Improved Sensitivity to Determine Antibiotic Residues in Chicken Meat by In-Line Solid-Phase Extraction Coupled to Capillary Electrophoresis-Tandem Mass Spectrometry.

Traditionally, capillary electrophoresis (CE) has been ruled out of many food safety applications, despite its inherent advantages, because its concentration sensitivity has been not high enough, mainly in relation to the monitoring of contaminants and residues, such as pesticides, veterinary medicines, environmental contaminants, toxins, etc. For this reason, researchers have proposed several strategies to overcome this limitation. So far, approaches based on chromatographic principles have been the most successful solutions. These approaches, known as in-line solid phase extraction, consist of the introduction of a small amount of stationary phase in the inlet section of the electrophoretic capillary (analyte concentrator, AC) to retain the analytes before separation takes place. In this chapter, this strategy is applied to CE coupled to tandem mass spectrometry (MS/MS) for the multiresidue detection of quinolone antibiotic residues in chicken meat. A previous sample treatment based on pressurized liquid extraction to obtain an optimum performance is also described.

Development of a QuEChERS-based extraction method for the determination of destruxins in potato plants by UHPLC-MS/MS.

Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with electrospray ionization has been proposed for the determination of fifteen natural destruxins (A, B, C, D, E, Ed, Ed1, A2, B2, D2, E2, Cl, DesmA, DesmB, and DH-A), secondary metabolites with insecticidal and phytotoxic activities produced by Metarhizium species fungus, which are being studied as biological agents in pest control. Therefore, procedures to control them in the food chain are required, starting with crops. As a consequence, in this study, a simple QuEChERS-based destruxin (dtx) extraction procedure has been developed and validated in four different parts of potato plant (tuber, root, stem and leaves) for the first time. For dtx A, the limits of detection obtained, ranged between 0.5 and 1.3 µg/kg, and for quantification, ranged between 1.7 and 4.2 µg/kg. Precision values were below 8.5%; and in all cases, recoveries were higher than 91%. Finally, the method has been applied in potato samples inoculated by EAMa 01/58-Su strain, where dtxs A and B were detected and quantified. In all cases, dtx B concentration was higher than dtx A.

Trace determination of 10 beta-lactam antibiotics in environmental and food samples by capillary liquid chromatography.

A sensitive and reliable method using capillary HPLC with UV-diode array detection (DAD) has been developed and validated for the trace determination of residues of 10 beta-lactam antibiotics of human and veterinary use, in milk, chicken meat and environmental water samples. The analytes included ampicillin, amoxicillin, penicillin V, penicillin G, cloxacillin, oxacillin, dicloxacillin, nafcillin, piperacillin and clavulanic acid. Legal levels are regulated by the EU Council regulation 2377/90 in animal edible tissues for these compounds. For food analysis, a solid-phase extraction (SPE) procedure consisting in a tandem of Oasis HLB and Alumina N cartridges was applied for off-line preconcentration and cleanup. For water analysis, the first step was only necessary. The limits of detection for the studied compounds were between 0.04-0.06 microg l(-1) for water samples and 0.80-1.40 microg l(-1) (or microg kg(-1)) in the case of foods derived from animals. Average recoveries for fortified samples at different concentration levels ranged between 82.9% and 98.2%, with relative standard deviations (RSDs) lower than 9%. The method showed the advantages of capillary HPLC for the detection of these widely applied antibiotics in different samples at very low concentration levels.

Capillary zone electrophoresis with diode-array detection for analysis of local anaesthetics and opium alkaloids in urine samples.

The present study describes the simultaneous determination of four drugs, two local anaesthetics (lidocaine and bupivacaine) and two opium alkaloids (noscapine and papaverine) by capillary zone electrophoresis (CZE) with solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Their recoveries ranged from 81 to 107% at the target concentrations of 2.0, 5.0 and 8.0 microgmL(-1) in spiked urine samples. Coefficients of variation of the recoveries ranged from 2.1 to 11.3% at these concentrations. The quantitation limits of the method were approximately 300 ngmL(-1) for the different compounds studied. The assay is very specific for these compounds and requires a short sample preparation procedure prior to the electrophoretic analysis.

Trace determination of beta-lactam antibiotics in environmental aqueous samples using off-line and on-line preconcentration in capillary electrophoresis.

A sensitive and reliable method using capillary zone electrophoresis with UV-diode array detection (CZE-DAD) has been developed and validated for trace determination of beta-lactam antibiotics in waste, well and river water matrices. Due to the lack of sensitivity of the UV-vis detection, a solvent extraction/solid-phase extraction (SPE) method applied for off-line preconcentration and cleanup of water samples, in combination with an on-line preconcentration methodology named large volume sample stacking (LVSS) have been applied. The analytes included nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G and amoxicillin. Average recoveries for water samples fortified with the studied beta-lactams at different concentration levels (1.0, 2.0 and 4.0 microg/L) were ranging between 94 and 99%, with relative standard deviations (RSDs) lower than 10%. The precision, calculated as intra-day and inter-day standard deviations fell within acceptable ranges (3.3-7.2%). The limits of detection were estimated to range between 0.08 and 0.80 microgL(-1) for the studied compounds. All the samples analyzed were negative for all the analytes at these levels of concentration and the method showed its usefulness for the detection of these widely applied beta-lactam antibiotics in different kinds of waters.

Setting up of recovery profiles: A tool to perform the compliance with recovery requirements for residue analysis.

The use of the recovery term has presented some confusion in Analytical Chemistry. Recent IUPAC recommendations propose to distinguish between two terms: recovery or recovery factor, Re, and apparent recovery, Re*. Apparent recovery includes recovery factor and a new recovery term proposed in this paper, named calibration recovery, Re(C), which depends of the type of systematic error due to the matrix effect (constant and/or proportional) and is related to the applied calibration methodology. This paper highlights the dependence of the calibration recovery on the sample analyte concentration and, for extension, of the apparent recovery, defines the recovery profile, and makes evident the need to determine a "fit for purpose" analyte concentration interval to comply with a regulated recovery requirements. An approach to estimate the calibration recovery and its associated uncertainty in relation to the above-mentioned dependence is presented. The usefulness of the proposed methodology has been shown in the quantification of a pesticide by GC-ECD for assessing dermal exposure.

Chemiluminescence determination of amikacin based on the inhibition of the luminol reaction catalyzed by copper.

A simple and sensitive method has been proposed for the amikacin sulphate determination. It is based on the inhibition of the chemiluminescence (CL) emission generated from the oxidation of luminol in alkaline medium by H2O2 catalyzed by Cu(II), due to the interaction caused by amikacin, which forms a robust complex with the catalyst. The optimization of the experimental and instrumental variables affecting this CL inhibition effect has been carried out using statistical models, based on the application of two-level full factorial and Box-Behnken designs. The performance characteristics of the proposed method have been established, showing that the method is efficient to determine amikacin sulphate in the linear range of 9.89-20 mg/L with a detection limit of 2.97 mg/L. It has been successfully applied to the amikacin sulphate determination in pharmaceutical formulations.

Quantitative determination of p-aminosalicylic acid and its degradation product m-aminophenol in pellets by ion-pair high-performance liquid chromatography applying the monolithic Chromolith Speedrod RP-18e column.

An ion-pair high performance liquid chromatographic method was developed for the simultaneous determination of p-aminosalicylic acid (PAS) and its degradation product m-aminophenol (MAP) in a newly developed multiparticular drug delivery system. Owing to the concentration differences of PAS and MAP, acetanilide and sulfanilic acid were used as internal standards, respectively. The separation was performed on a Chromolith SpeedROD RP-18e column, a new packing material consisting of monolithic rods of highly porous silica. The mobile phase composition was of 20 mm phosphate buffer, 20 mm tetrabutylammonium hydrogen sulphate and 16% (v/v) methanol adjusted to pH 6.8, at a flow-rate of 1.0 mL/min, resulting in a run-time of about 6 min. Detection was by UV at 233 nm. The method was validated and proved to be useful for stability testing of the new dosage form. Separation efficiency was compared between the new packing material Chromolith SpeedROD RP-18e and the conventional reversed-phase cartridge LiChroCART 125-4 (5 microm). A robustness test was carried out on both columns and different separation parameters (retention, resolution, run time, temperature) were determined.

An overview of qualimetric strategies for optimisation and calibration in pharmaceutical analysis using flow injection techniques.

Flow Injection analysis represents an attractive tool because of its great advantages, such as versatility, speed, high sampling rate and wide applicability in the field of pharmaceutical analysis. However, due to the inherent characteristics of the technique, the choice of the best set of operational and chemical conditions is complicated and the conventional univariate optimisation method present some limitations, mainly due to fact that the interdependence of variables is not considered. In relation to the calibration process, because of the transient character of the signals obtained using FIA manifolds coupled with different detection techniques, different strategies can be used in calibration to solve some problems related to the nature of the signal thereby improving the performance characteristics of the method. This paper offers an overview of different methodologies used in optimisation based on the use of statistically designed experiments and some strategies developed for calibration applied to the analysis of pharmaceuticals.

Application of an alkyl-diol silica precolumn in a column-switching system for the determination of meloxicam in plasma.

The group of LiChrospher alkyl-diol silica (ADS) phases that make part of the unique family of restricted-access materials, have been developed as special packings used in the liquid chromatographic integrated sample processing of biofluids. The advantage of these phases lies in the possibility of direct injection of untreated plasma. An on-line elimination of the protein matrix is achieved with a quantitative recovery together with an on-column enrichment. The present method describes a hand-operated on-line switching high-performance liquid chromatographic system for the determination of meloxicam. Spiked plasma samples were introduced on the ADS precolumn using a 0.05 M phosphate buffer, pH 6.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.05 M phosphate buffer-30% (v/v) acetonitrile (ACN)-25 mM t-butylamine (TBA) at a pH of 7.0, thus transferring the analyte to the analytical column LiChrocart 125-4 LiChrospher RP-8. The eluent was monitored by a UV-detector set at 364 nm. The developed column-switching method is fully applicable to plasma injections.

Comparison of morphine and hydromorphone analysis on reversed phase columns with different diameters.

A comparison of a reversed phase high-performance liquid chromatographic (HPLC) method performed on columns with different internal diameters is reported for the quantitative routine determination of morphine.HCl and hydromorphone.HCl in solutions used for intramuscular injection. The method is based on the ion-pairing properties of sodium dodecyl sulphate (SDS) with alkaloids on a reversed phase LiChrospher RP-18 packing material and UV-detection at 230 nm. The mobile phase consisted of an acetonitrile: water mixture 35:65 (v/v) containing 0.5% (w/v) SDS and 0.4% (v/v) acetic acid. Precision, linearity and limit of detection were compared on the 2, 3 and 4 mm i.d. x 125 mm columns. A robustness test for the determination of hydromorphone.HCl was also evaluated.

Determination of albumin in biological fluids by flow injection analysis using the peroxyoxalate chemiluminescent system in micellar medium.

A new sensitive analytical procedure for the determination of albumin is presented, based on the participation of a fluorescent derivative of the protein in the peroxyoxalate chemiluminescent system using imidazole as a catalyst. The chemiluminescent emission is detected in a flow injection analysis (FIA) system, employing micellar medium as carrier, whereas the derivatisation reaction is carried out off-line using fluorescamine as a label. The optimisation of the operational and chemical variables of the method such as concentration of reagents, flow-rates, injection volume, etc., involves the use of experimental designs, including Draper-Lin small composite design, scarcely used in analytical chemistry. The performance characteristics were established and a detection limit of about 0.1 fmol of albumin was obtained. The method has been validated and satisfactorily applied to the determination of albumin in human serum and urine.

Applying non-parametric statistical methods to the classical measurements of inclusion complex binding constants.

A study on using non-parametric statistical methods was carried out to calculate the binding constant of an inclusion complex and to estimate its associated uncertainty. First, a correct evaluation of the stoichiometry was carried out in order to ensure an accurate determination of the binding constant. For this purpose, the modified Benesi-Hildelbrand method had been previously applied. Then, four statistical methods (three non-parametric methods: two bootstrap approaches, the jackknife method and a parametric one: Fieller's theorem) were employed in order to compute the binding constant. The results obtained from applying these methods and the combination of the methods: jackknife after bootstrap and bootstrap after jackknife were compared. The best results in terms of accuracy were obtained from the application of a bootstrap method: the resampling residuals approach. These procedures were applied to the inclusion complex 2-hydroxil-propyl-beta-cyclodextrin-2,4-dichloro-phenoxyacetic, which shows photochemically-induced fluorescence.

Simultaneous quantification of chlorophenoxyacid herbicides based on time-resolved photochemical derivatization to induce fluorescence in micellar medium.

In this paper a sensitive and simple method for the resolution of mixtures of chlorophenoxyacid herbicides using photochemical derivatization induced fluorescence has been described. These compounds do not show any fluorescence, hence photolysed to induce fluorescence after direct irradiation with ultraviolet light in presence of a cationic surfactant (cetyltrimethylammonium chloride). Critical variables such as the surfactant concentration and the irradiation time have been optimised for each compound using Sequential Response Surface Methodology (SRSM) by applying Doehlert designs in order to obtain maximum fluorescence intensity. The difference shown between the optimised irradiation times for the formation of the photoproducts allowed us to propose a time-resolved photoactivation method, for the simultaneous determination of binary mixtures, based on the use of different linear calibration curves established at various irradiation times depending on the mixture to be resolved. Satisfactory recoveries were obtained in the analysis of several mixtures of these herbicides at different ratios in spiked waters.

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response.

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.

Optimizing analytical methods using sequential response surface methodology. Application to the pararosaniline determination of formaldehyde.

Sequential response surface methodology is a general procedure to re-optimize common analytical methods on the basis of the application of the response surface methodology and of a new approach to the steepest ascent method. This procedure, which is easy to apply, consists of estimating an analytical function relating the response with the experimental parameters by means of a second-degree polynomial. Thus, a 2nd order design covering the total experimental domain is used and when a maximum is obtained, the characteristics of the response surface are confirmed using a new design, which is obtained contracting the first one. In the proposed methodology, Box-Behnken designs are used because they offer advantages in comparison with second order designs more frequently used in the steepest ascent method (central composite designs), i.e. fewer experiments are needed, they are more efficient, they can be moved through the experimental domain and they can even be easily contracted or expanded.

Quimioluminiscencia: una interesante alternativapara la detección analítica en sistemas de flujo / Unfamiliar though exciting analytical detection in flowing streams: chemiluminescence

El estudio del tipo de interacción involucrada en la formación de dispersiones sólidas de tolbutamida con distintas proporciones de acetamida y propianamida, ha requerido del diseño y validación de un método analítico por cromatografía líquida de alta eficacia (CLAE) que permita cuantificar la proporción de los transportadores en mezclas físicas y en dispersión sólida. El método resultó ser lineal, preciso y exacto en el intervalo de concentración de 100-1,56 µg/mL para tolbutamida y 50-0,781 µg/mL para acetamida y propianamida (AU)

Micellar-enhanced photochemically induced fluorescence detection of chlorophenoxyacid herbicides. Flow injection analysis of mecoprop and 2,4-dichlorophenoxyacetic acid.

In this paper, a combination of a flow injection analysis (FIA) system with micellar-enhanced photochemically induced fluorescence (MEPIF) detection is presented as a powerful alternative for the rapid and sensitive analysis of chlorophenoxyacid herbicides. These compounds do not show native fluorescence but they can be photolysed into strongly fluorescence photoproducts after direct irradiation with ultraviolet light. The use of a cationic surfactant such as cetyltrimethylammonium chloride (CTAC) provides a considerable enhancement of photochemically induced fluorescence intensity and the nature of the technique allows a possible and easy adaptation to a FIA system. In this sense, parameters related to the nature of the analytical signal (pH, irradiation times, surfactant concentration) and to the FIA manifold (injection volume, flow rate and reactor length) have been optimised. Linear calibration graphs, with three replicates for each concentration value were established in the range of 0.2-5.0 mug ml(-1) for 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.1-5.0 mug ml(-1) for Mecoprop (MCPP). The IUPAC detection limits were 73.2 and 33.5 ng ml(-1) for 2,4-D and MCPP, respectively. Satisfactory recoveries were obtained in the analysis of these herbicides in spiked waters.

Detection in the liquid phase applying chemiluminescence.

Several chemiluminescence-based reactions are applicable to the determination of various bio-pharmaceutically important analytes, and they can be applied for monitoring chemiluminescence emission using flow injection, liquid chromatographic and capillary electrophoretic analysis, as well as for the development of chemiluminescence-based sensors or in immunoassays. As in general the emission intensity is linearly proportional to the concentration of any of the reagents, the technique allows the analysis of different species involved in the light-producing reaction, amongst which are the chemiluminescent reagent, oxidants, inhibitors, cofactors, catalysts, some fluorophore, etc. The present overview illustrates some important applications of the last decade on this rather unfamiliar luminescence technique to detectional challenges in the liquid phase. The required instrumentation is limited as no external light source is needed. Also, the technique opens perspectives for increasing detection sensitivity in miniaturized flowing streams. On the other hand, several drawbacks still limit full application, eg dependence of the emission signal upon a number of environmental factors forcing the analyst to make a compromise between separating and measuring conditions, a lack of selectivity in specific cases, the critical detection of the signal at strictly defined periods, especially in the case of sharp emission vs time profiles, and the development of detection devices in capillary electrophoresis.

Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma.

The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations. Plasma samples introduced to the ADS precolumn using a 0.1 M phosphate buffer, pH 7.0. After washing with the buffer the ADS column was backflushed with the mobile phase 0.01 M phosphate buffer-6% (v/v) 2-propanol-5 mM octanoic acid at a pH of 5.5, thus transporting the analytes to the chiral-HSA (human serum albumin) (100x4.0 mm) column where the separation of the ketoprofen enantiomers was achieved with a resolution factor of 1.4. The developed column-switching method was fully applicable to plasma injections.