Determination of nabam fungicide in crops by liquid chromatography with postcolumn reaction detection

The ethylenebisdithiocarbamate (EBDC) fungicide, nabam, was determined in several crop matrixes using liquid chromatography with postcolumn reaction detection. After separation by micellar liquid chromatography, nabam (EBDC sodium salt) was acid hydrolyzed to ethylenediamine and fluorigenically labeled with o-phthalaldehyde-mercaptoethanol (OPA-MERC). Standard curves were linear from the detection limit of ca 1 ng to 1000 ng. Nabam was recovered in high yield (89 plus or minus 7.7%) over a range of concentrations (0.1 to 20 ppm) from fortified samples of papaya, lettuce, cucumber, spinach, and applesauce using a simple extraction method. Efforts to convert the more popular EBDC fungicides, maneb and mancozeb, to nabam are discussed.     

Humic acid reactions with amino acids

Reactions between five humic acids extracted from soils with widely differing pedological histories and 17 amino acids commonly occurring in proteins were investigated in aqueous solutions at pH 3.0 and 6.5. The reactions were affected by: (a) nature of the humic acid: (b) pH of the system: and (c) length of contact. The principal reaction appeared to be the microbiological oxidative degradation of the amino acids leading to the formation of substantial amounts of ammonium. While there was no evidence that the humic acids per se interacted with the amino acids, they did not appear to interfere with the microbiological degradation of the amino acids.     

Determination of fluoroquinolones in milk samples by postcolumn derivatization liquid chromatography with luminescence detection

A liquid chromatography (LC) method with luminescence detection for the determination of eight quinolone antibiotics is reported. The system encompasses three consecutive steps: (a) chromatographic separation using reverse-phase mode (RP-LC), (b) postcolumn derivatization reaction, and (c) luminescence detection by monitoring fluorescence (FL) and time-resolved (TR) signals. The derivatization step is based on the reaction between quinolones and terbium(III) to form luminescent chelates, which were determined at lambda(ex) 340 and lambda(em) 545 nm (FL mode) or at lambda(ex) 281 and lambda(em) 545 nm (TR mode). Dynamic ranges of the calibration graphs, obtained with standard solutions of analytes and FL and TR modes, respectively, were 190-3500 and 316-2000 ng mL-1 for marbofloxacin, 8-3500 and 8.1-1500 ng mL-1 for ciprofloxacin, 6.2-3500 and 13-1500 ng mL-1 for danofloxacin, 7.4-3500 and 8.4-1500 ng mL-1 for enrofloxacin, 14-3500 and 20-2000 ng mL-1 for sarafloxacin, 12.5-3500 and 13.9-1200 ng mL-1 for difloxacin, 7.6-3500 and 13-3000 ng mL-1 for oxolinic acid, and 9-2000 and 130-3000 ng mL-1 for flumequine. Limit of detection values obtained using FL and TR modes, respectively, were 60 and 95 ng mL-1 for marbofloxacin, 2 and 2.4 ng mL-1 for ciprofloxacin, 1.9 and 3.9 ng mL-1 for danofloxacin, 2.2 and 2.5 ng mL-1 for enrofloxacin, 3.8 and 7 ng mL-1 for sarafloxacin, 4 and 4.2 ng mL-1 for difloxacin, 2.3 and 4 ng mL-1 for oxolinic acid, and 2.7 and 40 ng mL-1 for flumequine. The precision was established at two concentration levels of each analyte and expressed as the percentage of relative standard deviation with values ranging between 1.9 and 7.8%. The validation procedure for the analysis of samples was carried out using European Community recommendations, and the decision limit and detection capability were calculated for bovine whole milk. The method was applied to whole, semiskimmed, and skimmed milk samples spiked with the target analytes, and the recoveries ranged between 93.3 and 106.0%.     

Determination of phylloquinone and menaquinones in animal products with fluorescence detection after postcolumn reduction with metallic zinc

A high-performance liquid chromatographic (HPLC) method for the determination of phylloquinone and menaquinones in foods of animal origin is described. The K vitamers were quantified with a fluorescence detector after postcolumn reduction with metallic zinc using K1(25) as an internal standard. Extraction was done either with 2-propanol-hexane (meat and fish products) or with acid hydrolysis method (dairy products). Prior to quantification, sample extracts were purified by semipreparative HPLC; in addition, the fats of cheese and rainbow trout samples were removed with lipase hydrolysis. By this method the phylloquinone and menaquinones (MK-4 to MK-10) present in a few representative samples of different animal food groups were determined. HPLC-MS was used to confirm the identification of K vitamers. Long-chain menaquinones were found from bovine and pig livers as well as from various cheeses. The total vitamin K contents calculated as the sum of quantified K vitamers were in general low (mean content 10-100 ng/g); the highest amount was analyzed in chicken meat (600 ng/g).     

Supercritical fluid extraction of bioavailable amino acids from soils and their liquid chromatographic determination with fluorometric detection

A new procedure with supercritical CO2 modified with 0.5 mL of water and 0.75 mL of 0.1 M HCl in situ and 0.75 mL of water on-line at 15 MPa and 50 degrees C for 45 min was applied for the extraction of bioavailable amino acids from soil samples. Total extraction time was 60 min, but more favorable conditions are even possible for selected groups of amino acids. All analytes were trapped into 20 mL of methanol with satisfactory recovery (94-104%) and determined using high-performance liquid chromatography with fluorometric detection on a Zorbax Eclipse column (4.6 x 75 mm, 3.5 microm) with Na2HPO4 and acetonitrile/methanol/water as a mobile phase. Linear calibration curves were obtained (r > 0.999 except 0.99823 for Ile) with lower limits of detection (S/N = 3) in the range from 1.54 pg (Gly) to 13.5 pg (Cy2) or from 18.6 fmol (Ser) to 64.8 fmol (Lys). Validation and repeatability data are also given. Comparable results were obtained with a robust, commonly used extraction method (0.5 M ammonium acetate, 60 min in shaker, followed by filtration and lyophilization). Limiting values of artificial release of amino acids were also determined for each soil sample to eliminate any false results to ensure that all extracted amino acids originate from soil solution and exchangeable bound positions of soil samples.     

Liquid chromatographic determination of amprolium in poultry feed and premixes using postcolumn chemistry with fluorometric detection

Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.     

复合氨基酸微量元素螯合肥制备工艺研究

以废弃鸡毛水解液为复合氨基酸来源,研究其与微量元素铁、锌、锰、铜螯合的最佳工艺条件,并利用有机溶剂沉淀分离法制备固体复合氨基酸亚铁、锌、锰、铜螯合物,以螯合率为指标考察影响制备工艺的主要因素。结果表明,最佳工艺参数为:复合氨基酸与亚铁、锌、锰和铜螯合反应的最佳配位体摩尔比均为2∶1;最佳pH值范围分别为5.0、8.0、4.0～5.0和6.0～7.0;反应温度为室温(222～6℃);反应时间为30.min。在上述条件下,复合氨基酸与各微量元素的螯合率均超过95%;所得复合氨基酸螯合物可与0.2.mol/L磷酸二氢钾复配,克服了传统微肥完全不能与磷酸盐复配的缺陷。     

植物对氨基酸的吸收利用及氨基酸在农业中的应用

植物对氨基酸的吸收、转运、代谢以及氨基酸在肥料和农药上的应用国内外已有报道。已有研究证明,植物可直接吸收土壤中的氨基酸分子,其吸收后的转运、分配、代谢因氨基酸种类而异,产生的生理效应也不相同;氨基酸农药易被日光分解或被自然界微生物降解,在土壤中、植物体内不留残毒,其降解产物还可作为农作物的营养物质,提高农作物的品质和产量,施用这类农药,人畜安全,没有公害;氨基酸肥具有促进植株生长发育、增强抗逆性、改善土壤状况和提高作物产量的作用。     

Bound amino acids in humic acids from arable cropping systems

We investigated the varying concentrations of bound amino acids in humic acids (HA) extracted from soils under both crop rotation and continuous cropping of rye. The experiment was created in 1957. Since then, winter rye had been grown continuously and also the sequence of the 7 yr rotation had been started: potato, spring barley, alfalfa, alfalfa, oil seed rape, winter rye, and winter rye. Soils were fertilized with NPK and manure. Continuous cropping of rye increased total acidity of soils and the contents of carboxylic and phenolic groups in HA. The total amounts of the bound amino acids in HA from soils under crop rotation were higher than from continuous cropping of rye. Fertilization with NPK increased the contents of bound amino acids more than manure. Neutral amino acids dominated in all samples of HA, and basic amino acids had the lowest concentrations. In both types of cultivation, glutamic acids, glycine, alanine, valine, and lysine dominated. The proline contents in HA from continuous rye cropping were higher than in HA from soils under crop rotation. The concentrations of β‐alanine and lysine were higher in HA from crop rotation indicating a higher microbial biomass since these compounds are typical constituents of bacteria cell walls.     

Identification of amino acids in humic acid

Davidson, Sowden and Atkinson^l) in 1951 reported on the amino acids in the three soil organic matter fractions, and Stevenson, Marks, Varner and Martin~) in 1952 reported on the identification of eight amino acids from clay-adsorbed organic colloids in Brookstom slity clay loam. Also, some investigations on these amino acids have been carried out in our laboratory.     

Color characteristics of monascus pigments derived by fermentation with various amino acids

Various pigment colors were produced by Monascus fermentations with separate addition of 20 amino acids. The color characteristics and structures of the pigment derivatives were investigated. When each amino acid was added to the fermentation broth as a precursor, pigment extracts with different hue and chroma values were obtained depending on the content ratios of yellow, orange, and red colors in the fermentation broth. The yellow and orange pigments were identical regardless of amino acid addition. The red compounds varied on the basis of the type of amino acid added. LC-MS and (1)H and (13)C NMR structural analyses confirmed that the derivative pigments contained the moieties of the added amino acids. L, a, and b values of the CIELAB color system for the derivative pigments were measured. Values of hue and chroma were then calculated. The colors of the derivative pigments were in the range of orangish red to violet red. The hydrophilicities/hydrophobicities of the derivative pigments could be predicted from their log P values, which were estimated using computer programs.     

Enhanced photostability of monascus pigments derived with various amino acids via fermentation

The photostability of 18 amino acid derivatives from monascus pigment was tested under various physical and chemical conditions. Under sunlight, the half-life of derivatives was increased to 1.45-5.58 h, corresponding to a 6-25-fold improvement over a control red pigment (0.22 h). The degradation of pigment derivatives followed a first-order reaction, and the pigment stability increased with an increasing concentration while it decreased with both an increase and decrease in pH from 7. The stabilities of derivatives decreased in descending order in hexane, ethanol, propanol, methanol, ethyl ether, distilled water, chloroform, and acetonitrile. Pigment stability under UV light (365 nm) showed a pattern similar to stability after exposure to sunlight. After 30 days of incubation at 30 degrees C, more than 80% of the initial derivative contents remained while only 29% of the control red remained. The differences in degradation patterns that control red gradually changed to brown whereas the phenylalanine derivative remained a weak red were confirmed by HPLC analysis.     

Kinetics of interaction of vanillin with amino acids and peptides in model systems

Model systems were used to study the reaction kinetics of vanillin and pentalysine, lysine, glutathione, cysteine, aspartame, or phenylalanine (molar ratio 1:1) in phosphate buffer. The buffer pH was adjusted to the pK(a)(2) of the available alpha-amino group of each amino acid or peptide. Reductions of vanillin followed first-order kinetics at 55, 65, and 75 degrees C in the presence of each of the amino acids or peptides used. The reaction rates were accelerated as the temperature increased. The rate constants were highest for pentalysine followed by lysine, phenylalanine, glutathione/cysteine, and aspartame. The reduction of phenylalanine followed first-order kinetics, whereas the formation of its reaction product followed zero-order kinetics. The activation energy (E(a)) for the reaction ranged from 5.6 to 14.5 kcal/mol.     

Competition for amino acids between wheat roots and rhizosphere microorganisms and the role of amino acids in plant N acquisition

The direct uptake of organic nitrogen compounds from the soil solution by plant roots has been hypothesised to constitute a significant source of N to the plant particularly in N limiting ecosystems. The experiments undertaken here were designed to test whether wheat roots could out-compete the rhizosphere microflora for a pulse addition of organic N in the form of three contrasting amino acids, namely lysine, glycine and glutamate. Amino acids were added at a concentration reflecting reported soil solution concentrations (100 μM) and the uptake into either plant biomass or respiration or microbial biomass and respiration determined over a 24 h chase period. The results showed that the plant roots could only capture on average 6% of the added amino acid with the remainder captured by the microbial biomass. We therefore present direct in vivo evidence to support earlier work which has hypothesised that organic N may be of only limited consequence in high input agricultural systems. We suggest that this is a result of the higher concentrations of NO₃⁻ in agricultural soil solutions, the slow movement of amino acids in soil relative to NO₃⁻, the rapid turnover of amino acids by soil microorganisms, and the poor competitive ability of plant roots to capture amino acids from the soil solution.