Simultaneous purification of minor components in natural products using twin-column recycling chromatography with a step solvent gradient

The isolation of minor components from complex natural product matrices presents a significant challenge in the field of purification science due to their low concentrations and the presence of structurally similar compounds.This study introduces an optimized twin-column recycling chromatography method for the efficient and simultaneous purification of these elusive constituents.By introducing water at a small flowing rate between the twin columns,a step solvent gradient is created,by which the leading edge of concentration band would migrate at a slower rate than the trailing edge as it flowing from the upstream to downstream column.Hence,the band broadening is counterbalanced,resulting in an enrichment effect for those minor components in separation process.Herein,two target substances,which showed similar peak position in high performance liquid chromatography(HPLC)and did not exceed 1.8%in crude paclitaxel were selected as target compounds for separation.By using the twin-column recycling chromatography with a step solvent gradient,a successful purification was achieved in getting the two with the purity almost 100%.We suggest this method is suitable for the separation of most components in natural produces,which shows higher precision and recovery rate compared with the common lab-operated separation ways for natural products(thin-layer chromatography and prep-HPLC).

Determination of Benzo[a]pyrene in Edible Oil by High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FL)

In this study, an optimized high performance liquid chromatography-fluorescence detector (HPLC-FL) method for the determination of benzo[a]pyrene in edible oil was established. HPLC was performed with Thermo Fisher Scientific C18 column (250 mm×4.6 mm, 5 μm) as the chromatographic column and acetonitrile and water as the mobile phase, and the excitation wavelength and emission wavelength of fluorescence detector were 286 and 430 nm, respectively. The response was high, and the linear range was 0.5-10.0 ng/ml. The lowest limit of detection was 0.11 ng/ml, and the average recovery was 92.5%. This method is suitable for quantitative analysis of benzo[a]pyrene content in edible oil.

Efficient and selective extraction of uranium from seawater based on a novel pulsed liquid chromatography radionuclide separation method

The novel pulsed liquid chromatography radionuclide separation method presented here provides a new and promising strategy for the extraction of uranium from seawater.In this study,a new chromatographic separation method was proposed,and a pulsed nuclide automated separation device was developed,alongside a new chromatographic column.The length of this chromatographic column was 10 m,with an internal warp of 3 mm and a packing size of 1 mm,while the total separation units of the column reached 12,250.The most favorable conditions for the separation of nuclides were then obtained through optimizing the separation conditions of the device:Sample pH in the column=2,sample injection flow rate=5.698 mL/min,chromatographic column heating temperature=60℃.Separation experiments were also carried out for uranium,europium,and sodium ions in mixed solutions;uranium and sodium ions in water samples from the Ganjiang River;and uranium,sodium,and magnesium ions from seawater samples.The separation factors between the different nuclei were then calculated based on the experimental data,and a formula for the separation level was derived.The experimental results showed that the separation factor in the mixed solution of uranium and europium(1:1)was 1.088,while achieving the initial separation of uranium and europium theoretically required a 47-stage separation.Considering the separation factor of 1.50for the uranium and sodium ions in water samples from the Ganjiang River,achieving the initial separation of uranium and sodium ions would have theoretically required at least a 21-stage separation.Furthermore,for the seawater sample separation experiments,the separation factor of uranium and sodium ions was 1.2885;therefore,more than 28 stages of sample separation would be required to achieve uranium extraction from seawater.The novel pulsed liquid chromatography method proposed in this study was innovative in terms of uranium separation and enrichment,while expanding the possibilities of extracting uranium from seawater through chromatography.

Modified reversed-phase high performance liquid chromatography for the determination of leukotriene B_4 and 5-hydroxyeicosatetraenoic acid in peripheral neutrophils of rabbits

Modification was made on the reversed-phase high performance liquidchromatography（RP-HPLC）with Yue et al’s method as a base.The modified RP-HPLCwas used to detect leukotriene B4（LTB4）and 5-hydroxyeicosatetraenoic acid（5-HETE）.Itwas found that the modified method has the merits of simpler procedures,shortertesting time and more satisfactory efficacy.

Separation and analysis of six fractions in low temperature coal tar by column chromatography

The low temperature coal tar(CT)is taken as the raw material,and the extraction and column chromatography are used for detailed and accurate characterization in this paper.The n-heptane soluble fraction(CT-HS)and insoluble fraction(CT-HI)were obtained by n-heptane Soxhlet extraction.The extraction rate of CT-HS reached 92.79%(mass),which indicated that there are few heavy compounds in it.Further,different solvents(methylbenzene,benzene,ethyl acetate,methylbenzene-ethanol)were used to elute CT-HS by chromatographic column to obtain five fractions(saturates,aromatics,heteroatoms,phenolics and resins,named CT-SA,CT-AR,CT-HE,CT-PH,CT-RE,respectively).The yields of CTSA,CT-AR,CT-HE,CT-PH,CT-RE are 42.12%,10.43%,2.19%,9.50%and 6.63%(mass),respectively.Gas chromatography-mass spectrometry analysis of eluting components show that alkanes are the main components in CT,followed by polycyclic aromatics,and the corresponding fractions are CT-SA and CT-AR,respectively.The relative content of aliphatics in CT-SA is 76.93%,and the relative content of aromatics in CT-AR is 75.05%.This separation technology effectively separates and enriches different components in CT,and the activation energy required for the pyrolysis process of a single eluting fraction is lower than that of CT,which is expected to provide an important reference for the separation,analysis and conversion of complex oil products such as coal-oil co-processing products,coal tar and other complex heavy carbon oil products.

A High-Performance Liquid Chromatography Method for the Simultaneous Determination of Five Index Components in Danhong Injection

The purpose of this study was to establish a high-performance liquid chromatography (HPLC) method for the simultaneous determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection. The chromatographic method employed was as follows: the column was a Welch Ultimate XB-C18 column (250 mm × 4.6 mm, 10 μm), the mobile phase was a gradient elution of 0.4% formic acid aqueous solution (A) and acetonitrile (B), the detection wavelengths were 280 nm for sodium danshensu, protocatechuic aldehyde, and salvianolic acid B and 326 nm for 4-coumaric acid and rosmarinic acid, the sample volume was 10 μL, the flow rate was 1.0 mL/min, and the column temperature was 35°C. This method can realize the separation and determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid within 50 minutes. The linear relationships of the five peak areas and their concentrations are good (R2> 0.9997). The precision RSD values are all less than 1.0%. The reproducibility RSD values are all less than 1.3%. The stability RSD values are all less than 2.2%. The recovery values ranged from 92.4% to 99.4%. This method is simple, accurate, and reproducible. It can be used for the determination of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, salvianolic acid B, and 4-coumaric acid in Danhong injection.

Determination of Voriconazole in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry: Application in Therapeutic Drug Monitoring

A sensitive, accurate and robust Liquid Chromatography Tandem Mass Spectrometry method has been developed and validated to measure voriconazole trough levels in human plasma. The plasma samples were mixed with fluconazole as an Internal Standard and directed to protein precipitation and drug extraction. An aliquot of 1 μl was injected into the chromatographic system and separated by the Acquity BEH C18 column at a flow rate of 0.30 ml/min in a gradient mobile phase consisting of acetonitrile, Ultrapure water (UPW), methanol and formic acid. Voriconazole was detected by a Triple Quadrupole Detector (TQD) operating on Multiple Reaction Monitoring (MRM) and a positive ion mode Electrospray ionization (ESI) Q1 mass: 350.1 m/z, Q3 mass: 281.1 m/z. Method linearity of the calibration curve (0.10 - 8.00 μg/ml) indicated a correlation coefficient r ≥ 0.99. The intra and inter-assay accuracy was within 85% - 115% and the intra and inter-assay precision was ≤5.76%. Voriconazole recovery percentage was between 97.69 - 119.62%. The method was successively applied in routine voriconazole TDM.

Preparation Process and Thin Layer Chromatography Identification of Wufang Babu Poultice

[Objectives] To establish the process flow of preparation of Wufang Babu Poultice and the identification method of thin layer chromatography (TLC). [Methods] For the forming process of Wufang Babu Poultice, the preparation method of mixed pharmaceutical powder with suitable pharmaceutical excipients was adopted. Qualitative identification of medicinal materials in Wufang Babu Poultice (Strychni Semen, Rhei Radix Et Rhizoma, Dipsaci Radix, and Angelicae Sinensis Radix) was carried out by TLC. [Results] Mixed pharmaceutical powder mixed with glycerol, gelatin and other pharmaceutical excipients can be prepared for forming. The test solution chromatography of each medicinal material (Strychni Semen, Rhei Radix et Rhizoma, Dipsaci Radix, Angelicae Sinensis Radix) showed pigment spots of the same color at the same position as its corresponding control medicinal materials and reference chromatography, and the display was clear. [Conclusions] The preparation process is simple and feasible, and can be used as the forming process of Wufang Babu Poultice. The TLC determination method is simple to operate, has good specificity, and has no effect on negative results, and can be used for identification of Wufang Babu Poultice.

采用气相色谱-质谱指纹图谱鉴定铁木豆属木材

采用气相色谱-质谱指纹图谱分别鉴定平萼铁木豆、葱叶状铁木豆和马达加斯加铁木豆属木材。以二氯甲烷提取样品中的特定化合物,采用气相色谱-质谱法分析提取物,建立目标物色谱图。平萼铁木豆在保留时间25.1 min的最强吸收峰和22.3、22.6及23.4 min的次强吸收峰可作为平萼铁木豆的特征图谱,与其他两种铁木豆相区分。葱叶状铁木豆和马达加斯加铁木豆的最强吸收峰色谱保留时间均为16.3 min,葱叶状铁木豆的次强吸收峰色谱保留时间为18.5 min,另外还在色谱保留时间分别为19.4、19.8、24.3 min处有吸收峰,而马达加斯加铁木豆除在色谱保留时间17.7 min处有次强吸收峰外,还在色谱保留时间为23.4 min时有吸收峰。气相色谱-质谱法可用于平萼铁木豆、葱叶状铁木豆和马达加斯加铁木豆三类木材的鉴别。

基于GC-IMS和GC-MS技术结合化学计量法分析干燥方式对香椿挥发性成分的影响

为研究干燥方式对香椿挥发性风味物质的影响,利用气相色谱-离子迁移谱技术(gaschromatography-ion mobility spectrometry,GC-IMS)和气相色谱-质谱联用技术(gas chromatography-mass spectrometry,GC-MS)、相对香气活性值(relative odor activity value,ROAV)对真空冷冻干燥、热泵干燥、热风干燥、微波真空干燥的香椿样品进行挥发性成分分析,并进一步结合化学计量方法主成分分析(principal component analysis,PCA)和偏最小二乘判别分析(partial least squares-discriminant analysis,PLS-DA),探究样品间挥发性成分的差异性。结果表明,通过GC-IMS检测到78个峰,鉴定出59种化合物。通过GC-MS鉴定出94种挥发性化合物,主要挥发性成分是硫化物、烯烃类和醛类。经过干燥后总挥发性化合物的含量显著降低。通过ROAV法确定了(E,Z)-二-1-丙烯基二硫化物、2-巯基-3,4-二甲基-2,3-二氢噻吩、正己醛、2-己烯醛、丁香酚等为香椿样品中关键香气化合物。PCA和PLS-DA结果表明,不同干燥方式的样品与新鲜样品有明显的分离,彼此之间也有明显的区别。鲜样和真空冷冻干燥样品为一组,其他3个干燥香椿样品为一组。根据变量投影重要性(variable importance in projection,VIP)得分共筛选出10种(VIP>1)标志挥发性化合物。干燥方式对香椿风味特性影响显著,真空冷冻干燥组与鲜样最为接近,考虑实际应用热泵干燥为香椿最适宜的脱水方式。本研究为热加工过程中风味品质控制提供一定的理论参考。

辣椒EIL基因家族的鉴定及其在盐碱胁迫下的表达分析

EIL(Ethylene-insensitive 3-like)基因在参与植物乙烯信号通路的转导和生长发育过程中发挥着重要作用。为了解辣椒EIL基因家族成员信息,利用生物信息学手段对辣椒EIL基因家族成员的理化性质、蛋白质结构、系统进化、基因结构、保守域、启动子顺式作用元件及CaEILs基因在不同组织部位和不同非生物胁迫条件下的表达模式进行分析。同时使用实时荧光定量PCR(qRT-PCR)对辣椒叶片中CaEILs在盐碱胁迫下的表达模式进行研究。结果表明,辣椒9个CaEILs分布在6条染色体上,氨基酸数目、蛋白质理论分子质量和脂肪系数分别介于209~677、23.77~76.07 ku和63.10~87.75,且主要为酸性、亲水的不稳定性核蛋白。系统进化分析显示,CaEILs被分成了4个亚家组,9个CaEILs在根、茎、叶、花蕾、花中具有不同程度的表达,而低温、高温、高盐、干旱胁迫下均能诱导CaEILs的表达,其对以上非生物胁迫均具有不同程度的响应。此外,利用qRT-PCR对盐碱胁迫下辣椒叶片中CaEILs进行了检测,结果发现,随着处理时间的延长,CaEIL1、CaEIL2、CaEIL4、CaEIL5、CaEIL8的表达量整体呈上升趋势,而CaEIL3、CaEIL6、CaEIL7、CaEIL9的表达量整体呈下降趋势。

色谱技术在木犀草素中的研究进展

木犀草素是一种常见于多种药用植物中的类黄酮,具有抗氧化、抗炎、免疫调节等多种生物学作用。市售的含有木犀草素的药用植物及其制剂常用于治疗高血压、炎症性疾病,甚至癌症。然而,为了确定这些制剂的质量,应使用适当的分析方法。本研究主要对木犀草素的定量分析方法进行了全面综述,系统介绍了薄层色谱法(TLC)、高效薄层色谱法(HPTLC)、液相色谱法(LC)、气相色谱法(GC)和逆流色谱法(CCC)等色谱分析技术研究进展以及植物材料中木犀草素的测定条件。

阿莫西林胶囊在中国健康人体内的生物等效性研究

目的:研究阿莫西林胶囊在中国健康人体内的生物等效性。方法:以单中心、开放式、随机、双制剂、两周期、两序列交叉试验设计,共48例受试者(空腹试验和餐后试验各24例),口服0.25 g阿莫西林胶囊受试制剂或参比制剂。高效液相色谱—串联质谱(LC-MS/MS)分析方法测定给药后不同时间阿莫西林的血药浓度,并计算主要药代动力学参数,判定两制剂是否等效。结果:空腹试验显示,受试制剂和参比制剂阿莫西林的药物峰浓度(C_(max))分别为(5483.296±1321.102)ng/mL、(5611.291±1659.407)ng/mL,从时间0到t之间血药浓度—时间曲线下面积(AUC_(0-t))分别为(13255.3±1715.7)h·ng/mL、(13115.5±2091.7)h·ng/mL,从0时到无限时间(∞)的血药浓度—时间曲线下面积(AUC_(0-∞))分别为(13329.4±1718.8)h·ng/mL、(13192.7±2107.1)h·ng/mL,达峰时间(T_(max))均为1.38 h。餐后试验显示,受试制剂和参比制剂阿莫西林的C_(max)分别为(4218.072±780.598)ng/mL、(4156.713±877.752)ng/mL,AUC_(0-t)分别为(13073.9±1584.3)h·ng/mL、(12817.8±1575.5)h·ng/mL,AUC_(0-∞)分别为(13166.8±1606.0)h·ng/mL、(12914.8±1587.2)h·ng/mL,T_(max)均为3.00 h。两种试验制剂C_(max)、AUC_(0-t)、AUC_(0-∞)几何均值比值的90%置信区间(CI)均在可接受的生物等效性范围内(80%~125%)。结论:两种阿莫西林胶囊在中国健康志愿者体内吸收速度和吸收程度生物等效。

超高效液相色谱-串联质谱法同时测定蔬菜中氯酸盐和高氯酸盐

建立超高效液相色谱-串联质谱法测定蔬菜中氯酸盐和高氯酸盐。样品以少量水稀释分散后,加入乙腈提取,经PRIME HLB小柱净化,用Waters Torus DEA色谱柱(100 mm×2.1 mm,1.7μm)分离,以同位素内标法定量。在各自的线性范围内,氯酸盐、高氯酸盐的质量浓度与氯酸盐、高氯酸盐和相应同位素内标物的色谱峰面积比线性关系良好,相关系数均大于0.999。氯酸盐的检出限为3.0μg/kg,定量限为10μg/kg,高氯酸盐的检出限为1.5μg/kg,定量限为4.5μg/kg,平均回收率为88.9%~97.1%,相对标准偏差为2.9%~7.4%(n=6)。该方法简便、快速,适用于蔬菜中氯酸盐和高氯酸盐的同时快速检测。

离子色谱法检测水质中微量甘油

建立了离子色谱法测定水质中微量甘油的方法。在酸性条件下,使用高碘酸钠将甘油定量氧化成甲酸,再通过离子色谱法定量检测甲酸根含量,从而折算得到甘油准确含量。甘油衍生化样品用20 mmol/L KOH洗脱液淋洗,淋洗流量为1.0 mL/min, Dionex IonPac AS15阴离子交换色谱柱分离,抑制型电导检测器检测。衍生化后的甲酸根检出限为0.015 mg/L,对水质样品衍生化后的甲酸根离子在0.5、1.0、2.0、3.0 mg/L加标水平的回收率为96.26%~99.68%,该方法的相对标准偏差为0.39%。结果表明,该方法前处理条件温和、反应速度快、副产物少、灵敏度高、准确度高、重复性好,适用于水质中微量甘油含量的检测。

基于HPLC法及UPLC-MS法测定芫花饮片炮制前后7种成分的含量变化

目的:建立HPLC法及UPLC-MS法测定芫花中银椴苷、木犀草素、芹菜素、绿原酸、羟基芫花素、芫花素及芫花酯甲7种化学成分含量的方法,分析芫花炮制前后化学成分的变化。方法:采用HPLC法,测定银椴苷、木犀草素、芹菜素、绿原酸、羟基芫花素和芫花素6种成分的含量,色谱柱为Agilent Eclipse Plus C18(250 mm×4.6 mm,5μm),流动相为乙腈(A)-0.1%甲酸(B)梯度洗脱,流速为1.0 mL·min^(-1),进样量10μL。同时采用UPLC-MS方法测定芫花酯甲的含量,色谱柱为ACQUITY UPLC HSS T3 C18色谱柱(2.1 mm×100 mm,1.8μm),流动相为乙腈(A)-0.1%甲酸(B)梯度洗脱,流速0.3 mL·min-1,柱温35℃,进样量1μL。结果:7种成分的含量由高到低依次为芫花素>绿原酸>银椴苷>芹菜素>羟基芫花素>木犀草素>芫花酯甲。芫花炮制后7种成分的含量均发生一定程度的变化,其中绿原酸成分在炮制后含量有升高有降低,变化不明显;对于银椴苷和芹菜素成分,炮制后含量降低;对于木犀草素、羟基芫花素和芫花素3个成分,炮制后含量升高;对于芫花酯甲成分,炮制后含量降低。结论:该方法简便、灵敏、高效,为考察芫花饮片炮制前后的质量变化提供了技术支持。

电子舌和气相色谱-离子迁移谱技术分析炖煮时间对松茸鸡汤风味的影响

以雅江鲜松茸为试验材料,采用电子舌和气相色谱-离子迁移谱技术,使用主成分、正交偏最小二乘法判别分析,结合相对气味活度值分析炖煮时间对松茸鸡汤风味的影响。结果表明:鲜松茸对鸡汤滋味贡献很大,电子舌鲜味强度值从2.1提高到8.0。采用GC-IMS方法共检出45种化合物,加入松茸后醛类含量从47.88%下降到21.13%,由ROAV值(128.08)确认3-甲基-1-丁醇是松茸鸡汤的关键化合物。其中,伞花烃、3-辛醇、正辛醇是松茸鸡汤的特征化合物,使松茸鸡汤呈现独特的汽油味、苔藓清香、坚果香味、蘑菇气味和金属气味。炖煮5 min后,鸡汤中松茸特征风味最明显,达到最佳食用效果。研究结果为雅江松茸鸡汤风味特征研究提供参考。

益肝片的质量标准研究

目的:提升益肝片的质量标准。方法:采用薄层色谱法(TLC)鉴别益肝片中的板蓝根、虎杖;采用高效液相色谱法(HPLC)对益肝片中的(R,S)-告依春和大黄素进行含量测定。结果:通过TLC鉴别板蓝根、虎杖,相应斑点清晰、分离度好,阴性无干扰。(R,S)-告依春在进样量0.03~0.15μg范围内与峰面积线性关系良好(r=0.9998),平均加样回收率为97.05%,大黄素在进样量0.0495~0.297μg范围内与峰面积线性关系良好(r=1.0000),平均加样回收率为99.24%。结论:本质量标准能有效鉴别益肝片中的板蓝根、虎杖,也能有效测定(R,S)-告依春和大黄素的含量,且该法简便、可靠、重复性好,可用于益肝片的质量控制。

烫疗药提取工艺及质量标准提升研究

目的:优选烫疗药浸渍提取工艺,提升烫疗药质量标准。方法:以浸渍溶剂浓度、浸渍时间、浸渍温度、原料粉碎度为考察因素,以川续断皂苷Ⅵ含量和醇溶性浸出物为评价指标,通过L9(34)正交试验优选烫疗药最佳浸渍提取工艺;采用薄层色谱法(TLC)对烫疗药中防风、大黄、姜黄进行定性鉴别;采用高效液相色谱(HPLC)法对川续断皂苷Ⅵ进行定量测定。结果:筛选的最佳浸提工艺为将烫疗药处方饮片粉碎成细粉碎度,以50%的乙醇为溶剂,室温下浸渍5 d;川续断皂苷Ⅵ在19.30715~154.45720μg/mL范围内呈良好的线性关系(r=0.9999),平均加样回收率为102.37%,RSD=1.48%(n=6);定性鉴别中防风、大黄、姜黄斑点清晰,分离效果较好,具有鉴别特征。结论:优选的浸提工艺稳定可行;所建立的质量控制方法稳定性及重现性良好,专属性较强,可作为烫疗药的质量控制依据。

在线燃烧离子色谱法测定环氧树脂中的氯和溴

建立了在线燃烧离子色谱同时测定环氧树脂中氯和溴的分析方法.采用高温裂解炉与离子色谱的在线联用装置,通过对样品进行高温燃烧裂解和气化,以100 mg·L^(-1)过氧化氢为吸收液,将产生的卤化氢气体吸收并转化为无机阴离子,离子色谱法进行样品的测定,以氯和溴的峰面积外标法进行定量.对影响燃烧效果和测定结果准确度的因素如燃烧时间、燃烧温度、吸收液体积、称样量、氧气和氩气流速等条件进行了选择优化.在(0.10—2.50)mg·L^(-1)和(0.02—0.50)mg·L^(-1)范围内,氯和溴离子的线性相关系数(r^(2))大于0.999,该方法对于氯和溴的定量下限分别为0.55 mg·kg^(-1)和2.60 mg·kg^(-1).采用建立的方法分别对环氧树脂和EC680k低密度聚乙烯标准物质中的氯和溴进行测定,并与传统的氧弹燃烧-离子色谱法进行了比较.结果表明:在线燃烧离子色谱法对于环氧树脂中氯和溴测定结果的相对标准偏差分别为1.28%和2.29%,测定值与氧弹燃烧-离子色谱法基本一致,EC680k的测定结果与标准值符合,证明该方法具有良好的准确度和精密度.该方法准确度和灵敏度高、重复性较好,能够满足批量树脂类样品中氯和溴的含量筛查和多批次产品的质量控制.