Validation of the analytical procedure for the determination of polyaromatic hydrocarbons in smoke flavourings using high performance liquid chromatography coupled to an ultraviolet,diode array or fluorescence detector

High performance liquid chromatography (HPLC) coupled to an ultraviolet (UV), diode array or fluorescence detector (UV/DAD/FLD) has been used to set up an analytical procedure for the quantification of 16 EU priority polyaromatic hydrocarbons (PAHs) in smoke flavourings. The following parameters have been determined for the 16 EU priority PAHs: limit of detection, limit of quantification, precision (repeatability and intermediate precision), recovery and measurement uncertainty, using the concept of accuracy profiles. They were in close agreement with quality criteria described in the Commission Regulation (EC) no. 627/2006 concerning PAHs in smoke flavourings. Presented at the AOAC Europe Workshop, November 2006, Limassol, Cyprus     

Simultaneous determination of NSO-heterocycles, homocycles and their metabolites in groundwater of tar oil contaminated sites using LC with diode array UV and fluorescence detection

For monitoring groundwater at tar oil contaminated sites a simple method of analysis was developed for the simultaneous detection of several NSO heterocyclic compounds, homocyclic compounds, mobile two- and three-cyclic PAHs and selected metabolites. The groundwater samples are enriched using SPE with polymer material at pH 4. Chromatographic separation and detection is performed by LC with diode array UV or fluorescence detection. The recoveries of 25 selected compounds were mostly between 80-110% and the detection limits were 0.4-2.4 microg/L for UV detection and for the fluorescence detectable compounds 0.4-140 ng/L. The method was successfully applied to groundwater samples from a wood preserving facility. Especially benzo(b)thiophene showed an increasing dominance downgradient of the source. Detection of metabolites, such as 1-hydroxyiso-, 2-hydroxyquinoline and 2-hydroxy-4-methylquinoline, 2-naphthoic acid, and 1-indanone, indicating in situ biodegradation, was confirmed by LC-ESI-MS analysis.     

The Combination of Laser-Induced Fluorescence and Intensified Linear Diode Array Detection in Column Liquid Chromatography

Abstract

The frequency-doubled 514-nm argon-ion laser line providing excitation at 257 nm is combined with an intensified linear diode array (ILDA) detector to enable on-the-fly identification by laser-induced fluorescence (LIF) in conventional-size column liquid chromatography (LC). The potential of this detection system is demonstrated for the analysis of a standard mixture of polynuclear aromatic hydrocarbons (PAHs). Detection limits are at the 1.5 μg 1^?1 level (15 pg injected); the identitication limits are about one order of magnitude higher.     

Selective immunoclean-up followed by liquid chromatography for the monitoring of a biomarker of exposure to polycyclic aromatic hydrocarbons in urine at the ng l-1 level

A selective clean-up procedure using an immunosorbent (IS) was developed for the trace-level determination, in water and urine samples, of 3-benzo(a)pyrene-glucuronide (3-BP-G), a biomarker of exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). First, three sorbents used for the immobilization of antibodies were evaluated for their ability to limit the risk of non-specific interactions and to provide a high bonding density. The best sorbent, i.e. sepharose, was used for the immobilization of two different monoclonal antibodies. The most specific antibody for 3-BP-G was applied to the selective extraction from urine providing a clean extract, an easy and reliable quantification by comparison with a classical SPE process. The sensitivity of the fluorescence associated with the selectivity of the IS provides a limit of detection up to 1.2 ng l(-1) in urine for 3-BP-G.     

Critical study of and improvements in chromatographic methods for the analysis of type B trichothecenes

Various analytical methods used in the analysis of type B trichothecenes (deoxynivalenol, nivalenol, 3- and 15-acetyldeoxynivalenol) in cereals were compared and optimised in this work. These methods use either GC-electron-capture detection (ECD) of trimethylsilyl, trifluoroacetyl and heptafluorobutyryl derivatives or HPLC with UV or photodiode array detection of analytes. A new HPLC procedure using fluorescence detection prior derivatisation with coumarin-3-carbonyl chloride has been also tested. Five extraction solvents and two solid-phase extraction cartridges (silica, Florisil) plus a especial clean-up column (MycoSep 225) were compared in order to obtain the best recovery of the mycotoxins with minimal presence of coextractives in the chromatograms. The chosen extraction solvent was a mixture of acetonitrile-water (84:16, v/v). The MycoSep 225 column was chosen as the best alternative for clean-up of grain samples. For GC-ECD analysis, derivatisation of analytes with heptafluorobutyric anhydride prior the final determination was chosen as the most suitable procedure. HPLC-photodiode array (at 221 nm) analysis was more suitable for determination of type B trichothecenes than HPLC of the fluorescent coumarin-3-carbonyl derivatives. Recoveries obtained in spiked corn, rice and wheat are reported. The utility of the proposed methodology was assayed in cereal cultures of various Fusarium strains.     

Purity assessment of crystalline zearalenone

Commercially available solid zearalenone (ZON) to be used as a certified liquid calibrant (BCR-699) in a project funded by the European Commission within the Standard Measurement and Testing program was characterized and its purity determined. The degree of purity of the ZON was examined by UV spectrophotometer, liquid chromatography (LC) with diode array and fluorescence detection, 1H and 13C-NMR spectrometry, LC-mass spectrometry (LC/MS/MS), ion chromatography (IC), and differential scanning calorimetry (DSC). The diagrams obtained from DSC analysis and the UV spectrum showed no detectable impurities. Likewise, no impurities were observed by LC analysis with both diode array and fluorescence detection. IC determination revealed negligible contamination of ZON with chloride of 0.020 +/- 0.005% and nitrate of 0.016 +/- 0.006%. Zearalanone (ZAN) was identified as one of 2 minor (0.2%) impurities by LC/MS/MS. The 1H-NMR measurements revealed an additional peak, which has not been previously reported in the literature. It could be identified as part of the ZON spectrum as the signal arising from the phenolic proton attached to C4'. The manufacturer states an additional contamination with 0.2% methylene chloride, which could be confirmed to an extent of 0.1% by 1H-NMR. Minor impurities, whose structures remain unknown, were discovered at 3.5 and < 1 ppm. Total percentage of impurities based on NMR measurement was estimated not to exceed 1%. A purity of 99.5% with a tolerance of +/- 0.5% was finally attributed to the ZON studied in this project.     

Solid-Phase Extraction,Clean-pp and Liquid Chromatography for Routine Multiresidue Analysis of Neutral and Acidic pesticides In Natural Waters in One Run

Abstract

Solid-phase extraction using C₁₈ silica cartridges, liquid chromatography analysis and UV diode array detection were investigated for the routine trace-level determination of neutral pesticides over a wide range of polarity. Detection limits below the 0.1 μg/1 range were easily obtained in drinking water. If neutral and acidic pesticides over a wide range of polarity have to be determined in the same run, samples have to be acidified to obtain good recoveries of extraction. The effect of the sample matrix was studied and detection limits in the 0.1 μg/1 range were obtained in drinking water except for the more polar ones which are in the interfering peak of humic and fulvic acids. For surface water, a clean-up step using a Florisil cartridge has to be included in the procedure that allows detection limits in the range 0.05–0.3 μg/1.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Determination of polycyclic aromatic hydrocarbons in sediment by liquid chromatography-atmospheric pressure photoionization-mass spectrometry.

We describe a method for the simultaneous determination of 12 kinds of polycyclic aromatic hydrocarbons (PAHs) in sediment based on liquid chromatography-atmospheric pressure photoionization-mass spectrometry (LC/APPI/MS). The method consists of PAH extractions by ultrasonics, clean-up by a solid-phase extraction procedure and determination by LC/APPI/MS. The limits of the determination for PAHs in sediment using the proposed method ranged from 0.06 to 0.9 mg/kg. PAHs were detected by this method in sediment samples on the mg/kg level.     

Improving analysis of apolar organic compounds by the use of a capillary titania-based column: Application to the direct determination of faecal sterols cholesterol and coprostanol in wastewater samples

This article reports a new procedure for the direct determination of faecal sterols coprostanol and cholesterol in wastewater samples as tracers of human sewage contamination. The method combines in-tube solid-phase microextraction (IT-SPME) for analyte enrichment and capillary liquid chromatography (LC) for separation with diode array detection for identification and quantification. A titania-based polymeric capillary column and a conventional octadecyl silica (ODS) capillary column were evaluated and compared for their ability to separate the analytes. The titania-based column allowed the separation of the analytes in much shorter chromatographic times and with better chromatographic profiles, which in turn resulted in better detectability. In addition, IT-SPME allowed the direct injection into the chromatographic system of sample volumes as large as 200 μL, thus making unnecessary off-line clean-up and concentration steps. In such a way, the tested compounds could be directly analysed in less than 10 min, the limits of detection (LODs) being 10 and 1.2 μg/L for coprostanol and cholesterol, respectively. The reliability of the proposed method was tested by processing several wastewater samples.     

Determination of the anthraquinones aloe-emodin and aloin-A by liquid chromatography with mass spectrometric and diode array detection

Methods using liquid chromatography/mass spectrometry (LC/MS) and LC with diode array detection (DAD) in the UV range (LC/UV) were developed for the determination of low levels of the anthraquinones aloe-emodin and aloin-A (barbaloin) in aloe-based products. The methods were used to analyze several commercial products (liquids, semisolids, and solids) for the 2 anthraquinones. The wavelengths used for quantification of aloin-A, aloe-emodin, and emodin (internal standard) by DAD were 357, 257, and 289 nm, respectively. The on-column sensitivities were 0.25 and 0.05 ng by LC/UV and 0.01 and 0.025 ng by LC/MS for aloin-A and aloe-emodin, respectively. The methods are simple and sensitive and provide reproducible results; therefore, they are suitable for the determination of these anthraquinones in various aloe-based products.     

New micromethod combining miniaturized matrix solid-phase dispersion and in-tube in-valve solid-phase microextraction for estimating polycyclic aromatic hydrocarbons in bivalves

Miniaturized matrix solid-phase dispersion (MSPD) was developed for the extraction of common polycyclic aromatic hydrocarbons (PAHs) from bivalve samples (100mg, dry weight). Additional clean-up and analyte enrichment was accomplished by in-tube solid-phase microextraction (SPME). For this purpose the extracts collected after MSPD were diluted with water and injected into a capillary column coated with the extractive phase. This capillary column was connected to the analytical column by means of a switching valve. Separation and quantification of the PAHs were carried out using a monolithic LC column and fluorescence detection. Since the in-tube SPME device allowed the processing of large volumes of the extracts (2.0 mL) excellent sensitivity was achieved, thus making solvent evaporation operations unnecessary. The overall recoveries ranged from 10% to 28% for the studied compounds. The relative standard deviation (RSD) ranged from 2% to 10% for intra-day variation (n=3), and the limits of detection (LODs) were < or =0.6 ng/g (dry weight). The proposed procedure was very simple and rapid (total analysis time was approximately 20 min), and the consumption of organic solvents and extractive phases was drastically reduced. The reliability of the proposed MSPD/in-tube SPME method was tested by analysing several bivalves (mussels and tellins) as well as a standard reference material (SRM).     

High-performance liquid chromatographic strategies for the determination and confirmation of anticoagulant rodenticide residues in animal tissues

A comprehensive approach to the analysis of anticoagulant rodenticide residues in animal tissues based on high-performance liquid chromatography (HPLC) has been developed. Residues of warfarin, coumatetralyl, difenacoum, brodifacoum, bromadiolone, diphacinone and chlorophacinone were extracted with chloroformacetone (1:1, v/v). Extracts were cleaned-up by an integrated gel permeation and adsorption chromatographic procedure which divided the rodenticides into two groups. Residues were then determined and confirmed using normal-phase, ion-pair and weak ion-exchange HPLC techniques. Ion-pair gradient separation resolved all seven rodenticides in a single chromatographic analysis. UV detection methods were employed for all seven rodenticides. Use of a diode array detection system permitted additional confirmation of residues down to 0.1 mg kg-1 by matching UV spectra and derivatives of spectra. Sensitive fluorescence detection was possible for the coumarin-based rodenticides but not for diphacinone and chlorophacinone. Post-column pH-switching fluorescence detection methods were shown to be superior to other methods of fluorescence detection of coumarin-based rodenticides. Recoveries from spiked liver tissue were around 90% at levels from 0.05 to 1 mg kg-1. Detection limits of around 0.002 mg kg-1 for most rodenticides and of 0.01 mg kg-1 for warfarin could be achieved with animal tissue extracts.     

Evaluation of matrix solid‐phase dispersion extraction for the determination of polycyclic aromatic hydrocarbons in household dust with the aid of experimental design and response surface methodology

A simple, fast, and inexpensive procedure for sample preparation based on matrix solid‐phase dispersion was developed for the determination of Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons in indoor dust samples. Parameters that affect the extraction efficiency such as type of dispersant, elution solvent, and solvent volume were evaluated and optimized with the aid of experimental design and response surface methodology. Analysis was performed by HPLC coupled with UV‐Vis diode array detector (UV‐DAD). For verification, a GC coupled with a mass spectrometer in SIM mode was also applied. Recoveries obtained were from 53 to 120% for all target analytes with detection limits ranging from 0.2 to 10 ng/g and 0.2 to 2 ng/g for LC‐UV‐DAD and GC‐MS, respectively. The optimized method was used for the analysis of 11 household dust samples collected from private houses.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal. Received: 23 July 1999 / Revised: 9 September 1999 / Accepted: 16 September 1999     

Comparison of LC–UV, LC–ELSD and LC–MS Methods for the Determination of Sesquiterpenoids in Various Species of Artemisia

Simple and specific analytical methods for the quantitative determination of sesquiterpenoids from various species of Artemisia plant samples were developed. By LC–UV, LC–ELSD, the separation was achieved by reversed-phase chromatography on a C18 column with water and acetonitrile both containing 0.025% trifluoroacetic acid as the mobile phase. In the LC–MS system, trifluoroacetic acid was replaced by 0.1% formic acid. The wavelength used for quantification of sesquiterpenoids with a diode array detector was 205 nm. The limits of detection by LC–MS was found to be 5, 10, 25, 50, 50 ng mL^?1. The limits of detection by LC–UV and LC–ELSD were found to be 5.0, 3.0, 100, 100, 7.5 μg mL^?1, by LC–UV and 50, 25, 30, 100 and 75 μg mL^?1 by LC–ELSD. LC–mass spectrometry coupled with electrospray ionization (ESI) interface is described for the identification and quantification of sesquiterpenoids in various plant samples. This method involved the use of the [M + H]⁺ ions of sesquiterpenoids in the positive ion mode with extractive ion monitoring.     

Simultaneous determination of two serine proteinases by hydrophobic interaction chromatography

Summary An LC clean-up procedure based upon a complexation between polycyclic aromatic hydrocarbons (PAHs) and silica with chemically bonded 2,4-dinitroaniline has been combined with GC/MS. The LC pre-separation makes it possible to obtain a relatively clean fraction of PAHs free from alkanes, alkylbenzenes and naphthalenes, PCBs, chlorinated pesticides and many other interfering compounds. This fraction has been analyzed using capillary GC and mass selective detector (MSD). Substantial improvement of the MS spectra of PAHs with three or more fused benzene rings is achieved.     

Capillary electrophoresis with a UV light-emitting diode source for chemical monitoring of native and derivatized fluorescent compounds

We report the utilization of a high power UV light-emitting diode for fluorescence detection (UV-LED-IF) in CE separations. CE-UV-LED-IF allows analysis of a range of environmentally and biologically important compounds, including PAHs and biogenic amines, including neurotransmitters, amino acids, proteins, and peptides, that have been derivatized with UV-excited fluorogenic labels, e.g., o-phthalic dicarboxaldehyde/beta-mercaptoethanol (OPA/beta-ME). The 365 nm UV-LED was used as a stable, low cost source for detection of UV-excited fluorescent compounds. UV-LED-IF was used with both zonal CE separations and MEKC. Native fluorescence detection of PAHs was accomplished with detection limits ranging from 10 nM to 1.3 microM. Detection limits for OPA/beta-ME-labeled glutamic acid and aspartic acid were 11 and 10 nM, respectively, for off-line labeling, and 47 and 47 nM, respectively, for on-line labeling, comparable to UV-laser-based systems. Analysis of OPA/beta-ME-labeled proteins and peptides was performed with 28 and 47 nM detection limits for BSA and myoglobin, respectively.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.