Hollow Fiber Liquid Based Microextraction of Nalidixic Acid in Urine Samples Using Aliquat 336 as a Carrier Combined with High-Performance Liquid Chromatography.

A new and simple hollow fiber liquid-phase microextraction was used in conjunction with HPLC for the extraction and quantitative determination of nalidixic acid (NA) in human urine samples. This study considers this technique an alternative to common methods of hollow fiber microextraction based on pH gradient and electromembrane extraction of NA in human urine samples. In this research, the drug was extracted from the source phase (donor phase) into a modified organic phase with Aliquat 336 (hydrophobic ion-pair reagent) as a carrier able to impregnate pores of the hollow fiber. In this study, the effects of several factors were tested on the extraction efficiency of the drug. Under optimum conditions, the linearity of the method was observed over the range 0.05-2.0 µg mL(-1) with a correlation coefficient of r = 0.9983. The results of tests on the method determined a good sensitivity with a limit of detection of 0.008 µg mL(-1). The intra-day relative standard deviation (n = 9) for 0.8 µg mL(-1) was 6.2%, and the inter-day relative standard deviation (n = 5) for 0.8 µg mL(-1) was 5.6%. This new strategy was successfully applied to analyze a real urine sample with satisfactory results.

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.

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography.

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15-100 micrograms ml-1, with a correlation coefficient greater than 0.999 and detection limits in the 0.2-0.7 ng ml-1 range. Intra- and inter-day precision values of about 0.8-1.2% RSD (n = 11) and 1.3-2.0% RSD (n = 30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml-1.

False-positive EMIT II opiates from ofloxacin.

The cross-reactivities of four quinolone antibiotics--ofloxacin, norfloxacin, ciprofloxacin, and nalidixic acid--toward the EMIT II Opiates enzyme immunoassay were examined. Drug-free urine was spiked with the individual drugs up to 5,000 mg/L. Only ofloxacin showed potential for causing a false-positive opiates immunoassay screening result (apparent morphine > 300 micrograms/L). The method produced a positive opiate result at an ofloxacin concentration of 200 mg/L, a 0.16% cross-reactivity. Three hospitalized patients taking therapeutic doses of ofloxacin all gave false-positive EMIT II Opiates urine screening results. Three patients taking norfloxacin and three patients taking ciprofloxacin gave true-negative urine screening results. False-positive results were also obtained from the urines of two volunteers who each consumed a single 400-mg ofloxacin pill.

Determination of the chemotherapeutic quinolonic and cinolonic derivatives in urine by high-performance liquid chromatography with ultraviolet and fluorescence detection in series.

An HPLC method with ultraviolet and fluorimetric detection has been established for the separation and determination of six quinolonic and cinolonic antibiotics. A Nova-Pak C18 column (150 x 3.9 mm) and a Waters 486 UV and a Waters 470 fluorescence detector have been used. The influence of variables such as mobile-phase composition and flow-rate, has been studied. An acetonitrile-aqueous solution of oxalic acid 4x10(-4) M (28:72, v/v) has been selected as optimum. The wavelength for the photometric detection of the six antibiotics was 265 nm. For the fluorimetric detection two pairs of excitation/emission wavelengths, 260/360 or 270/440 nm, were selected for the determination of nalidixic acid, 7-hydroxymethylnalidixic acid and oxolinic acid, and for the determination of pipemidic acid and cinoxacin, respectively. The analytical parameters and detection and quantification limits of the method have been determined. The proposed method has been applied for the determination of the six compounds in urine, applying different procedures depending on their concentration, the results being very acceptable.

Interference of ofloxacin with determination of urinary porphyrins.

The second-generation quinolone ofloxacin interferes with the screening test of porphyrins. We observed a 20-fold increase in the porphyrin concentration measured in urine of an ofloxacin-treated patient, compared with drug-free normal urine. Two other fluorinated 4-quinolones tested, norfloxacin and ciprofloxacin, had a less marked effect (a twofold increase), whereas the first-generation quinolone, nalidixic acid, did not affect the measured porphyrin concentration at all. The interference is probably due to the overlap in the emission fluorescence spectra of ofloxacin and urinary porphyrins at approximately 600 nm. To avoid a false-positive diagnosis of porphyria, we suggest using HPLC to separate ofloxacin (10-min retention time) from urinary porphyrins (which only start to elute at 12 min). Nonetheless, given a threefold increase in urinary porphyrins observed in the urine of an ofloxacin-treated patient, we also discuss a possible interference of the drug with the metabolism of porphyrins.

Probenecid inhibits the renal clearance and renal glucuronidation of nalidixic acid. A pilot experiment.

The aim of this pilot study was to demonstrate the possible inhibitory effect of probenecid on the renal glucuronidation and on the renal clearance of nalidixic acid in a human volunteer. Under acidic urine conditions, hardly any nalidixic acid is excreted unchanged (0.2%). It is excreted as acyl glucuronide (53.4%), 7-hydroxymethylnalidixic acid (10.0%), the latter's acyl glucuronide 30.9%, and 7-carboxynalidixic acid (4.2%). Under probenecid co-medication the renal glucuronidation of nalidixic acid is reduced from 53% to 16%; the renal clearance of both nalidixic acid and 7-hydroxymethylnalidixic acid are reduced (p < 0.001); the intrinsic t1/2 of the metabolite 7-hydroxymethylnalidixic acid increased from 0.48 h to 4.24 h. The amount of acyl glucuronidation of 7-hydroxymethylnalidixic acid was not altered. The in vitro protein binding of both acyl glucuronides was increased, while no effect on the unconjugated compounds was seen. Nalidixic acid had no effect on the maximal renal excretion rate of probenecid acyl glucuronide.

Bioequivalence study of nalidixic acid tablets: in vitro-in vivo correlation.

The USP dissolution test was used to select seven products with a wide range of dissolution characteristics for in vivo examination. The bioequivalence of seven (500 mg) products was evaluated in two crossover urinary excretion experiments. In each study three products were compared with the innovator product (Negram-Winthrop-Breon) in 12 subjects according to a 4 x 4 latin square design. Significantly different, lower bioavailability was observed in two products in relation to that of the innovator product. Linear in vitro-in vivo correlations were found between the cumulative amount excreted at 24 h and the log of the amount dissolved at 30 min and between log of the cumulative amount excreted up to 24 h and the log of the amount dissolved at 45 min.

Direct gradient reversed-phase HPLC analysis and preliminary pharmacokinetics of nalidixic acid, 7-hydroxymethylnalidixic acid, 7-carboxynalidixic acid, and their corresponding glucuronide conjugates in humans.

A gradient reversed-phase high pressure liquid chromatographic analysis was developed for the direct measurement of nalidixic acid with its acyl glucuronide, 7-hydroxymethylnalidixic acid with its acyl and ether glucuronides, and 7-carboxynalidixic acid in human plasma and urine. The glucuronides and 7-carboxynalidixic acid were not present in plasma after an oral dose of 1,000 mg nalidixic acid. The acyl glucuronides of 7-carboxynalidixic acid were not present in plasma and urine. The acyl glucuronides are stable in urine at pH 5.0-5.5. The subject's urine must therefore be acidified by the oral intake of 4 x 1 g of ammonium chloride per day. With acidic urine, hardly any nalidixic acid was excreted unchanged (0.2%). It was excreted as acyl glucuronide (53.4% of dose), 7-hydroxymethyl-nalidixic acid (10.0%), the latter's acyl glucuronide (30.9%), and 7-carboxynalidixic acid (4.2%).

The effect of infinitesimal drug dilutions on the pharmacokinetics of nalidixic acid and atenolol.

1. Ten healthy subjects received two treatments: a single 1 g oral dose of nalidixic acid (NA) followed 1 h later by either an infinitesimal dilution of the drug (NA 7CH) or by succussed water which served as placebo. The study was repeated 18 months later in 10 different subjects. 2. A further 10 healthy subjects received three treatments: a single 100 mg oral dose of atenolol (AT) followed 3 h later by either placebo or a dilution of AT (AT 7CH) or of bisoprolol (BI 7CH). The homoeopathic preparations were administered by the sublingual route. 3. In the first NA experiment NA 7CH significantly shortened the elimination half-life of NA from 8.6 +/- 2.2 (placebo) to 6.4 +/- 1.6 h (NA 7CH). In the second NA experiment none of the pharmacokinetic parameters was modified significantly by the administration of NA 7CH. Neither AT 7CH nor BI 7CH modified the pharmacokinetics of AT.

A preliminary report on the derivatization-gas chromatographic determination of nalidixic acid and 3,7-dicarboxynalidixic acid in urine.

A preliminary study on the gas chromatographic analysis of urine spiked with nalidixic acid and 3,7-dicarboxynalidixic acid is described. The method is based on the transfer of an ion-pair of NA or CNA with tetradecyldimethylbenzylammonium chloride (benzalkonium chloride) from alkaline aqueous solution into methyl isobutyl ketone (MIBK), the organic phase, where these salts are derivatized with pentafluorobenzyl bromide. The derivatives formed by this process are chromatographed on an analytical column packed with 1.5% OV-101 and detected with a flame-ionization detector. Several parameters affecting the transfer and/or derivatization of NA or CNA were investigated. These parameters include the phase transfer catalyst employed, the organic solvent used, the concentration of the acid and base added, the amount of derivatizing agent required, and reaction time and temperature.

Dose-dependent pharmacokinetic study of pefloxacin, a new antibacterial agent, in humans.

A dose-dependent pharmacokinetic study of pefloxacin was performed after four intravenous infusions and four orally administered doses. After intravenous infusion, the pharmacokinetic profiles of the plasma concentrations showed a biphasic decline, with half-lives (mean +/- SD) of 8.55 +/- 4.20 min and 11.50 +/- 1.75 h, respectively. Intravenous infusion and oral administration yielded similar results. The pharmacokinetic parameters remained constant in the dose range of 200-800 mg from the plasma and urine data.

Pefloxacin in rabbits: protein binding, extravascular diffusion, urinary excretion and bactericidal effect in experimental endocarditis.

The serum protein binding, extravascular diffusion and urinary excretion of pefloxacin were studied in rabbits. The effect of furosemide on the urinary excretion of pefloxacin was investigated. In an experimental model of Escherichia coli endocarditis, diffusion into heart valves and infected vegetations and bactericidal effect of pefloxacin were also studied. We observed a serum protein binding of 25%. Extravascular concentrations found were within the range of the minimal inhibitory concentrations for most susceptible strains. Pefloxacin appeared to be reabsorbed by renal tubules (fractional excretion: 61 +/- 21%). Furosemide significantly increased the renal excretion of pefloxacin through a tubular process. We observed a good penetration of pefloxacin into normal heart valves and infected vegetations. Pefloxacin reduced the colony counts in infected vegetations after seven im injections of the drug (given as 15 mg/kg/12 h).

Bioavailability of nalidixic acid from uncoated tablets in humans--Part I: Correlation with the dissolution rates of the tablets.

Bioavailabilities of three commercially available tablets and two trial tablets of nalidixic acid were determined. The correlation between in vivo bioavailability parameters and in vitro dissolution rates derived by eight methods (oscillating basket I and II, beaker I, II, and III, rotating basket, paddle and rotating flask) is discussed. Nalidixic acid and its metabolite, 7-hydroxynalidixic acid, were determined in serum by HPLC with a strong anion-exchange resin column. All parameters for nalidixic acid bioavailability showed significant differences among the tablets tested. The relationship between Cmax and AUC was not linear but concave. However, Cmax decreased linearly in proportion to the delay of Tmax. The rank order of tablets according to bioavailabilities was just the same as the rank order obtained when comparing dissolution rates determined by any of the above methods, with the exception of one tablet which had a poor disintegrating ability. The parameters for the rate of bioavailability (Cmax and Tmax) showed good linearity with 1/T50 determined by all of the methods. However, AUC showed significant correlation with neither T50 nor 1/T50.

Quantitation of norfloxacin, a new antibacterial agent in human plasma and urine by ion-pair reverse-phase chromatography.

A specific and sensitive high-performance liquid chromatographic method for the analysis of norfloxacin in human plasma and urine is described. Norfloxacin was extracted from the sample matrix using dichloromethane under neutral conditions, followed by back extraction into dilute phosphoric acid for chromatographic analysis on a reverse-phase column with a mobile phase consisting of methanol, phosphate buffer, and ion-pairing reagent (pH 3.0) at a flow rate of 2.0 mL/min. The ability of this method to distinguish intact norfloxacin from its metabolites was demonstrated. The method is linear, quantitative, and reproducible for both plasma analysis (0.05-2.5 microgram/mL) and urinalysis (1.0-500 micrograms/mL) using peak area ratios (norfloxacin-internal standard) for quantitation. The stability of norfloxacin and its metabolites in dilute phosphoric acid was studied. To assess the presence of norfloxacin conjugates in the urine of dosed individuals, the effects of urine hydrolysis on drug quantitation were examined. Urine and plasma levels of norfloxacin at selected time points following the administration of single drug doses are presented.

[Nalidixic acid: urinary levels during short-term therapy with reduced doses]. / Nalidixinsäure: Harnspiegel bei Kurzzeittherapie mit reduzierten Dosen.

The acute inflammation of the lower afferent urinary tract is a superficial mucosal infection so long as structural and functional anomalies are excluded. A single or brief administration of antibacterially active substances with high urine concentrations can shorten uncomplicated attacks of infection and diminish the symptoms. The investigation of the urinary concentration of biologically active metabolites after administration of a daily dose of 2 g nalidixic acid revealed values which ensure that the minimum inhibitory concentrations in the afferent urinary tract are exceeded. The different responsiveness of the fluorimetric and microbiological methods applied for determination of the biologically active substances in urine led to results which were well correlated.

Pharmacokinetics of norfloxacin in healthy volunteers and patients with renal and hepatic damage.

The pharmacokinetics of norfloxacin were studied in six healthy volunteers, and three patients each with moderate renal and hepatic damage. A new specific and sensitive high performance liquid chromatography method was set up to measure plasma and urine concentrations of norfloxacin. The mean urinary concentrations after a single oral dose of 400 mg norfloxacin exceeded many times the MIC and MBC values of most of the bacterial strains responsible for urinary tract infections. Results in the patients with hepatic and renal damage indicated slight and not statistically significant differences in comparison with healthy volunteers.

High-performance liquid chromatography of pefloxacin and its main active metabolites in biological fluids.

We describe a high-performance liquid chromatographic (HPLC) method for the analysis of pefloxacin, a new antibacterial agent, in plasma and urine following administration of a therapeutic dose in humans. HPLC assay of pefloxacin and its two main active metabolites in urine is also described. The applicability of the methods to pharmacokinetic studies of pefloxacin in humans is demonstrated.