Rapid high-performance liquid chromatographic method for Vitamin C determination in human milk versus an enzymatic method

Vitamin C is an antioxidant that can be considered a possible biomarker of oxidative stability in human milk. A high-performance liquid chromatographic method was developed and validated for determining the total Vitamin C (ascorbic acid and dehydroascorbic acid) and ascorbic acid levels in human milk. This method was then compared with an enzymatic method (a Colorimetric technique) for quantifying ascorbic acid levels. Repeatability and reproducibility were acceptable for all methods. However, the high-performance liquid chromatography (HPLC) technique provided more satisfactory results than the enzymatic method due to this last method detected 37% less ascorbic acid and does not determine the total Vitamin C because of the enzymatic method cannot reduce the dehydroascorbic acid (DHA) to ascorbic acid. Furthermore, the HPLC method has the added advantages that it requires less reagents and material, and is simpler and less time consuming than the enzymatic method. In conclusion, the drawbacks of this enzymatic method would justify its substitution for a HPLC method.     

抗坏血酸在高粘度聚乙烯醇胶体溶液中分散均一性考察

目的:考察了采用实验室常用的磁力搅拌法和改进的机械搅拌法后,聚乙烯醇胶体溶液中抗坏血酸的分布均一度。接着,在机械搅拌法的基础上,研究了抗坏血酸粉末与抗坏血酸溶液在胶中分散的均一性。方法:分散混合方法:机械搅拌法,磁力搅拌法;均一性检验方法:以单位质量胶载药量RSD值作为指标;统计学分析方法:t检验。结果:采用磁力搅拌法不能够较好地混合抗坏血酸与PVA胶体溶液;而机械搅拌法RSD值控制在10%以内,说明胶中抗坏血酸均一分布,符合生产要求。T检验表明两种方法具有显著性差异。药物溶于水后分散更为均一。结论:解决药物在高粘度溶液介质中均匀分散的问题。     

Kinetics of fatty acid binding ability of glycated human serum albumin

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.     

曲酸的发酵生成与检测(综述)

本文概述曲霉发酵合成曲酸所需的各种营养因素及其对产酸力的影响,发酵工艺与菌种诱变,合成机理,曲酸的鉴定和检测方法,并对发酵工艺的发展趋势提出一些看法。     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

一种快速筛选产琥珀酸厌氧菌的方法

为筛选得到高产琥珀酸的厌氧菌株,建立了一种快速半定量分析发酵液中琥珀酸的方法。首先通过高效液相色谱法(HPLC)分析出产琥珀酸厌氧菌发酵液中主要含有琥珀酸,乳酸和乙酸,然后将发酵液经过纸层析展开后,发现琥珀酸可以和副产物乳酸,乙酸完全分开,最后用半定量的方法求出琥珀酸的含量。结果表明纸层析法简单经济,可以作为产琥珀酸厌氧菌的初筛方法。     

A simple and rapid electrophoretic method for the separation of glucuronic acid and iduronic acid

A new electrophoretic method using Titan III cellulose acetate plates has been developed for the separation and quantitation of glucuronic acid and iduronic acid. This method is quite simple, and glucuronic acid and iduronic acid can be separated within 50 min. This method was applied to the analyses of uronic acids in chondroitin sulfates A and C, and dermatan sulfate.     

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.     

端基法测定聚唾液酸平均聚合度

建立了端基法测定聚唾液酸平均聚合度的方法.采用间苯二酚比色法和丙二腈荧光法分别测定聚唾液酸中唾液酸总量和还原端唾液酸残基含量,两者之比即为平均聚合度.研究表明,在pH 9.5的硼酸缓冲液中,80℃水浴下聚唾液酸还原端唾液酸残基与丙二腈反应25 min后生成荧光物质,其荧光强度与还原端唾液酸残基含量呈线性正相关关系,线性范围在1～ 20 mg/L之间,变异系数和检出限分别为3.7％和0.36 mg/L.端基法测定大肠杆菌发酵液中聚唾液酸的平均聚合度为45.76,与高效液相凝胶色谱法比较,误差为3.2％.该法可以简便快速地测定发酵液中聚唾液酸的平均聚合度,有利于聚唾液酸生产过程分析及产品性能评估.     

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.     

微生物法生产丙烯酸的研究进展

目前,丙烯酸工业生产都是利用石油产品丙烯氧化得到,石油的不可再生性迫使人们寻找新的替代方法,利用微生物来生产丙烯酸是个有希望的选择。该文回顾了近期利用微生物法生产丙烯酸的研究和进展,介绍了利用不同原料生产丙烯酸的方法,并对微生物法生产丙烯酸的可行性进行了展望。     

两种用于测定A/C群脑膜炎球菌多糖疫苗中乳糖含量方法的比较分析

为探索一个灵敏、简便、快速的定量检测A/C群脑膜炎球菌多糖疫苗中乳糖含量的方法,对两种化学法进行了分析和比较。两种化学方法分别采用间苯二酚显色法和苦味酸显色法。间苯二酚显色法为套用唾液酸测定法,苦味酸显色法是对乳制品中乳糖含量检测方法的改良。两种方法的相关系数均大于0.99,线性良好。用间苯二酚法测定样品的回收试验检测结果平均值和变异系数分别为100.4%和3.09%,用苦味酸法测定样品的回收试验检测平均值和变异系数分别为99.9%和1.43%。这两种方法可以初步应用于疫苗中乳糖含量检测。     

Determination of Fructose in the Presence of a Large Excess of Glucose

A modification of the cysteine-sulfuric acid method of Dische and Devi¹¹⁾ is described. The modified procedure is based on the observation that fructose and glucose give different time course of color development. By this modified procedure, fructose in the presence of 100-fold excess of glucose can be determined with an error of about 10%.

Modifications of the anthrone-sulfuric acid method and the phenol-sulfuric acid method are described. By employing the principle of two-point determination of Mokrasch and by modifying the conditions for color development, fructose in the presence of 100-fold excess of glucose can be determined with an error of about 15% by the modified anthrone-sulfuric acid method. The modified phenol-sulfuric acid method also gave the same order of sensitivity and specificity to fructose as the modified anthrone-sulfuric acid method.     

Use of water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for the fluorescent determination of uronic acids and carboxylic acids

Reaction between glucuronic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group.     

Quantitative determination of pamoic acid in dog and rat serum by automated ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography

Pamoic acid is used as a counter ion to obtain long-acting pharmaceutical formulations of certain basic drugs. In order to investigate the pharmacokinetics of pamoic acid, a simple, sensitive and reliable method has been established for the quantitative determination of pamoic acid in serum from dog and rat. The method uses ion-pair solid-phase extraction followed by ion-pair reversed-phase high-performance liquid chromatograpy. The influence on recovery of the addition of different agents (tetrabutylammonium acetate, methanol, sodium hydroxide) to the serum samples prior to solid-phase extraction was studied and the analytical method was validated. The method was found to be valid for accurate, precise and selective determination of pamoic acid in the tested concentration range of 5–200 ng/ml serum. The overall performance of the HPLC method was found to be satisfactory for the purpose of determining concentrations of pamoic acid in serum samples from pharmacokinetic studies with pamoic acid in dogs and rats.     

生物法合成丙烯酸的研究进展

丙烯酸是一种重要的化工原料,被广泛应用于涂料、超吸附材料等领域。目前丙烯酸的获得主要通过丙烯氧化,但由于石油资源日渐枯竭以及生产过程造成的环境问题,利用生物质资源生产丙烯酸已成为研究热点。介绍了丙烯酸的性质及其在工业上的应用,并详细综述了生物法制备丙烯酸的研究进展。根据丙烯酸生产中是否应用传统的化工过程,将其分为半生物合成和全生物合成。半生物法主要包括乳酸化学法脱水以及丙烯腈、丙烯酰胺的生物转化;全生物法主要包括乳酸生物法脱水、3-羟基丙酸途径、糖直接发酵法以及DMSP(二甲基巯基丙酸内盐)途径。由于乳酸发酵的工艺成熟、原料易得,因此对乳酸脱水进行了重点介绍,其中生物法脱水符合可持续发展的要求,对其进行了详细介绍。同时还分析了各种方法的优缺点,探讨了利用生物质资源生产丙烯酸的研究趋势。     

A colorimetric method for the quantitation of uronic acids and a specific assay for galacturonic acid.

A method of quantitating uronic acids and uronic acids from pectin in particular is described. The method uses carbazole in 80% sulfuric acid with borate ions added. The assay is carried out at 60 degrees C. This assay has some cross reactivity with aldose sugars and must be timed precisely. A further method that is specific for galacturonic acid is also described. This method uses concentrated sulfuric acid and carbazole only. Of the biological substances tested, only formaldehyde and glyceraldehyde showed a reactivity of more than 10% that of galacturonic acid on a weight to weight basis.     

High-performance liquid chromatographic determination of phenylacetic acid in human plasma extracted with ethyl acetate

This paper describes a high-performance liquid chromatographic method with ultraviolet detection for measuring plasma phenylacetic acid. This simple and reliable method consists of an acid hydrolysis of conjugated phenylacetic acid before extraction with an organic solvent: washed ethyl acetate saturated in sodium chloride. The recovery of extraction was estimated by internal standardization with phenylpropionic acid, and validated by addition of phenylacetic acid standards. A preliminary application to plasma phenylacetic acid in patients suffering from depression is described.     

The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE-DAD

A sensitive on-line DPPH-CE-DAD method was developed and validated for both screening and determining the concentration of seven antioxidants of Reduning injection. The pH and concentrations of buffer solution, SDS, β-CD and organic modifier were studied for the detection of DPPH and seven antioxidants. By on-line mixing DPPH and sample solution, a DPPH-CE method for testing the antioxidant activity of the complex matrix was successfully established and used to screen the antioxidant components of Reduning injection. Then, antioxidant components including caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid were quantified by the newly established CE–DAD method. Finally, the total antioxidant activity and the multiple active components were selected as markers to evaluate the quality of Reduning injection. The results demonstrated that the on-line DPPH-CE-DAD method was reagent-saving, rapid and feasible for on-line simultaneous determination of total pharmacological activity and contents of multi-components samples. It was also a powerful method for evaluating the quality control and mechanism of action of TCM injection.     

游离整体细胞法工业化生产L-天冬氨酸

利用游离整体细胞催化技术在工业规模上进行Ｌ－天冬氨酸的生产,发现游离整体细胞催化技术不仅缩短了工艺流程,减少了设备投资,而且反应效率（生产效率）、生产能力、生产成本等方面均优于传统的固定化细胞法。