The automated determination of glucosamine,galactosamine, muramic acid,and mannosamine in soil and root hydrolysates by HPLC

Amino sugars make a significant contribution to soil organic N and are mainly of microbial origin. The most important amino sugars in soil are glucosamine, galactosamine, muramic acid, and mannosamine. A method was developed for the simultaneous determination of these four amino sugars by high‐performance reverse‐phase liquid chromatography in standard solutions, soil and root hydrolysates. Pre‐column derivatization with o‐phthaldialdehyde (OPA) was used in an automated sample injector with thermostatic regulation of the reagent at 4 °C. The separation of the four amino sugars was fully satisfactory and was not disturbed by other fluorescent components in the soil and root hydrolysates.     

Ultrasensitive determination of jasmonic acid in plant tissues using high-performance liquid chromatography with fluorescence detection

An ultrasensitive and selective high-performance liquid chromatographic method for the volatile signaling hormone, jasmonic acid, has been developed based on precolumn derivatization with 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide). The derivatization reaction was carried out at 60 °C for 30 min in the presence of phosphoric acid. The formed jasmonic acid derivative was eluted using a mobile phase of methanol/pH 6.50 ammonium formate buffer/tetrahydrofuran (67:30:3, v/v/v) in 10 min on a C(18) column and detected with fluorescence detection at excitation and emission wavelengths of 495 and 505 nm, respectively. The detection limit (signal-to-noise ratio = 4) reached 1.14 × 10(-10) M or 2.29 fmol per injection (20 μL), which is the lowest of the existing methods. The proposed method has been successfully applied to the direct determination of trace jasmonic acid in the crude extracts of soybean leaves from soybean mosaic virus-infected and normal plants with recoveries of 95-104%.     

Rapid and sensitive determination of phenol in honey by high-performance liquid chromatography with fluorescence detection

The objective of this research was to develop a novel high-performance liquid chromatographic (HPLC) method involving a simple sample preparation procedure for the rapid, low-cost, and sensitive quantitation of phenol in honey at levels of regulatory and practical importance. After proper dilution of honey with water, the samples were analyzed by a gradient HPLC system, using a reversed-phase column with fluorescence detection at excitation and emission wavelengths of 270 and 300 nm, respectively. The eluents applied were water-acetonitrile-85% orthophosphoric acid (10:10:0.01, v/v/v) and water-85% orthophosphoric acid (20:0.01, v/v). The retention time of phenol was found to be 14.1 min, and the limit of quantitation for phenol in honey was set at 5 microg/kg. Overall recovery was 98%. The proposed method has been successfully applied to real sample analysis.     

Shifts in amino sugar and ergosterol contents after addition of sucrose and cellulose to soil

An incubation experiment with organic soil amendments was carried out with the aim to determine whether formation and use of microbial tissue (biomass and residues) could be monitored by measuring glucosamine and muramic acid. Living fungal tissue was additionally determined by the cell-membrane component ergosterol. The organic amendments were fibrous maize cellulose and sugarcane sucrose adjusted to the same C/N ratio of 15. In a subsequent step, spherical cellulose was added without N to determine whether the microbial residues formed initially were preferentially decomposed. In the non-amended control treatment, ergosterol remained constant at 0.44 μg g⁻¹ soil throughout the 67-day incubation. It increased to a highest value of 1.9 μg g⁻¹ soil at day 5 in the sucrose treatment and to 5.0 μg g⁻¹ soil at day 33 in the fibrous cellulose treatment. Then, the ergosterol content declined again. The addition of spherical cellulose had no further significant effects on the ergosterol content in these two treatments. The non-amended control treatment contained 48 μg muramic acid and 650 μg glucosamine g⁻¹ soil at day 5. During incubation, these contents decreased by 17% and 19%, respectively. A 33% increase in muramic acid and an 8% increase in glucosamine were observed after adding sucrose. Consequently, the ratio of fungal C to bacterial C based on bacterial muramic acid and fungal glucosamine was lowered in comparison with the other two treatments. No effect on the two amino sugars was observed after adding cellulose initially or subsequently during the second incubation period. This indicates that the differences in quality between sucrose and cellulose had a strong impact on the formation of microbial residues. However, the amino sugars did not indicate a preferential decomposition of microbial residues as N sources.     

Relationships between microbial indices in roots and silt loam soils forming a gradient in soil organic matter

Perennial rye grass (Lolium perenne) was grown in a greenhouse pot experiment on seven soils to answer the question whether the microbial colonisation of roots is related to existing differences in soil microbial indices. The soils were similar in texture, but differed considerably in soil organic matter, microbial biomass, and microbial community structure. Ergosterol and fungal glucosamine were significantly interrelated in the root material. This ergosterol was also significantly correlated with the average ergosterol content of bulk and rhizosphere soil. In addition, the sum of fungal C and bacterial C in the root material revealed a significant linear relationship with microbial biomass C in soil. The colonisation of roots with microorganisms increased apparently with an increase in soil microbial biomass. In the root material, microbial tissue consisted of 77% fungi and 23% bacteria. In soil, the fungal dominance was slightly, but significantly lower, with 70% fungi and 30% bacteria. Fungal glucosamine in the root material was significantly correlated with that in soil (r=0.65). This indicates a close relationship between the composition of dead microbial remains in soil and the living fraction in soil and root material for unknown reasons.     

Fluorometric determination of benzylideneacetone in fragrance products by liquid chromatography with post-column derivatization

A method is described for the liquid chromatographic (LC)-fluorometric determination of benzylideneacetone in fragrance products. Benzylideneacetone is first separated from other fragrance ingredients by LC and then reacted post-column with a methanolic solution of isonicotinic acid hydrazide and aluminum nitrate. The reactants are maintained at 65 degrees C for about 1.5 min to quantitatively form the fluorescent isonicotinoyl hydrazone derivative of benzylideneacetone. The aluminum ion forms a complex with the hydrazone to enhance the fluorescence of the derivative. The amount of benzylideneacetone is determined by measuring the intensity of the fluorescence emitted by the hydrazone derivative and comparing that value with those obtained for derivatized standards. Recovery studies were conducted by spiking commercial fragrances with benzylideneacetone at concentrations of 0.01, 0.05, and 0.1% (w/v). Recoveries ranged from 98 to 104% with a mean recovery of 100.2% and a standard deviation of 2.4%.     

Determination of biogenic amines in squid and white prawn by high-performance liquid chromatography with postcolumn derivatization

A simple method was developed for the determination of biogenic amines in aquatic food products using a reverse-phase high-performance liquid chromatography with postcolumn automatic o-phthalaldehyde derivatization and fluorescence detection. The linearity, repeatability, and recovery of the method for seven amines (tyramine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine) were evaluated. This optimized method was applied to detect biogenic amines in squid and white prawn during refrigerated storage. Sensory analysis, pH value, and total volatile base nitrogen value were combined to evaluate the freshness quality of these two raw materials. Agmatine and cadaverine in squid, cadaverine, and putrescine in white prawn were the most obviously changed amines during the storage at two different temperatures, and these biogenic amines could be the effective quality indicators for the freshness of the raw aquatic materials.     

Amino acid composition of soil peptides chromatographed by high performance liquid chromatography on C18 and C8 columns

Summary Low molecular weight fractions (LMW; <5000 daltons) of organic matter were isolated from three soils by a mild extraction procedure and gel-permeation chromatography. The peptides present in the LMW soil extracts were separated on a Whatman Partisphere C18 and a Beckman Ultrapore C8 column by reverse-phase high performance liquid chromatography (HPLC). The peptide fractions were collected, acid hydrolyzed, and analyzed for amino acid composition. The C8 bonded-phase column gave better separation of the LMW soil peptide material than the C18 column. The total quantities of amino acids released from LMW peptides by acid hydrolysis were greater than the quantities released by immobilized protease hydrolysis (Warman and Isnor 1990). Total soil N present in the form of LMW peptides in these three soils ranged from 4 to 15%. The total recovery of peptide amino acid-N showed little difference between the C18 and C8 columns for two of the soils tested.     

高效液相色谱法测定土壤中化学武器降解产物二苯砷酸

二苯氰砷(diphenylcyanoarsine)和二苯氯砷(di-phenylchloroarsine)是一战和二战期间被大量制造和使用的一类呕吐剂和糜烂剂,在日制化学武器系统中俗称"红弹"。战后,大量化学武器被遗弃于中国、欧洲和日本,且大部分通过海洋倾倒和陆地填埋方     

Determination of microbial biomass and fungal and bacterial distribution in cattle faeces

As an important component of organic fertilizers, animal faeces require methods for determining diet effects on their microbial quality to improve nutrient use efficiency in soil and to decrease gaseous greenhouse emissions to the environment. The objectives of the present study were (i) to apply the chloroform fumigation extraction (CFE) method for determining microbial biomass in cattle faeces, (ii) to determine the fungal cell-membrane component ergosterol, and (iii) to measure the cell-wall components fungal glucosamine and bacterial muramic acid as indices for the microbial community structure. Additionally, ergosterol and amino sugar data provide independent control values for the reliability of the microbial biomass range obtained by the CFE method. A variety of extractant solutions were tested for the CFE method to obtain stable extracts and reproducible microbial biomass C and N values, leading to the replacement of the original 0.5 M K₂SO₄ extractant for 0.05 M CuSO₄. The plausibility of the data was assessed in a 28-day incubation study at 25 °C with cattle faeces of one heifer, where microbial biomass C and N were repeatedly measured together with ergosterol. Here, the microbial biomass indices showed dynamic characteristics and possible shifts in the microbial community. In faeces of five different heifers, the mean microbial biomass C/N ratio was 5.6, the mean microbial biomass to organic C ratio was 2.2%, and the mean ergosterol to microbial biomass C ratio was 1.1‰. Ergosterol and amino sugar analysis revealed a significant contribution of fungi, with a percentage of more than 40% to the microbial community. All three methods are expected to be suitable tools for analysing the quality of cattle faeces.     

高效液相色谱法同时测定土壤中环丙氨嗪和三聚氰胺

本试验研究建立了同时测定土壤中环丙氨嗪和三聚氰胺残留量的高效液相色谱法.红壤、潮土等5种土壤样品经氨水/甲醇 (5/95,v/v)超声提取3次,浓缩处理后上机检测.环丙氨嗪和三聚氰胺的标准曲线在0.1 ～ 15.0 μg/ml浓度范围内线性关系良好,绝对系数(R2)分别为1.0000和0.9998;在0.5 ～ 5 mg/kg添加范围内,环丙氨嗪和三聚氰胺在土壤中的平均回收率分别为87.2% ～ 101.1% 和 75.3% ～ 101.6%,变异系数分别为3.3% ～ 8.1%、1.6% ～ 9.9%,最低检测限分别为0.05 mg/kg、0.07 mg/kg.与国际上气相/液相色谱-质谱连用法相比,操作简单,经济方便易于普及.     

Simultaneous determination of free amino acid content in tea infusions by using high-performance liquid chromatography with fluorescence detection coupled with alternating penalty trilinear decomposition algorithm

In this paper, a novel application of alternating penalty trilinear decomposition (APTLD) for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) has been developed to simultaneously determine the contents of free amino acids in tea. Although the spectra of amino acid derivatives were similar and a large number of water-soluble compounds are coextracted, APTLD could predict the accurate concentrations together with reasonable resolution of chromatographic and spectral profiles for the amino acids of interest owing to its "second-order advantage". An additional advantage of the proposed method is lower cost than traditional methods. The results indicate that it is an attractive alternative strategy for the routine resolution and quantification of amino acids in the presence of unknown interferences or when complete separation is not easily achieved.     

Prolonged simulated acid rain treatment in the subarctic: Effect on the soil respiration rate and microbial biomass

Humus chemistry and respiration rate, ATP, ergosterol, and muramic acid concentration as measures of chemical properties, microbial activity, biomass, and indicators of fungal and bacterial biomass were studied in a long-term acid rain experiment in the far north of Finnish Lapland. The treatments used in this study were dry control, irrigated control (spring water, pH 6), and two levels of simulated acid rain (pH 4 and pH 3). Originally (1985–1988), simulated acid rain was prepared by adding both H₂SO₄ and HNO₃ (1.9:1 by weight). In 1989 the treatments were modified as follows. In subarea 1 the treatments continued unchanged (H₂SO₄+HNO₃ in rain to pH 4 and pH 3), but in subarea 2 only H₂SO₄ was applied. The plots were sampled in 1992. The acid application affected humus chemistry by lowering the pH, cation exchange capacity, and base saturation (due to a decrease in Ca and Mg) in the treatment with H₂SO₄+HNO₃ to pH 4 (total proton load over 8 years 2.92 kmol ha^-1), whereas the microbial variables were not affected at this proton load, and only the respiration rate decreased by 20% in the strongest simulated acid rain treatment (total proton load 14.9 kmol ha^-1). The different ratios of H₂SO₄+HNO₃ in subareas 1 and 2 did not affect the results.     

A simple and economic method for simultaneous determination of 11 antibiotics in manure by solid-phase extraction and high-performance liquid chromatography

Purpose

A simple and highly efficient economic method for the analysis of 11 antibacterial drugs including two tetracyclines, three quinolones, four sulfonamides, chloramphenicol and tylosin, in livestock manure, was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC).

Materials and methods

The analytes were successively extracted by EDTA-McIlvaine solution and organic solvent mixture. The extracts were degreased with n-hexane and cleaned through SPE on a hydrophile-lipophile balance (HLB) cartridge. All compounds were determined on a C18 reverse phase column with gradient elution.

Results and discussion

Recoveries calculated from spiked samples of animal manures ranged from 62.65 to 99.16 % for 11 antibiotics with relative standard deviations of less than 10.0 %. Limits of detection ranged from 0.1 to 1.9 μg kg^?1, and limits of quantification ranged from 0.3 to 5.9 μg kg^?1.

Conclusions

The results show that SPE-HPLC is an inexpensive and practical method for rapid detection of multiple antibiotics in animal manure.

     

Free and HF-HO-extractable amino acids determined by high performance liquid chromatography in a loamy sand soil

Summary Amino acids were extracted from fertility plots of a loamy sand soil with 0.05 M HF-HCl and with a 10% ethanol solution (free amino acids) and analysed by reverse-phase high performance liquid chromatography (HPLC). The total quantities of amino-N compounds analysed were 4.4 g/g soil for the acid treatment and 22.6 g/g soil for the 10% ethanol extract. Glycine and glutamic acid were the most abundant of 15 amino-N compounds in the HF-HCl extracts, whereas glutamic acid and ornithine + NH⁺ ₄ were found in the highest concentration in the 10% ethanol extracts. The HF-HCI pretreatment is used to increase the efficiency of the extraction of soil organic matter. Although this pretreatment removed some amino acids, the acids extract less than 1% of the total amino-N content of the crude soil extracts. The pretreatment, therefore, was not overly destructive. Comparisons between the amino acids extracted from the fertility plots were not conclusive, except for glycine, which was greater in concentration in the higher fertilizer N plots of the same crop rotation.     

Reverse-phase liquid chromatographic determination of lysine in complete feeds and premixes, using manual precolumn derivatization

A sample portion is hydrolyzed with 6N HCl for 23 h and cooled, the pH is adjusted to 7.7 with NaOH, and the solution is diluted with pH 7.7 borate buffer. An aliquot of the sample extract is derivatized with 9-fluorenylmethyl chloroformate (9-FMC). Lysine is separated from other amino acids by isocratic reverse-phase liquid chromatography (LC) using fluorescence detection: 260 nm excitation and 313 nm emission. The mobile phase is acetonitrile-0.1M acetic acid (pH 4.2) buffer (53 + 47). Linearity is satisfactory over a range of 0.4-24 micrograms/mL. Results from 9 feed samples (1.1-2.7% lysine) analyzed by both the LC method and an amino acid analyzer were not significantly different statistically. Recovery of standard lysine, spiked just before derivatization on these same 9 samples (in duplicate), was 100.9% with a coefficient of variation (CV) of 2.4%. A study of within-day and between-day method precision resulted in CVs of 1.1 and 1.8%, respectively. The variation of results was negligible when the method was tested for ruggedness on 7 factors.     

The effect of lime on microbial activity in spruce (Picea abies L.) forests

Summary The use of lime increased heat output and decreased the C:N ratio (global indicators of biological activity) in the organic horizons of a spruce forest (Picea abies L.) soil. These changes were still present after 18 years. During the same period, the muramic acid content increased slightly, while the concentration of both ergosterol and glucosamine decreases. The ratios of ergosterol or glucosamine to muramic acid decreased significantly after 3 years in the plots that had been irrigated and limed, and after 8 years in the limed non-irrigated plots.     

Determination of neutral sugars in soil by capillary gas chromatography after derivatization to aldononitrile acetates

We have quantified ribose, rhamnose, arabinose, xylose, fucose, mannose, glucose, and galactose in soil by gas chromatography (GC) simultaneously after converting to aldononitrile acetate derivatives. A recommended single-hydrolytic step by 4 M trifluoroacetic acid (TFA) at 105 °C for 4 h was more effective for releasing soil neutral sugars from non-cellulosic carbohydrates and better suited to our purification procedure compared with the sulphuric acid hydrolysis. Linearity of the GC detection for each neutral sugar was in the range of 10-640 μg ml⁻¹ and the recovery of neutral sugars from the spiked soil samples ranged from 76% to 109.7%. The coefficients of variation of the neutral sugars in four soils were lower than 2.0% for the instrument and 4.6-7.6% for the whole determination procedures. Compared with the trimethylsilyl (TMS) derivatization, the recovery of our newly modified method was more satisfactory and the reproducibility of ribose was improved significantly. Moreover, the aldononitrile acetate derivative was more stable than TMS derivative. Therefore, it is a promising approach suitable for a routine use in the quantitative analysis of soil neutral sugars, since it is fast, sensitive, and reproducible.     

Sulfamethazine and sulfathiazole determination at residue levels in swine feeds by reverse-phase liquid chromatography with post-column derivatization

Twenty g sample, to which sulfamerazine has been added as internal standard, is extracted with 0.3N HCl + 1.5% diethylamine in 25% methanol. The sample extract is chilled (to aid clarification), centrifuged, and filtered. The sulfonamides are separated from each other and from co-extracted materials on a C-18 reverse-phase column and detected at 450 nm following post-column derivatization with dimethylaminobenzaldehyde. Two isocratic mobile phases have been tested: (1) acetonitrile-2% acetic acid (17 + 83), with an analysis time of 13 min; and (2) acetonitrile-methanol-2% acetic acid (4 + 16 + 80), with an analysis time of 20 min but an improved analysis for some samples. As many as 40 samples have been analyzed at one time unattended with the aid of an autosampler. A total of about 1500 field samples have been assayed using the method. Method sensitivity is 0.1 ppm for either analyte in a hog finishing fed. Linearity for each of the analytes is satisfactory over a range of 0.4-25 ppm in spiked feeds. Coefficients of variation range from 13% at 0.5 ppm to 2% at 13 ppm as tested over a period of time in naturally contaminated samples. The absolute recovery of sulfamerazine varies with sample matrix, but, in the presence of sulfamerazine as internal standard, recovery has been 96.7-99.7% over the range of 0.1-10 ppm. Sulfamerazine and sulfamoxole were tested for their suitability as internal standards. Sulfamerazine is a good internal standard for sulfamethazine; neither is ideal for sulfathiazole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct and simultaneous analysis of sinigrin and allyl isothiocyanate in mustard samples by high-performance liquid chromatography

A reversed-phase HPLC method for the simultaneous determination of the glucosinolate sinigrin and its major degradation product allyl isothiocyanate (AITC) was developed and used for direct analysis of aqueous extracts from Oriental mustard (Brassica juncea L.) related materials (ground and cracked seeds, powders, and bran) and from soil samples. The lowest detection limit was 0.1 microg/mL for both sinigrin and AITC). The developed method was used to trace the degradation of sinigrin to AITC in aqueous extracts. One of the major advantages of this method is the complete estimation of sinigrin content. The simultaneous analysis of both sinigrin and AITC in a single run avoided the underestimation caused by separate analyses.