Determination of acidic non-steroidal anti-inflammatory drugs in aquatic samples by liquid chromatography-triple quadrupole mass spectrometry combined with carbon nanotubes-based solid-phase extraction.

A solid-phase extraction (SPE) method based on multi-walled carbon nanotubes (CNT) was developed for the determination of 12 acidic non-steroidal anti-inflammatory drugs (NSAIDs) in surface waters and tap water. Pristine and functionalised CNTs were evaluated as sorbent materials. Batch experiments were used to optimise sorption and desorption conditions (sorbent type and amount, adsorption time, pH). The adsorption equilibrium was reached after 8 to 48 h duration, which increased with the pH of solution. Non-agglomerated pristine CNTs (20 mg) showed the most optimal adsorption (94 to 100%) for all of the analytes after a 30-min contact period in acidified water solutions (100 mL). The compounds retained at those conditions were recovered by 40 to 95% by using 5% ammonium hydroxide in methanol as the desorbing solution at ambient conditions. A comprehensive liquid chromatography coupled to triple quadrupole mass spectrometry (LC-QqQ-MS/MS) was used for the analysis of real water samples. The method showed sufficient recovery (65-125%) and good precision (2-14% relative standard deviation (RSD)). The limits of detection and quantification ranged between 0.01 and 1.3 ng L-1 and 0.04 and 3.9 ng L-1. Only diclofenac and ibuprofen were found in the analysed surface water samples from Latvia (n = 10) and Norway (n = 14). Diclofenac was found at 1.7-8.4 ng L-1 concentration in two samples of surface waters, whereas the concentrations of ibuprofen ranged between 1.0 and 9.2 ng L-1 in seven samples collected in Norway and 3.9-17 ng L-1 in three samples from Latvia.

Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10 µm OT columns suitable for small molecules, peptides and proteins.

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 µm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 µg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-µg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is "fit-for-all", well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

Determination of airborne trialkyl and triaryl organophosphates originating from hydraulic fluids by gas chromatography-mass spectrometry. Development of methodology for combined aerosol and vapor sampling.

Methodology for personal occupational exposure assessment of airborne trialkyl and triaryl organophosphates originating from hydraulic fluids by active combined aerosol and vapor sampling at 1.5L/min is presented. Determination of the organophosphates was performed by gas chromatography-mass spectrometry. Combinations of adsorbents (Anasorb 747, Anasorb CSC, Chromosorb 106, XAD-2 and silica gel) with an upstream cassette with glass fiber or PTFE filters and different desorption/extraction solvents (CS(2), CS(2)-dimethylformamide (50:1, v/v), toluene, dichloromethane, methyl-t-butyl ether and methanol) have been evaluated for optimized combined vapor and aerosol air sampling of the organophosphates tri-isobutyl, tri-n-butyl, triphenyl, tri-o-cresyl, tri-m-cresyl and tri-p-cresyl phosphates. The combination of Chromosorb 106 and 37 mm filter cassette with glass fiber filter and dichloromethane as desorption/extraction solvent was the best combination for mixed phase air sampling of the organophosphates originating from hydraulic fluids. The triaryl phosphates were recovered solely from the filter, while the trialkyl phosphates were recovered from both the filter and the adsorbent. The total sampling efficiency on the combined sampler was in the range 92-101% for the studied organophosphates based on spiking experiments followed by pulling air through the sampler. Recoveries after 28 days storage were 98-102% and 99-101% when stored at 5 and -20 degrees C, respectively. The methodology was further evaluated in an exposure chamber with generated oil aerosol atmospheres with both synthetic and mineral base oils with added organophosphates in various concentrations, yielding total sampling efficiencies in close comparison to the spiking experiments. The applicability of the method was demonstrated by exposure measurements in a mechanical workshop where system suitability tests are performed on different aircraft components in a test bench, displaying tricresyl phosphate air concentrations of 0.024 and 0.28 mg/m(3), as well as during aircraft maintenance displaying tri-n-butyl phosphate air concentrations of 0.061 and 0.072 mg/m(3).

Determination and quantification of urinary metabolites after dietary exposure to acrylamide.

It is known that heat-treated carbohydrate-rich foods may contain high levels of acrylamide (AA) and up to 4000 microg kg-1 in potato crisps and 2000 microg kg-1 in French fries have been reported. In order to obtain more information on the human exposure to and metabolism of AA, a method for the determination of known urinary metabolites from the dietary exposure of AA using solid-phase extraction and liquid chromatography with positive electrospray MS/MS detection was developed. The validated assay range was from 8.6 to 342.9 microg l-1. The urinary metabolites were synthesized and their structures determined by NMR and MS. To test the method, a pilot study was conducted in which all urine during 48 h starting with 24 h fasting was collected. The two urinary metabolites, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (MA-GA3) and N-acetyl-S-(3-amino-3-oxopropyl)cysteine (MA-AA), were found to be above the detection limit. Fasting during 1 day caused about a 50% decrease in the total level of the metabolites, but after 1 day of a normal diet, the metabolite levels increased back to pre-fasting levels. The total amount of AA in the form of urinary metabolites excreted over the period was estimated to be about 40 microg AA day-1 for the average non-smoker.

Determination of rotenone in river water utilizing packed capillary column switching liquid chromatography with UV and time-of-flight mass spectrometric detection.

Fast and sensitive packed capillary column switching liquid chromatography methodology has been developed for the determination of the pesticide rotenone in river water. Sample volumes of up to 1 ml are loaded onto a 23 x 0.25 mm, 5 microm Kromasil C18 packed capillary precolumn using a noneluting solvent composition of water-acetonitrile (99:1, v/v) at flow-rates up to 100 microl/min prior to solute backflushing onto a 200 x 0.32 mm, 3.5 microm Kromasil C18 packed capillary analytical column using a mobile phase of water-acetonitrile (30:70, v/v) at a flow-rate of 5 microl/min. The method was evaluated using river water samples spiked with rotenone in the concentration range 0.5-50 ng/ml using UV detection. The within-assay precision was between 5.0 and 7.7% relative standard deviation (RSD, n = 6) and the between assay precision was between 7.5 and 8.9% RSD (n = 6). The method was linear within the investigated mass range displaying a calibration curve correlation factor of 0.997. The mass limit of detection was 10 pg corresponding to a concentration limit of detection of 10 pg/ml, using time-of-flight mass spectrometry.

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes.

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.

Brominated flame retardants in laboratory air.

During the development of a method for determination of brominated flame retardants in human plasma and serum using solid-phase extraction, several brominated flame retardants were found in the procedural blanks. The contaminants originated most probably from the laboratory air. The brominated flame retardants were found to be adsorbed on glass surfaces and to be acquired using solid-phase sampling. 2,4,6-Tribromophenol, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) were the most abundant brominated flame retardants in our laboratory air, however, large differences in contamination with respect to sampling time and place were observed.

Brominated flame retardants in plasma samples from three different occupational groups in Norway.

Brominated flame retardants (BFRs) are widely used in plastics, textile coatings, electrical appliances and printed circuit boards to prohibit the development of fires. In order to investigate how exposure to BFRs is related to specific occupations, samples were obtained from Norwegian individuals working at an electronics dismantling facility, in the production of printed circuit boards, or as laboratory personnel. Nine BFRs were quantified in the plasma samples: 2,4,4'-tribromodiphenyl ether (BDE-28), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153), 2,2',4,4',5,6'-hexabromodiphenyl ether (BDE-154), 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE-183), 2,4,6-tribromophenol (TriBP) and tetrabromobisphenol A (TBBP-A). The BFRs were extracted from plasma using solid-phase extraction (SPE). The plasma lipids were decomposed by treatment with concentrated sulfuric acid directly on the SPE column, prior to the elution of the BFRs. Following diazomethane derivatization, the samples were analysed by gas chromatography-electron capture mass spectrometry. The subjects working at the electronics dismantling plant had significantly higher plasma levels of TBBP-A and BDE-153 compared to the other groups, and the heptabrominated congener BDE-183 was only detected in plasma from this group. TriBP was generally the most abundant BFR present, and the plasma concentrations were in the range 0.17-81 ng g-1 lipids. BDE-47 was the dominant BDE congener in all the individual samples and the levels were in the range 0.43-14.6 ng g-1 lipids. The total amounts of the seven BDEs were 8.8, 3.9 and 3.0 ng g-1 lipids for the group of electronics dismantlers, circuit board producers and laboratory personnel, respectively. Generally, large variations in the individual concentration levels were found within the groups, especially in the group of electronics dismantlers, where the relative standard deviations for BDE concentrations were in the range 23-164%. The levels of BFRs were not correlated to age or the level of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153). The present work indicates that the population in Norway is exposed to several BFRs, probably with food as a major source. The elevated level of higher brominated BDEs and TBBP-A in the plasma from the workers at the dismantling plant suggests an additional occupational exposure for these individuals. Thus, human exposure to BFRs seems to originate from a combination of different sources; however, further studies investigating plasma samples from a larger number of individuals are necessary for a more complete assessment of human exposure pathways to these environmental contaminants.

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography-electron-capture mass spectrometry.

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC-ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene-divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2-25, 0.4-40, 4-200 and 4-200 pg g(-1) plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo-o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93-107%. The repeatability of the method was in the range 4-30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g(-1) plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.

Determination of 1-(2-methoxyphenyl)piperazine derivatives of isocyanates at low concentrations by temperature-programmed miniaturized liquid chromatography.

A temperature-programmed packed capillary LC method with large-volume injection on-column focusing has been developed for screening and determination of 1-(2-methoxyphenyl)piperazine derivatives of airborne toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, hexamethylenediisocyanate and methylenebisphenyl-4,4-diisocyanate, based on sampling methods described in MDHS 25/3. Injection volumes up to 100 microl were successfully loaded onto the 250x0.32 mm I.D. capillary column packed with 3 microm Hypersil ODS particles. The isocyanate derivatives were loaded at 10 degrees C and eluted by a three-step temperature program starting at 10 degrees C for 10 min, followed by a temperature ramp of 2.5 degrees C min(-1) to 45 degrees C and then 9.9 degrees C min(-1) to 90 degrees C. The mobile phase consisted of acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (45:55, v/v). The isocyanate derivatives were dissolved in acetonitrile-acetate buffer (3% triethylamine, pH 4.5) (30:70, v/v) to achieve sufficient focusing. The concentration limit of detection of the individual derivatives utilizing an "U" shaped flow cell with a 8.0 mm light path and an injection volume of 100 microl was 44, 87, 43 and 210 pg ml(-1) for toluene-2,6-diisocyanate, hexamethylenediisocyanate, toluene-2,4-diisocyanate and methylenebisphenyl-4,4-diisocyanate, respectively. Within the investigated concentration range, 10-500 ng ml(-1), the linear calibration curves gave correlation coefficients ranging from 0.994 to 0.998. The repeatability of the method with regard to retention time and peak height ranged from 0.3 to 1.1% and 1.1 to 2.3% (n=9) relative standard deviation, respectively. The average recovery of the method, with regard to toluene-2,4-diisocyanate, was 97.7+/-1.6% (n=9).

Nonaqueous electrochromatography on continuous bed columns of sol-gel bonded large-pore C18 material: separation of retinyl esters.

A nonaqueous electrochromatographic reversed-phase separation method for retinyl esters using continuous bed columns has been developed. The packing material 7 microm Nucleosil 4,000 angstroms C18 was sol-gel bonded in 180 microm I.D. capillaries. The mobile phase used was 2.5 mM lithium acetate in N,N-dimethylformamide-acetonitrile-methanol (2+7+1, v/v). At 350 V/cm and 30 degrees C, this mobile phase composition gave rise to an electroosmotic flow of 1 mm/s. No Joule heating nor bubble formation were observed even at 625 V/cm (17 microA). With a 36 cm L(eff) column complete separation of the commercially available and synthesized standards (all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate) was obtained within 10 min. The within-day and between-day variations of retention times of all-trans-retinyl palmitate were <0.3% relative standard deviation (RSD) (n=3) and <2% RSD (n=6), respectively. The within-day and between-day variations of peak areas were both <2% (both n=3). The columns were used for more than 1 month without degradation. Liver extracts from arctic seal were analyzed.

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection.

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (microLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 microl injection volumes with on-column focusing. A 350x0.32 mm capillary column packed with Kromasil 100-C18 of 5 microm particle size was used with a mobile phase of acetonitrile-10 mM ammonium acetate (85:15, v/v) at a flow of 5 microl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90 degrees C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile-water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 microm poly(vinylidene difluoride) syringe filter before microLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 microg/kg. The within-day and between-day repeatabilities were in the range 4-18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.

Temperature effects on packed-capillary liquid chromatography of the X-ray contrast agent iodixanol.

The effect of varying the operating temperature from 6 to 90 degrees C on the chromatographic performance of the exo-exo and exo-endo isomers of the X-ray contrast agent lodixanol in packed-capillary reversed-phase liquid chromatography shows increasing interconversion rates between the two isomeric conformers with increasing temperature. At 90 degrees C, Iodixanol elutes as one sharp peak due to an increased interconversion rate between the two isomeric conformers. Consequently, increased sensitivity is achieved. Temperature programming from 6 to 40 degrees C is utilized to optimize the resolution and determination of the exo-exo and exo-endo isomers. Temperature programming provides a significant decrease in the retention times in comparison with the isothermal separations while still preserving baseline separation of the isomers.

Purity testing of organometallic catalysts by packed capillary supercritical fluid chromatography.

A packed capillary column supercritical fluid chromatography system with flame ionization detection has been used for purity testing of candidates for homogeneous catalysis such as methyl tricarbonyl pentamethylcyclopentadienyl tungsten [Cp*W(CO)3Me], methyl tricarbonyl cyclopentadienyl tungsten [CpW(CO)3Me], tetramethyl pentamethylcyclopentadienyl iridium (Cp*IrMe4), trimethyl (1,4,7-trimethyl-1,4,7-triazocyclononane) rhodium (CnRhMe3), trimethylphosphine hydride dicarbonyl cyclopentadienyl molybdenum [eta5-CpMoH(CO)2PMe3] and triphenylphosphine hydride dicarbonyl cyclopentadienyl molybdenum [eta5-CpMoH(CO)2PPh3]. A mass limit of detection of 240 pg was found for eta5-CpMoH(CO)2PMe3 when using a 60-nl injection volume and pure CO2 as mobile phase on a 5 microm Kromasil C18 column. The stability of the catalysts in solution has been examined. After 24 h more than 70% of eta5-CpMoH(CO)2PMe3 and 50% of eta5-CpMoH(CO)2PPh3 had decomposed. Due to the instability of the compounds the purity testing had to take place rapidly after sample dissolution.

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection.

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C8 non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of +/-1% acetonitrile and +/-0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050-5.0 micromol/l with fluorescence detection and 0.12-5.0 micromol/l with ultraviolet detection. The limits of quantitation were 0.025 micromol/l for citalopram and paroxetine, 0.050 micromol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 micromol/l for the paroxetine metabolites by fluorescence detection, and 0.10 micromol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4-10.6% and 3.1-20.3%, respectively. Recoveries were in the 63-114% range for citalopram, fluoxetine and paroxetine, and in the 38-95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.

Nonaqueous electrochromatography on C30 columns: separation of retinyl esters.

A nonaqueous packed capillary electrochromatographic reversed-phase method for separation of retinyl esters has been developed. The retinyl esters all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate were separated on a 180 microm ID column packed with 5 microm C30 particles with a mobile phase consisting of 2.5 mM lithium acetate in N,N-dimethylformamide-methanol (99:1, v/v). With this mobile phase, the electroosmotic flow was 0.8 mm/s at 650 V/cm and 40 degrees C, and the separation was completed in less than 30 min on 30 cm columns. To obtain electrostable frits of the hydrophobic C30 material both the preparation of the frits and the conditioning of the column prior to electroconditioning were of importance. Selectivity changes were introduced by replacing up to 70% v/v of the N,N-dimethylformamide by acetonitrile. The increase in the amount of acetonitrile was, however, accompanied by a significant increase in analysis time. Liver extracts obtained from arctic seal were analyzed.

Capillary gas chromatography coupled with microplasma mass spectrometry for organotin speciation.

Gas chromatography was coupled with microplasma mass spectrometry for selective detection of organotin compounds. The microplasma ion source was a capacitively coupled radiofrequency helium plasma, which was located inside the high vacuum area of the mass spectrometer. Only 1-3 ml min-1 of helium carrier gas from the gas chromatograph was necessary for sustaining the plasma while 0.15-1.5 ml min-1 of hydrogen was added as reagent gas. Hydrogen was applied for prevention of carbon deposition and served to minimize the interactions between tin and the fused-silica inner surface of the microplasma ion source. Both carbon and tin were detected as positively charged atomic ions, which were expelled from the microplasma ion source and directly focused by electrostatic lenses towards the quadrupole mass analyzer. Tin exhibited high selectivity to carbon (> 10(4)) and a detection limit of 3.5 pg s-1.

Determination of low levels of an antioxidant in polyolefins by large-volume injection temperature-programmed packed capillary liquid chromatography.

Sub-ambient column temperatures, promoting strong interactions between the analyte and the stationary phase material, were utilized to focus large volumes of the polyolefin antioxidant Irganox 1076 [benzenepropanoic acid, 3.5-bis(1,1-dimethylethyl)-4-hydroxy-, octadecyl ester] on the column inlet, using pure acetonitrile as sample solvent and mobile phase. Injection volumes up to 100 microl were successfully employed on a 50 cm x 320 microm I.D. capillary column packed with 5 microm Kromasil 100 ODS particles. Irganox 1076 was eluted after completed injection by temperature programming, using a temperature program from 7 to 90 degrees C, in 3 degrees C min(-1). UV detection, using a low-dispersion "U"-shaped flowcell, was performed at 280 nm. The method was applied for the determination of Irganox 1076 that was extracted from low-density polyethylene (0.6 ppm, w/w). Both Soxhlet and microwave-aided solvent extractions were performed, using chloroform and acetonitrile as solvents, respectively. The microwave-aided extraction with acetonitrile was found to give approximately the same yield as the standard Soxhlet reference method. Consequently, small volumes of acetonitrile could be used both as extraction solvent, sample solvent and mobile phase, simplifying the analysis process. The mass limit of detection of the method was found to be 3.3 ng, corresponding to a concentration limit of detection of 33 ng ml(-1), utilizing an injection volume of 100 microl. The within and between day precision of retention times displayed relative standard deviations below 1.2%.

Characterization of metabolites of benz(j)aceanthrylene in faeces, urine and bile from rat.

1. The excretion of benz[j]aceanthrylene (B[j]A) and the biotransformation products found in faeces, urine and bile of rat exposed to [3H]-labelled B[j]A have been studied. 2. About 95% of the administered radioactivity was excreted within 7 days, 79% via faeces and 16% via urine, and most of the radioactivity in urine and faeces was excreted within 2 days. 3. The B[j]A metabolites excreted between days 1 and 2, including those excreted in bile during the first 5.5 h in a separate experiment, were further characterized by HPLC, UV and electrospray/atmospheric pressure chemical ionization mass spectrometry. 4. In faeces, bile and urine, hydroxylated B[j]A metabolites predominated. The major metabolites in faeces were B[j]A-1,2-dihydrodiol-8-hydroxy and B[j]A-1,2-dihydrodiol-10-hydroxy. These metabolites were found as conjugated metabolites in the bile. The glucuronide conjugate of B[j]A-1,2-dihydrodiol-10-hydroxy was also a major metabolite in urine. Two sulphate conjugates of oxidized B[j]A were detected in bile, a sulphate conjugate of a B[j]A-dihydrodiol-phenol and B[j]A-1,2-dihydrodiol-10-sulphate. Trans-B[j]A-1,2-dihydrodiol was detected in urine, faeces and bile. 5. These findings support the hypothesis that epoxidation at the cyclopenta ring is an important biotransformation pathway for B[j]A in vivo. In addition to the characterized metabolites, a large fraction of polar compounds, possibly glutathione conjugates, was also excreted in urine and bile.

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection.

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.