Mette Krogh

Development of a simple in-vial liquid-phase microextraction device for drug analysis compatible with capillary gas chromatography, capillary electrophoresis and high-performance liquid chromatography

A simple, inexpensive and disposable device for liquid-phase microextraction (LPME) is presented for use in combination with capillary gas chromatography (GC), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). 1-4 ml samples of human urine or plasma were filled into conventional 4-ml vials, whereafter 15-25 microl of the extraction medium (acceptor solution) was filled into a short piece of a porous hollow fiber and placed into the sample vial. The drugs of interest were extracted from the sample solutions and into the small volumes of acceptor solution based on high partition coefficients and were preconcentrated by a factor of 30-125. For LPME in combination with GC, the porous hollow fiber was filled with 15 microl n-octanol as the acceptor solution. Following 30 min of extraction, the organic acceptor solution was injected directly into the GC system. For LPME in combination with CE and HPLC, n-octanol was immobilized within the pores of the hollow fiber, while the internal volume of the fiber was filled with either 25 microl of 0.1 M HCl (for extraction of basic compounds) or 25 microl 0.02 M NaOH (for acidic compounds). Following 45 min extraction, the aqueous acceptor solution was injected directly into the CE or HPLC system. Owing to the low cost, the extraction devices were disposed after a single extraction which eliminated the possibility of carry over effects. In addition, because no expensive instrumentation was required for LPME, 10-30 samples were extracted in parallel to provide a high number of samples per unit time capacity.

Liquid-phase microextraction as a sample preparation technique prior to capillary gas chromatographic-determination of benzodiazepines in biological matrices

Liquid-phase microextraction (LPME) and gas chromatography were applied to determine diazepam and the main metabolite N-desmethyldiazepam in human urine and plasma. The analytes were extracted from 3.0-3.5 ml sample volumes directly into 25 microl of extraction solvent. The microextraction device consisted of a porous hollow fiber of polypropylene attached to two guiding needles inserted through a septum and a 4 ml vial. The hollow fiber filled with extraction solvent was immersed in sample solution. The extraction device was continuously vibrated at 600 rpm for 50 min. An aliquot (1 microl) of the extraction solvent with preconcentrated analytes was injected directly into the capillary gas chromatograph. Thirty samples were extracted simultaneously on the vibrator, providing a high sample capacity. The limits of detection were from 0.020 to 0.115 nmol/ml for diazepam and N-desmethyldiazepam in plasma and urine using a nitrogen-phosphorus detector (NPD).

Solid-phase microextraction for the determination of the free concentration of valproic acid in human plasma by capillary gas chromatography

The potential of solid-phase microextraction in the bioanalysis of drugs is demonstrated. The free concentration of valproic acid in human plasma was determined by equilibrium dialysis at room temperature. To the dialysate was added an internal standard and the pH was adjusted to 2.5. The polymethylsiloxane-coated fused-silica fibre of the solid-phase microextraction device was inserted into the dialysate for 3 min. The sorbed analytes were then thermally desorbed at 210 degrees C in the split-splitless injection port of the gas chromatograph, separated on a Nukol capillary column and detected with a flame ionization detector. The method was shown to be highly reproducible with a detection limit of 1 microgram/ml of free valproic acid in human plasma.

Analysis of drug seizures of heroin and amphetamine by capillary electrophoresis

Abstract Capillary electrophoresis has been used to separate heroin and amphetamine from structurally related compounds and commonly occurring adulterants in drug seizures. The method was based on micellar electrokinetic chromatography with a running buffer of pH 9.0 containing sodium dodecyl sulphate as surfactant and acetonitrile as organic modifier. The drugs were dissolved in running buffer containing crystal violet. Crystal violet was used to calculate relative migration times for drug identification and as internal standard for quantitative analysis. Both qualitative and quantitative analysis was shown to be reproducible. Because of the speed and resolving power of the method it is a powerful alternative to the high-performance liquid chromatographic and gas chromatographic methods in current use for the analysis of illicit drugs.

Automated determination of 'Ecstasy' and amphetamines in urine by SPME and capillary gas chromatography after propylchloroformate derivatisation.

The determination of amphetamines and their methylenedioxylated analogs in urine by propylchloroformate derivatisation and automated solid-phase microextraction is described. The urine sample was adjusted to pH 10.8 and added propylchloroformate reagent and an internal standard. Derivatisation resulted in water-stable carbamates which were automatically extracted by solid-phase microextraction. A fiber coated with polydimethylsiloxane was inserted into the urine matrix and agitated for 16 min. The fibre with the extracted carbamates was injected into the heated split-splitless injection port of the gas chromatograph where the analytes were evaporated at 300 degrees C, separated on a methylsilicone capillary column and detected by either a nitrogen phosphorous detector or by mass spectrometry. The method was shown to be highly reproducible and robust with respect to variations in the urine matrices. The detection limits were 5 ng/ml(-1) of methamphetamine, MDMA and MDEA and 15 ng/ml(-1) of amphetamine and MDA in urine. The method is a solvent free, automated alternative to traditional methods for determination of the amphetamine and their methylendioxylated analogs in urine.

Aqueous alkylchloroformate derivatisation and solid-phase microextraction: determination of amphetamines in urine by capillary gas chromatography

The alkylchloroformate derivatisation and solid-phase microextraction of amphetamine and methamphetamine directly in urine samples prior to capillary gas chromatographic analysis is described. The alkylchloroformate reagent was added to the urine sample, which was adjusted to pH 10.8, and an internal standard was added. The resulting products were water-stable carbamates that were extracted without organic solvent. The polydimethylsiloxane coated fibre was inserted into the modified sample and agitated for 14 min. The fibre with the extracted derivatisation products was injected into the capillary gas chromatograph. The extracted carbamates were evaporated at 300 degrees C in the split-splitless injection port of the gas chromatograph, separated on a methylsilicone capillary column and detected by either a nitrogen-phosphorus detector or by mass spectrometry. The method was shown to be reproducible with a detection limit of 50 ng/ml of amphetamine and methamphetamine in urine.

Solvent-modified solid-phase microextraction for the determination of diazepam in human plasma samples by capillary gas chromatography.

This paper describes microextraction and gas chromatographic analysis of diazepam from human plasma. The method was based on immobilisation of 1.5 microliters of 1-octanol on a polyacrylate-coated fiber designed for solid-phase microextraction. The solvent-modified fibre was used to extract diazepam from the samples. The plasma sample was pre-treated to release diazepam from the protein binding. The fibre was inserted into the modified plasma sample, adjusted to pH 5.5 an internal standard was added and the mixture was carefully stirred for 4 min. The fibre with the immobilised solvent and the enriched analytes was injected into the capillary gas chromatograph. The solvent and the extracted analytes were evaporated at 300 degrees C in the split-splitless injection port of the gas chromatograph, separated on a methylsilicon capillary column and detected with a nitrogen-phosphorus detector. The method was shown to be reproducible with a detection limit of 0.10 nmol/ml in human plasma.

Solid-phase microextraction/capillary gas chromatography for the profiling of confiscated ecstacy and amphetamine

SummaryImpurity profiling of ecstacy and amphetamine seizures was accomplished by solid-phase microextraction (SPME) combined with capillary gas chromatography (GC). Samples were dissolved in 0.1 M aqueous acetate buffer (pH 5.0) as the only manual operation and subsequently subjected to SPME-GC. Ecstacy tablets were analyzed by head-space SPME to avoid contamination of SPME fibers with insoluble tablet components, while illicit amphetamine powders were exposed to immersed SPME. A SPME fiber of polydimethylsiloxane-divinylbenzene was found to provide excellent extraction of both polar and non-polar impurities. For both illicit ecstacy and amphetamine, complex impurity profiles were obtained by SPME providing a high information content. For ecstacy, profiles (relative peak areas) were repeatable within 2.2 to 12.6% RSD (n=6) while similar data on amphetamine varied between 2.0 and 10.9% RSD (n=6). No carry-over was observed although each fiber was used for 50 to 100 extractions.

Automated analysis of free and total concentrations of three antiepileptic drugs in plasma with on-line dialysis and high-performance liquid chromatography

A fully automated method for determination of the free and total concentration of drugs with a varying degree of protein binding is described. The antiepileptic drugs phenytoin, carbamazepine and phenobarbitone were chosen to demonstrate the utility of this technique. The method was based on the ASTED system and combined on-line equilibrium dialysis at 37 degrees C with concentration of the dialysate on a trace enrichment column and HPLC determination with UV detection. The dialysis cell was a modification of the ASTED dialysis cell and 22% of the free concentration of the drugs were recovered in the recipient channel of the dialyser after 10 min of dialysis at 37 degrees C. The free concentration, the total concentration as well as the drugs protein binding could be determined. The method was shown to be well suited for routine monitoring of the free and the total concentrations of the drugs in plasma from epileptic patients.

Automated sample preparation by on-line dialysis and trace enrichment. Analysis of morphine, 6-monoacetylmorphine, codeine, ethylmorphine and pholcodine in plasma and whole blood by capillary gas chromatography and capillary gas chromatography-mass spectrometry.

A fully automated sample preparation method for the determination of five opiates in human plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to gas chromatography and gas chromatography-mass spectrometry. Dialysis and trace enrichment on a polymer column was shown to be a highly reliable method for sample preparation. The method can be used, after minor modification, to determine other basic drugs in plasma and whole blood. The method demonstrates the potential of the automated sequential trace enrichment of dialysate (ASTED) system for automated sample preparation.