Liquid chromatographic determination of polycyclic aromatic hydrocarbons in fish and shellfish

A simple and accurate analytical method for determination of polycyclic aromatic hydrocarbons (PAHs) in fish and shellfish is presented, which is considered to be useful for routine analyses and for screening purposes. The procedure involves alkaline digestion, extraction with n-hexane, silica gel column chromatography, and liquid chromatographic (LC) determination with fluorometric detection. During development of the analytical method for determination of PAHs, it was found that benzo[a]pyrene, a representative PAH, was decomposed easily by the analytical procedure, and this tendency was investigated for the experimental conditions used. Benzo[a]pyrene was decomposed by the coexistence of alkaline conditions, light, and oxygen; by peroxides in aged ethyl ether; and by oxygen when absorbed on silica gel. Thus, to obtain good recoveries and precise analytical results, these decomposition conditions must be avoided. The following precautions are recommended: protection from light through all analytical steps; addition of Na2S to alkaline digestion mixture as an antioxidant; complete removal of peroxides from ethyl ether just before use; quick column chromatography on silica gel; and prevention of air from contact with adsorbent. When this simple method was applied to fish and shellfish samples, very good recoveries of PAHs from fortified fish samples were obtained, and no serious interferences were observed in fish and shellfish extracts.     

Determination of dioxins and furans in foods and biological tissues: review and update

Determination of trace residues of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) in various matrixes is carried out by a limited number of laboratories in the United States, Canada, and other countries. Current methods for analysis of foods and biological tissues include a combination of preparation, extraction, cleanup, isolation, determination, and identity confirmation procedures. Soxhlet, liquid/liquid, solid-phase, and column extraction procedures are used as well as treatment with acid or base before solvent extraction. Cleanup and isolation steps include sulfuric acid partitioning; adsorption chromatography on Florisil, silica gel, or alumina; gel permeation chromatography; multi-stage column chromatography on sulfuric acid silica and alkali silica; carbon column chromatography; and liquid chromatography fractionation with size exclusion, normal-phase, and reverse-phase columns. Activated carbon and multistage chromatographic columns are widely used in cleanup schemes. Isomer-specific identification and quantitation of PCDD and PCDF congeners at parts-per-trillion levels or lower are carried out by high resolution (capillary) gas chromatography (HRGC) and multiple ion detection mass spectrometry. In addition to chemical methods, bioassay procedures have been recommended (e.g., use of monoclonal antibodies, for immunoassay determination of PCDDs and PCDFs).     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Extraction, gas-liquid chromatographic detection, and quantitation of hexachlorophene residues from plant tissues high in lipid content

A method has been developed for the extraction, cleanup, derivatization, detection, and quantitation of hexachlorophene (HCP) residues from 2 types of plant storage tissue high in lipid content. Wet soybean or peanut tissue was homogenized and extracted with ethyl ether and chromatographed on silica gel to remove the neutral lipids. The cleaned up sample was methylated with diazomethane and the dimethoxyhexachlorophene was eluted from a second silical gel column and chromatographed on a 6' glass column packed with 3% OV-1 or 3% SE-30 on Gas-Chrom Q. The instrument detection limit for the 63Ni electron capture detector was less than 0.1 ng for dimethoxyhexachlorophene and about 1 ppb HCP residue in plant issue. Recovery of 10-420 ppb HCP added to tissue averaged 90.9 +/- 5.7%. Interfering substances were removed, column life was increased, peak sharpness was increased, and tailing of the parent compound was decreased by using appropriate column chromatography.     

Determination of volatile N-nitrosamines in frankfurters containing minced fish and surimi

A method was developed to quantitatively measure volatile N-nitrosamines, particularly N-nitrosodimethylamine (NDMA), in cured minced fish or surimi-meat frankfurters. This method is free from artifact formation. First, 2 dry solid-phase extraction columns are prepared. Solvent is passed through the top column containing the fish-meat into a second column containing acid Celite. The eluate from the Celite column is then passed through a third column containing silica gel. Nitrosamines are eluted from the acid Celite column and then from the silica gel column into the same receiver. Recovery of the internal standard, N-nitrosoazetidine, added at the 10 ppb level, was 86.5%. In addition, a few samples of nitrite-treated salmon (lox) were also tested for N-nitrosamines. The results show that the method is applicable to samples containing nitrite-treated fish and fish-derived products.     

High pressure liquid chromatographic determination of aflatoxins in corn.

A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.     

Polycyclic aromatic hydrocarbon profile analysis of high-protein foods, oils, and fats by gas chromatography.

A method is described for the determination of polycyclic aromatic hydrocarbons (PAHs) with 3-7 rings in (I) meat, poultry, fish, and yeast; and (II) oils and fats. The extraction of PAHs from group I is incomplete, and, therefore, group I samples must be dissolved homogeneously by saponification in 2N methanolic potassium hydroxide. The PAHs are concentrated by liquid-liquid extraction (methanol-water-cyclohexane, N,N - dimethylformamide - water-cyclohexane) and by column chromatography on Sephadex LH 20. The PAHs are separated by high-performance gas-liquid chromatography (GLC) with columns containing 5% OV-101 on Gas-Chrom Q and estimated by integration of the flame ionization detector signals in relation to an internal standard (3,6-dimethylphenanthrene and/or benzo(b)chrysene). The sensitivity is significantly higher than that obtained with ultraviolet spectroscopic methods. The reproducibility and margin of error were tested with meat samples fortified with 11 PAHs and with samples of sunflower oil. The method was further applied to meat, smoked fish, yeast, and unrefined sunflower oil. All samples investigated contained more than 100 PAHs (characterized by mass spectrometry) of which only the main components were determined: phenanthrene, anthracene, fluorene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene + benzo (j)fluoranthene + benzo(k) fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, dibenz(a,j)anthracene, dibenz(a,h)anthracene + indeno(1,2,3,-cd)pyrene, benzo(ghi)perylene, anthanthrene, and coronene. In contrast to other methods, the GLC profile analysis allows the recording of known and unknown PAH peaks simultaneously and also allows a compilation of all PAHs.     

High-pressure liquid chromatography of benzo(a) pyrene and benzo (ghi) perylene in oil-contaminated shellfish.

A high-pressure liquid chromatographic procedure is described for the determination of benzo(a) pyrene and benzo(ghi) perylene. These polynuclear aromatics are extracted with acetonitrile and partitioned into petroleum ether, the petroleum ether is removed, and the residue is saponified. The compounds are purified and isolated by passing the residue through a silica gel column and a high-pressure liquid chromatographic column, and detected by their ultraviolet absorption. Recoveries of standards through the procedure averaged 104%.     

Rapid, semimicro method for determination of polycyclic aromatic hydrocarbons in shellfish by automated gel permeation/liquid chromatography

A simple, rapid, easily automated method is described for the determination of polycyclic aromatic hydrocarbons (PAHs) in shellfish such as American lobster (Homarus americanus) and blue mussel (Mytilus edulis). PAHs are extracted from small amounts (1-8 g) of tissue by saponification in 1N ethanolic potassium hydroxide followed by partitioning into 2,2,4-trimethylpentane. This solution is evaporated just to dryness by rotary evaporation and the residue is dissolved in cyclohexane-dichloromethane (1 + 1) for gel permeation chromatography (GPC) on Bio-Beads SX-3. The GPC procedure is ideal as a screening method in the range 25-18 000 ng PAHs/g tissue. If individual PAH measurements are required, the appropriate GPC fraction is collected and PAHs are separated by reverse phase liquid chromatography (LC) with fluorometric detection. Individual PAHs at concentrations as low as 0.25-10 ng/g can be determined. Recoveries of added fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene were quantitative, with relative standard deviations ranging from 0.0 to 16.9%.     

Gas chromatographic profile analysis of basic nitrogen-containing aromatic compounds (azaarenes) in high protein foods

A method is described for the determination of basic nitrogen-containing polycyclic aromatic compounds (N-PACs, azaarenes) in meat. The enrichment procedure includes liquid-liquid partition (dimethylformamide-water-cyclohexane), extraction of N-PACs by sulfuric acid, reextraction after neutralization by cyclohexane or, alternatively, by nonadsorbing ion exchange chromatography. Further purification is performed by column chromatography on Sephadex LH 20 using a closed system to avoid sample contamination by laboratory pollutants. N-PACs are analyzed by capillary gas chromatography and measured by comparing to the corresponding peak areas of an internal standard (e.g., 10-azabenzo(a)pyrene). The limit of detection of this method ranges from 0.1 to 0.4 ng for benzacridines, dibenzacridines, and their methyl derivatives. The results of a collaborative study, stimulated by IUPAC, are reported: Coefficients of variation for the various azaarenes were 4.0-13.6% for the check analysis and 10.4-25.4% for a spiked ham sample. Consequently, IUPAC suggests this procedure as a recommended method.     

Improved cleanup for liquid chromatographic analysis and fluorescence detection of aflatoxins M1 and M2 in fluid milk products

A rapid method is described for extraction and cleanup of raw and processed milk for determination of aflatoxins M1 and M2 by using a C18 Sep-Pak/silica gel cleanup column combination. Aflatoxins are separated by normal phase liquid chromatography and their concentrations are determined by fluorescence detection in a silica gel-packed flow cell. Recoveries ranged from 99 to 103% with coefficients of variation less than 2% for M1 levels of 0.117-1.17 ng/mL added to raw milk. Similar recoveries were obtained for M2. The coefficient of variation for analysis of 5 subsamples of naturally contaminated milk was less than 1%. Agreement with the official method is satisfactory. Each sample requires less than 25 mL solvent and 10 min actual handling time. Sample chromatograms show no interferences in the M1-M2 elution region and no late-eluting peaks, which permits spacing injections at 13-20 min intervals. Aflatoxin levels as low as 0.03 ppb may be determined by this procedure. Extracts have also been analyzed by thin layer chromatography.     

Determination of aloesin and aloeresin A for the detection of aloe in beverages

This work describes a sensitive high-performance liquid chromatography (HPLC) method for the quantification of aloesin and aloeresin A in alcoholic beverages containing aloe as a flavoring agent. The compounds were prepared from Aloe ferox juice. Sephadex LH20 and ion-exchange resin AG1X2 column chromatography were used for aloesin. Aloeresin A was obtained by Sephadex LH20 and silica gel column chromatography followed by purification on Discovery DSC-18 solid-phase extraction tubes. A 98 mg amount of aloesin (>99% purity) and 34 mg of aloeresin A (>98% purity) were recovered from 2.5 g of aloe juice. The HPLC method was validated, and intra- and interday performances were established. In-house validation was carried out by analyzing samples of beverages with and without aloe as a flavoring agent.     

Determination of monocyclic and polycyclic aromatic hydrocarbons in fish tissue

An analytical method is presented in which fish tissue is analyzed for neutral monocyclic and polycyclic aromatic hydrocarbons (AHs) and aromatic sulfur heterocycles (ASHs) by capillary column gas chromatography (CGC) with photoionization detection. The sample enrichment procedure includes saponification with aqueous KOH, acidification of the digestates, and extraction of the aromatic compounds into cyclopentane-dichloromethane. Adsorption chromatography on tandem segments of potassium silicate and silica gel removes 99% of the coextracted lipid. Final enrichment by gel permeation chromatography eliminates residual biogenic material and potentially interfering alkanes. Relatively volatile monoaromatics are included among the analytes by virtue of the efficiency of the complementary enrichment steps, the use of small quantities of only low-boiling solvents, and the selectivity of the detector. Most targeted compounds (AHs ranging in size from C3-alkylbenzenes through benzo[g,h,l]perylene and ASHs within the same size range) can be determined in 5 g (wet weight) samples of fish tissue at concentrations as low as 20 ng/g. Comparisons are made of recoveries of selected AHs under ordinary and gold fluorescent lighting conditions.     

Improved separation method for highly purified lutein from Chlorella powder using jet mill and flash column chromatography on silica gel

We investigated an improved method for the separation of high-purified lutein from a commercially available spray-dried Chlorella powder (CP) using fine grinding by jet mill and flash column chromatography on a silica gel. Saponification and extraction of lutein were enhanced 2.3-2.9-fold in jet mill-treated CP (mean particle size, 20 microm) as compared to untreated CP (mean particle size, 67 microm). The carotenoid extract was dissolved in ether-hexane (1:1 v/v) and subjected to flash column chromatography on silica gel. A mixture of alpha- and beta-carotene was eluted with hexane, followed by elution with hexane-acetone-chloroform (7:2:1 v/v). Lutein (dark-orange band) was collected after the elution of an unknown colorless compound (detected based on UV absorbance). The purity of lutein in this fraction was over 99%, and the yield was 60%. The present study provides key information for obtaining highly purified lutein using flash column chromatography on a silica gel.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

长江三角洲地区污泥中多环芳烃的污染研究

To ascertain the contaminated conditions of polycyclic aromatic hydrocarbons （PAHs） in sludge and to evaluate the risk of application of this sludge for agricultural purposes, 44 sludge samples obtained from 15 cities in the Yangtze River Delta area of China were investigated using capillary gas chromatography/mass spectrometry （GC/MS） after ultrasonic extraction and silica gel cleanup. PAHs＇ contents ranged from 0.0167 to 15.4860 mg kg^-1 （dry weight, DW） and averaged 1.376 mg kg^-1, with most samples containing 〈 1.5 mg kg^-1. Pyrene （PY）, fluoranthene （FL）, benzo[b]fluoranthene （BbF）, indeno[1,2,3-cd]pyrene （IND）, benzo[a]pyrene （B[a]P）, and benzo[g,h,i]perylene （BghiP） were the most dominant compounds, ranging from 0.1582 to 0.2518 mg kg^-1. Single PAH, such as naphthalene （NAP, 2-benzene rings）, phenanthrene （PA, 3-benzene rings）, PY （4-benzene rings）, and FL （3-benzene rings）, had high detection rates （76.1%-93.5%）. The distribution patterns of PAHs were found to vary with the sludge samples; however, the patterns showed that a few compounds with 2- and 3-benzene rings were commonly found in the samples, whereas those with 4-, 5-, and 6-benzene rings were usually less commonly found. All the 44 sludge samples were within the B[a]P concentration limit for sludge applied to agricultural land in China （〈 3.0 rag kg^-1）. The probable sources of PAH contamination in the sludge samples were petroleum, petroleum products, and combustion of liquid and solid fuel. The concentrations and distributions of the 16 PAHs in sludge were related to sludge type, source, and treatment technology, together with the physicochemical properties.     

Rapid magnetic solid-phase extraction based on magnetic multiwalled carbon nanotubes for the determination of polycyclic aromatic hydrocarbons in edible oils

In this study, magnetic multiwalled carbon nanotubes were fabricated by a simple method and applied to magnetic solid-phase extraction (MSPE) of eight heavy molecular weight polycyclic aromatic hydrocarbons (PAHs) including chrysene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene and benzo[g,h,i]perylene from edible oil samples. Several parameters affecting the extraction efficiency were investigated, including the type and volume of desorption solvent, extraction and desorption time, washing solution and the amount of sorbent. Under the optimized conditions, a simple and effective method for the determination of PAHs in edible oils was developed by coupling with gas chromatography-mass spectrometry (GC-MS). The whole pretreatment process was rapid, and it can be accomplished within 10 min. The limits of quantitation for the target PAHs were found to be 0.34-2.9 ng/g. The recoveries in oil sample were in the range 87.8-122.3% with the RSDs less than 6.8% (intraday) and 9.6% (interday). This method was successfully applied to the analysis of PAHs in seven kinds of edible oils from local markets.     

High pressure liquid chromatographic determination of polynuclear aromatic hydrocarbons in oysters.

A high pressure liquid chromatographic (HPLC) procedure is described for determining 13 polynuclear aromatic hydrocarbon (PNA) compounds in oysters at the 2 ppb level. These compounds are extracted from shellfish with acetonitrile and partitioned into petroleum ether; the petroleum ether is removed and the residue is saponified. The aromatic compounds are isolated by passing the saponifeid residue through silica gel and further purified and fractionated by muStyragel gel permeation chromatography. The in-ividual PNAs are then quantitatively determined by using a reverse phase HPLC column coupled to fluorescence, spectrophotometric, and 254 nm absorbance detectors in series. Recoveries from spiked samples generally were greater than 80%.     

Gas chromatographic determination of mecarbam and its metabolites mecarboxon, diethoate, and diethoxon in cottonseeds

A gas chromatographic method is described for determining residues of mecarbam and 3 of its metabolites, mecarboxon, diethoate, and diethoxon, in cottonseeds. For mecarbam analysis, following Soxhlet extraction with chloroform (after blending), the oily extract is partitioned with propylene carbonate and cleaned up on a silica gel column. Metabolites are extracted by the same method, followed by cleanup of mecarboxon on a silica gel column or diethoxon on an alumina column; cleanup of diethoate can be performed on either column. All 4 compounds are determined using a flame photometric detector equipped with a phosphorus filter. Average recoveries for cottonseed samples fortified with 0.03-1.0 ppm mecarbam ranged from 80 to 88%. Average recoveries were 81-88% for mecarboxon and 90-92% for diethoate (alumina column) and diethoxon from samples fortified with 0.05-1.0 ppm. Average recovery of diethoate from samples cleaned up on the silica gel column were 84-88% in the range of 0.05-0.2 ppm. Values obtained for mecarbam residues in field-treated samples are also presented.     

Determination of seven glycidyl esters in edible oils by gel permeation chromatography extraction and liquid chromatography coupled to mass spectrometry detection

A method based on a gel permeation chromatography (GPC) extraction procedure combined with an additional cleanup by solid-phase extraction (SPE) on silica gel and liquid chromatography-mass spectrometry (LC-MS) detection has been validated for the analysis of seven glycidyl esters (GEs) including glycidyl laurate, myristate, palmitate, stearate, oleate, linoleate, and linolenate in various edible oils. This method was conjointly developed and validated by two different laboratories, using two different detection systems, a LC time of flight MS (LC-ToF-MS) and a LC triple-quadrupole MS (LC-MS/MS). The extraction procedure allowed targeting low contamination levels due to a highly efficient matrix removal from the 400 mg oil sample loaded on the GPC column and is suitable for routine analysis as 24 samples can be extracted in an automated and reproducible way every 12 h. GPC extraction combined with SPE cleanup and LC-MS/MS detection leads to a limit of quantification in oil samples between 50 and 100 μg/kg depending on the type of glycidyl ester. Recoveries ranged from 68 to 111% (average = 93%). Quantification was performed by automated standard addition on extracts to compensate matrix effects artifacts. To control recoveries of each sample four isotopically labeled GEs ((13)C(3) or (13)C(4)) were included in the method.