Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2

It was found that the bioactive compound, dibutyl phthalate, was produced by a new soil isolate Streptomyces albidoflavus 321.2. Once this active compound was recovered by ethyl acetate from the fermented broth, being possible to isolate 13.4 mg/l, it was purified by paper, silica gel column, thin layer and gas chromatography. Structure was determined by analysing UV, IR and GC-MS spectra. During analysis, such active compound showed strong activity against gram-positive and gram-negative bacteria, as well as unicellular and filamentous fungi. The antimicrobial activity of the compound was reversed by the amino acid proline. No acute toxicity was observed.     

4-hydroxyestrone,isolation and identification in human urine

Portions of pregnancy and midcycle urines were submitted to hot acid hydrolysis, extracted with benzene/ethyl acetate and the extracts washed with ascorbic acid buffer. From the remaining organic phase the catecholestrogens were removed with borate buffer and further purified on Sephadex LH-20 columns. After derivatisation 4-hydroxyestrone was separated from the isomeric 2-hydroxy compound by gas chromatography. The mass spectra of the 4-hydroxyestrone peak were identical with that of authentic 4-hydroxyestrone. After treatment of the extracts with sodium borohydride 4-hydroxyestradiol-17β was identified by GC-MS. By the addition of trace amounts of tritiated 4-hydroxyestrone a recovery of 40% was calculated. On the basis of this recovery and the peak heights of the gas chromatograms an excretion of 4 μg (midcycle) and 40 μg (pregnancy) of 4-hydroxyestrone/24 h was estimated.     

植物组织中糖与糖醇乙酰化及毛细管气相色谱分析

介绍了一种用1-甲基咪唑为溶剂和催化剂、盐酸羟胺和乙酸酐为肟化和乙酰化试剂,对植物样品中糖与糖醇进行乙酰化衍生化后的气相色谱分离和质谱鉴定的分析方法。并对糖与糖醇乙酰化影响较大的反应温度、反应时间、反应物组成和反应物浓度等条件进行了比较研究,确定了糖与糖醇乙酰化各步反应的最佳反应温度和反应时间,分析了各组分间相互作用及其用量对衍生化效率的影响。对多种糖与糖醇乙酰化产物进行毛细管气相色谱分离、FID检测及GC-MS结构鉴定。研究证明, 在合适条件下应用此方法对糖与糖醇进行乙酰化,反应完全,产物单一,能得到理想的分离、检测和定量分析效果。适用于微量植物组织中多种单糖、双糖及其糖醇的定量分析。     

Identification of Fmoc-beta-Ala-OH and Fmoc-beta-Ala-amino acid-OH as new impurities in Fmoc-protected amino acid derivatives.

During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a beta-Ala insertion mutant of the mother peptide. The source of the beta-Ala was identified as contamination of the Fmoc-Ala-OH raw material with Fmoc-beta-Ala-Ala-OH. Further studies also demonstrated the presence of beta-Ala in other Fmoc-amino acids, particularly in Fmoc-Arg(Pbf)-OH. In this case, it was due to the presence of both Fmoc-beta-Ala-OH and Fmoc-beta-Ala-Arg(Pbf)-OH. It is concluded that beta-Ala contamination of Fmoc-amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc-amino acid derivatives. The beta-Ala is often present as Fmoc-beta-Ala-OH and/or as a dipeptide, Fmoc-beta-Ala-amino acid-OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of beta-Ala-related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated beta-Ala contamination as a problem in the manufacture of the drug substance.     

乌桕桕脂中甾醇和其他成分分离鉴定

通过柱层析、薄层层析等化学方法,从乌桕桕脂中分离得到甾醇及脂肪酸酯类化合物。经气相色谱和气-质连用分析,鉴定出下列化合物：β—谷甾醇、菜油甾醇、r7—豆甾烯醇,肉豆蔻酸甲酯、肉豆蔻酸乙酯、棕榈酸甲酯、硬酯酸甲酯和硬酯酸乙酯。     

Purification and identification of N-glycolylneuraminic acid (Neu5Gc) from the holothuroidea Gumi, Cucumaria echinata.

N-Glycolylneuraminic acid (Neu5Gc), precious sialic acid which could not be synthesized by a chemical method, occurrs in the body of holothuroidea, Gumi Cucumaria echinata. Gumi contains 85% of total sialic acid, as Neu5Gc, in the body. Neu5Gc was purified from dry powder of the body using Dowex 1-x8 (HCOO* form) anion exchange chromatography after mild acid hydrolysis with 0.1 N trifluoroacetic acid. Using GC-MS and 1H-NMR spectroscopy, the purified Neu5Gc was correctly identified to be Neu5Gc. The purity of Neu5Gc was more than 99%. This is the first report of purification and identification of Neu5Gc from holothuroidea by using anion exchange chromatography, GC-MS, and 1H-NMR.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

印度獐牙菜挥发油化学成分的研究

研究了印度獐牙菜(Swertia chitayita Buch-Ham．)挥发油的化学成分。采用水蒸气蒸馏法提取、毛细管气相色谱-质谱联用技术对印度獐牙菜挥发性化学成分进行了分离,经计算机质谱谱库检索对分离的化合物进行鉴定,并用已知烃类进行标定,共确定了其中63种化合物,所鉴定出的化合物含量占全挥发油的81．36％;用峰面积归一化法测定了各化学成分的相对含量。研究结果表明,印度獐牙菜的主要化学成分为：十六烷酸乙酯(19．54％)、4-(苯甲基)哌啶(11．72％)、油酸乙酯(7．82％)、丁基化羟基甲苯(6．70％)、亚油酸乙酯(5．80％)、丁二酸二乙酯(3．21％)、3a,6a-二氢-2(3H,4H)环戊二烯并[b]呋喃酮(2．13％)等。     

Fatty Acid Metabolism in Microorganisms

During the investigation on the metabolism of azelaic acid by Micrococcus sp., it was found that the bacterium produced a large amount of keto acid (α-ketoglutaric acid) under the restricted condition for nitrogen source. The acid was identified as α-ketoglutaric acid by physico-chemical and biological methods. The mechanism of the production of α-ketoglutaric acid from azelaic acid was investigated. From the result, it was suggested that α-ketoglutaric acid production proceeded thrpugh the further oxidation of acetic acid produced from azelaic acid and that the production might be functioned by TCA cycle enzymes of the bacterium. Similarly, α-ketoglutaric acid was found to be produced remarkably from other various fatty acids.     

The sesquiterpene α-copaene is induced in tomato leaves infected by Botrytis cinerea

Abstract

Explorative experiments were done to investigate the possibility that tomato plants infected by Botrytis cinerea have a different emission of volatile organic compounds (VOC) than healthy plants. This was done by headspace analysis of volatiles emitted by detached leaves of infected and healthy plants. Principal component analysis (PCA) of GC-FID chromatograms revealed clearly separated clusters between infected and control leaves and identification of an interesting compound. In further analysis by GC-MS, the significantly distinctive component (p≤0.05) was identified as the sesquiterpene α-copaene. In previous work on herbivore damage, α-copaene was not distinctive, which may suggest that α-copaene may be specifically associated to fungal infections in tomato.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)₂ residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.     

Phenotype differentiation of three E. coli strains by GC-FID and GC-MS based metabolomics

Two mutants of E. coli with deletion of sdhAB and ackA-pta genes respectively and their wild-type strains were subjected to gas chromatography-flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS) metabolomics analysis. Intracellular metabolites of the three strains were profiled by GC-FID firstly. Methodological evaluation of the employed platform indicated that the limit of detection ranges were from 0.2 to 12.5ng for some representative metabolites and the corresponding recoveries were varied from 68.7 to 122.7%. Secondly, multivariable data analysis was applied to the acquired data sets. As expected, the three phenotypes could be easily differentiated, and the perturbed metabolite pools in the genetically modified strains were screened. Lastly, the metabolites playing key roles in the differentiation were further identified by GC-MS. It was confirmed that succinic acid and aspartic acid were similarly affected in the modified strains. But proline content was altered contrarily. Additionally, deletion of sdhAB gene also affected the growth property of relevant mutant greatly. The potential mechanism was postulated accordingly.     

Purification and spectroscopic properties of pyrene fatty acids

Pyrene fatty acids are routinely purified by silica based column chromatography and analyzed on thin-layer silica plates (H.-J. Galla et al., Chem. Phys. Lipids, 23 (1979) 239-251). Although pyrene decanoic acid runs as a single spot on thin-layer chromatography (TLC), gas-liquid chromatography (GC) of the methyl ester derivatives of a representative sample revealed four separate peaks with the major component only 92% of the total. High performance reverse phase liquid chromatography (HPLC) was used to purify pyrene decanoic acid and separate the contaminants. After two passes on a C18 reverse phase HPLC column, pyrene decanoic acid is 99.98% pure by GC analysis. Absorption, fluorescence, and NMR spectra were recorded for pyrene decanoic acid and the major impurities. The results indicate that one impurity is a C10 fatty acid with an altered aromatic moiety. Two other impurities are pyrene derivatives but their acyl chains probably are not decanoic acid.     

Characteristics of lipid fractions of larvae of the black soldier fly <Emphasis Type="Italic">Hermetia illucens</Emphasis>

The lipid fraction of larvae of the black soldier fly Hermetia illucens was shown to contain lauric acid (38.43 wt %) and its esters, azelaic and sebacic acids, and azelaic acid dibutyl ester. The dominant compound in the group of identified glycerides was lauric acid monoglyceride (0.70 wt %). Glycerides were also represented by triglycerides and diglycerides of lauric acid. Sterols were represented primarily by phytosterols (over 75%), the major of which was alpha-sitosterol (45%). The identified lipid complex composition is apparently determined by the biological characteristics of the fly Hermetia illucens and ensures antibacterial defence of larvae and stability of lipids at changing ambient temperature.     

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks

Background

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system.

Results

A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount.

Conclusions

The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization.

     

The Purity of Fluorescent Dyes: a Report Delivered to the Scientific Session of the Annual Meeting of the Biological Stain Commission

The Biological Stain Commission occasionally has been requested to certify fluorochromes as biological stains. Although formal certification is unlikely in the near future, the Commission is nevertheless concerned with the quality of these reagents. Commercial samples of fourteen fluorochromes were investigated for the presence of fluorescent organic impurities using reverse phase thin layer chromatography. Our findings suggest that some fluorochrome dyes are pure, but most are impure. Most fluorochromes vary in purity among vendors and among batches sold by single vendors. Impurities may be present at such high concentration that little of the presumed compound is present. Some impurities behave quite differently from the nominal dye. This may either create confusion or it might be useful. In the latter case, however, the impurity may occur only in a single batch. Impurities result from problems related to organic syntheses, separations, and economics. Solving those problems is often expensive, and what is expensive may not be performed. Fortunately, knowledge of synthetic chemistry often permits identification of fluorochromes likely to be impure. Moreover, predictions of likely staining effects of particular impurities can be made if appropriate structure-activity models are available. Possible actions by the Commission aimed at limiting the problems resulting from impurities of fluorescent dyes are noted.     

Comparison of Volatile Compounds from Chungkuk-Jang and Itohiki-Natto

Volatile compounds from two South-East Asian fermented soybean foods, Chungkuk-jang (CKJ) and Itohiki-natto (natto), were analysed by gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), and GC-sniffing. A total of 112 compounds were identified. A large amount of ethanol was detected from CKJ, while acetone and methyl isobutyrate were major components of natto. The characteristic odor compounds of CKJ were some ethyl esters of short chain fatty acids, diallyl disulfide, and several natto-like odor compounds were identified as ammonia, 2,5-dimethylpyrazine, and 2-methylbutanoic acid.     

化学药物杂质研究进展

药物杂质的分析和控制是用药安全有效的基础。从杂质谱分析、杂质检测、杂质限度的制定等方面综述了近年来国内外化学药 物杂质研究进展,旨在为化学药物杂质的研究提供有效思路和方法。     

四合木茎叶石油醚提取物化学成分分析

利用GC-MS法对四合木茎叶的石油醚提取物进行了成分鉴定。鉴定出40种化合物,包括烷烃类化合物8种、羧酸类化合物11种、酯类化合物9种、萜类化合物2种、甾体类化合物2种、醇类化合物4种,占样品总量的80.5%。其中相对含量最高的3种成分依次为油酸(15.33%)、β-谷甾醇(9.12%)和十八碳二烯酸甲酯(7.24%)。β-谷甾醇可能是四合木具有抗虫性的主要成分。