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%.     

Determination of quinomethionate (6-methylquinoline-2,3-diyldithiocarbonate) residues in crops by in situ fluorometry.

A simple method is described for the quantitative determination of quinomethionate (6-methylquinoline- 2,3 - diyldithiocarbonate) in crops. The pesticide residue is extracted with acetonitrile and partitioned in petroleum ether. After separation from the co-extractives by thin layer chromatography (TLC), the fluorescence is measured directly on a silica gel TLC plate. An average of 89% recovery is obtained at the 0.05 ppm level in apples, peaches, pears, and tomatoes.     

Determination of polybrominated biphenyl residues in dairy products.

A method for the determination of polybrominated biphenyls (PBBs) in dairy products is described. Fat is extracted from the products by the official AOAC method. The PBB residues are separated from the fatty material by gel permeation chromatography prior to gas-liquid chromatographic (GLC) quantitation. An additional cleanup using petroleum ether elution through a miniature Florisil column is necessary for thin layer chromatographic (TLC) confirmation. Recoveries of PBBs from samples fortified at levels from 0.1 to 0.5 ppm ranged from 94 to 104% with an average of 99%. GLC sensitivity permits the estimation of PBB residue levels as low as 0.007 ppm. Routine TLC confirmation is limited by sensitivity to greater than or equal to 0.2 ppm.     

Determination of aflatoxicol and aflatoxins B1 and M1 in eggs

Procedures from 2 methods, one for aflatoxins B1 and M1 in eggs and one for aflatoxicol in milk, blood, and liver, have been combined to determine the 3 toxins in eggs. The sample is blended with sodium chloride-saturated water and this mixture is then blended with acetone. After separation from the solid residue, the aqueous acetone extract is defatted with petroleum ether. The toxins are next partitioned into chloroform and separated from interferences on a silica gel column. Aflatoxicol is determined by fluorescence measurement after separation on a C18 reverse phase liquid chromatographic column, and aflatoxins B1 and M1 are determined by fluorescence densitometry after separation on a silica gel thin layer chromatographic plate. In a recovery study with eggs, mean recoveries of aflatoxicol added at levels of 0.1, 0.05, and 0.025 ng/g were 87, 77, and 78%, respectively. Mean recoveries of aflatoxins B1 and M1 added at a level of 0.1 ng/g were 75 and 87%, respectively, and at an added level of 0.05 ng/g were 86 and 75%. The within-laboratory precision (repeatability) ranged from 2 to 13%.     

Free radical scavenging active components from Cedrus deodara

An activity-directed fractionation and purification process was used to identify the antioxidant components of Cedrus deodara. Dried heartwood powder of C. deodara was first defatted with petroleum ether and then extracted with chloroform. The chloroform extract showed strong antioxidant activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. This fraction was then subjected to separation and purification using silica gel column chromatography. Three compounds with potent antioxidant activity were isolated in significant yields and identified by spectroscopic methods ((1)H NMR, (13)C NMR, IR, and MS). They were identified as (-)-matairesinol, (-)-nortrachelogenin, and a dibenzylbutyrolactollignan (4,4',9-trihydroxy-3,3'-dimethoxy-9,9'-epoxylignan). This is the first report of the occurrence of these compounds in C. deodara.     

Critical assessment of various techniques for the extraction of carotenoids and co-enzyme Q10 from the Thraustochytrid strain ONC-T18

A variety of techniques for extracting carotenoids from the marine Thraustochytrium sp. ONC-T18 was compared. Specifically, the organic solvents acetone, ethyl acetate, and petroleum ether were tested, along with direct and indirect ultrasonic assisted extraction (probe vs bath) methods. Techniques that used petroleum ether/acetone/water (15:75:10, v/v/v) with 3 h of agitation, or 5 min in an ultrasonic bath, produced the highest extraction yields of total carotenoids (29-30.5 microg g-1). Concentrations up to 11.5 microg g-1 of canthaxanthin and 17.5 microg g-1 of beta;-carotene were detected in extracts stored for 6 weeks. Astaxanthin and echinenone were also detected as minor compounds. Extracts with and without antioxidants showed similar carotenoid concentration profiles. However, total carotenoid concentrations were approximately 8% higher when antioxidants were used. Finally, an easy-to-perform and inexpensive method to detect co-enzymes in ONC-T18 was also developed using silica gel TLC plates. Five percent methanol in toluene as a mobile phase consistently eluted co-enzyme Q10 standards and could separate the co-enzyme fractions present in ONC-T18.     

Determining higher fatty acid levels in plant materials 1

The higher fatty acids (HFA) are important plant constituents that are implicated in the grass tetany hazard in livestock. A method is given whereby a technician can analyze 12 forage samples daily for HFA content.

The method consists of saponifying the plant material in ethanol and KOH and extracting the subsequently acidified HFA with petroleum ether. The petroleum ether phase is evaporated and the HFA residue is dissolved in ethanol and then titrated with standardized isobutanolic KOH in the absence of O₂ by using a N₂ atmosphere. A standard plant sample, analyzed over a 37‐day period, had a mean of 136 mmol H⁺ /kg ±4.5, where a 0.1 mmol H⁺ / l palmitic standard was determined with an accuracy of 99 ± 1.9%.     

PAHs and Petroleum Markers in the Atmospheric Environment of Alexandria City,Egypt

To evaluate the chemical fingerprint of hydrocarbons in airborneorganic matter in the arid environment of Alexandria City, Egypt,the compositions of aliphatic and aromatic compounds were determined in suspended particulate material collected from a street undergoing heavy traffic in central Alexandria and in bulkdeposition samples collected from a site representing an area increasingly influenced by human and industrial activities. Qualitative and quantitative characterizations of individual compounds were based on gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. More than 100 organic compounds are quantified in each sample, including n-alkanes, isoprenoids, polycyclic aromatic hydrocarbons(PAHs), sulfur-bearing heterocyclics, steranes/diasteranes, terpanes and aromatic steroids. The use of hydrocarbon profilesand ratios for identifying sources and processes is discussed.The molecular distribution of alkanes revealed that the mainsource of these compounds is from petroleum contamination withtrace input of vascular plant wax. The PAH profiles, especiallythe relative abundance of alkyl-PAHs and sulfur-containing heterocyclics, showed that PAHs are chiefly derived from trafficsources. The results further indicated that diesel vehicles aremore important PAH sources than gasoline vehicles. In addition,the source fingerprint of fossil fuel biomarkers such as steranes, terpanes and aromatic steroids agreed well with thefingerprint of unburned lubricating oil, which are probably contributed to vehicle exhaust emissions.     

Chemical composition of volatile extract and biological activities of volatile and less-volatile extracts of juniper berry (Juniperus drupacea L.) fruit

Volatile chemicals in a dichloromethane extract from a steam distillate of juniper berry fruit (Juniperus drupacea L.) and its two column chromatographic fractions (eluted with hexane and ethyl ether) were analyzed by gas chromatography/mass spectrometry. The major compounds in the dichloromethane extract were alpha-pinene (23.73%), thymol methyl ether (17.32%), and camphor (10.12%). A fraction eluted with hexane contained alpha-pinene (44.24%) as the major constituent. A fraction eluted with ethyl ether had thymol methyl ether (22.27%) and camphor (19.65%) as the main components. Three samples prepared from the distillate and two additional samples prepared by petroleum ether and ethanol extraction directly from juniper berry fruits exhibited clear antioxidant activities with dose response in both 1,2-diphenyl picrylhydrazyl and beta-carotene assays. All samples except the hexane fraction showed comparable activities to that of the synthetic antioxidant t-butyl hydroquinone at a level of 200 microg/mL in the two testing systems. The extracts of dichloromethane, petroleum ether, and ethanol exhibited appreciable antimicrobial activities against six microorganisms with minimum inhibitory concentrations ranging from 0.5 mg/mL (volatile extract against Candida albicans ) to 1.2 mg/mL (ethanol extract against Aspergillus niger ). The results of the present study suggest that this fruit could be a natural antioxidant supplement for foods and beverages.     

Capillary column gas chromatographic determination of dicamba in water, including mass spectrometric confirmation

A sensitive method is described for determining dicamba at low micrograms/L levels in ground waters by capillary column gas chromatography with electron-capture detection (GC-EC); compound identity is confirmed by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. Dicamba residue is hydrolyzed in KOH to form the potassium salt. The sample is then extracted with ethyl ether which is discarded. The aqueous phase is acidified to pH less than 1 and extracted twice with ethyl ether. The combined ethyl ether extracts are concentrated, and the residue is methylated using diazomethane to form the corresponding dicamba ester. The derivatized sample is cleaned up on a deactivated silica gel column. The methylated dicamba is separated on an SE-30 capillary column and quantitated by electron-capture or mass spectrometric detection. Average recoveries (X +/- SD) for ground water samples fortified with 0.40 microgram/L of dicamba are 86 +/- 5% by GC-EC and 97 +/- 7% by GC-MS detections. The EDL (estimated detection limit) for this method is 0.1 microgram dicamba/L water (ppb).     

Determination of trace volatile organic compounds in fish tissues by gas chromatography

Several volatile organic compounds associated with petroleum fuels (mainly alkylated benzenes) were extracted from spiked fish tissue samples with a stream of air, trapped on charcoal, eluted with a solvent, and analyzed by gas chromatography. These volatile compounds are among the most water-soluble components of crude oils and petroleum products, and they have been associated with tainting in fish tissues. Recoveries for these compounds were about 90% when spiked directly either onto traps or into fish tissues although naphthalene desorbed poorly from the charcoal; recoveries of this compound were about 50%. Relative standard deviations (RSD) for most recoveries of spiked samples were in the 2-10% range based on 6 samples analyzed in duplicate. However, when live fish were contaminated experimentally by adding the aromatic compounds to the aquarium water, the RSDs were higher (10-30%).     

Electrofocusing of methanolic extracts for identification of individual flavonol biomolecules in Camellia species

An effort has been made to isolate individual catechin compounds from green tea leaves in their pure form by electrophoresis. In the present study total polyphenol extraction was carried out initially and estimated through spectrophotometric and HPLC methods. Extracted polyphenol was separated on 0.7% agarose gel and visualized at 360 nm. Fragmented individual compounds were gel eluted with methanol and confirmed as (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) by HPLC. The method developed describes a suitable method for the isolation of valuable molecules in tea.     

Gas chromatographic-mass spectrometric determination of six sulfonamide residues in egg and animal tissues

A gas chromatographic-mass spectrometric method using selected ion monitoring mode for simultaneous determination of 6 sulfonamides in egg and edible animal tissues has been developed. Sulfonamides are extracted from a sample with acetonitrile. The extract is passed through a silica cartridge column and concentrated. Diazomethane in ether is added to methylate sulfonamides. After evaporation, the residue is dissolved in methylene chloride and cleaned up by silica gel column chromatography. The methylene chloride eluate containing sulfonamide-methyl derivatives is evaporated to dryness, redissolved in ether and partitioned between 6N hydrochloric acid. The acid phase is made alkaline, extracted with ether, and the ether solution, after concentration, is analyzed by gas chromatography-mass spectrometry in selected ion monitoring mode. Average recoveries from egg and silver salmon fortified at 1 and 0.2 ppm levels with 6 sulfonamides are 99.2 and 84.3%, respectively; coefficients of variation are 7.03 and 11.20%, respectively. Detection limits are 0.01-0.05 ppm.     

Isolation and identification of insecticidal components from Citrus aurantium fruit peel extract

Three active components were identified by bioassay-guided fractionation of bitter orange ( Citrus aurantium L.) fruit peel petroleum ether extract. Silica gel fractionation of the extract yielded a fraction that inflicted up to 96% mortality to adults of the olive fruit fly Bactrocera oleae (Gmelin) three days post-treatment. Subsequent HPLC purification of the active fraction resulted in the isolation of three components, eluted in fractions F 222, F 224, and F 226, that induced adult mortality. Considering the data obtained from UV, FTIR, MS, and (1)H NMR spectra, they were identified as 7-methoxy-8-(3'-methyl-2'-butenyl)-2 H-1-benzopyran-2-one (osthol), 4-methoxy-7 H-furo[3,2- g]benzopyran-7-one (bergapten), and 4-(( E)-3'-methyl-5'-(3',3'-dimethyloxiran-2'-yl)pent-2'-enyloxy)-7 H-furo[3,2- g][1]benzopyran-7-one (6',7'-epoxybergamottin). Our results are in concordance with those reported in the literature and were further verified by direct comparison to authentic components. 6',7'-Epoxybergamottin was toxic when tested individually, while bergapten and osthol were found to act synergistically to 6',7'-epoxybergamottin.     

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 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.     

Biochemical Characterization of γ‐75 k Secalins of Rye I. Amino Acid Sequences

The prolamin fraction of the rye cultivar Danko was reduced with dithioerythritol and separated by reversed‐phase HPLC on C₁₈ silica gel. Two major γ‐75k secalins, P1 and P2, were collected, purified by rechromatography, derivatized with 4‐vinylpyridine, and digested in parallel with α‐chymotrypsin, thermolysin, and trypsin. The different enzymatic hydrolyzates were preparatively separated by two‐step reversed‐phase HPLC on C₁₈ silica gel, and the resulting peptides were characterized by sequence analysis and, in parts, by mass spectrometry. By means of overlapping peptides and by comparison with a known DNA sequence of a γ‐75k secalin (gSec2A) derived from a wheat translocation line (2RS. 2BL), 84% of the P1 sequence and 35% of the P2 sequence could be assigned. Heterogeneity at several sequence positions demonstrated that both protein preparations were not pure and contained at least two or three components. The sequence of the C‐terminal domain of P1 was almost completely determined except for one of the 148 residues which could not be identified. The partially determined sequences of P2 were highly homologous with those of P1. The results revealed a close relationship between P1, P2, and gSec2A and a high degree of homology with γ‐gliadins of wheat including eight cysteine residues in homologous positions. The partially sequenced N‐terminal domain of P1 was similar to that of gSec2A and consisted of repetitive sequences rich in glutamine, proline, and aromatic amino acids. Differences from γ‐gliadins were found in the strongly increased number of residues, in the more frequent modifications of the repetitive motifs, and in the presence of a cysteine residue at position 12. The partial amino acid sequence of the N‐terminal domain of P2 was in agreement with that of P1, besides a few exceptions in single positions and in the presence of a second cysteine residue.     

Separation of normal paraffins and evaluation of petroleum contamination in foods

A method for separating n-paraffins from petroleum hydrocarbons in foods was developed. The method consists of 5 initial steps: digestion of sample with alkali, silica gel column chromatography, molecular sieve adsorption, destruction of the sieve with HCl, and oxidation with KMnO4. Recoveries of n-paraffins added to 55 g oyster at a level of 0.36 ppm ranged from 80% for normal pentadecane to 100% for n-paraffins over 18 carbon atoms. This method also facilitated the analysis of iso-paraffins such as pristane (2,6,10,14-tetramethylpentadecane) and phytane (2,6,10,14-tetramethylhexadecane), and other hydrocarbons, which are thought to be good marker compounds for the estimation of petroleum pollution.     

GC-ITMS determination and degradation of captan during winemaking

Captan and its metabolite tetrahydrophthalimide (THPI) were determined in grapes, must, and wine by GC-ITMS. Pesticides were extracted with acetone/petroleum ether (50:50 v/v). Because of the high selectivity of the ITMS detector, no interferent was found and cleanup was not necessary. Recoveries from fortified grapes, must, and wines ranged between 90 and 113% with a maximum coefficient of variation of 11%. Limits of quantitation were 0.01 mg/kg for both compounds. In model systems, captan and its metabolites, THPI, cis-4-cyclohexene-1,2-dicarboxylic acid, and 1,2,3,6-tetrahydrophthalamic acid, were determined by HPLC. The degradation of captan during winemaking was studied. Captan degraded in must, giving 100% THPI, and at the end of fermentation, only THPI was found in wine. The degradation of captan to THPI was due to the acidity in must and wine. This metabolite was present at low levels on grapes, and, unlike captan, it had no negative effect on the fermentative process. Model systems showed that the mechanism of disappearance of captan in grapes was due to photodegradation and codistillation.     

Gas-liquid chromatographic determination of kepone residues in finfish, shellfish, and crustaceans.

Finfish, shellfish, and crustacean samples are extracted with isopropanol and benzene; the extract is filtered and then concentrated. The extract, dissolved in hexane, is treated with oleum and extracted with aqueous alkali. The aqueous phase is acidified and extracted with petroleum ether-ethyl ether (1 + 1). The Kepone residue is determined by electron capture gas-liquid chromatography (GLC). Recoveries obtained by 8 laboratories from 15 species of finfish fortified at 0.02-0.23 ppm ranged from 37 to 107% with a mean +/- relative standard deviation of 79.4 +/- 14.5%. For oysters fortified at 0.01-0.10 ppm, recoveries range from 63 to 129% with a mean of 78.8 +/- 20.8%. For crustaceans fortified at 0.05-0.26 ppm, recoveries ranged from 52 to 110% with a mean of 78 +/- 16.4%. The approximate limits of quantitation for finfish and for shellfish and crustaceans are 0.02 and 0.05 ppm, respectively, under the GLC conditions used in this study.