烟叶中β -胡萝卜素的分离纯化和测定

采用直接柱色谱的方法,用石油醚单一溶剂作为洗脱液,从霉烟或碎烟末浸膏中提取了β- 胡萝卜素纯品。通过 1H-NMR和 13 C-NMR谱图鉴定了β -胡萝卜素的结构。用高效液相色谱法(HPLC)测得β -胡萝卜素纯品的纯度为98%,测定方法准确性高、重现性好,β-胡萝卜素纯品的红外光谱图和熔点均与文献值相符。利用该方法提取β -胡萝卜素纯品,不仅收率达到80%,纯度好,而且单一溶剂作为洗脱液很容易回收再利用,因此说该方法是一种有价值的生产天然β-胡萝卜素的方法,具有良好的工业应用前景。     

食用香料甲基2-甲基-3-呋喃基二硫醚的分离提纯及结构鉴定

 用气相色谱法分离提纯食用香料甲基 2 甲基 3 呋喃基二硫醚 ,纯度 (质量分数 )达到 99.5%。分离柱为4%FFAP +1 %H3PO4ChromosorbW /AW DMCS(80目～ 1 0 0目 )不锈钢柱。用红外光谱、气相色谱 /质谱和核磁共振谱进行了结构鉴定。     

藏药镰形棘豆中2',4'-二羟基查尔酮对照品的制备和鉴定

采用乙醇提取、乙酸乙酯萃取、普通硅胶柱色谱和反相硅胶柱色谱分离纯化2',4'-二羟基查尔酮,采用紫外光谱、红外光谱、核磁共振波谱、质谱等技术鉴定结构,应用薄层色谱法、高效液相色谱-二极管阵列检测法、高效液相色谱-蒸发光散射检测法、超高效液相色谱-质谱联用法进行纯度检测。结果从镰形棘豆中分离得到纯度大于99.0%的2',4'-二羟基查尔酮。表明该制备方法简便,制备量大,所制得的2',4'-二羟基查尔酮纯度高,可作为镰形棘豆药材及其复方药的质量控制的对照品。     

土豆叶中茄尼醇的高速逆流色谱法分离纯化及质谱解析

采用超微回流提取方法提取土豆叶中的茄尼醇,用高速逆流色谱(HSCCC)对粗提物中的茄尼醇进行分离纯化。以正己烷-甲醇(体积比为10∶7)作为两相溶剂系统,以其下相为流动相,上相为固定相,经过一步HSCCC从60 mg茄尼醇粗提物中分离得到了5 mg 纯度为98.7%的茄尼醇;对分离得到的茄尼醇进行大气压化学电离质谱解析,研究了茄尼醇的大气压化学电离质谱的一级电离规律和二级质谱裂解规律。     

大孔阴离子树脂分离纯化微生物转化液中5-氟尿苷的研究

探寻从微生物转化液中分离纯化5-氟尿苷(5-FUR)的工艺条件。先后尝试了大孔吸附树脂、大孔弱碱性阴离子交换树脂以及硅胶柱色谱的分离方法,首先利用大孔弱碱性阴离子交换树脂吸附,然后用1mol/L NaCl动态洗脱得到含量较高的两种含氟化合物,最终通过硅胶柱色谱,采用正己烷-乙酸乙酯(含10%醋酸)为流动相进行洗脱,得到了纯度为100%的纯品。在目标化合物和杂质理化性质极其相似,含量极其悬殊的条件下,成功去除含量极高的杂质,实现了5-FUR的分离纯化,圆满地达到了预期的目的。     

高效液相色谱法制备胡黄连中胡黄连甙Ⅱ的对照品

运用高效液相色谱法(HPLC)制备了胡黄连中胡黄连甙Ⅱ的对照品,色谱条件为C 18制备柱(300 mmX20 mm i.d., 10 μm),流动相为甲醇-水(体积比为1∶1),流速8 mL/min,265　nm紫外检测,进样量100　μL。用HPLC和毛细管区带电泳法(CZE)测定了自制对照品的纯度,结果表明,自制对照品的纯度在99.0%以上,稳定性好, 且2种方法测定的结果具有可比性。此制备方法克服了以前用经典柱色谱方法分离 纯化过程中,胡黄连甙Ⅱ不稳定,操作繁琐和纯度低的缺点。     

硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物

采用硅胶柱色谱结合高速逆流色谱法分离纯化了荷花中3种黄酮类化合物。荷花粗提物先经过硅胶柱色谱初步分离,得到黄酮含量高的组分,再经过高速逆流色谱分离,以乙酸乙酯-乙醇-水-乙酸(4:1:5:0.025, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相,在主机转速800 r/min、流速2.0 mL/min、检测波长254 nm条件下,从150 mg样品中一次性分离制备得到6.1 mg槲皮素-3-O-β-D-葡萄糖醛酸苷(I), 14.8 mg杨梅素-3-O-β-D-葡萄糖苷(II)和20.2 mg紫云英苷(III),经高效液相色谱检测其纯度分别为97.0%、95.4%、96.3%,并通过质谱和核磁共振氢谱、碳谱鉴定各化合物的结构。该方法简便、快速、节省溶剂,可以对荷花中的黄酮类化合物进行快速有效的分离纯化,具有较好的实用价值,为荷花资源的进一步开发应用提供了参考依据。     

反相高效液相色谱法测定油橄榄中山楂酸含量

以枇杷叶为原料采用高效液相制备色谱技术制备了纯度达98.91%的山楂酸对照品,并用作制备标准曲线。油橄榄样品用乙醇-水(80+20)溶液90℃索氏回流提取24 h,在KromasailC_(18)色谱柱(250 mm×4.6 mm,5μm)上分离,以甲醇-1%(体积分数)乙酸(88+12)混合溶液为流动相,流量1.0 mL·min~(-1),在215 nm波长处进行测定。此法用于油橄榄样品中山楂酸含量的检测,并在实样基础上加入山楂酸标准作方法的回收及精密度试验,测得平均回收率为98%,相对标准偏差(n=5)为1.21%。     

固相萃取-高效液相色谱法同时测定羊肉制品中9种杂环胺

建立了同时测定羊肉制品中9种杂环胺的固相萃取-高效液相色谱(SPE-HPLC)分析方法。样品经2 mol/L NaOH超声提取、80 mL二氯甲烷液-液萃取,利用阳离子交换柱(MCX柱)净化和富集后进行HPLC分析。采用反相C18色谱柱,以乙腈和0.01 mol/L磷酸溶液(用三乙胺调整pH 3.6)为流动相对杂环胺进行梯度洗脱,使用二极管阵列检测器(DAD)分别在228 nm(2-氨基-9H-吡啶并［2,3-b］吲哚(AaC)、2-氨基-3-甲基-9H-吡啶并［2,3-b］吲哚(MeAaC))、253 nm(2-氨基-3-甲基咪唑并［4,5-f］喹啉(IQ)、1-甲基-9H-吡啶并［3,4-b］吲哚(Harman)、2-9H-吡啶并［3,4-b］吲哚(Norharman))、263 nm(2-氨基-3,8-二甲基咪唑并［4,5-f］喹喔啉(MeIQx)、2-氨基-3,4,8-三甲基咪唑并［4,5-f］喹喔啉(4,8-DiMeIQx)、3-氨基-1-甲基-5H-吡啶并［4,3-b］吲哚(Trp-p-2))、321 nm(2-氨基-1-甲基-6-苯基咪唑并［4,5-b］吡啶(PhIP))处进行检测。实验结果表明,9种杂环胺分离效果良好,回收率为50.27%～94.77%(n=6),相对标准偏差为0.08%～4.42%;通过全波长扫描,确定检出限(以信噪比(S/N)=3计)为1.6～41.0 μg/L。该方法操作简单,结果准确,重现性好,可用于同时检测羊肉制品中9种杂环胺的含量。     

高速逆流色谱在分离纯化木蝴蝶活性成分中的线性放大

利用高速逆流色谱分离纯化中草药木蝴蝶乙酸乙酯粗提物中的黄酮类活性成分,并将分离规模从分析型线性放大到制备型,以获得大量的活性成分,为进一步的药物筛选提供物质基础。实验在分析型高速逆流色谱上对分离参数进行了系统优化,并将优化条件放大到制备型高速逆流色谱上对911.6 mg木蝴蝶乙酸乙酯粗提物进行分离,得到5种化合物,经高效液相色谱、电喷雾电离质谱和核磁共振氢谱、碳谱分析鉴定,分别为白杨素(160.9 mg,纯度为97.3%)、黄芩素(130.4 mg,纯度为97.6%)、黄芩素-7-O-葡萄糖苷(314.0 mg,纯度为98.3%)、黄芩素-7-O-双葡萄糖苷(179.1 mg,纯度为99.2%)和一种新的白杨素双葡萄糖苷(21.7 mg,纯度为98.8%)。该放大过程不仅将处理量提高了53倍,还保持了在分析型设备上的分离度和分离时间。该工作为天然产物的研究提供了一个高效的分离纯化方法。     

Gas chromatographic-mass spectrometric determination of two new antimycotic agents, 1-[(5-chloro-2-benzofuranyl)(2-chlorophenyl)methyl]-1H-imidazole and 1-[(5-bromo-2-benzofuranyl)phenylmethyl]-1H-imidazole, in rabbit plasma following topical administration: a preliminary comparison with bifonazole

A method for the analysis of the antimycotic drugs 1-[(5-chloro-2-benzofuranyl)(2-chlorophenyl)methyl]-1H-imidazole, 1-[(5-bromo-2-benzofuranyl)phenylmethyl]-1H-imidazole and bifonazole in rabbit plasma, employing gas chromatography-mass spectrometry with selected-ion monitoring, was developed. The procedure involved single-step purification of the biological matrix via liquid-liquid extraction on Extrelut columns and use of a carrier substance to minimize the negative effects of adsorption sites during the gas chromatographic process. The limits of detection ranged from 0.1 to 1.4 ng/ml, starting from a 200-microliter sample. The method was applied to a preliminary evaluation of percutaneous absorption of both drugs in the rabbit after a single administration, in comparison with bifonazole.     

Microfluidic reactions using [11C]carbon monoxide solutions for the synthesis of a positron emission tomography radiotracer

Microfluidic technology has been used to perform [(11)C]carbonylation reactions using solutions containing [(11)C]CO in the form of the complex, copper(i)tris(3,5-dimethylpyrazolyl)borate-[(11)C]carbonyl (Cu(Tp*)[(11)C]CO). The synthesis of the model compound [(11)C]N-benzylbenzamide and the known tracer molecule [(11)C]trans-N-[5-(2-flurophenyl)-2-pyrimidinyl]-3-oxospiro[5-azaisobenzofurane-1(3H),1'-cyclohexane]-4'-carboxamide ([(11)C]MK-0233), a ligand for the neuropeptide Y Y5 receptor, have been performed using this technique. Following semi-preparative HPLC purification and reformulation, 1262 ± 113 MBq of [(11)C]MK-0233 was produced at the end of the synthesis with a specific activity of 100 ± 30 GBq μmol(-1) and a >99% radiochemical purity. This corresponds to a decay corrected radiochemical yield of 7.2 ± 0.7%. Using a 3 mL vial as the reaction vessel, and following semi-preparative HPLC purification and reformulation, 1255 ± 392 MBq of [(11)C]MK-0233 was produced at the end of the synthesis with a specific activity of 100 ± 15 GBq μmol(-1) and a >99% radiochemical purity. This corresponds to a decay corrected radiochemical yield of 7.1 ± 2.2%.     

Separation and purification of glucosinolates from crude plant homogenates by high-speed counter-current chromatography

Glucosinolates are anionic, hydrophilic plant secondary metabolites which are of particular interest due to their role in the prevention of cancer and other chronic and degenerative diseases. The separation and purification of glucosinolates from a variety of plant sources (e.g. seeds of broccoli, arugula and the horseradish tree), was achieved using high-speed counter-current chromatography (HSCCC). A high-salt, highly polar system containing 1-propanol-acetonitrile-saturated aqueous ammonium sulfate-water (1:0.5:1.2:1), was run on a semi-preparative scale and then transferred directly to preparative scale. Up to 7 g of a concentrated methanolic syrup containing about 10% glucosinolates was loaded on an 850-ml HSCCC column, and good separation and recovery were demonstrated for 4-methylsulfinylbutyl, 3-methylsulfinylpropyl, 4-methylthiobutyl, 2-propenyl and 4-(rhamnopyranosyloxy)benzyl glucosinolates. Multiple injections (5 to 6 times) were performed with well-preserved liquid stationary phase under centrifugal force. Pooled sequential runs with broccoli seed extract yielded about 20 g of its predominant glucosinolate, glucoraphanin, which was produced at > 95% purity and reduced to powdered form.     

Use of a fast protein electrophoretic purification procedure for N-terminal sequence analysis to identify S-locus related proteins in stigmas of Brassica oleracea.

In the cruciferous plant Brassica oleracea L. (cabbage), the S-locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self-fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which another member is expressed, the S-locus related gene (SLR1gene). Our aim was to determine whether the SLR1-gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1-proteins by Con A-peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N-terminal amino acid determination. By comparison of the N-terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1-cDNA, we demonstrate the expression of SLR1-like proteins in stigmas of B. oleracea.     

Analysis of Processing Effects on Glucosinolate Profiles in Red Cabbage by LC-MS/MS in Multiple Reaction Monitoring Mode

Red cabbage (Brassica oleracea L. var. capitata) continues to receive increasing attention on its health-promoting properties because of its high glucosinolate content. Glucosinolates are an unstable active substance; however, there are few studies on their changes in different cooking processes. In this study, we investigated the effects of processing methods (boiling, steaming, microwave heating, frying, stir-frying) and boiling time on glucosinolates in red cabbage. Ten glucosinolates, including 4-methoxyglucobrassicin, neoglucobrassicin, glucoalyssin, glucobrassicin, glucoraphanin, glucoiberin, progoitrin, gluconapin and sinigrin, in red cabbage were detected. Decreases of 32.36%, 24.83%, 25.27%, 81.11% and 84.29% for total glucosinolates were observed after boiling, microwaving, steaming, frying and stir-frying. Indole glucosinolates were more efficiently lost compared to aliphatic glucosinolates after boiling, while microwaving, steaming, frying and stir-frying also resulted in a greater reduction in indole glucosinolates than aliphatic glucosinolates. Glucoalyssin, glucoerucin and sinigrin were more thermal sensitive than other glucosinolates. It was confirmed that microwaving and steaming retained higher levels of glucosinolates than other methods and may be better for cooking red cabbage.     

Rapid quality control analysis of (13)C-enriched substrate synthesis by isotope ratio mass spectrometry

There is a growing interest in the use of (13)C-enriched substrates to investigate metabolic processes in humans. The non-invasive nature of (13)C breath tests makes them attractive to clinicians, particularly because they can be safely used in children. The availability of suitable (13)C-enriched substrates can limit the application of this biotechnology. We have used isotope ratio mass spectrometry to assay the chemical purity and isotopic enrichment of substrates that were synthesised to study gut transit and colonic fermentation. Lactose ureide and lactose [(13)C]ureide were synthesised by acid-catalysed condensation of lactose and urea or (13)C urea, respectively. Glucose ureide and glucose [(13)C]ureide were synthesised by similar methods but required an additional purification step to remove urea of crystallisation. Substrates were analysed by standard analytical techniques and combustion isotope ratio mass spectrometry for carbon and nitrogen content and (13)C-enrichment. Monitoring the C/N ratio proved to be a sensitive assay of chemical purity. Analysis of the percentage composition of C and N (and hence O + H) suggested that lactose ureide crystallises as the dihydrate. It was synthesised with approximately 99% chemical purity and with the theoretical enrichment. Glucose ureide was synthesised with approximately 98% chemical purity but with lower than theoretical enrichment.     

Extraction and analysis of intact glucosinolates--a validated pressurized liquid extraction/liquid chromatography-mass spectrometry protocol for Isatis tinctoria, and qualitative analysis of other cruciferous plants

Glucosinolates have attracted significant interest due to the chemopreventive properties of some of their transformation products. Numerous protocols for the extraction and analysis of glucosinolates have been published, but limited effort has been devoted to optimize and validate crucial extraction parameters and sample preparation steps. We carried out a systematic optimization and validation of a quantitative assay for the direct analysis of intact glucosinolates in Isatis tinctoria leaves (woad, Brassicaceae). Various parameters such as solvent composition, particle size, temperature, and number of required extraction steps were optimized using pressurized liquid extraction (PLE). We observed thermal degradation of glucosinolates at temperatures above 50 degrees C, and loss of >60% within 10min at 100 degrees C, but no enzymatic degradation in the leaf samples at ambient temperature. Excellent peak shape and resolution was obtained by reversed-phase chromatography on a Phenomenex Aqua column using 10mM ammonium formate as ion-pair reagent. Detection was carried out by electrospray ionisation mass spectrometry in the negative ion mode. Analysis of cruciferous vegetables and spices such as broccoli (Brassica oleracea L. var. italica), garden cress (Lepidium sativum L.) and black mustard (Sinapis nigra L.) demonstrated the general applicability of the method.     

Isolation,identification, biological estimation,and profiling of glucosinolates in Isatis indigotica roots

Abstract

Four main glucosinolates (progoitrin, epiprogoitrin, gluconapin, and neoglucobrassicin) were isolated and identified from Isatis indigotica roots (Isatidis Radix). Among these, neoglucobrassicin was the first indole glucosinolate isolated from this herb, and its structure was elucidated by NMR, MS, and X-ray diffraction analysis. The glucosinolate components of Isatidis Radix have been profiled by combined use of a conventional preparative column chromatography method and a new UHPLC-QTOF-MS/MS method, and validated by simultaneous analysis of intact and desulfurized GLS. A total of 16 glucosinolates (GLS) including 12 aliphatic GLS, two aromatic GLS, and two indole GLS were identified on the basis of their UV profiles, MS/MS data, and/or by comparison with reference substances. Among these nine GLS were reported from I. indigotica for the first time. The total GLS, four isolated GLS, and (R,S)-goitrin (a degraded product of progoitrin and epiprogoitrin) have been estimated in vitro for anti-influenza viral, anti-bacterial, and anti-inflammatory activities. All the glucosinolate samples were inactive against H3N2 virus and four pathogenic bacteria, and two previously unnoticed GLS (gluconapin and neoglucobrassicin) showed significantly anti-inflammatory activity against the respiratory burst of mouse macrophages.     

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.     

Determination of riboflavin in pharmaceutical products by automatic discrete-sample analysis

A procedure for the determination of iron, copper, nickel, cobalt, manganese and chromium down to 0.01 μg g^-1 in sodium calcium silicate glass, sodium borosilicate glass, sodium carbonate and calcium carbonate is described. The analytical procedure depends on the separation at pH 6 of the metal diethyldithiocarbamates into isobutyl methyl ketone, and their determination by flameless atomic absorption spectrometry, with a Massmann-type graphite furnace. The limiting factors on the detection limits attainable are discussed and related to the purity of the acids used for sample solution, sample contamination during chemical separation and the sensitivity of the analytical technique.