Gunnar Thorsén

New interface for coupling flow-injection and capillary electrophoresis

Abstract A new interface for coupling flow injection analysis (FIA) and capillary electrophoresis (CE) has been developed. An FIA system is connected to a flow-through channel of the interface. One end of a capillary is positioned in a flow-through channel inside the interface together with a platinum electrode. The other end and a second platinum electrode are immersed in a liquid reservoir situated outside the interface. A constant high voltage is applied across the capillary. A sample plug injected in the FIA part of the system is carried by an electrolyte stream towards the interface and when it passes by the first capillary end, a minor fraction of the sample is introduced into the capillary and gets separated. A UV spectrophotometric detector is placed close to the second capillary end. The FIA-CE interface enables multiple injections so that a high sampling throughput can be achieved (up to 150 samples per hour). The interface was tested by running a standard mixture of nine common anions. The repeatability was approximately 2% (r.s.d.). Rapid, qualitative screening of samples can be performed. Real samples such as tap and rain water were also analyzed. The new interface allows mechanized sample handling prior to the CE separation step.

Physiological effects of diclofenac, ibuprofen and propranolol on Baltic Sea blue mussels

Pharmaceuticals are constantly dispersed into the environment and little is known of the effects on non-target organisms. This is an issue of growing concern. In this study, Baltic Sea blue mussels, Mytilus edulis trossulus, were exposed to diclofenac, ibuprofen and propranolol, three pharmaceuticals that are produced and sold in large quantities and have a widespread occurrence in aquatic environments. The mussels were exposed to pharmaceuticals in concentrations ranging from 1 to 10,000 microg l(-1). The pharmaceuticals were added both separately and in combination. Mussels exposed to high concentrations of pharmaceuticals showed a clear response compared to controls. Firstly, they had a significantly lower scope for growth, which indicates that the organisms had a smaller part of their energy available for normal metabolism, and secondly, they had lower byssus strength and lower abundance of byssus threads, resulting in reduced ability to attach to the underlying substrate. Mussels exposed to lower concentrations showed tendencies of the same results. The concentration of diclofenac and propranolol was quantified in the mussels using both liquid chromatography coupled to mass spectrometry (LC-MS). The measurements showed a significantly higher concentration in the organisms as compared to the water the mussels were exposed to; the uptake reached concentrations two orders of magnitudes higher than found in sewage treatment plant effluents. This study showed that common pharmaceuticals are taken up and negatively affect the physiology of a non-target species at levels of two to three orders of magnitudes higher than found in sewage treatment plant effluents.

SALDI-MS signal enhancement using oxidized graphitized carbon black nanoparticles.

The signal intensity of low-molecular-weight compounds analyzed using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS) was significantly enhanced when oxidized graphitized carbon black (GCB) particles were used as the desorption/ionization surface. The surface of oxidized GCB contains more carboxylic acid groups than non-oxidized GCB. Carboxylic acid groups enhance the efficiency of the ionization process and the desorption of more hydrophobic compounds. A common pharmaceutical compound, propranolol, was successfully extracted from Baltic Sea blue mussels and quantified using oxidized GCB as the SALDI surface, whereas deuterated propranolol was used as the internal standard. The calibration curve showed a wide linear dynamic range of response (0.1–20 µg/mL) and good reproducibility (RSD < 10%). It was not possible to detect propranolol in Baltic Sea blue mussels when non-oxidized GCB was used as the SALDI surface.

Enantiomeric determination of amino compounds with high sensitivity using the chiral reagents (+)- and (−)-1-(9-anthryl)-2-propyl chloroformate

New chiral precolumn reagents, (+)- and (-)-1-(9-anthryl)-2-propyl chloroformate (APOC), are introduced for the chiral separation of amino acids and small peptides in capillary electrophoresis. Chiral separation of 17 amino acids and four small peptides as their diastereomeric 1-(9-anthryl)-2-propyl carbamate derivatives have been achieved by micellar electrokinetic chromatography. The detection limit for the derivatives is in the femtomole range with UV detection and in the attomole range with laser-induced fluorescence (LIF) detection. LIF detection was used to determine the enantiomeric excess of four APOC-derivatised peptides. The use of the new, anthracene-based reagents in conjunction with argon ion LIF makes enantiomeric determinations at ppm levels feasible. In this paper determinations below promille levels are performed without overloading the separation system.

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection.

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/-)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids D-alanine, D-glutamine, and D-aspartic acid have been observed in cerebrospinal fluid, and D-alanine and D-glutamic acid in urine. To the best of our knowledge no measurements of either D-alanine in cerebrospinal fluid or D-glutamic acid in urine have been presented in the literature before.

Screening and Quantification of Pesticides in Water Using a Dual-Function Graphitized Carbon Black Disk

A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS.

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 microM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.

Optimisation of resolution in micellar electrokinetic chromatography by multivariate evaluation of electrolytes

A novel approach to multivariate evaluation of separation electrolytes for micellar electrokinetic chromatography is presented. An initial screening of the experimental parameters is performed using a Plackett-Burman design. Significant parameters are further evaluated using full factorial designs. The total resolution of the separation is calculated and used as response. The proposed scheme has been applied to the optimisation of the separation of phenols and the chiral separation of (+)-1-(9-anthryl)-2-propyl chloroformate-derivatized amino acids. A total of eight experimental parameters were evaluated and optimal conditions found in less than 48 experiments.

μ-Trap for the SALDI-MS Screening of Organic Compounds Prior to LC/MS Analysis

A procedure for rapidly screening and quantitatively analyzing organic molecules is presented, in which a miniaturized solid-phase extraction (SPE) cartridge containing 0.6 mg of graphitized carbon black (the GCB-mu-trap) is used for sample pretreatment. Then surface-assisted laser desorption ionization time-of-flight mass spectrometry (SALDI-TOF-MS) screening is followed by liquid chromatography/mass spectrometry (LC/MS) for robust quantitative analysis of samples containing analytes of interest. Liquid samples with volumes up to 100 mL were extracted using the GCB-mu-trap, and SALDI screening was performed by transferring a few particles of the GCB 4 sorbent from the mu-trap onto a stainless steel plate. Analytes were then simply ionized and desorbed by irradiating the GCB 4 particles without any further pretreatment. GCB 4 was found to be an excellent surface for the SALDI analysis of small molecules, providing spectra with very clean backgrounds. The small size of the cartridge (micropipet filter tip) results in enrichment of the analytes on a small surface area, affording low SALDI-TOF-MS detection limits. Furthermore, the removal of just a few particles from the mu-trap does not significantly affect the subsequent quantitative determination. This approach offers considerable reductions in analytical costs by eliminating unnecessary SPE-LC/MS analyses.

Determination of benzothiazole and benzotriazole derivates in tire and clothing textile samples by high performance liquid chromatography-electrospray ionization tandem mass spectrometry.

A high performance liquid chromatography-tandem mass spectrometry method utilizing electrospray ionization in positive and negative mode has been developed for the separation and detection of benzothiazole and benzotriazole derivates. Ultra-sonication assisted solvent extraction of these compounds has also been developed and the overall method demonstrated on a selected clothing textile and an automobile tire sample. Matrix effects and extraction recoveries, as well as linearity and limits of detection have been evaluated. The calibration curves spanned over more than two orders of magnitude with coefficients of correlation R(2)>0.99 and the limits of detection and the limits of quantification were in the range 1.7-58pg injected and 18-140pg/g, respectively. The extraction recoveries ranged between 69% and 102% and the matrix effects between 75% and 101%. Benzothiazole and benzotriazole derivates were determined in the textile sample and benzothiazole derivatives determined in the tire sample with good analytical performance.