

Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.

The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently.

Liquid chromatographic determination of flumequine, nalidixic acid, oxolinic acid, and piromidic acid residues in catfish (Ictalurus punctatus).

A peer-verified, liquid chromatographic (LC) method for simultaneous determination of residues of flumequine (FLU), nalidixic acid (NAL), oxolinic acid (OXO), and piromidic acid (PIR) in catfish muscle is presented. Sample workup involves homogenizing tissue with acetone, defatting with hexane, and extracting quinolones into chloroform. Sample is purified further by partitioning into base and then subsequently back-extracting into chloroform after acidifying the aqueous phase. After solvent is evaporated, the residue is diluted with mobile phase, and analytes are introduced into an LC system where separations are made with a 5 microns, reversed-phase polymer column and an isocratic, buffered acetonitrile-tetrahydrofuran mobile phase. Determinations are made by UV detection at 280 nm for PIR and by fluorescence detection (excitation at 325 excitation and emission at 365 nm) for the other 3 analytes. Each quinolone was used to fortify catfish muscle at 5, 10, 20, 40, and 80 ng/g. The following recoveries and relative standard deviation (RSD) values represent an average of the 5 levels for each analyte: FLU, 79.7% (RSD = 5.7%); OXO, 80.8% (RSD = 6.3%); PIR, 75.0% (RSD = 5.9%); and NAL, 87.1% (RSD = 10%). Assay of 5 levels (base incurred catfish, plus 4 dilutions with control catfish) of catfish muscle incurred with the 4 quinolones gave the following averages: FLU: base, 198 ng/g (RSD = 2.3%); dilutions, 98.0 ng/g (RSD = 4.2%), 61.6 ng/g (RSD = 4.4%), 21.6 ng/g (RSD = 2.8%), 9.24 ng/g (RSD = 8.7%); OXO, base, 257 ng/g (RSD = 6.9%); dilutions, 146 ng/g (RSD = 5.5%), 95.0 ng/g (RSD = 4.1%), 30.7 ng/g (RSD = 3.8%), 13.7 ng/g (RSD = 4.6%); PIR, base, 22.1 ng/g (RSD = 4.2%); dilutions, 13.7% ng/g (RSD = 6.7%), 6.49 ng/g (RSD = 15%), 2.65 ng/g (RSD = 15%); and NAL, base, 75.1 ng/g (RSD = 3.8%); dilutions, 42.3 ng/g (RSD = 5.1%), 24.1 ng/g (RSD = 6.3%), 8.59 ng/g (RSD = 4.8%). A second multiresidue analysis of the 4 quinolones was performed by an outside analyst. Average recoveries from catfish fortified at 5, 10, 20, and 40 ng/g were FLU, 75.9% (RSD = 4.0%); OXO, 84.0% (RSD = 5.5%); NAL, 85.6% (RSD = 8.9%); and PIR, 66.2% (RSD = 8.7%).

Determination and confirmation of identities of flumequine and nalidixic, oxolinic, and piromidic acids in salmon and shrimp.

A previously published liquid chromatographic (LC) method for determining residues of flumequine (FLU) and nalidixic (NAL), oxolinic (OXO), and piromidic (PIR) acids in catfish tissue was applied to salmon and shrimp muscle. Identities of all 4 residues in salmon and shrimp were confirmed by gas chromatography/mass spectrometry (GC/MS). The tissue is homogenized with acetone, the acetone extract is defatted with hexane, and the quinolones are extracted into chloroform. The extract is further purified by first partitioning into base and then back-extracting from a solution acidified to pH 6.0. Analytes are determined by LC with simultaneous UV and fluorescence detection. Muscle tissue was fortified with each quinolone at 5, 10, 20, 40, and 80 ng/g. Average recoveries and relative standard deviations (RSDs) for salmon, which represent an average of the 5 levels for each analyte, ranged from 75.9 to 90.8% and from 2.25 to 6.40%, respectively. Average recoveries and RSDs for shrimp ranged from 81.3 to 91.2% and from 7.34 to 10.7%, respectively. Identities of OXO, FLU, NAL, and PIR were confirmed in extracts of salmon and shrimp tissue fortified at 10 ng/g by determination of decarboxylated quinolones by GC/MS. Four diagnostic ions were monitored for OXO, FLU, and PIR, and 5 ions were monitored for NAL. All ion relative abundances were within 10% of those calculated for standard decarboxylated quinolones. Optimum conditions for decarboxylation and GC/MS confirmation are given.

Liquid chromatographic determination of nalidixic acid in pharmaceutical preparations.

A simple, rapid, and environmentally safe liquid chromatographic (LC) method was developed for the qualitative and quantitative determination of pharmaceutical preparations of nalidixic acid. The new method was applied to commercial preparations of tablets and a suspension of nalidixic acid and found to be satisfactory for both quantitative and qualitative determinations. Previous LC methods either used chloroform to extract, which we were trying to eliminate, or used a mobile phase of about pH 2.5, which will destroy the column coating. The LC system for the new method uses sulfanilic acid as internal standard, a mu-Bondapak C18 column, and a mobile phase of methanol, 0.0045M dibasic potassium phosphate, and 0.0072M hexadecyltrimethylammonium bromide. The detection wavelength is 254 nm. The sample is dissolved in methanol, and an aliquot is injected through a 20 microL injection loop. Average recoveries ranged from 99.4 to 101.3%.

Simultaneous quantitative determination of metronidazole and nalidixic acid in tablets by difference spectroscopy.

A difference spectrophotometric procedure has been developed for simultaneous determination of metronidazole (MDZ) and nalidixic acid (NA) in tablets. The method comprised the measurement of the absorbance of a solution of the tablet extract in 0.1 M NaOH relative to that of an equimolar solution in 0.1 M HCl at 292 nm for NA and 325 nm for MDZ. The presence of identical isosbestic points for pure drug solutions and tablet extracts indicated the non-interference of excipients in the absorption at these wavelengths. Compliance with Beer's law was observed in the concentration ranges 5-25 micrograms ml(-1) for MDZ and 15-35 micrograms ml(-1) for NA these wavelengths.