Lab-on-a-Chip-Surface Enhanced Raman Scattering Combined with the Standard Addition Method: Toward the Quantification of Nitroxoline in Spiked Human Urine Samples.

The emergence of antibacterial resistance and the development of new drugs lead to a continuous change of guidelines for medical treatments. Hence, new analytical tools are required for the detection of drugs in biological fluids. In this study, the first surface enhanced Raman scattering (SERS) detection of nitroxoline (NTX) in purified water and in spiked human urine samples is reported. Insights concerning the nature of the molecule-metal interaction and its influence on the overall SERS signal are provided. Furthermore, three randomly collected urine samples originating from a healthy volunteer were spiked to assess the limit of detection (LOD), the limit of quantification (LOQ), and the linear dynamic range of the lab-on-a-chip SERS (LoC-SERS) method for NTX detection in human urine. The LOD is ∼3 µM (0.57 mg/L), LOQ ∼ 6.5 µM (1.23 mg/L) while the linear range is between 4.28 and 42.8 µM (0.81-8.13 mg/L). This covers the minimum inhibitory concentration (MIC) values of the most commonly encountered uropathogens. Finally, seven clinical samples having an "unknown" NTX concentration were simulated. The LoC-SERS technique combined with the standard addition method and statistical data analysis provided a good prediction of the unknown concentrations. Additionally, it is also demonstrated that the predictions carried out by multicurve resolution alternating least-squares (MCR-ALS) algorithm provides reliable results, and it is preferred to a univariate statistical approach.

An immunochromatographic assay for rapid and direct detection of 3-amino-5-morpholino-2-oxazolidone (AMOZ) in meat and feed samples.

BACKGROUND: Furaltadone (FTD) is a type of nitrofuran and has been banned in many countries as a veterinary drug in food-producing animals owing to its potential carcinogenicity and mutagenicity. FTD is unstable in vivo, rapidly metabolizing to 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ); thus AMOZ can be used as an indicator for illegal usage of FTD. Usually, for the determination of nitrofurans, the analyte is often a derivative of the metabolite rather than the metabolite itself. In this study, based on the monoclonal antibody (mAb) against AMOZ, a competitive immunochromatographic assay (ICA) using a colloidal gold-mAb probe for rapid and direct detection of AMOZ without a derivatization step in meat and feed samples was developed. RESULTS: The intensity of red color in the test line is inversely related to the analyte concentration and the visual detection limit was found to be 10 ng mL⁻¹. The performance of this assay was simple and convenient because the tedious and time-consuming derivatization step was avoided. The ICA detection was completed within 10 min. The ICA strips could be used for 7 weeks at room temperature without significant loss of activity. The AMOZ spiked samples were detected by ICA and confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good agreement. CONCLUSION: The proposed ICA provides a feasible tool for simple, sensitive, rapid, convenient and semi-quantitative detection of AMOZ in meat and feed samples on site. To our knowledge, this is the first report of the ICA for direct detection of AMOZ.

Oxidation of trimethoprim by ferrate(VI): kinetics, products, and antibacterial activity.

Kinetics, stoichiometry, and products of the oxidation of trimethoprim (TMP), one of the most commonly detected antibacterial agents in surface waters and municipal wastewaters, by ferrate(VI) (Fe(VI)) were determined. The pH dependent second-order rate constants of the reactions of Fe(VI) with TMP were examined using acid-base properties of Fe(VI) and TMP. The kinetics of reactions of diaminopyrimidine (DAP) and trimethoxytoluene (TMT) with Fe(VI) were also determined to understand the reactivity of Fe(VI) with TMP. Oxidation products of the reactions of Fe(VI) with TMP and DAP were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reaction pathways of oxidation of TMP by Fe(VI) are proposed to demonstrate the cleavage of the TMP molecule to ultimately result in 3,4,5,-trimethoxybenzaldehyde and 2,4-dinitropyrimidine as among the final identified products. The oxidized products mixture exhibited no antibacterial activity against E. coli after complete consumption of TMP. Removal of TMP in the secondary effluent by Fe(VI) was achieved.

Square wave voltammetric determination of nitrofurantoin in pharmaceutical formulations on highly boron-doped diamond electrodes at different boron-doping contents.

The influence of the boron-doping levels in boron-doped diamond film electrodes on the electrochemical response of nitrofurantoin (NFT) and the development of an electroanalytical procedure for NFT determination were investigated. The investigations were carried out using the techniques of cyclic voltammetry and square wave voltammetry on diamond film electrodes with different boron-doping levels (i.e., 5000, 10,000 and 20,000 mg L(-1)). The level of boron-doping in the diamond film electrodes influenced the electrochemical reduction of NFT. The appropriate cyclic voltammetric response of NFT was obtained with Britton-Robinson buffer at pH 4 and for diamond films doped with 10,000 and 20,000 mg L(-1) of boron. These two films were selected for the development of the electroanalytical procedure. The use of square wave voltammetry with the optimized parameters demonstrated a good linear relationship between the peak current and the NFT concentration for a wide range of concentration. The lower limit of detection for the electrodes doped with 10,000 and 20,000 mg L(-1) of boron were 2.69 x 10(-8) mol L(-1) (6.40 microg L(-1)) and 8.15 x 10(-9) mol L(-1) (1.94 microg L(-1)), respectively, while the lower limits of quantification were 8.96 x 10(-8) mol L(-1) (21.33 microg L(-1)) and 2.72 x 10(-8) mol L(-1) (6.47 microg L(-1)), respectively. The applicability of the proposed procedure was tested using a commercial pharmaceutical formulation of NFT, and the results were compared with the procedure recommended by the British Pharmacopeia. The proposed procedure was sensitive, accurate and precise for analysis of NFT and did not require complex preparations or renovations of the electrode surface. This presents the advantage of eliminating mercury waste and minimizing the adsorptive problems related to the use of other electrodic solid surfaces.

Milk secretion of nitrofurantoin, as a specific BCRP/ABCG2 substrate, in assaf sheep: modulation by isoflavones.

Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1-calves fed forage with isoflavones; G2-calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3-calves fed forage without isoflavones; G4-calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C(max) in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter-individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.

Simultaneous determination of sulphamethoxazole and trimethoprim in powder mixtures by attenuated total reflection-Fourier transform infrared and multivariate calibration.

A partial least-squares calibration (PLS) procedure in combination with infrared spectroscopy has been developed for simultaneous determination of sulphamethoxazole (SMZ) and trimethoprim (TMP) in raw material powder mixtures used for manufacturing commercial pharmaceutical products. Multivariate calibration modeling procedures, interval partial least squares (iPLS) and synergy partial least squares (siPLS), were applied to select a spectral range that provided the lowest prediction error in comparison to the full-spectrum model. The experimental matrix was constructed using 49 synthetic samples and 15 commercial samples. The considered concentration ranges were 400-900 mg g(-1) SMZ and 80-240 mg g(-1) TMP. Spectral data were recorded between 650 and 4000 cm(-1) with a 4 cm(-1) resolution by Fourier transform infrared spectroscopy coupled with attenuated total reflectance (ATR-FTIR) accessory. The proposed procedure was compared with conventional procedure by high performance liquid chromatography (HPLC) using 15 commercial samples containing SMZ and TMP. The results showed that PLS regression model combined to ATR-FTIR is a relatively simple, rapid and accurate procedure that could be applied to the simultaneous determination of SMZ and TMP in routine quality control of powder mixtures. A root mean square error of prediction (RMSEP) of 13.18 mg g(-1) for SMZ and 6.03 mg g(-1) for TMP was obtained after selection of better intervals by siPLS. Using the proposed procedure it is possible to analyze each sample in less than 3 min considering two replicates (excluding the grinding step). Accuracy was checked by comparison to HPLC method and agreement better than 98.8% was achieved.

Sequential determination of norfloxaxin and levofloxacin in the presence of other fluorquinolones using synchronous scanning room-temperature phosphorimetry and Th (IV) as the selective signal inducer.

The selective determination of norfloxacin in mixtures with other fluorquinolones was achieved by synchronous scanning solid surface room-temperature phosphorimetry (SSRTP) and Th(NO(3))(4) as selective phosphorescence inducer. The method also allowed the determination of levofloxacin in a sequential way. The optimization of experimental conditions was made through an univariate approach, in order to find the best conditions for norfloxacin phosphorescence, followed by a 2(3) factorial design in order to verify interaction among relevant variables, to check robustness for each variable and to perform final adjustment of parameters. Absolute limit of detection (ALOD) for norfloxacin was 12ng with a linear signal response extending up to 400ng. Under the same experimental conditions set for norfloxacin, the ALOD for levofloxacin was 13ng with linear signal response up to 450ng. Accuracy of the method, using Th (IV) as selective phosphorescence inducer, was evaluated through the analysis of commercial and simulated pharmaceutical formulations with recoveries between 94.4 and 101% for norfloxacin and 95.9 and 103.8% for levofloxacin. The use of Cd (II), a traditional phosphorescence inducer for fluorquinolones, did not allow selective determination of norfloxacin. Further studies indicated the potential application of the method in urine samples.

Determination of sulfonamides and trimethoprim using high temperature HPLC with simultaneous temperature and solvent gradient.

A fast HPLC method for the analysis of eight selected sulfonamides (SA) and trimethoprim has been developed with the use of high temperature HPLC. The separation could be achieved in less than 1.5 min on a 50 mm sub 2 microm column with simultaneous solvent and temperature gradient programming. Due to the lower viscosity of the mobile phase and the increased mass transfer at higher temperatures, the separation could be performed on a conventional HPLC system obtaining peak widths at half height between 0.6 and 1.3 s.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens.

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days --3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) -- to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg(-1) furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 microg kg(-1) minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Simultaneous determination of trimethoprim and sulphamethoxazole in veterinary formulations by chromatographic multivariate methods.

A comparative chromatographic study was developed for the simultaneous quantitative resolution of trimethoprim (TMP) and sulphamethoxazole (SMX) in veterinary formulations. Multi-wavelength chromatograms were recorded by using diode array detector (DAD) system at the five-wavelength set consisting of 220, 230, 240, 250 and 260 nm. In the first step, five different calibration equations at the above wavelengths for each drug were obtained by using the relationship between concentration and peak area. These calibration graphs were used for the quantitative evaluation of TMP and SMX in samples. These single-wavelength applications were called traditional LC method. In the second step, principal component regression (PCR) and partial least-squares (PLS) calibrations were applied to the above mentioned multi-wavelength chromatograms. The amount of two investigated drugs in samples was determined by the constructed PCR and PLS calibrations. The experimental results obtained from each single-wavelength calibration graph were compared with those obtained by the chemometric approaches and chromatographic multivariate approaches give successful results more than traditional LC method.

Flow-injection chemiluminescence determination of ofloxacin and levofloxacin in pharmaceutical preparations and biological fluids.

A novel, rapid and sensitive analytical method is described for determination of ofloxacin and levofloxacin by enhanced chemiluminescence (CL) with flow-injection sampling. The method is based on the CL reaction of the Ce(IV)-Na2S2O4-ofloxacin/levofloxacin-H2SO2 system. The enhanced CL mechanism was developed and the optimum conditions for CL emission were investigated. The CL intensity was correlated linearly (r = 0.9988) with the concentration of ofloxacin (or levofloxacin) in the range of 1.0 x 10(-8) - 1.0 x 10(-7) g ml(-1) and 1.0 x 10(-7) - 6.0 x 10(-6) g ml(-1). The detection limit (S/N = 3) is 7 x 10(-9) g ml(-1). The relative standard derivation (RSD, n = 11) is 2.0% for ofloxacin at 4 x 10(-7) g ml(-1) and for levofloxacin at 6 x 10(-7) g ml(-1). This method has been successfully applied for the determination of ofloxacin and levofloxacin in pharmaceutical preparations and biological fluids with satisfactory results.

Behavior of fluoroquinolones and trimethoprim during mechanical, chemical, and active sludge treatment of sewage water and digestion of sludge.

The behavior and fate of three fluoroquinolones (norfloxacin, ofloxacin, and ciprofloxacin), one sulfonamide (sulfamethoxazole), and trimethoprim were investigated at a sewage treatment plant in Umeå, Sweden, in 2004. This plant uses conventional mechanical, chemical, and activated sludge methods to treat the sewage water and digest the sludge; the dewatered digested sludge is pelleted (dry weight > 90% of total weight). Raw sewage water and particles as well as effluents and sludge from specific treatment areas within the plant were sampled. In addition to quantifying the antibiotics within the plant, we characterized the sample matrixes to facilitate evaluation of the results. Of the five substances examined, only norfloxacin, ciprofloxacin, and trimethoprim were present in concentrations higher than their limits of quantification. Norfloxacin and ciprofloxacin sorbed to sludge in a manner that was independent of changes in pH during sewage treatment, and more than 70% of the total amount of these compounds passing through the plant was ultimately found in the digested sludge. The results suggest that fluoroquinolones undergo thermal degradation during pelleting, but more studies are needed to confirm this. Trimethoprim was found in the final effluent at approximately the same concentration and mass flow as in the raw sewage, and could not be quantified in any solid sample. Predicted environmental concentrations, based on consumption data for Umeå municipality, correlated well with the results obtained, especially when the predicted concentrations were corrected to account for the amount of each active substance excreted in urine. The results obtained were compared to those of previous studies of these three substances' behavior and fate and were found to be similar, although some of the other plants studied employed the various treatment steps in different orders.

Determination of methenamine, methenamine mandelate and methenamine hippurate in pharmaceutical preparations using ion-exchange HPLC.

An ion-exchange column high-performance liquid chromatography (HPLC) method has been developed for the determination of methenamine in methenamine and methenamine hippurate pharmaceutical preparations. The HPLC method uses a Zorbax SCX-300 column with acetonitrile-0.1M sodium perchlorate monohydrate (pH 5.8) (70:30, v/v) as the mobile phase at the flow rate of 1 mL/min. UV-detection was at 212 nm. The linear concentration plots for methenamine were linear over the concentration range of 0.25-50mM for methenamine and methenamine mandelate standards. The intra-day RSD precision was <1.25%, and for inter-day, <1.85%. The peaks for mandelic acid, hippuric acid and the other ingredients from placebo tablets do not interfere with the analysis for methenamine. The accuracy of this method was shown to be 99-101% by measuring the recovery of methenamine from spiked placebo tablets. The assay of methenamine from methenamine hippurate tablets and from a urinary antiseptic tablet containing methenamine were in the range of 98-102%. This HPLC method is a fast, simple and straightforward method for the analysis of methenamine in pharmaceutical preparations.

Antibiotic resistance in bacteria from shrimp farming in mangrove areas.

Shrimp farming is a sufficiently large and mature industry to have an effective range of antimicrobial agents for most bacterial diseases in shrimp culture. However, at present, there exists great concern over the widespread use of antibiotics in aquaculture, which may result in residue of antibiotics in water and mud, and subsequently, the development of antibiotic resistance in bacteria in the environment. There is limited understanding about the effect of antibiotic residues on bacteria resistance in shrimp farming environment. Therefore, a study was conducted to investigate bacterial resistance to Norfloxacin (NFXC), Oxolinic Acid (OXLA), Trimethoprim (TMP) and Sulfamethoxazole (SMX), which were found in four shrimp farming locations in mangrove areas in Vietnam. Findings indicate that there is a relatively high incidence of bacteria resistance to these antibiotics observed in most of the studied sites, particularly to antibiotics with concentration of 0.1 microg/ml. Yet the relation between concentration of antibiotic residues and incidence of antibiotic resistance is not clearly defined. Among individual antibiotics, the incidence of resistance to TMP and SMX was higher than the others. Identification of bacteria isolated from mud samples by DNA analyzer shows that Bacillus and Vibrio are predominant among bacteria resistant to the antibiotics. The result of the study also indicates that these antibiotics in media degraded more rapidly due to the presence of resistant bacteria.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

The occurrence of nitrofuran metabolites in the tissues of chickens exposed to very low dietary concentrations of the nitrofurans.

The global problem of food products contaminated by residues of the banned carcinogenic nitrofuran drugs has prompted research into how such residues accumulate in tissues. In the study described here, two aspects have been investigated where the nitrofurans accumulate in tissues from chickens exposed to either a dietary or an environmental source of contamination. Twenty groups of broilers were fed a diet containing one of the nitrofurans: furazolidone, nitrofurazone, nitrofurantoin or furaltadone at concentrations of 30, 100, 300, 1000 and 3000 microg kg(-1). At the lowest concentration of furazolidone contamination (0.01% of the therapeutic dose) tissue bound AOZ metabolite residues were detected in liver (1.1 +/- 0.2 microg kg(-1)) and in muscle (0.33 +/- 0.03 microg kg(-1)). Similar results were obtained for AMOZ (0.6 +/- 0.2 microg kg(-1) in liver), the tissue bound metabolite of furaltadone. There was no appreciable accumulation of nitrofurantoin in chicken muscle. The AHD metabolite was not detected in muscle from birds fed nitrofurantoin at either 30 or 100 microg kg(-1). For nitrofurazone the concentrations of the SEM metabolite were higher in muscle than in liver for all dietary concentrations. The potential for a contaminated environment to cause nitrofuran residues in chickens was investigated. Six chickens were placed in a pen that was previously occupied by birds fed a diet containing 3000 microg kg(-1) of furazolidone. After 24 hours' exposure of the chickens to the litter in the pen, AOZ residues of 0.13 +/- 0.04 and 0.10 +/- 0.03 microg kg(-1) were detected in liver and muscle, respectively. The results of both experiments have implications for the poultry industry in trying to eliminate nitrofurans from their production systems, and for regulatory analysts faced with the detection of low concentrations of the drugs, both in tissues and in feedingstuffs.

[Nonlinear partial least squares method for simultaneous derivative spectrophotometric determination of two components in compound sulfamethoxazole].

A nonlinear model of consistency with derivative absorption in multiple components systems has been given. Model data were estimated by partial least squares method. A method for the simultaneous derivative spectrophotometric determination of two components with nonlinear partial least squares method was established. The method has been applied to the simultaneous determination of two components in compound sulfamethoxazole tablets. The average recoveries of sulfamethoxazole and trimethoprim in synthetic samples were 99.8% and 100.1%, and relative standard deviations were 1.3% and 1.6%, respectively. The results by nonlinear PLS method are significantly better than those by linear PLS method. The results for actual compound formulation agreed with those obtained by standard method.

Novel source of semicarbazide: levels of semicarbazide in cooked crayfish samples determined by LC/MS/MS.

Nitrofuran antibiotics were previously used in animal healthcare but are now prohibited. Semicarbazide is a breakdown product of 5-nitrofurazone and protein-bound semicarbazide is used as a marker residue for the illegal use of 5-nitrofurazone. However, the presence of the prohibited semicarbazide has been reported in some food items of animal origin. A novel observation is reported that semicarbazide can be detected in Finnish crayfish samples, i.e. crustacea, never medicated with nitrofurazone. The origin of the semicarbazide is presently unknown. Positive identification was undertaken by liquid chromatography coupled with tandem mass spectrometry detection. The level of semicarbazide was determined as the protein-bound form as well as the total amount of semicarbazide in the sample. The average levels of total semicarbazide and the protein-bound form were 4.2 and 0.5 ng g(-1) fresh crayfish meat, respectively. All the tested samples (n = 18) contained traces of semicarbazide, the highest amount being 12 ng g(-1) fresh crayfish meat.

Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects. High-performance liquid chromatographic (HPLC) separation of the analytes was achieved in less than 7 min. The compounds were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on an aminopropyl column. Recoveries were not dependent on the concentration level. The mean recoveries were as follows: trimethoprim 79.0%, sulfadiazine 80.5%, N(4)-acetylsulfadiazine 91.0%, sulfamerazine 78.6%, sulfadimidine 77.2% and sulfamethoxazole 82.8%. Linearity was established over a concentration range of 5 to 5000 microg/kg with correlation coefficients greater than 0.99. The method had a limit of quantitation (LOQ) of 5 microg/kg manure.