Chlorinated compounds and phenols in tissue, fat, and blood from rats fed industrial waste site soil extract: methods and analysis

A method was developed to analyze rat tissue, fat, and blood for some of the chlorinated compounds found in an extract of soil from an industrial waste site. Extraction with hexane and then with ethyl ether-hexane (1 + 1) was followed by concentration over steam, and gas chromatographic analysis with an electron capture detector. Volatile compounds were analyzed in a glass column coated with 6% SP-2100 plus 4% OV-11 on Chromosorb W. Semivolatile compounds, chlorinated compounds, and pesticides were analyzed in a 70 m glass capillary column coated with 5% OV-101. Phenols were analyzed in a glass column packed with 1% SP-1240 DA on Supelcoport. However, the most efficient means of separation was to use the same glass column for volatile compounds, a DB-5 fused silica capillary column for semivolatile compounds, pesticides, and phenols, and the same 1% SP-1240 DA glass column for separation of beta-BHC and pentachlorophenol. Recoveries ranged from 86.3 +/- 9.1% (mean +/- standard deviation) to 105 +/- 10.4%. Sensitivities for semivolatile chlorinated compounds, pesticides, and phenols were about 4 ng/g for fat, 1 ng/g for tissue, and 0.2 ng/mL for blood. Sensitivities for volatile compounds were about 4-fold higher (16, 4, and 0.8, respectively). Sensitivities for dichlorobenzenes and dichlorotoluenes were 8 ng/g for fat, 2 ng/g for tissue, and 0.4 ng/mL for blood.     

Simplified extraction and cleanup for multiresidue determination of pesticides in lanolin

In the proposed method, a light petroleum solution of lanolin (wool fat) is adsorbed on diatomaceous earth in an Extrelut column, and the pesticides are eluted with acetonitrile saturated with light petroleum. After evaporation to a small volume, the extract is subjected to solid-phase extraction (SPE) on a C-18 column. The acetonitrile eluate is evaporated to dryness and the residue is taken up in light petroleum. Organophosphorus pesticides are determined by temperature-programmed gas chromatography (GC) on a wide-bore column using a flame photometric detector in the phosphorus mode. Organochlorine pesticides are determined after miniaturized Florisil cleanup by classic GC on an OV-17/QF-1 packed column, using an electron capture detector. This procedure is more rapid and straightforward than the time-consuming AOAC extraction method, 29.014. Cleanup was better and the results obtained were comparable. Recoveries for 13 organochlorine and organophosphorus pesticides, frequently found in lanolin, ranged from 80 to 90%.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue

A method for isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and oxytetracycline-fortified fish muscle tissue samples (0.5 g) were blended with octadecylsllyl (C18, 40 microns, 18% load, endcapped) derivatized silica packing material (2 g) containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetate. A column made from the C18/fish tissue matrix was first washed with hexane (8 mL), following which the oxytetracycline was eluted with acetonitrile-methanol (1 + 1, v/v) containing 0.06% w/v each of butylated hydroxyanisole and butylated hydroxytoluene. The eluate contained oxytetracycline analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array set at 365 nm). Standard curves for oxytetracycline isolated from fortified samples were linear (0.998 +/- 0.002) with an average absolute percentage recovery of 80.9 +/- 6.6% for the concentration range (50, 100, 200, 400, 800, 1600, and 3200 ng/g) examined. The interassay variability was 11.3 +/- 5.2% with an intra-assay variability of 1.1%.     

Separation of Delta5- and Delta7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.     

Liquid chromatographic determination of melamine in beverages

A liquid chromatographic method is described for the determination of melamine in beverages. Melamine is separated by column chromatography using cation and anion exchange resin and determined by ion-pair liquid chromatography using an ODS column and a mixture of acetonitrile and 0.05M phosphate buffer (pH 3.0) containing 0.005M sodium 1-laurylsulfate (1 + 4, v/v) as mobile phase. Recoveries of melamine ranged between 90.3 +/- 7.8 and 102.1 +/- 5.6% at levels of 0.6 to 2.4 ppm in 4 kinds of beverages. The quantitation limit was 2.5 micrograms melamine in 50 mL beverage. The method was applied to the migration test of melamine from melamine-formaldehyde resin products to the beverages.     

High pressure liquid chromatographic determination of zearalenone in chicken tissues

A method is reported for the extraction and analysis of zearalenone in chicken fat, heart muscle, and kidney tissue by using high pressure liquid chromatography (HPLC). Zearalenone is extracted with acetonitrile, cleaned up with hexane, and extracted further with ethyl acetate. Zearalenone is determined by HPLC using a reverse phase radial compression separation system, an ultraviolet absorbance detector, and a mobile phase of acetonitrile-water (60 + 40) (v/v). Recoveries of zearalenone added at levels from 50 to 200 ng/g are in the range 82.6-95.1%.     

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.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

Purification of fusaproliferin from cultures of Fusarium subglutinans by preparative high-performance liquid chromatography

Thirty-six Fusarium strains were grown on cracked yellow corn and evaluated for optimum fusaproliferin production, with Fusarium subglutinans E-1583 producing the highest levels (1600 microg/g). Three solvent systems were tested for extracting fusaproliferin from the cultures of F. subglutinans E-1583. Methanol gave the highest fusaproliferin recovery, followed by methanol/1% aqueous NaCl (55:45, v/v) and acetonitrile/methanol/H(2)O (16:3:1, v/v/v). Hexane partitioning was effective in removing many impurities from the crude fusaproliferin extracts prior to the liquid chromatography step. Fusaproliferin samples were further purified by high-performance liquid chromatography (HPLC) with a C18 preparatory column using a mobile phase of acetonitrile/H(2)O (80:20, v/v). The purity of the fusaproliferin was verified by analytical HPLC, GC/MS, (1)H NMR spectroscopy, and electrospray ionization (ESI) MS. The isolated fusaproliferin was shown to be free of impurities and can be used as a standard for routine analysis. Fusaproliferin was shown to be temperature-sensitive when samples were stored at room temperature (20-24 degrees C) for more than several days. After 30 days at 4 degrees C, approximately 8% of the fusaproliferin had been transformed to deacetyl-fusaproliferin; however, samples stored at -20 degrees C for 1 year contained only trace amounts of the deacetylated form.     

Analysis of O,S-dimethyl hydrogen phosphorothioate in urine, a specific biomarker for methamidophos

A rugged and sensitive method was developed to monitor urinary concentrations of O,S-dimethyl hydrogen phosphorothioate (O,S-DMPT), a specific biomarker of exposure to the organophosphate insecticide methamidophos. After pH adjustment and C18 solid phase extraction column cleanup, the urine was lyophilized at a low temperature to prevent loss of possibly highly volatile and unstable O,S-DMPT metabolite. The dried residue was derivatized using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and 1% tert-butyldimethylchlorosilane (MTBSTFA + 1% TBDMCS) in acetonitrile. After it was filtered, the derivatized product was analyzed and quantified by gas chromatography using a pulse flame photometric detector specific for phosphorus compounds. The limit of detection for this method was 0.004 ppm with a limit of quantitation of 0.02 ppm of urine. The mean recovery value for O,S-DMPT from 17 urine samples fortified at varying concentrations was 108% with a standard deviation of 12%.     

Determination of zeranol/zearalenone and their metabolites in edible animal tissue by liquid chromatography with electrochemical detection and confirmation by gas chromatography/mass spectrometry

A sensitive method is described for the determination and confirmation of zeranol and zearalenone, as well as their isomers and metabolites, in edible animal tissue. The analytes are extracted from tissue with methanol, hydrolyzed enzymatically, cleaned up by acid-base partitioning, determined by liquid chromatography (LC) with electrochemical (EC) detection, and confirmed by gas chromatography/mass spectrometry (GC/MS). LC analysis is performed by isocratic elution with a buffered mobile phase using a Nova-Pak reverse-phase C18 column with amperometric EC detection at +0.90 V. Capillary GC/MS analysis of the trimethylsilyl derivatives provides mass spectral confirmations.     

Liquid chromatographic determination of sulfamoyldapsone in swine tissues

A liquid chromatographic (LC) method is described for the quantitative determination of sulfamoyldapsone (2-sulfamoyl-4,4'-diaminodiphenyl sulfone) in swine muscle, liver, kidney, and fat. Sulfamoyldapsone was extracted from tissues with acetonitrile saturated with n-hexane. The extract was washed with n-hexane saturated with acetonitrile, concentrated, and cleaned up by alumina column chromatography. Sulfamoyldapsone was separated on an ODS column by using acetonitrile-methanol-water (6 + 18 + 76) and was detected at 292 nm. Overall average recovery of sulfamoyldapsone added to tissues at levels of 0.1 and 0.5 microgram/g was 93.3% +/- 6.0. Detection limit was 0.02 microgram/g in these tissues.     

Purification of cyclopiazonic acid by liquid chromatography

A purification procedure for cyclopiazonic acid has been developed, using sequential preparative and semi-preparative liquid chromatography. Crude cyclopiazonic acid (324 mg) was extracted from a 1 L fermentation medium with chloroform-methanol (80 + 20), dried, dissolved in chloroform, and chromatographed on an oxalic acid/silica preparative column with chloroform-methanol (99 + 1) as the eluant. A semi-preparative oxalic acid/silica column and chloroform-methanol (99.5 + 0.5) were then used for rechromatography of the partially purified cyclopiazonic acid. This second chromatographic treatment yielded fractions from which cyclopiazonic acid was readily crystallized (106.7 mg; 33% recovery). Analytical chromatography was developed using an amino column in an ion-exchange mode, with a methanol-phosphate buffer eluant. Response was linear from 10 to 800 micrograms/injection of standard solutions. Cyclopiazonic acid chemically binds sodium from soda-lime vials.     

气相色谱法测定水产品中三氯杀螨醇残留量

为拓展食品中农药残留监控范围,本文建立了水产品中三氯杀螨醇的气相色谱测定方法。试验基质为鳗鲡、罗非鱼、对虾、玻纹巴菲蛤,取其可食用组织的均匀试样1g,用正己烷超声萃取,用浓硫酸和弗罗里硅土净化,二氯甲烷／正己烷混合液淋洗层析柱。洗脱液收集和旋转蒸发浓缩后,用气相色谱法测定其中的三氯杀螨醇,外标法定量。测定仪器为HP6890N型气相色谱仪,配HP7683B型自动进样器、HP-5型毛细管气相色谱柱（30m×0．32mm×0．25μm）和。Ni微电子捕获检测器。三氯杀螨醇浓度线性范围是0．0025-0．4μg·mL-1（r=0．9998,P〈0．001）,方法定量检测下限为0．01μg·g-1。用未检出三氯杀螨醇的鳗鲡等4种试样,添加3个水平的三氯杀螨醇,分别为0．01、0．10、O．50μg·g-1,每种试样每个添加量测定6份。结果显示,加标回收率在71％-111％范围内,批内变异系数为3．2％～8．5％（n=6）,批间变异系数为2．5％～7．1％（n=4）。定量限点加标试样的回收率为73％～94％,批内变异系数为5．9％-7．7％,峰高信噪比〉10。本方法试样用量少,前处理简便,可操作性强,适合测定水产品可食部分的三氯杀螨醇残留量。     

Multiresidue screen for organophosphorus insecticides using gel permeation chromatography--silica gel cleanup.

A multiresidue screen for quantitative determination of 43 organophosphorus insecticides in 5 g of plant and animal tissues is described. The organophosphorus insecticides are extracted with methanol-dichloromethane (10 + 90, v/v) and cleaned up using automated gel permeation chromatography with hexane-ethyl acetate (60 + 40) eluant and in-line silica gel minicolumns. Concentrated extracts are analyzed by gas chromatography with flame photometric detection. The method recovers 43 organophosphorus insecticides in the range of 72 to 115%. Analysis of fortified bovine liver (n = 5) shows an average 95.9 +/- 4.8% recovery at the 0.05 micrograms/g level and 93 +/- 3.8% at the 0.5 micrograms/g level. Analysis of fortified bovine rumen content (n = 5) shows an average 98 +/- 4.2% recovery at the 0.1 micrograms/g level and 98.7 +/- 2.8% at the 1 micrograms/g level. Method detection limits ranged from 0.01 to 0.05 micrograms/g for the compounds studied using a nominal 5 gram sample.     

Isolation and identification of steroidal saponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto)

A new furostanol pentaoligoside and spirostanol tetraoligoside were isolated for the first time from yam tubers (Dioscorea pseudojaponica Yamamoto) from Taiwan, together with four known yam saponins, methyl protodioscin, methyl protogracillin, dioscin, and gracillin. Their structures were characterized as 26-O-beta-D-glucopyranosyl-22alpha-methoxyl-(25R)-furost-5-en-3beta,26-diol, 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside, and (25R)-spirost-5-en-3beta-ol 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-([alpha-L-rhamnopyranosyl-(1-->4)]-O-[alpha-L-rhamnopyranosyl-(1-->4)])-beta-D-glucopyranoside. The structural identification was performed using LC-MS and 1H and 13C NMR. The methanol extract of yam tubers was fractionated by XAD-2 column chromatography using a methanol/water gradient elution system to yield furostanol and spirostanol glycoside fractions. Preparative high-performance liquid chromatography, employing a C18 column and a mobile phase of methanol/water (69:31, v/v), was used to separate each furostanol glycoside, whereas a mobile phase of methanol/water (79:21, v/v) was used to resolve the individual spirostanol glycosides. The conversions from steroid saponins to diosgenin after acid hydrolysis were around 68 and 90% for furostanol and spirostanol glycosides, respectively.     

Matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in beef fat

A multiresidue technique is presented for the extraction and quantitative gas chromatographic screening of 9 insecticides (lindane, heptachlor, aldrin, heptachlor epoxide, p,p'-DDE, dieldrin, endrin, p,p'-TDE, and p,p'-DDT) as residues in beef fat. Beef fat was fortified by adding the 9 insecticides, plus dibutyl chlorendate as internal standard, to 0.5 g portions of beef fat and blending with 2 g C18 (octadecylsilyl)-derivatized silica. The C18/fat matrix blend was fashioned into a column by adding the blend to a 10 mL syringe barrel containing 2 g activated Florisil. The insecticides were then eluted from the column with 8 mL acetonitrile, and a 2 microL portion of the acetonitrile eluate was then directly analyzed by gas chromatography with electron capture detection. Unfortified blank controls were treated similarly. The acetonitrile eluate contained all of the pesticide analytes (31.25-500 ng/g) and was free of interfering co-extractants. Correlation coefficients for the 9 extracted pesticide standard curves (linear regression analysis, n = 5) ranged from 0.9969 (+/- 0.0021) to 0.9999 (+/- 0.0001). Average relative percentage recoveries (85 +/- 3.4% to 102 +/- 5.0%, n = 25 for each insecticide), inter-assay variability (6.0 +/- 1.0% to 14.0 +/- 6.7%, n = 25 for each insecticide), and intra-assay variability (2.5-5.1% n = 5 for each insecticide) indicated that the methodology is acceptable for the extraction, determination, and screening of these residues in beef fat.     

Analysis of sulfonamides in animal feeds by liquid chromatography with fluorescence detection

Two analytical methodologies for the simultaneous analysis of eight sulfonamide antibiotics in animal feeds were developed. Analytes were extracted in a simple and rapid procedure by manual shaking with an ethyl acetate/ultrapure water mixture (99:1, v/v) without further sample cleanup. Mean recoveries ranging from 72.7% to 99.4% with relative standard deviations below 9% were achieved from spiked animal feed samples. Determination was carried out by high-performance liquid chromatography using fluorometric detection with precolumn derivatization. The separation of the derivatized compounds was performed using two different chromatographic columns: a conventional C(18) column and a recently available core-shell particle Kinetex C(18) column. Both methods were validated in-house in six different feed matrices, and the two approaches were compared. The experiments showed that the method using the Kinetex column was superior with regard to speed of analysis and precision, both under repeatability and intermediate reproducibility conditions. The limits of detection and quantification were also greatly improved, below 0.10 and 0.34 μg/g, respectively. Finally, this novel approach was successfully applied to the analysis of real feed samples.     

Separation and purification of tricin from an antioxidant product derived from bamboo leaves

Tricin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) occurs in its glycosidic form in rice bran and other grass species such as wheat, barley, and maize. Tricin is considered sufficiently safe for clinical development as a cancer chemopreventive agent, therefore it can be used for cancer prevention. This study established a new method for the preparation of tricin from bamboo leaves as an alternative to traditional methods such as chemical synthesis via the Baker-Venkata-Raman reaction between acetylsyringic acid and phloroacetophenone. Tricin was prepared from an antioxidant product that was derived from bamboo leaves (AOB) by extraction with aqueous ethanol. A concentrated solution of this product was obtained and then processed by polystyrene (AB-8) resin column chromatography and preparative high-performance liquid chromatography (HPLC) with 30% (v/v) acetonitrile in 1% (v/v) acetic acid as the mobile phase. The collected tricin-rich fraction was further sequentially purified by dialysis membrane separation and drowning-out crystallization methods. The purity was assessed by analytical HPLC with 25% (v/v) acetonitrile in 1% (v/v) acetic acid as the mobile phase, and the chemical confirmation was evaluated by infrared, mass, nuclear magnetic resonance, and ultraviolet spectroscopies. Tricin (3.09 g) was prepared from 174 g of a crude column chromatography fraction obtained from 5 L of AOB concentrated solution. The present method is appropriate for preparing quantities of pure tricin, which can be used for the quantification of tricin in various plant herbs and further for pharmaceutical/biomedical applications and evaluation.     

Simultaneous analysis of nivalenol and deoxynivalenol in cereals by liquid chromatography

A sensitive method is described for determination of nivalenol (NIV) and deoxynivalenol (DON) in cereals by using reverse phase liquid chromatography and UV detection at 222 nm. The sample is extracted with acetonitrile-water (85 + 15) and an aliquot is purified by passage through a combined column of cation exchange resin and alumina-carbon (20 + 1). Analysis at this stage is possible with some samples but the method recommends passing an aliquot through a carbon minicolumn after evaporation and solubilization in methanol. Interference from coextracted compounds at this point is negligible. Recoveries of both NIV and DON from spiked extracts taken through the full method were in the range 83-94%. The relative standard deviation, based on 5 replicate determinations from each of 2 corn samples, was approximately 5% for both NIV and DON. With a 10 microL injection, the minimum contamination (3 X signal/noise ratio) able to be detected in cereal samples was about 0.015 micrograms NIV/g and 0.05 micrograms DON/g. The cleaned up extracts are also suitable for analysis by gas chromatography.