Zearalenone in cereal grains

Zearalenone, a secondary metabolite with estrogenic properties, is produced by severalFusarium species that colonize cereal grains in the field and in storage. Recently, there have been reports of zearalenone contamination in corn, oats, barley, wheat, and grain sorghum. Corn and grain sorghum were examined for contamination due to obvious mold damage. Wheat, corn, and sorghum have been examined to determine the incidence of zearalenone in grains moving through commercial channels and stored on farms and at country elevators. Other grains such as oats and barley were analyzed because of associated estrogenic disturbances in farm animals. Stepsin procedures for the determination of zearalenone are extraction of a representative sample, partial purification of the extract by column chromatography, alkali treatment, or liquid-liquid partitioning, and subsequent measurement of the isolated toxin. Zearalenone is measured in partially purified extracts by thin layer chromatography (TLC), gas liquid chromatography (GLC), and high pressure liquid chromatography (HPLC). Confirmation of zearalenone contamination can be accomplished by gas chromatography-mass spectroscopy (GC-MS). Multitoxin screening procedures have been deveoped for zearalenone in combination with one or more of the following mycotoxins: aflatoxin, T-2 toxin, diacetoxyscirpenol, patulin, ochratoxin, penicillic acid, citrinin, penitrem A, and sterigmatocystin.     

Isolation of altenuisol and altertoxins I and II,minor mycotoxins elaborated byalternaria

A method for the isolation of milligram quantities of 3 minorAlternaria mycotoxins, altenuisol (AS), altertoxin I (AT-I) and altertoxin II (AT-II) was developed. Crude toxin preparation was first subjected to a preparative high pressure liquid Chromatograph step using PrepLC/System 500 and a silica gel column. The 3 minor toxins were eluted from the column with 25% ethyl acetate. Further purification of these toxins was achieved by passing the partially purified toxins through a Sephadex LH-20 column (2.5 X 25 cm) twice. In a solvent system of hexane/methylehe chloride/methanol (1:1:1, v/v/v), AT-I was eluted from the Sephadex column first, followed by AT-II and AS. The distribution of variousAlternaria mycotoxins in different fractions obtained from the Sephadex step and some of the physicochemical properties of AT-I and AT-II are described.     

Isolation and purification of deoxynivalenol and a new trichothecene by high pressure liquid chromatography

Deoxynivalenol (3,7,15-trihydroxy-12,l3-epoxytrichothec-9-ene-8-one) was extracted from corn with methanol/water (80:20, v/v) and purified by liquid:liquid partitioning and by preparative high pressure liquid chromatography (HPLC). This procedure was used to prepare mg quantities of toxin from field-inoculated corn for reference standards. Analysis of the isolated deoxynivalenol by analytical HPLC, gas liquid chromatography (GLC) and gas liquid chromatography/mass spectroscopy (GLC/MS) indicated the presence of a second compound similar to deoxynivalenol. This compound comigrates with deoxynivalenol on thin layer chromatography plates in chloroform/methanol (90:10, v/v), but can be separated by HPLC on a reverse-phase C₈ column with methanol/water (10:90, v/v). GC/MS of the compound and the trimethylsilyl ether derivative gave parent ions of m/e 280 and 424, respectively. These data and NMR data indicate that the compound is 3,15-dihydroxy-12,13-epoxytrichothec-9-ene-8-one, a previously unreported trichothecene.     

Determination of surfactant mixtures in shampoos and detergents by HPLC

A technique for separating 4 nonionic, 7 anionic and 4 amphoteric surfactants with n-dodecyl groups was studied by high performance liquid chromatography (HPLC) and applied to the determination of these surfactants in commercial shampoos and household detergents. Conditions used for the separation were: column packing and size, TSK-LS 410 (5μ) and 6 mm i.d. × 500 mm (2 connected, 250 mm columns); mobile phase, water/methanol (25/75, v/v) containing 0.25 M sodium perchlorate adjusted to pH 2.5 with phosphoric acid; column temp., 50 C; detector, RI. Surfactants in shampoos and detergents were clearly distinguished from each other and determined without column chromatographic pretreatment, e.g., ion-exchange chromatography.     

Lipid composition of oats (Avena sativa L.)

Compositions of lipids extracted from a sample of Hinoat oat by seven solvent systems and that extracted with chloroform/methanol (2:1 v/v) from six selected cultivars representing high and low lipid contents are reported. Lipid components (steryl esters, triglycerides, partial glycerides, free fatty acids, glycolipids and phospholipids) were separated by silicic acid column chromatography and thin layer chromatography and quantitated by GLC analysis of fatty acids and phosphorus determinations. Twelve oat cultivars were examined for the fatty acid composition of lipid extracted with n-hexane. Lipids extracted from Hinoat by different solvent systems ranged from 5.6 to 8.8%. Quantitative distribution of lipid components extracted with chloroform/methanol from six cultivars containing 4.6 to 11.6% lipid showed a significant correlation (γ=0.99) between the total lipid and the neutral lipid content. Phospholipid content was similar in all cultivars, but glycolipids showed a two-fold increase in high lipid oats. Triglycerides contained less palmitic and more oleic acid than the glycolipids or phospholipids. Nine glycolipids and 11 phospholipids have been identified, and the polar lipid composition of Hinoat oat is presented.     

Lipids of seven cereal grains

Grain samples of representative varieties of barley, corn, oats, rye, sorghum, triticale, and wheat grown commercially in the north central US were analyzed. Chemical constituents of the varieties studied are presented to provide an overview of their characteristics. Lipids of the milled grain samples were solvent extracted, classified by silicic acid column chromatography, and separated by thin layer chromatography. Fatty acid composition of the total lipid was determined by gas liquid chromatography and the fatty acid content was determined by saponification and extraction. Total lipid content of the grains ranged from 2.3% for ‘Polk’ wheat to 6.6% for ‘Chief’ oats. Lipid composition varied considerably. The row crops, corn and sorghum, have a high neutral lipid and low glycolipid content. The small grain varieties have a more balanced distribution among neutral lipids, glycolipids, and phospholipids. Fatty acid composition of the total lipid was similar for all grains. Minor qualitative differences were noted among the lipid classes of the 7 cereals.     

Purification of homoharringtonine and removal of residual solvents by spray drying

Homoharringtonine was purified from Cephalotaxus koreana by a combination of extraction, synthetic adsorbent treatment, low-pressure chromatography, and high performance liquid chromatography (HPLC). A crude extract was obtained by methanol extraction of biomass, followed by liquid-liquid extraction using chloroform. The waxy compounds were efficiently removed by adsorbent (active clay P-1) treatment. The extract was purified to greater than 52% with an 86.4% step yield by silica gel low-pressure chromatography. High performance liquid chromatography steps, which were composed of an HPLC step with silica column and an HPLC step with ODS column, were applied to give 98% purity with high yield. Amorphous homoharringtonine, with a fine particle size, was simply made by dissolving homoharringtonine in methylene chloride/methanol (98/2, v/v), followed by spray drying. Residual solvents, methylene chloride and methanol, could be reduced to 250 ppm and 1,160 ppm by spray drying and successive drying in a vacuum oven.     

Comparative study of cellulose isolated by totally chlorine‐free method from wood and cereal straw

Highly purified cellulose preparations were obtained by pretreatment of dewaxed barley straw, oil palm frond fiber, poplar wood, maize stems, wheat straw, rice straw, and rye straw with 2.0% H₂O₂ at 45°C and pH 11.6 for 16 h, and sequential purification with 80% acetic acid–70% nitric acid (10/1, v/v) at 120°C for 15 min. The purified cellulose obtained was relatively free of bound hemicelluloses (2.3–3.2%) and lignin (0.4–0.6%) and had a yield of 35.5% from barley straw, 39.6% from oil palm frond fiber, 40.8% from poplar wood, 36.0% from maize stems, 34.1% from wheat straw, 23.4% from rice straw, and 35.8% from rye straw. The weight‐average molecular weights of the purified cellulose ranged from 39,030 to 48,380 g/mol. The thermal stability of the purified cellulose was higher than that of the corresponding crude cellulose. In comparison, the isolated crude and purified cellulose samples were also studied by Fourier transform IR and cross‐polarization/magic‐angle spinning ¹³C‐NMR spectroscopy. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 322–335, 2005     

Thin layer chromatographic analysis of possible aflatoxins within grain dusts

The National Institute for Occupational Safety and Health is interested in assessing the hazards to grain workers associated with respirable grain dusts of all types. One of these hazards could involve the occurrence of mycotoxin producing fungi either upon or within grains. Aflatoxins, types of mycotoxins produced by bothAspergillus flavus andA. parasiticus, are hepatocarcinogens, mutagens, teratogens and toxins. Here, we report an attempt to determine whether settled and/or airborne dusts from barley, corn, flax, oats and Durum as well as spring wheats contain aflatoxins. These dusts were collected at port grain terminals in the Superior-Duluth regions of the United States. The dusts were extracted with and chromatographed upon thin layer plates in a variety of solvents which have been approved for the separation of aflatoxins. Two acceptable aflatoxin B₁ confirmatory tests were employed to verify suspected aflatoxin B₁ within the extracts. Each dust contained a chloroform-soluble, blue fluorescent compound(s) which possessed an Rf similar to that of aflatoxin B₁ upon chromatography of chloroform extracts in chloroform/95% methanol. Methylene chloride/H₂O) extracted a blue fluorescent compound(s) from each dust, and the compound(s) possessed Rf intermediate between those of aflatoxin B₁ and B₂ upon chromatography in acetone/methylene chloride. The methylene chloride/H₂O extracted compounds failed to turn yellow upon spraying with 25% sulphuric acid in methanol and subsequent viewing with an ultraviolet source. Our results confirm those of Sorenson et al., who reported that aflatoxins were absent from airborne grain dusts collected from the Superior-Duluth areas of the United States in the fall of 1977. In conclusion, we stress the need for extracting, detecting, and identifying aflatoxins by a variety of analytical procedures including thin layer and high performance liquid chromatography and “approved” confirmatory tests.     

Chemistry of Color Fixation in Crude,Physically Refined and Chemically Refined Rice Bran Oils upon Heating

Compared to other vegetable oils, rice bran oil (RBO) has a characteristic dark color which further deepens upon heating or frying of foods in the oil. Darkening of the oil during heating has been studied. The dark color‐causing material in crude, chemically refined and physically refined rice bran oils was separated using a silica gel column for a hexane‐eluted oil fraction and a methanol eluted fraction. The methanol eluted fraction for all the above three types of RBO produced a dark color upon heating, hence the physically refined RBO methanol fraction was investigated further and contained monoglycerides (23.4 %) and diglycerides (67.4 %) of linoleic + linolenic acids in its methanol fraction as analyzed by column chromatography and HPLC which decreased in concentration after heating. The linoleic acid level of 37.7 % in the methanol fraction was reduced significantly to 18 % after heating (52.3 % reduction). The IR and NMR spectra were similar to those of a monoglyceride/diglyceride with NMR spectra indicating a lower amount of olefinic protons for the heated sample. These results showed that the darkening of RBO was due to the oxidation and polymerization of monoglycerides/diglycerides containing linoleic acid/linolenic acid.     

Horse erythrocyte gangliosides: Preparation of the major hematoside NeuNG1-Lac-Cer

A simple method for the isolation of hematoside NeuNG1-Lac-Cer from horse erythrocytes is described. An aliquot of the crude ganglioside fraction was labeled by tritiated sodium borohydride after mild periodate oxidation. The compounds obtained were used as radioactive tracers in column chromatography. Gangliosides were applied onto a silicic acid column and eluted stepwise by solvents of steadily increasing polarity. The major ganglioside, NeuNG1-Lac-Cer, was eluted in a high yield by the solvent mixture chloroform/methanol/water (60∶35∶8, v/v/v).     

Recovery of Sinapic Acid from the Waste Effluent of Mustard Protein Isolation by Ion Exchange Chromatography

Sinapic acid present in the waste stream of yellow mustard protein isolation was purified by strong base Dowex (1 × 8, Cl^?) ion exchange chromatography. The ratio of loading volume to resin bed volume was 19:1. Approximately 80 % of sinapic acid was adsorbed. The column was washed with two bed volumes of water to remove remaining undesirable components. Approximately 75 % of sinapic acid adsorbed by the resins in the column was eluted by ten bed volumes of a solution containing 0.9 M acetic acid and methanol (4:6, v/v). Up to 15 adsorption and regeneration cycles resulted in only a slight, 3–5 %, reduction in ion exchange capacity, indicating that this is a viable approach to the recovery and purification of sinapic acid. The recovery of this valuable nutraceutical ingredient improves the economic viability of an integrated extraction process for this Canadian oilseed crop.     

Determination of ascorbyl palmitate by high performance liquid chromatography

An HPLC method for the determination of ascorbyl palmitate in vegetable oil and lard has been developed. Chromatographic conditions consist of a diamine column, a mobile phase of 70:30 (v/v) methanol:0.02M monobasic potassium phosphate buffer, pH 3.5, and UV detection. Samples were extracted with methanol. An overall average recovery value of 96.7% was obtained for ascorbyl palmitate in five representative vegetable oils and lard.     

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

Two procedures to remove polar contaminants from a crude brain lipid extract by using prepacked reversed-phase columns

Two procedures were developed using prepacked, reversed-phase columns (Bond Elut) for the separation of lipids from water-soluble contaminants. A crude lipid extract from brain tissue, was diluted stepwise with a methanol/water (method 1) or a methanol/saline (method 2) mixture and, with each step, was passed through the column. As the polarity of the solvent was increased, all lipids became bound to the column, while the water-soluble compounds remained in the eluate. After three subsequent dilutions and column elutions, the eluate containing the more polar contaminants was discarded. The bound lipids were then eluted with a small volume of chloroform/methanol (1∶2, v/v). Alternatively two fractions were eluted, the first fraction eluted with methanol/water (12∶1, v/v), contained gangliosides, phosphatidyl-serine, phosphatidylinositol, phosphatidic acid and sulfatides. The second fraction, eluted with chloroform/methanol (1∶2, v/v), contained all remaining phospholipids, cerebrosides and cholesterol. For both methods a quantitative recovery of cholesterol and phospholipids was obtained. In method 2, when water was replaced by saline in the dilution solvent mixture, gangliosides were also bound and quantitatively recovered.     

气相色谱-质谱法测定粮谷中丁草胺的残留量

大米、糙米、玉米、大麦、小麦试样用正己烷提取,经凝胶色谱和固相萃取柱净化,气相色谱-质谱仪检测,外标法定量.添加水平在0.01～0.50 mg/kg时,回收率范围为80.6%～98.5%;相对标准偏差为8.6%～15.5%;测定低限为0.010 mg/kg.     

从葡萄皮红中分离纯化锦葵花素葡萄糖苷的研究

利用柱层析和重结晶方法对葡萄皮红中锦葵花素葡萄糖苷的分离纯化工艺进行了研究。结果表明,Amberlite XAD-16大孔树脂柱层析、活性炭/硅藻土混合柱层析和聚酰胺柱层析的最佳洗脱浓度分别为20%乙醇水溶液、15%甲醇-氯仿和30%乙醇水溶液。将产品按固液比为1∶5(m/v)的比例加入甲醇溶解,再加入20 mL正丁酮,冷冻36 h,得到的单体含量可达95%。该工艺成本低廉,原料来源广泛,产品纯度高,具有广阔的应用前景。     

Extraction and Demulsification of Oil From Wheat Germ,Barley Germ,and Rice Bran Using an Aqueous Enzymatic Method

An aqueous enzymatic method was developed to extract oil from wheat germ. Wheat germ pretreatment, effect of various industrial enzymes, pH, wheat germ to water ratio, reaction time and effect of various methods of demulsification, were investigated. Pretreatment at 180 °C in a conventional oven for 4 min reduced the moisture 12.8–2.2 % and significantly increased the oil yield. Adding a combination of protease (Fermgen) and cellulase (Spezyme CP) resulted in a 72 % yield of emulsified oil from wheat germ (both commercial and laboratory milled wheat germ). Using the same oil extraction conditions optimized for wheat germ, yields of 51 and 39 % emulsified oil were obtained from barley germ (laboratory milled), and rice bran, respectively. Three physical demulsification methods (heating, freeze-thawing, and pH adjustment) and enzymatic methods (Protex 6L, Protex 7L, Alcalase, Fermgen, Lysomax and G-zyme 999) were compared. After demulsification with Protex 6L, free oil yields of 63.8 and 59.5 % were obtained with commercial wheat germ and with laboratory milled wheat germ, respectively. Using the same demulsification conditions optimized for wheat germ, yields of 45.7 % emulsified oil and 35 % free oil were obtained for barley germ and rice bran, respectively.     

Separation and determination of C21 dicarboxylic acid and xylenesulfonate hydrotropes in mixtures with dodecylbenzenesulfonate

Column chromatographic procedures have been developed for separating binary mixtures of commercial sodium dodecylbenzenesulfonate (DDBS) with the hydrotropes sodiumm-xylenesulfonate (SXS) and 8-[5(6)-carboxy-4-hexyl-cyclohex-2-enyl] octanoic acid (DAC) and ternary mixtures of these three materials. The materials are separated on a specially purified silica gel column. The C₂₁ dicarboxylic acid (DAC) is eluted with 2:3 (v/v) chloroform/acetone in the presence of SXS, with 3:2 (v/v) chloroform/acetone in its absence. The DDBS is eluted with 1:9 (v/v) isopropyl alcohol/acetone in the presence of SXS, with methanol in its absence. The SXS is eluted with methanol in the presence of DAC, with 8:2 (v/v) isopropyl alcohol/acetone in its absence. The presence of SXS in mixtures with DDBS in greater than equimolar amounts interferes with the Hyamine 1622, mixed indicator titration for DDBS. Visiting Scholar from Xinjiang Chemical Institute, Scientific Academy of the People’s Republic of China.