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Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC-ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD20% for 86% of the compounds), reproducibility (%RSD40% for 96% of the compounds) and the accuracy (80-120% for 88% of the compounds) were satisfactory. Evaluation of the CCbeta values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 microg/l) that tested positive for this compound. UPLC-ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.

Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC(50) of 17beta-testosterone and the EC(50) of the compound, of 5alpha-dihydrotestosterone, methyltrienolone, and 17beta-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

Capillary zone electrophoresis was coupled on-line via a tricoaxial sheath flow interface with a benchtop quadrupole mass spectrometer (CZE-MS) equipped with a (pneumatically assisted) electrospray ionization source. Experimental parameters were optimized for operation in the negative-ion scan mode. The system was applied to the fingerprinting of an alkylsulphate detergent, to the identification of an impurity in a chloramine-T disinfectant, to the characterization of a complex polyethoxylated alkylphosphate emulgator, to the analysis of phenoxy acid herbicides and to the impurity profiling of MCPP. The optimized system was found to fully maintain the CZE separation performance, showing up to 1 million theoretical plates for the low-molecular-mass analytes studied. The sensitivity in the full-scan mode was typically in the picogram range and sufficient for the separation and identification of minor impurities in these industrial chemicals, down to the 0.1% level. In addition, the feasibility of on-column transient isotachophoresis (ITP-CZE-MS) under high electroosmotic flow conditions has been demonstrated for sample preconcentration and subsequent identification of trace impurities.

Capillary zone electrophoretic (CZE) methods were developed for the separation of phenoxy acid herbicides and related impurities (including positional isomers) originating from production processes. In addition, chiral separations of phenoxypropionic acid herbicides were achieved by adding a suitable cyclodextrin-type chiral selector to the electrophoresis buffer. The selectivity of the separation can be controlled and fine-tuned by using different types of cyclodextrins. The presence of specific impurities could be confirmed after comparison of the electropherograms obtained with CZE systems having different selectivities. The CZE method shows good precision, linearity and long-term stability. The methods developed have been successfully applied to the analysis of real production samples and the determination of their enantiopurity. Both the identification and determination of the impurities compare with the results obtained with chromatographic methods. The CZE methods, however, are more flexible and simpler and will be more economical. The methods have potential for automated quality control assays.

A capillary zone electrophoretic method for the separation of positional isomers of phenylenediamine is presented. The phenylenediamines were baseline resolved using a fused-silica capillary. However, the analysis of samples containing both an excess of p-phenylenediamine and minor traces of the meta and ortho isomers, and also a high salt content, required a non-polar (DB-1) bonded phase in order to reduce the electroosmotic flow. The method can be used for purity testing of p-phenylene diamine and for the determination of impurities in hydrolysates of synthetic polymers.

Time-resolved luminescence of the terbium(III)-acetylacetone chelate has been used as an indirect detection method for capillary zone electrophoresis. Three different modes were investigated: dynamic quenching of the background signal by selected anions; ligand displacement of acetylacetone by other complexing agents; and electrophoretic displacement of acetylacetonate by anions in general. The latter two modes required the use of a post-capillary reactor; in this study, a coaxial flow-type reactor was used for this purpose. The results of dynamic quenching were good for the determination of nitrite, yielding detection limits of 3 · 10−9 M (0.2 ppb). In addition, the feasibility of ligand and electrophoretic displacement was demonstrated. However, improvement of the post-capillary reactor and substitution of the xenon lamp by a focused laser beam is required to exploit fully the potential of the ligand and electrophoretic displacement options.

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.

Quantitative aspects of indirect UV absorbance detection in capillary zone electrophoresis were studied using the separation of sodium alkylsulphate surfactants as a model system. Nearly uniform response factors, excellent reproducibility of electrophoretic mobilities and linearity of the detection signal can be obtained when veronal buffer is used as the UV-absorbing background electrolyte. The feasibility of the system for the analysis of commercial mixtures of primary sodium alkyl sulphates is demonstrated.

The separation process in capillary zone electrophoresis is governed by many experimental parameters, such as the choice of the electrolyte, field strength, ionic strength, pH, temperature and additives (including organic modifiers). Positional isomers of substituted benzoic acids were used as model compounds in order to study the contributions of pH, electrolyte, ionic strength, addition of alcohols, the choice of the counter ion and the temperature to the electrophoretic separation and the magnitude of the electroosmotic flow. The results thus obtained were compared critically with those of previous studies, in which only the modifier content had been varied. Obviously, pH was the most effective parameter in optimizing the resolution. However, thermostating of the capillary at elevated temperatures turned out to be a very effective additional feature; the resolution was increased and the analysis time was shortened considerably. Other options, such as the addition of alcohols or the choice of the counter ion, will also increase the resolution but also increase the analysis time. The improvement in the concentration sensitivity was studied using the technique of sample stacking. Volumes of up to 15 nl could be introduced in this way without serious separation problems, yielding a detection limit of 10−6M.

The use of beta-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of beta-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 beta-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCalpha and CCbeta values of less than 0.2 and less than 0.5 microg/l respectively, for all beta-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCbeta values of less than 10.0 and less than 5.0 microg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of beta-agonists.

A liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) method was developed using the latest high-resolution LC column technology, the ultra performance liquid chromatography (UPLC (TM)), and electrospray ionization (ESI) in the positive ion mode. Gradient UPLC separation conditions were optimized for a group of 22 analytes comprising 17 glucocorticosteroids, specific designer steroids such as tetrahydrogestrinone (THG) and specific beta(2)-agonists such as formoterol. The UPLC/TOFMS separation obtained required 5.5 min only for all the substances tested. Even the critical pair of dexamethasone and betamethasone isomers was almost completely resolved. Thanks to the over 10,000 full-width at half maximum (FWHM) mass resolution and high mass accuracy features of TOFMS 50 mDa window accurate mass chromatograms could be reconstructed for the individual analytes. Sensitive screening in human and calf urine samples fortified at the glucocorticosteroids minimum required performance limit (MRPL) of 30 mu g L-1 (human urine, sports doping) and 2 mu g L-1 (calf urine, veterinary control) could be obtained. The potential of UPLC/TOFMS for confirmatory analysis was shown by determining the accurate mass of all compounds and fragment ions upon in-source collision-induced dissociation (CID) at different energies. The exact mass measurement errors for all glucocorticosteroids were found to be within 6 ppm. Considering veterinary control, limits of detection (LOD) and limits of quantification (LOQ) were determined for most of the analytes in calf urine and found to range from 0.1 to 3.3 and from 0.4 to 4.4 mu g L-1, respectively. The method can be easily extended with other banned substances of interest, as demonstrated by the addition of 21 beta(2)-agonists to the original analyte mixture in urine, without causing any interferences. (c) 2006 Elsevier B.V. All rights reserved.

The usefulness of several grades of carbon dioxide for online supercritical fluid extraction/capillary gas chromatography has been studied. If trace-level work has to be performed, no single grade can be recommended for all modes of detection studied. Good-quality carbon dioxide is available for use with flame ionization and thermionic detectors. With electron-capture detection, however, distinct problems still exist.

The design of an automated cartridge exchange module for on-line sample handling in liquid chromatography is described. When combined with a low-cost purge pump, a solvent selection valve and an auto-sampler, a fully automated sample handling system is obtained. Samples are sorbed on a disposable cartridge packed with 40 micron octyl-bonded silica, purged for clean-up and eluted on-line to the analytical column. Unattended operation of the system is demonstrated for various examples, i.e., the determination of anti-epileptic drugs in serum, an anti-cancer drug in plasma, barbiturates in urine, phenylurea herbicides in river water and caffeine in a soft drink.

A small pre-column packed with a mercury(II)-8-hydroxyquinoline phase is used for the selective trace enrichment and clean-up of 2-mercaptobenzimidazole, a thiol model compound. Strong covalent bond formation between tha analyte and the metal-loaded sorbent allows the introduction of a wash step with methanol—water (1:1). This results in a superior selectivity to that observed with the frequently used reversed-phase packing materials. Desorption of the pre-concentrated thiol from the pre-column is effected with an eluent containing cysteine as a displacer. The elution profiles are ineffecient, however, so that peak compression of the analyte on the top of the C18 separation column is necessary. With UV obsorbance detection at 254 nm, the detection limit is 1 ppb. The excellent selectivity of the on-line pre-column sample handling is demonstrated in the analysis of river water and industrial waste water samples.

The influence of precolumn design on the efficiency of liquid chromatographic separation systems which involve the use of an on-line trace-enrichment step has been studied. For most practical purposes, precolumns having an inner diameter of 2–4.6 mm and a length of l0–2 mm can be recommended; these allow sampling flow-rates of up to at least 10 ml min -1 at a back pressure of 1–10 MPa. Within this range, the precolumn geometry does not appear to be critical in the on-line trace enrichment of either strongly retained or more weakly retained analytes. A new precolumn design is presented which allows rapid slurry packing with a micro-spatula or via syringe loading, and easy replacement of low-cost precolumn cartridges.

The use of strongly acidic cation exchangers as catalysts in post-column packed-bed reactors has been studied. The main aim was their application in the liquid chromatographic determination of carbohydates, i.e. , in the conversion of non-reducing oligosaccharides, especially sucrose, into more easily detectable products with reducing properties. Three types of polystyrene-based ion exchangers with different percentages of cross-linking (4 and 8%) and one silica-based ion exchanger were compared. Studies on the influence of flow-rate and reaction temperature on reactor performance were carried out so as to allow the determination of peak broadening in the reactor and of the rate of hydrolysis of the saccharides. In both instances the 4% cross-linked polystyrene resin gave the best results.

Amphetamine-type drugs with a wide polarity range have been screened in both human and horse urine using on-line pre-concentration on pre-columns packed with hydrophobic and cation-exchange sorbents in series and gradient microbore high-performance liquid chromatography. The underivatized amphetamines were identified by UV detection at 210 nm. The method has potential for the automated liquid chromatographic screening of amphetamines in urine, e.g., for doping control.