Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC

Since the discovery of multiple bioactivities for agarobiose oligomers, a quantitative method has been in great need to monitor the agarobiose oligomers. This report demonstrates that agarobiose oligomers can be separated with high resolution in HPLC after introducing a-naphthylamine into compounds. Agarobiose oligomers ranged from biose to decaose were isolated by Sephadex column. HPLC analysis indicated that each oliomer could be quantified with good linearity and a low detection limit of 0.1-4 mug/ml. The chromatographic profiles of agaro-oligosaccharides with different hydrolysis modes (hydrochloride, citric acid, solid acid, and hydroxyl radical degradation) showed that agarobiose could be obtained more than 57.8% using solid acid mediated hydrolysis, while hydrochloride acid could degrade agar into a series of agaro-oligosaccharides from biose to decaose. The yield of oligosaccharides was low if hydrolyzed by citric acid. The Fenton degradation can increase the speed of hydrolysis, but the product was complex. (C) 2004 Elsevier B.V. All rights reserved.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (> 16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

A modified HPLC method for analysis of PSP toxins in algae and shellfish from China

Improvements to an established HPLC method are introduced. The modified method is more efficient for separation and detection of the toxins responsible for paralytic shellfish poisoning (PSP). The PSP toxin content of two strains of Alexandrium tamarense and approximately forty shellfish samples collected from different locations in China have been analyzed with this HPLC method. Only one shellfish sample, collected from Lianyungang, China, contained PSP toxins.

An efficient combination of supercritical fluid extraction and high-speed counter-current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker-Gawler. The optimization of parameters was carried out using an orthogonal test L-9 (3)(4) including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55 degrees C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6-aldehydo-isoophiopogonone A, and 6-formyl-isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6-aldehydo-isoophiopogonone A (98.3% purity) and 13.5 mg of 6-formyl-isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI-MS and NMR analysis.

Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC

We developed an HPLC method for analysis of the monosaccharide composition of fucoidans. The fucoidan was hydrolyzed into monosaccharides with 2 mol/L trifluoroacetic acid. Using ribose as the internal standard, the monosaccharide derivatives, obtained with 1-Phenyl-3-methyl-5-pyrazolone (PMP), were separated by reverse-phase HPLC using a gradient elution process, and monitored by ultraviolet detection at 245 nm. In the concentration range of 0.1-2.0 mmol/L, the peak area of each monosaccharide had a good linear relationship with its concentration (r(2)> 0.998). The average recoveries of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose were 86.2%, 95.1%, 62.5%, 102.0%, 94.8%, 66.6%, and 105.1%, respectively. This method was accurate and had good reproducibility and could be used to determine the monosaccharide contents of fucoidans.

藏药达乌里秦艽中环烯醚萜苷的HPLC测定

采用HPLC法同时测定了藏药达乌里秦艽中龙胆苦苷(gentiopicroside)、獐牙菜苦苷(swertiamarin)、獐牙菜苷(sweroside)和落干酸(loganic acid)4种主要环烯醚萜苷类成分,利用在线质谱(MS)进行了定性分析,并与同属藏药麻花艽进行了比较。色谱柱为Eclipse XDB-C_8(4.6 mm×150 mmi.d.,5μm),流动相A:体积分数5%乙腈(含10 mmol甲酸)水溶液,流动相B:体积分数95%乙腈水溶液,以流动相A在0～15 min内由100%线性减至90%,15～20 min内保持90%不变线性梯度洗脱。流速1.0 mL/min,检测波长240 nm,柱温30℃。结果表明,4种成分均达到了基线分离,具有较好的线性关系和回收率。本法可作为达乌里秦艽药材有效成分的测定方法,为其有效成分的进一步开发利用提供依据。

RP-HPLC测定几种花类药材黄酮含量

目的：为充分利用花类药材，建立马蔺花、桃花、月季等15种花类药材中黄酮含量测定的RP—HPLC方法，并比较它们的含量。方法：花类药材用甲醇提取，反相高效液相色谱法分析药材中槲皮素、山柰酚、异鼠李素3种黄酮苷元。结果：采用KromasilC_18柱（4．6mm×250／mm，5μm），以甲醇-0．1％磷酸（50：50）为流动相，可使3种黄酮苷元达到基线分离，其中马蔺花含槲皮素1．536％，桃花含山柰酚0．572％，茶花含异鼠李素0．029％，皆为听测样品中最高。结论：本实验首次采用RP—HPLC法测定这15种花类药材的总黄酮含量，本方法操作简便，快速，准确，可作为黄酮的定量分析方法。

青海栽培和野生唐古特大黄蒽醌类成分的HPLC对比分析

采用HPLC法测定青海栽培唐古特大黄中的5种蒽醌含量,并和野生唐古特大黄药材进行了比较.结果表明,二、三、四年龄栽培唐古特大黄中芦荟大黄素、大黄酸、大黄素、大黄酚、大黄素甲醚5种蒽醌总量分别为1.21%,2.01%,1.62%,其中三、四年龄栽培唐古特大黄已达到<药典>规定的药用标准;野生大黄的总蒽醌含量远高于栽培大黄为3.64%.

藏药提宗龙胆和线叶龙胆花中四种苦苷类成分的HPLC测定

应用HPLC分析方法同时测定藏药提宗龙胆和线叶龙胆两种植物花中落干酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷4种苦苷类成分的含量.采用Econosphere C_(18)色谱柱(250×4.6 mm,5 μm),以甲醇-0.5%乙酸为流动相进行梯度洗脱,流速为1.0 mL/min;检测波长为245 nm.结果表明,除提宗龙胆中未检出獐牙菜苦苷外,其它成分均在两种植物中存在,但含量存在一定的差异.

藏药短管兔耳草中松果菊苷和麦角甾苷的RP-HPLC分析

建立了藏药短管兔耳草中松果菊苷和麦角甾苷含量的高效液相色谱分析法.采用Waters XTerra RP18色谱柱(150mm×4.6 mm,5μm),以甲醇-1%冰醋酸溶液(28:72,V:V)为流动相,流速1.0 mL/min,检测波长330 nm,柱温30℃,在20 min内分离检测了该两种化合物.松果菊苷和麦角甾苷进样量分别在0.077～4.950μg(r=0.999 9)和0.085～5.450μg(r=0.999 9)内呈良好线性,平均加样回收率分别为98.35%和92.50%,RSD分别为2.35%和2.86%.所建立的方法简便、快捷、结果准确可靠,重现性好,可用于藏药短管兔耳草的质量控制,并为兔耳草属植物中苯丙素苷类化合物的分离分析提供一定的参考.

抱茎獐牙菜中当药黄素的HPLC测定研究

[目的]采用反相高效液相色谱法对抱茎獐牙菜中当药黄素的含量进行测定。[方法]色谱柱为Kromasil C18（250min×4．60mm，5μm），流动相为甲醇-浓度0．02％磷酸水溶液，梯度洗脱，流速为1ml／min，检测渡长为260nm。[结果]当药黄素在0．92-4．60μg范围内线性关系良好，r=0．99950平均加样回收率为98．73％，RSD为0.31％。[结论]抱茎獐牙菜中当药黄素在花的部位含量最高。该测定方法适应性广，可用于测定抱茎獐牙菜中的当药黄素。

HPLC测定藏药帕朱胶囊中胡椒碱

帕朱胶囊是藏医最常用的治疗胃寒性疾病药物之一。收载于《中华人民共和国卫生部药用标准•藏药》（第一册），主要由寒水石、河子、石榴子、胡椒和荜茇等药物组成；功能主治健胃散寒，除痰、破痞瘤，养荣强壮。现代临床试验表明，帕朱胶囊对中晚期肿瘤患者有改善细胞免疫及体液免疫功能的作用，同时还可以减轻患者疼痛症状。尽管帕朱胶囊长期以来被广泛使用，但是却没有很好的质量控制标准。我们采用超临界CO2萃取方法提取帕朱胶囊的可溶性成分，并用GC—MS法对提取部位进行化学成分分析，发现其主要成分为胡椒碱，含量占提取物的44，2％。因此，本实验用HPLC法对帕朱胶囊中所含胡椒碱进行测定，以期建立胡椒碱的科学检测方法，提高产品质量控制标准。

藏药独一味RP-HPLC色谱指纹图谱的建立及其质量评价

[目的]建立独一味药材HPLC指纹图谱，研究不同地区独一味药材的质量，为其质量控制提供有效的方法。[方法]采用RP-HPLC方法梯度洗脱，测定10批不同地区的独一味样品。[色谱备件]Kromasil C18柱（250mm×4．60mm，5μm），流动相为甲醇-0．4％的磷酸水溶液（55：45），梯度洗脱程序为0～70min，甲醇的体积分数由1％线性增加至15％；70～100min内，甲醇由15％线性增加至17％；100～130min内，甲醇由17％线性增加至20％；130～150min，甲醇由20％线性增加至100％；流速为1ml/min，检测波长为254n／n，柱温为30℃。[结果]确定了独一味药材中的7个共有峰。根据聚类分析结果，将独一味药材分为3类。[结论]该研究建立的方法重复性好，可用于不同地区独一味药材的质量评价，结合含量测定可用于独一味药材的质量的全面控制。

HPLC-APCI-MS法分析测定白刺籽油中游离脂肪酸

利用荧光衍生试剂1,2-苯并-3,4-二氢咔唑-9-乙基对甲苯磺酸酯(BDETS)作为脂肪酸柱前衍生化试剂,采用梯度洗脱在Eclipse XDB-C_8色谱柱上对游离脂肪酸(FFA)(油酸、亚油酸、软脂酸和硬脂酸)衍生物进行分离.利用柱后在线的串联质谱以大气压化学电离源(APCI)正离子模式实现了各组分的质谱定性.荧光检测的激发和发射波长分别为λ_(ex)=333 nm,λ_(em)=390 nm.脂肪酸的线性回归系数大于0.9990,检出限为3.38～6.59 nmol/L.建立的方法具有良好的重现性.利用此方法对超临界CO_2提取的唐古特白刺籽油中几种游离脂肪酸进行了分析.结果表明白刺籽油中含有大量的游离不饱和脂肪酸.

抱茎獐牙菜提取物的HPLC指纹图谱研究

目的：用高效液相色谱法建立抱茎獐牙菜乙醇提取物的指纹图谱分析方法。方法：采用Phenomenex Kromasil C18色谱柱（4．6mm×250mm，5μm）；甲醇-0．1％磷酸为流动相，线性梯度洗脱，检测波长为260nm。结果：共有20个共有峰。结论：此方法准确、重复性好，为有效地控制抱茎獐牙菜药材提取物的质量提供了科学的依据。