高效液相色谱法测定菌群降解纤维素产物中的糖、有机酸和醇

采用硫酸除钙和调节pH的样品预处理方法,利用高效液相色谱对菌群降解纤维素发酵液中的糖、有机酸和醇3类物质进行分析,待测组分与培养基成分能够得到有效分离。从菌群降解纤维素发酵液中检出纤维二糖、葡萄糖、乙醇、丁醇、甘油、乙酸与丁酸并进行了定量分析,7种组分的检出限范围为0.10~2.00 mg/L,线性相关系数均大于0.9996,线性范围为0.020~1.000 g/L,回收率为85.41%~115.60%,相对标准偏差(RSD)为0.22%~4.62%(n=6)。该方法准确可靠,可实现对菌群降解纤维素发酵液中糖、醇和有机酸的同时准确测定。     

高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物

建立了高效液相色谱(HPLC)同时测定生物质乳酸发酵液中有机酸及糖类的分析方法。使用Bio-Rad Aminex HPX-87H色谱柱,以5 mmol/L的H2SO4为流动相,在柱温55 ℃,流速0.6 mL/min条件下,采用示差折光检测器进行检测。结果表明,该方法可在17 min内实现发酵液中各种有机酸和糖类化合物等的完全分离与定量,6种有机酸和3种糖类化合物在0.15～5.19 g/L范围内的线性关系良好,回归方程的线性相关系数在0.9998以上。将该法用于米根霉发酵液的检测,两个水平的加标回收率为96.91%～103.11%,相对标准偏差(n=6)为0.81%～4.61%。该法适用于微生物发酵液中多种有机酸和糖类的快速、高效分离和定量测定。     

Ion-exclusion chromatography determination of organic acid in uridine 5'-monophosphate fermentation broth

Simultaneous determination of organic acids using ion-exclusion liquid chromatography and ultraviolet detection is described. The chromatographic conditions are optimized when an Aminex HPX-87H column (300 × 7.8 mm) is employed, with a solution of 3 mmol/L sulfuric acid as eluent, a flow rate of 0.4 mL/min and a column temperature of 60°C. Eight organic acids (including orotic acid, α-ketoglutaric acid, citric acid, pyruvic acid, malic acid, succinic acid, lactic acid and acetic acid) and one nucleotide are successfully quantified. The calibration curves for these analytes are linear, with correlation coefficients exceeding 0.999. The average recovery of organic acids is in the range of 97.6% ～ 103.1%, and the relative standard deviation is in the range of 0.037% ～ 0.38%. The method is subsequently applied to obtain organic acid profiles of uridine 5'-monophosphate culture broth fermented from orotic acid by Saccharomyces cerevisiae. These data demonstrate the quantitative accuracy for nucleotide fermentation mixtures, and suggest that the method may also be applicable to other biological samples.     

毛细管离子色谱-串联质谱法测定热纤梭杆菌发酵液中的低相对分子质量有机酸

建立了一种测定热纤梭杆菌发酵液中的低相对分子质量有机酸的毛细管离子色谱-串联质谱方法。探索优化了色谱和质谱的检测条件,在最佳分析条件下同时检测8种有机酸。离子色谱以KOH水溶液作为流动相进行梯度洗脱;用IonSwift MAX-100毛细管柱进行分离;在喷雾电压为3.0 kV、喷雾气压强为2 000 kPa、成源温度为275℃的条件下,选择离子监测(SIM)模式下运行质谱。结果表明,柠檬酸和异柠檬酸两种同分异构体能够得到很好的分离,8种有机酸在一定浓度范围内具有良好的线性关系,相对标准偏差为1.45%~5.99%,相关系数为0.9696~0.9986,平均加标回收率为89.0%~110.0%,8种有机酸的检出限为0.01~0.50 mg/L。该方法进样量少,灵敏度高,重现性好,能够满足实际样品的检测要求,可用于嗜热厌氧菌发酵液中低分子量有机酸的测定。     

Growth-induced changes in the proteome of Helicobacter pylori

Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.     

Analysis of carboxylic acid metabolites from the tricarboxylic acid cycle in Bacillus subtilis cell extract by capillary electrophoresis using an indirect photometric detection method

With a growing interest in metabolome analysis, there is a need for developing robust methods for analysis of intracellular metabolites profiles in real samples like e.g., bacteria cell. Due to their weak absorbance properties, tri- and dicarboxylic acids from TCA cycle (citric, isocitric, 2-oxoglutaric, succinic, fumaric, malic) as well as carboxylic acid metabolites from glycolysis pathway, urea cycle and metabolism of amino compounds (formic, pyruvic, lactic, acetic, glutamic) were analyzed by capillary electrophoresis (CE) with indirect UV detection. Using 4 mM 2,6-pyridinedicarboxylic acid as a highly UV absorbing carrier electrolyte, 0.2 mM cetyltrimethylammonium bromide, 10% ethylene glycol and 10% acetonitrile, pH 3.5, carboxylic acids metabolites were analyzed in Bacillus subtilis cell extract from two different cultures: glucose and malate. CE with an electrokinetic injection mode achieved limits of detection in the range of 13-54 ppb (1.12-10(-7) - 5.96-10(-7) M). The reproducibility and linearity of method was investigated with RSD for migration time less than 1.3% and acceptable correlation coefficients. The optimized CE method was used to compare metabolome content of cell extract derived from two different culture media containing either glucose or malate as a carbon source. The changes in carboxylic acid metabolites profile were observed depending from used culture medium. Carboxylic acid concentrations ranged: in cell extract from malate culture from 59 to 0.5 microM for lactate and citrate, respectively, and in cell extract from glucose culture from 133 to 0.5 microM for glutamate and citrate, respectively. Appropriate concentrations of carboxylic acid in the single bacterium cell were estimated at mM and sub-mM levels.     

高效液相色谱法测定克鲁维酵母菊芋发酵液中的乙醇,糖和有机酸类代谢成分

建立了高效液相色谱法同时分析菊芋发酵液中的乙醇和有机酸的方法.采用HPLC有机酸分析柱, 流动相为0.01 mol/L H2SO4,流速为0.5 mL/min, 以紫外和示差折光检测器作为双通道检测手段,同时对克鲁维酵母菊芋发酵液中的柠檬酸、α-酮戊二酸、葡萄糖、丙酮酸、果糖、琥珀酸、乳酸、甘油、乙酸、乙醇进行了定量分析,本方法的回收率为95.8%～109.6%;RSD为0.33%～4.0%,结果表明,本方法简单、快速、准确,适用于监控克鲁维酵母发酵产物并指导整个发酵过程条件的优化.     

Simultaneous determination of low-molecular-weight organic acids and chlorinated acid herbicides in environmental water by a portable CE system with contactless conductivity detection

This report describes a method to simultaneously determine 11 low-molecular-weight (LMW) organic acids and 16 chlorinated acid herbicides within a single run by a portable CE system with contactless conductivity detection (CCD) in a poly(vinyl alcohol) (PVA)-coated capillary. Under the optimized condition, the LODs of CE-CCD ranged from 0.056 to 0.270 ppm, which were better than for indirect UV (IUV) detection of the 11 LMW organic acids or UV detection of the 16 chlorinated acid herbicides. Combined with an on-line field-amplified sample stacking (FASS) procedure, sensitivity enhancement of 632- to 1078-fold was achieved, with satisfactory reproducibility (RSDs of migration times less than 2.2%, and RSDs of peak areas less than 5.1%). The FASS-CE-CCD method was successfully applied to determine the two groups of acidic pollutants in two kinds of environmental water samples. The portable CE-CCD system shows advantages such as simplicity, cost effectiveness, and miniaturization. Therefore, the method presented in this report has great potential for onsite analysis of various pollutants at the trace level.     

Time-resolved selected ion flow tube mass spectrometric quantification of the volatile compounds generated by E. coli JM109 cultured in two different media

Preliminary measurements have been made of the volatile compounds emitted by the bacterium E. coli JM109 cultured in the commonly used media Dulbecco's modified Eagle's medium (DMEM) and lysogeny broth (LB) using selected ion flow tube mass spectrometry, SIFT-MS, as a step towards the real time, non-invasive monitoring of accidental infections of mammalian cell cultures. In one procedure, the culture medium alone and the E. coli cells/medium combination were held at 37 °C in bottles sealed with septa for a given time period, usually overnight, to allow the bacterium to proliferate, after which the captured headspace was analysed directly by SIFT-MS. Several compounds were seen to be produced by the E. coli cells that depended on the liquid medium used: when cultured in DMEM, copious amounts of ethanol, acetaldehyde and hydrogen sulphide were produced; in LB ammonia is the major volatile product. In a second procedure, to ensure aerobic conditions prevailed in the cell culture, selected volatile compounds were monitored by SIFT-MS in real time for several hours above the open-to-air E. coli/DMEM culture held at close to 37 °C. The temporal variations in the concentrations of some compounds, which reflect their production rates in the culture, indicate maxima. Thus, the maxima in the ethanol and acetaldehyde production are a reflection of the reduction of glucose from the DMEM by the vigorous E. coli cells and the maximum in the hydrogen sulphide level is an indication of the loss of the sulphur-bearing amino acids from the DMEM. Serendipitously, emissions from DMEM inadvertently infected with the bacterium C. testosteroni were observed when large quantities of ammonia were seen to be produced. The results of this preliminary study suggest that monitoring volatile compounds might assist in the early detection of bacterial infection in large-scale bioreactors.     

阀切换-离子色谱法测定1,2-环氧丁烷产品中有机酸

建立了阀切换-离子色谱法测定1,2-环氧丁烷（1,2-BO）产品中有机酸（甲酸、乙酸和丙酸）的方法。样品经乙醇稀释后,通过阀切换技术,有机酸被IonPac TAC-ULP1捕集柱捕集,然后进入IonPac AS11分析柱分离,并由电导检测器检测。结果表明：甲酸、乙酸和丙酸均能实现较好地分离,在各自的范围内其色谱峰面积呈现良好的线性关系。实际样品的加标回收率为92.5%~111.8%,相对标准偏差小于5.6%（n=3）,检出限为0.60~4.80 μg/L。该方法具有前处理简单、快速、准确等特点,适用于不溶于水的有机体系中有机酸的分析。     

Novel Determination of Organic Acids in Diesel and Motor Oil by Ion Chromatography

A robust analytical method is required for the determination of low-molecular weight organic acids, which are potential causes of refinery and internal combustion engine corrosion. The ion chromatographic method developed in this study allows the determination of acetic acid and formic acid in diesel oil mixtures with a motor oil volume fraction of up to 10%. The hydrophobic matrix is automatically removed in-line through a matrix elution step with organic solvent and nonaqueous anion-exchange analyte extraction. Acetic acid and formic acid, as the smallest and most acidic aliphatic naphthenic acids, were determined. Gradient anion-exchange chromatography on high-capacity columns in combination with suppressed conductivity detection allowed their selective and sensitive determination. Typical recovery values were from 82 to 107% for each acid in the matrices evaluated with reproducibility less than 5% for acid fortified samples.     

Comparative Analysis of the Phenolic Profile of Lycium barbarum L. Fruits from Different Regions in China

Lycium barbarum L. (LB) fruits have high nutritive values and therapeutic effects. The aim of this study was to comprehensively evaluate the differences in phenolic composition of LB fruits from different geographical regions. Different methods of characterization and statistical analysis of data showed that different geographic sources of China could be significantly separated from each other. The highest total phenolic compound (TPC) content was observed in LB fruits from Ningxia (LBN), followed by those from Gansu (LBG) and Qinghai (LBQ). The Fourier transform infrared (FTIR) spectra of LB fruits revealed that LBQ had a peak at 2972 cm⁻¹ whereas there was no similar peak in LBG and LBQ. A new HPLC method was established for the simultaneous determination of 8 phenolic compounds by quantitative analysis of multiple components by a single marker (QAMS), including 4 phenolic acids (chlorogenic acid, caffeic acid, 4-hydroxycinnamic acid, and ferulic acid), 1 coumarin (scopoletin), and 3 flavonoids (kaempferol-3-O-rutinoside, rutin, and narcissoside). It was showed that rutin was the most dominant phenolic compound in LBQ, although the average content of 4 phenolic acids was also high in LBQ, and scopoletin was the richest in LBG. UHPLC-Q-TOF-MS was used to qualitatively analyze the phenolics, which showed LBN was abundant in phenolic acids, LBQ was rich in flavonoids, and coumarins were the most plentiful in LBG. In conclusion, this study can provide references for the quality control and evaluation of phenolics in LB fruits and their by-products.     

Production of fumaric acid using rice bran and subsequent conversion to succinic acid through a two-step process

The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated. Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source. The effective concentration of rice bran to produce fumaric acid was 5 g/L. A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing appro × 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.     

High-performance liquid chromatography determination of phenyllactic acid in MRS broth

Phenyllactic acid (PLA) is an organic acid produced by some strains of lactic acid bacteria (LAB) and concentrations higher than 7.5 mg/ml inhibit growth of moulds and yeasts. Since PLA can be used to select LAB, a rapid, simple and cheap method for its determination is desirable. Typical methods for its analysis in broth are time-consuming, analytically complicated, and have poor recoveries. Herein we propose a simple and rapid method that does not require extraction, but only microfiltration of broth before injection in HPLC. The improved chromatographic conditions allow separation and quantification of PLA with a recovery of 98.7%. The method is highly reproducible with an intraday repeatability of the total peak area of 2.00%, while an interday repeatability of 2.69%.     

离子色谱法测定水中4种有机酸

建立了水样中甲酸、乙酸、丙烯酸和甲基丙烯酸的离子色谱测定方法,采用IonPac AS11-HC阴离子分离柱,以KOH为淋洗液,采用浓度梯度洗脱,可同时测定上述4种有机酸。方法对实际水样中甲酸、乙酸、丙烯酸和甲基丙烯酸的平均加标回收率在80.2%~103.3%,相对标准偏差在5.8%之内,检出限分别为:0.005,0.003,0.005和0.004mg/L;定量下限分别为:0.020,0.012,0.020和0.016mg/L。方法准确、简便、环保,能够满足实际水样的测定需要,且水样中常见的阴离子不会对目标污染物的测定产生影响。     

Quantification of small molecule organic acids from Mycobacterium tuberculosis culture supernatant using ion exclusion liquid chromatography/mass spectrometry

Quantitative analysis of cellular small molecule organic acids of intermediary metabolism can provide critical insight into bacterial metabolic pathways. The concentration of these metabolites in culture supernatant varies at different growth stages or under particular environmental conditions reflecting both the energy and the biosynthetic needs, yielding metabolic information about the microorganism. The method described here utilizes ion exclusion chromatography with formic acid, coupled with a mass-selective detector using selective ion monitoring with negative mode electrospray ionization (SIM ES-), to detect and quantify several small organic acids in culture supernatants. The microM limits of quantitation (LOQs) were found to be 5.5 +/- 0.9 for pyruvate, 7.0 +/- 0.4 for malate, 2.5 +/- 0.5 for succinate, 12.7 +/- 0.8 for lactate, and 6.6 +/- 0.2 for fumarate. The method was used to detect and quantify these acids in the culture supernatants from Mycobacterium tuberculosis. Supernatant samples were spiked with stable-isotope-labeled internal standards, and the organic acids were quantified by isotope ratiometry.     

Novel approach for the rapid determination of water-soluble organic acids in wine by co-electroosmotic flow capillary zone electrophoresis

An innovative protocol for the fast analysis of some organic acids in red wine by co-electroosmotic capillary zone electrophoresis and indirect UV detection using hexadimethrine bromide (HDB) as coating agent was proposed. The adsorption of HDB onto the capillary wall provided a stable electroosmotic flow and separation of small anions was carried out using background electrolytes containing no polymer additive. Low RSD% values (<3.6%) in terms of migration times and effective mobilities were obtained from the analysis of a mixture of nitrate and nitrite and of a mixture of organic acids. An experimental design approach was used to investigate the effects of temperature, separation voltage, and percentage of methanol added to the running buffer solution on the separation of the analytes. A faster method allowing the separation of the organic acids involved in the malolactic fermentation of wine was developed. Using a running electrolyte consisting of 35% (v/v) methanol in a solution of 22 mM benzoic acid at pH 6.10 adjusted with 1.0 M TRIS-base buffer, the separation of tartaric, malic, succinic, acetic, and lactic acids was feasible in less than 210 s. Application of the method to the quantification of the above-mentioned organic acids in Italian red wine samples is reported.     

HPLC‐DAD methodology for the quantification of organic acids,furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo‐diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250×4.6 mm, 5 μm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan‐3‐ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans‐resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n=5) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rosé and fortified wines.     

Use of high performance liquid chromatography in defining the abnormalities in the free amino acid patterns in the cerebrospinal fluid of patients with aseptic meningitis.

Free amino acids were quantitatively determined in cerebrospinal fluid (CSF) and plasma samples from patients with aseptic meningitis by a newly developed high performance liquid chromatographic (HPLC) method. The method of analysis was based on precolumn derivatization of orthophthaladehyde in the presence of 2-mercaptoethanol and detection was made at Eex = 340 nm and Eem = 450 nm. The method was sensitive and the limit for detection was less than 1 pmol for most of the amino acids. It took 45 min to separate 26 amino acids with highly reproducible results, giving a coefficient of variance for retention times and integrated areas less than 0.4% and 2%, respectively, after five replicate runs. The results accumulated in 10 patients were compared statistically with 11 age-matched healthy controls. Among the amino acids almost all the neurotransmitter candidates, such as aspartic acid, glutamic acid, glutamine, glycine, tyrosine, phenylalanine and gamma-aminobutyric acid (GABA), were significantly increased in the patients' CSF, whereas arginine and threonine were low. No change was observed in plasma amino acids in patients as compared to healthy controls. The higher levels of most of the neurotransmitters, especially GABA, aspartic acid and glutamic acid, could be used diagnostically in assessing the progression and remission in aseptic meningitis.     

Measurements of low-molecular-mass carboxylic acids in atmospheric aerosols by capillary electrophoresis

Capillary electrophoresis (CE) methods for the determination of low-molecular-mass (LMM) carboxylic acids in airborne particular matter have been developed. The separations of 22 LMM carboxylic acids, including acids derived from the oxidation of biogenic hydrocarbons, are performed using a background electrolyte consisting of 3.0mM 2,6-naphthalenedicarboxylic acid and 18.0mM 2,2-bis (hydroxymethyl)-2,2',2"-nitrilotriethanol (Bis-tris) in 16% (v/v) 1-propanol within 10 min. Using a combination of a buffer mixed with an organic solvent and electroosmotic flow modifier, a minimum of peak overlaps is achieved with migration time variation of less than 1% and peak area ratio (relative to an internal standard) variation of less than 5% within 1 day. The detection limits for the aliphatic LMM acids that can be determined by this method are in the range of 30-140 micro g/L. Furthermore, a simple method for efficient extraction of LMM organic acids from particulate atmospheric matter collected on quartz fiber filters using high-volume samplers is developed. Combining the extraction procedure with a reduction of the extract to approximately 0.2 mL allows for the measurement of LLM in atmospheric particulate organic matter at concentrations well below 1 ng.m(-3). Repeat analysis of filters collected in tunnels, urban, suburban, and forested areas demonstrate that the procedure allows for measurements of aliphatic and aromatic LMM acids within a variability of 10-25%.