Quantification of moxifloxacin in urine using surface-enhanced Raman spectroscopy (SERS) and multivariate curve resolution on a nanostructured gold surface.

A simple procedure is proposed for the determination of the antibiotic moxifloxacin in urine using nanostructured gold as surface-enhanced Raman scattering signal enhancer. The standard addition method in conjunction to multivariate curve resolution-alternating least squares was applied to eliminate the matrix effect and to isolate the spectral contribution of the analyte. Even in the presence of unexpected interferences in the urinary media, it was possible to extract and quantify the analyte response, reaching, in this way, the so-called second-order advantage from first-order data. Moreover, although a saturation phenomenon of the metallic surface was observed, the results of the proposed methodology presented important advantages such as high sensitivity and simpler experimental procedures. The moxifloxacin was determined at levels of 0.70 and 1.50 µg mL(-1) in urine diluted to 1.0% (corresponding to 70.0 and 150 µg mL(-1) in the original samples) with relative errors of 4.23 and 8.70%, respectively. The limit of detection (0.085 µg mL(-1)) and limit of quantification (0.26 µg mL(-1)) values indicated that the quantification can be accomplished in urine up to 24 h after the administration of a single 400-mg dose.

In vivo metabolic investigation of moxifloxacin using liquid chromatography/electrospray ionization tandem mass spectrometry in combination with online hydrogen/deuterium exchange experiments.

RATIONALE: Tuberculosis is a leading cause of death from an infectious disease and moxifloxacin is an effective drug as compared to other fluoroquinolones. To date only two metabolites of the drug are known. Therefore, the present study on characterization of hitherto unknown in vivo metabolites of moxifloxacin using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is undertaken. METHODS: In vivo metabolites of moxifloxacin have been identified and characterized by using LC/ESI-MS/MS in combination with an online hydrogen/deuterium (H/D) exchange technique. To identify in vivo metabolites, blood, urine and faeces samples were collected after oral administration of moxifloxacin to Sprague-Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation, liquid-liquid extraction followed by solid-phase extraction and LC/MS/MS analysis. RESULTS: A total of nine phase I and ten phase II metabolites of moxifloxacin have been identified in urine samples including N-sulphated, glucuronide and hydroxylated metabolites which are also observed in plasma samples. In faeces samples, only the N-sulphated metabolite is observed. The structures of metabolites have been elucidated based on fragmentation patterns, accurate mass measurements and online H/D exchange LC/MS/MS experiments. Online H/D exchange experiments are used to support the identification and structural characterization of drug metabolites. CONCLUSIONS: A total of 19 in vivo metabolites of moxifloxacin have been characterized using LC/ESI-MS/MS in combination with accurate mass measurements and online H/D exchange experiments. The main phase I metabolites of moxifloxacin are hydroxylated, decarbonylated, desmethylated and desmethylhydroxylated metabolites which undergo subsequent phase II glucuronidation pathways.

Liquid chromatography-tandem mass spectrometry method for determination of phencynonate in rat blood and urine.

A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl ether-dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm x 2.1mm i.d., 3 microm). The mobile phase was composed of methanol-water (85:15, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat blood and 1-500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57-79.49% in blood and 56.85-64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.

Urinary concentrations and bactericidal activities of newer fluoroquinolones in healthy volunteers.

Eleven healthy male subjects participated in a crossover study to compare the urine concentrations and bactericidal activities of newer fluoroquinolones against common uropathogens. Each volunteer received a single oral dose of gatifloxacin (400 mg), levofloxacin (250 mg), moxifloxacin (400 mg) and trovafloxacin (200 mg), and a urine sample was obtained at 2, 6, 12 and 24 h after the dose. Urine concentrations were highest with gatifloxacin and levofloxacin and lowest with trovafloxacin. Each drug concentration was studied against a levofloxacin susceptible and moderately-susceptible strain of Escherichia coli (minimal inhibitory concentration, MICs: 0.125 and 4 mg/l), K. pneumoniae (MICs: 0.125 and 4 mg/l), Pseudomonas aeruginosa (MICs: 0.5 and 4 mg/l) and Enterococcus faecalis (MICs: 0.25 and 4 mg/l). The duration of urine bactericidal activity (UBA) was based upon the median bactericidal titre at each time period. Both gatifloxacin and levofloxacin exhibited prolonged (> or = 6 h) UBA against all of the study isolates. Moxifloxacin exhibited prolonged UBA against both isolates of E. coli, K. pneumoniae and E. faecalis but not against either strain of P. aeruginosa. Prolonged UBA was not observed for trovafloxacin against the moderately-susceptible strains with the exception of E. faecalis. Furthermore, UBA was not observed for trovafloxacin against the susceptible strain of P. aeruginosa. Although these newer fluoroquinolones exhibited similar in vitro activity against these uropathogens, only those compounds with the highest urinary concentrations (gatifloxacin and levofloxacin) produced prolonged UBA against both strains of P. aeruginosa. The findings from this study suggest that both microbiological activity and urinary concentrations are important parameters to consider when choosing a fluoroquinolone for empirical treatment of urinary tract infections (UTIs).

Spectrofluorimetric study of the interaction between europium(III) and moxifloxacin in micellar solution and its analytical application.

A sensitive spectrofluorimetric method has been developed for the determination of moxifloxacin (MOX) using europium(III)-MOX complex as a fluorescence probe in the presence of an anionic surfactant, sodium dodecyl benzene sulfonate (SDBS). The fluorescence (FL) intensity of Eu(3+) was enhanced by complexation with MOX at 614 nm after excitation at 373 nm. The FL intensity of the Eu(3+)-MOX complex was significantly intensified in the presence of SDBS. Under the optimum conditions, it was found that the enhanced FL intensity of the system showed a good linear relationship with the concentration of MOX over the range of 1.8 × 10(-11)-7.3 × 10(-9) g mL(-1) with a correlation coefficient of 0.9998. The limit of detection of MOX was found to be 2.8 × 10(-12) g mL(-1) with relative standard deviation (RSD) of 1.25% for 5 replicate determination of 1.5 × 10(-8) g mL(-1) MOX. The proposed method is simple, offers higher sensitivity with wide linear range and can be successfully applied to determine MOX in pharmaceutical and biological samples with good reproducibility. The luminescence mechanism is also discussed in detail with ultraviolet absorption spectra.

Multiple species metabolism of PHA-568487, a selective alpha 7 nicotinic acetylcholine receptor agonist.

The quinuclidine PHA-0568487(1) is an agonist of the alpha 7 nicotinic acetylcholine receptor that was designed to mitigate the bioactivation associated with the core scaffold and subsequently remove associated liabilities with in vivo tolerability. The drug metabolites of 1 in nonclinical species were identified in plasma and urine of rats, dogs and monkeys receiving oral administrations of 1. The in vitro biotransformation of 1 was subsequently investigated in multiple species employing cryopreserved hepatocytes, hepatic subcellular fractions and recombinantly-expressed human P450 enzymes. In addition, in vitro metabolism of synthetically prepared metabolite precursors were instrumental in the elucidation of several secondary metabolites. The results indicated that the principal biotransformation of 1 was oxidation of the benzo[1,4]dioxane moiety (M8, M10) followed by subsequent oxidation to a range of secondary metabolites (M1-7, M9, M11, M13-15, and M17-18). The carboxylic acids M1 and M2 resulting from the oxidative cleavage of the dioxane ring were the principal metabolites observed in the plasma, urine and hepatocyte incubations across all species (M1 & M2). Quinuclidine oxidation was another pathway of importance, yielding an N-oxide (M12) which was also observed in all species.P450 2D6 and FMO1 catalyze the oxidation of the quinuclidine nitrogen. The N oxidation of the quinuclidine moiety is consistent with previously published accounts of this scaffold's metabolism and, interestingly, may implicate the uncommon quinuclidine moiety as an entity directing the metabolism of this scaffold (e.g., 1) via FMO1 and P450 2D6 oxidation.

Validation of a MLC method with fluorescence detection for the determination of quinolones in urine samples by direct injection.

A sensitive and robust method was developed and validated for the routine identification and quantification of five quinolones in urine samples directly injected into a micellar liquid chromatographic system without any pre-treatment step. Since the simultaneous elution of the five compounds was not resolved, two mobile phases have been proposed: (a) for ciprofloxacin and levofloxacin 0.15M sodium dodecyl sulphate, 12.5% propanol and 0.5% triethylamine at pH 3.0 as the mobile phase and the detector at excitation wavelength 285 nm and emission wavelength 465 nm; and (b) for lomefloxacin, ofloxacin and moxifloxacin 0.05 M sodium dodecyl sulphate, 12.5% propanol and 0.5% triethylamine at pH 3.0 as the mobile phase and the detector at excitation wavelength 295 nm and emission wavelength 485 nm. Using these conditions, and in accordance with the food and drug analysis (FDA) guideline, the limit of quantification was 1 ng/mL, and the relative standard deviation and accuracy of the inter-day assay were 1.0-8.4% and 0.11-1.5%, respectively. Detection of the urinary excretion of four quinolones was followed up at 12h after the healthy volunteers had taken the drug. No potential interference from metabolites was observed. This procedure permits the rapid and reproducible measurement of low levels of quinolones in a small amount of urine.

Influence of activated charcoal on the pharmacokinetics of moxifloxacin following intravenous and oral administration of a 400 mg single dose to healthy males.

AIMS: To evaluate the extent to which enterohepatic recycling circulation contributes to moxifloxacin bioavailability in healthy, males by administration of activated charcoal and to evaluate the efficacy of activated charcoal administration in decreasing systemic concentrations of moxifloxacin in the event of overdose. METHODS: Nine healthy males, mean age 34 years (range 23-45 years) participated in a single centre, randomized, nonplacebo-controlled, three way crossover study. The pharmacokinetics of moxifloxacin in plasma and urine were determined for up to 96 h following a 400 mg single dose randomly administered on three separate occasions with a minimum washout phase of 1 week. Treatment A was 400 mg moxifloxacin IV as a 1 h infusion, treatment B was 400 mg moxifloxacin IV as a 1 h infusion with oral activated charcoal (5 g directly before the start of the infusion, 5 g immediately after the end of the infusion, and 10 g at 2, 4 and 8 h after the start of the infusion), treatment C was 400 mg oral moxifloxacin with activated charcoal (10 g 15 min before and at 2, 4 and 8 h after drug administration). The subjects underwent a series of clinical and laboratory tests. RESULTS: Single 400 mg doses of moxifloxacin (PO and/or IV) were safe and well tolerated. The bioavailability of moxifloxacin was significantly decreased when given with charcoal (AUC = 35.5 (IV reference) vs 5.40 (PO) vs 28.5 (IV) mg l(-1) h). Concurrently peak concentrations were lowered C(max) = 3.38 (IV reference) vs 0.62(PO) vs 2.97 (IV) mg l(-1)) by approximately 85% (P < 0.05) following oral administration and by 20% after IV treatment (P < 0.05). Bioavailability amounted to 15.4% (95% confidence interval 9.6, 25.0%) for treatment B while it was 80.4% (95% confidence interval 76.3.6, 84.6%) for treatment C. Terminal half-lives were not affected. The kinetics of urinary excretion corroborated these findings. CONCLUSIONS: The results of this study show that moxifloxacin undergoes pronounced enteric recycling after systemic uptake. In addition, these findings confirm that activated charcoal may be useful in treating moxifloxacin overdose by preventing its absorption.

The chemistry, toxicology, and identification in rat and human urine of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one: a reactive metabolite in felbamate bioactivation.

4-Hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one has been proposed to be a reactive metabolite of the anti-epileptic drug felbamate [Thompson et al. (1996) Chem. Res. Toxicol. 9, 1225-1229]. 4-Hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one exists in equilibrium with 3-oxo-2-phenylpropyl aminooate, which is known to eliminate to generate 2-phenylpropenal. Thus, this species is postulated to be a latent form of the ultimate reactive metabolite, 2-phenylpropenal. The chemistry of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one is proposed to parallel that of 4-hydroxycyclophosphamide, the bioactivated form of cyclophosphamide that undergoes ring-opening to aldophosphamide and subsequent elimination to afford 2-propenal (acrolein). The work presented here reports the chemical synthesis of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one and demonstrates that under buffered conditions it exists in equilibrium with 3-oxo-2-phenylpropyl aminooate. The rate-limiting step in the decomposition of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one is the irreversible beta-elimination from 3-oxo-2-phenylpropyl aminooate to 2-phenylpropenal. We have found the half-life of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one to be 4.6 +/- 0.4 h under in vitro conditions that mimic the physiological setting. As a consequence of the relatively long half-life of 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one, we have sought evidence for the significance of this pathway in experimental and clinical conditions. We report here the observation of this metabolite in the urine of rats being treated with 3-hydroxy-2-phenylpropyl aminooate, the esterase-mediated metabolite of felbamate, and in the urine of patients undergoing felbamate therapy. In addition, we have shown that 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one is toxic to cultured cells in a time-dependent manner, most likely as a result of its decomposition to 2-phenylpropenal. Taken together, the data support the hypothesis that 4-hydroxy-5-phenyl-1,3-oxazaperhydroin-2-one represents a "time-release" form of 2-phenylpropenal capable of traveling to distal sites from its locus of bioactivation and thereby mediates felbamate associated toxicities.