Determination of quinolones and fluoroquinolones in hospital sewage water by off-line and on-line solid-phase extraction procedures coupled to HPLC-UV.

In this work, the development of two solid-phase extraction procedures (off-line and on-line formats) for the identification and quantification of several (fluoro)quinolones in hospital sewage water by HPLC-UV is described. Both procedures are based on the use of C18 and anion exchange (SAX) sorbents for the preconcentration and clean-up steps, respectively, and all variables influencing both steps were optimised. In the off-line format, after its pH was adjusted to 2.5, sample was preconcentrated on a C18 cartridge and eluted with 4 mL of methanol/ammonia (94/6). The methanolic extract must be diluted up to 10 mL with water to allow quantitative retention of the analytes on the SAX cartridge. In the on-line format, the addition of 2.5% of NH4Cl to the sewage water sample (pH = 2.5) was necessary to increase the breakthrough volumes of the analytes in the C18 precolumn. Quantitative transfer of the (fluoro)quinolones from the C18 precolumn to the SAX precolumn was accomplished by pumping 2 mL of a mixture methanol/water (40/60, pH = 9.2) at 2 mL min(-1). Elution of the analytes from the SAX precolumn by means of the chromatographic mobile phase required the inclusion of an additional isocratic step at the beginning of the gradient program. Both off-line and on-line solid phase extraction procedures coupled to HPLC-UV were applied to the analysis of a sewage water sample collected in the sewer system at the output of the St Dimphna Hospital (Geel, Belgium). The fluoroquinolone ciprofloxacin was found in this sample and quantified at 5.8 +/- 0.4 microg L(-1) (off-line method) and 5.6 +/- 0.5 microg L(-1) (on-line method). The analysis of spiked samples containing the seven (fluoro)quinolones studied provided quantitative recoveries in all cases with low RSD values (from 6 to 12%), and all the analytes could be identified by means of their UV spectra with match factors varying from 950 to 985 depending on the (fluoro)quinolone.

Study of the evolution and degradation products of ciprofloxacin and oxolinic acid in river water samples by HPLC-UV/MS/MS-MS.

The results of a degradation study of the (fluoro)quinolone antibiotics ciprofloxacin and oxolinic acid in river water samples are presented in this paper. The decomposition of these compounds at ambient temperature was monitored during five months by HPLC-UV, and two consecutive degradation processes (photo- and bio/chemical-degradation) were observed in both cases although with different degradation rates. Ciprofloxacin was completely degraded after 3 months whereas 80% of oxolinic acid remained unaltered after five months of storage. The analysis of the degradation compounds formed was carried out using MS and tandem MS-MS, allowing the identification of four new ciprofloxacin transformation products not previously described in the literature. Possible degradation pathways for this antibiotic in river water are proposed.

Detection of coexisting protein conformations in capillary zone electrophoresis subsequent to transient contact with sodium dodecyl sulfate solutions.

Non-native conformations of proteins were generated by temporary contact with aqueous solutions of sodium dodecyl sulfate (SDS) and separated from the native state with capillary zone electrophoresis (CZE) in alkaline borate buffer deficient of SDS. Nine proteins at concentrations of 2.0 or 3.0 mg.L(-1) were compared in terms of their susceptibility to SDS. For superoxide dismutase and ferritin the tendency of unfolding was modest with < 25% of the protein being transformed to the non-native state at 10 mmol.L(-1) SDS. Highest susceptibility was observed for albumin, myoglobin (Mb), and hemoglobin with > 75% in the non-native state even at 2.0 mmol.L(-1) SDS. The influence of varying SDS concentrations on the conformational state of Mb was tested. Increasing the SDS concentration, circular dichroism revealed a reduction in alpha-helix, an increase in random coil, and an introduction of beta-sheet, which is absent in native structure. Modifications in the secondary structure were in agreement with distinct changes in the shape of the non-native Mb peak in CZE and make a gradual unfolding/refolding process with several coexisting molten globules instead of two-state transition of conformations most plausible for Mb. CZE was found to contribute to a further understanding of holo-Mb transformation towards a population of non-native conformations (i) by means of calculated peak area ratios of native to non-native states, which showed sigmoid transition, (ii) by detecting the release of the prosthetic heme group, and (iii) by changes in the effective electrophoretic mobility of the Mb-SDS peaks. Reconstituted holo-Mb forms differed in the Soret band around 410 nm, indicating diversity in the conformation of the heme pocket.

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection.

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L(-1)) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L(-1)) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.

Stability of fluoroquinolone antibiotics in river water samples and in octadecyl silica solid-phase extraction cartridges.

Intensive use of antibiotics in human and veterinarian medicine and in industrial farming (food additives) has resulted in the transport of important quantities of the active ingredients to environmental waters. Environmental analysis usually requires sample storage for certain periods of time and, consequently, it is of great importance to know the stability of antibiotics in these kinds of sample. Thus, in this work the stability in river water of oxolinic acid (Oxo) and ciprofloxacin (Cip), taken as representatives of fluoroquinolone and quinolone antibiotics respectively, has been evaluated. The stability of these compounds in river water has been studied both in containers and on C(18) solid-phase extraction cartridges (SPE) under different storage conditions (time, light, and temperature). Data analysis revealed that Cip and Oxo have different degradation profiles with different degradation kinetics in river water. It was also concluded that these antibiotics are stable both in the containers and on SPE cartridges for at least 2 weeks at ambient temperature, and stability can be increased substantially if samples are stored at low temperatures (4 and -18 degrees C).

Capillary zone electrophoresis of metal-binding proteins in formic acid with UV- and mass spectrometric detection using cationic transient capillary isotachophoresis for preconcentration.

A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.

Trace enrichment of (fluoro)quinolone antibiotics in surface waters by solid-phase extraction and their determination by liquid chromatography-ultraviolet detection.

A new and simple analytical methodology for the simultaneous analysis of acidic and zwitterionic (fluoro)quinolones in surface waters at trace concentration level is presented. The method is based on the preconcentration of these analytes by a solid-phase extraction procedure and their subsequent quantification by liquid chromatography using ultraviolet detection. The breakthrough volumes of the selected (fluoro)quinolones in four different sorbents--C18, styrenedivinylbenzene (SDB), C18-cation-exchange and SDB-cation-exchange--have been evaluated and varied between 25 and 150 ml depending on the antibiotic and the sorbent used. An exhaustive study of the influence of sample pH on the preconcentration step has been carried out in order to find a suitable procedure for extraction of acidic and zwitterionic FQs in one single step. Under optimum conditions, it was possible to percolate up to 250 ml of water solution onto both C18 and SDB-cation-exchange cartridges with quantitative recoveries for all the analytes tested. However, matrix components of the surface water samples analysed negatively affected the recoveries of the analytes in the SDB-cation-exchange cartridge and thus, C18 cartridges were finally selected for the analysis of the (fluoro)quinolones in lake and river water. The limits of detection achieved with this procedure varied between 8 and 20 ng l(-1) proving its suitability for the determination of the (fluoro)quinolones in water samples at a realistic environmental concentration level.

Determination of haemoglobin A(1c) by liquid chromatography using a new cation-exchange column.

The use of a new cation-exchange column, ProPac SCX-10, for the determination of haemoglobin A(1c) (HbA(1c)) by high-performance liquid chromatography is described. After optimization of the analytical method for the separation of the various isoforms of haemoglobin with the ProPac SCX-10 column, the method was applied to the determination of HbA(1c) in blood from 59 volunteers. Three of the 59 had previously been diagnosed as diabetics. Interference studies for carbamylation, acetylation and pre-HbA(1c) were carried out via "in-vitro" experiments. No interference due to carbamylation was observed at the urea values normally found in uremic patients undergoing dialysis. No interference from pre-HbA(1c) was detected either. The method is able to separate haemoglobin A (alpha(2)beta(2)), haemoglobin S (haemoglobin from sickle cell anaemia patients) and haemoglobin A(2) (alpha(2)delta(2)) without interference. The method of Hampel was applied to detect outliers. A value of 3.29+/-0.44% (2sigma) for HbA(1c) was obtained in the analysis of 56 blood samples from non-diabetics. This average value is lower than that reported by most of the methods currently used in routine analyses.

Comparative study of coulometric and amperometric detection for the determination of macrolides in human urine using high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) method using chromatographic conditions optimised in a previous work was applied for the separation of three macrolide antibiotics roxithromycin (Rox), oleandomycin (Ole) and rosamicin (Ros) and further determination of two of them, roxithromycin (Rox) and oleandomycin (Ole), in human urine samples. A comparative study of the behaviour of these macrolides under the two types of electrochemical detection (EC) widely coupled with HPLC, that is coulometric (EC-C) and amperometric (EC-A), was carried out by applying the same multiresidue method. From the assays performed using both detectors the comparison was made taking relevant criteria such as detection limits, linearity, recovery and precision values into account. As a result of this comparison, the coulometric detector appears slightly more suitable than the amperometric one for macrolide analysis.

Development of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometry method for the analysis of fluoroquinolone antibiotics.

The applicability of a capillary zone electrophoresis-electrospray ionisation tandem mass spectrometric (CZE-ESI-MS-MS) method for the separation of nine fluoroquinolones was investigated. Method optimisation involved systematic trouble-shooting starting with the type and duration of capillary pre-washing and conditioning, the choice of both the CE run buffer, MS sheath liquid, CE run potential, ESI spray voltage, sheath gas flow-rate, MS capillary voltage and CE capillary and MS capillary temperatures. Another extremely important factor was found to be the degree to which the CE capillary protrudes into the ESI chamber as well as whether or not sheath gas and spray voltage are employed during the CE injection or not. The importance of the latter has, to our knowledge, not been addressed elsewhere. Nine fluoroquinolones have been separated and detected in a single run by this technique.

Multiresidue determination of (fluoro)quinolone antibiotics in swine kidney using liquid chromatography-tandem mass spectrometry.

New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.

Validation of an analytical procedure for the determination of oxidative hair dyes in cosmetic formulations.

A high range and variety of cosmetic formulations that contain oxidative hair dyes and matrix-forming compounds have been industrially developed over recent years and are now available on the international market. Member states of the European Union are responsible for conducting analyses of cosmetic products as deemed necessary by law and European regulation enforcement. Therefore, inspection authorities as well as the cosmetics trade and industry need validated analytical methods for the identification, characterization, and/or quality control of specific active ingredients or formulations with the aim of implementing the European Union Cosmetic Directives (76/768/ECC, 95/17/EC). In this frame, we validated a candidate reference method that enables the identification and quantification of hair dye-forming compounds. This method consists of a separation by RP-HPLC coupled with a DAD after a liquid-liquid extraction procedure for separating matrix components from the dye-forming compounds. The validation of the method includes common criteria such as the repeatability of the analysis and the establishment of figures of merit, as well as statistical evaluations and quality assurance in order to follow the recommendations of the Eurachem guide for analytical measurements.

Optimization and validation of an analytical procedure for the determination of oxidative hair dyes in commercial cosmetic formulations.

A method has been developed and validated for the analysis of commonly used intermediates of oxidative hair dyes in commercial cosmetic formulations, including both liquid and cream forms, in dark and blonde shades. The commercial formulations are submitted to extraction by an organic solvent, and the resulting aqueous phase is analyzed by reverse-phase HPLC with a gradient elution and detection with DAD and/or ESI-MS-MS. A spectra library containing 200-400 nm spectra of the target substances and their HPLC retention times has been recorded for the identification. The quantification of the target substances is also performed after spiking of the commercial formulations, using an external calibration. The recoveries obtained are very good for all selected intermediates. The whole procedure has been found to be highly suitable for the identification and quantification of dye intermediates. Also implemented has been a database containing (a) the retention times, (b) the spectral, MS, and MS/MS characteristics of the intermediates, (c) acidity constant values of some intermediates of interest experimentally determined and compared to the available NIST values, (d) the chromatographic conditions used, (e) the behavior towards extraction of dye intermediates, and (f) matrix compounds.

Recent developments in quantification methods for metallothionein.

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.