Determination of Organic Acids in Root Exudates by High Performance Liquid Chromatography: I. Development and Assessment of Chromatographic Conditions *1

Methods for determining nine low molecular weight organic acids in root exudates were developed by using reversed phase high performance liquid chromatography with UV (ultraviolet) detection at 214 nm. The mobile phase was 18 mmol L -1 kH 2PO 4 adjusted to pH 2.25 with phosphoric acid and the flow rate was 0.3 mL min -1 . The analytical column was a reversed phase silica based C 18 column (Shim pack CLC ODS). The root exudates were collected through submerging the whole root system into aerated deionized water for 2 hours. The filtered exudate solutions were concentrated to dryness by rotary evaporation at 40 °C, dissolved in 10 mL mobile phase. The chromatographic conditions of organic acid determination were analyzed. The results showed that there was a high selectivity and sensitivity in the organic acid determination by reversed phase high performance liquid chromatography. Coefficients of variation for organic acid determination were lower than 10% except lactic acid. The recoveries were consistently between 80.1% to 108.3%. Detection limits were approximately 0.05 to 4.5 mg L -1 for organic acids except succinic acid with the detection limit of 7.0 mg L -1 . Phosphorus deficiency may contribute to the release of organic acids in soybean root exudates especially malic, lactic and citric acids.

MODELLING OF MECHANICAL MECHANISM OF CHROMATOGRAPHIC SYSTEM AND THEORETICAL EQUATIONS SHOWING DYNAMICAL CHARACTERISTICS OF CHROMATOGRAPHY

A simple model of chromatographic mechanical mechanism is present, and then a scrics of theoretical chromatographic equations and fundamental Formulae are derived. These theoretical equations and formulae not only reserve thermodynamic characteristics in the current fundamental chromatographic formulae, but also introduce one or more kinetic parameter, so it is possible to make the macroscopic-control on the effect of kinetic characteristics on chromatographic system.

Gas Chromatographic Method for Identification and Quantification of Commonly Used Residual Solvents in Pharmaceuticals Products

Background: Impurities are not expected in the final pharmaceutical products. All impurities should be regulated in both drug substances and drug products in accordance with pharmacopeias and ICH guidelines. Three different types of impurities are generally available in the pharmaceutical’s product specification: organic impurities, inorganic impurities, and residual solvents. Residual solvents are organic volatile chemicals used or generated during the manufacturing of drug substances or drug products. Purpose: The aim of this study is to develop a cost-effective gas chromatographic method for the identification and quantification of some commonly used solvents—methanol, acetone, isopropyl alcohol (IPA), methylene chloride, ethyl acetate, tetrahydrofuran (THF), benzene, toluene, and pyridine—in pharmaceutical product manufacturing. This method will be able to identify and quantify the multiple solvents within a single gas chromatographic procedure. Method: A gas chromatography (GC) equipped with a headspace sampler and a flame ionization detector, and a column DB 624, 30-meter-long × 0.32-millimeter internal diameter, 1,8 μm-thick, Brand-Agilent was used to develop this method. The initial GC oven temperature was 40°C and held for 5 minutes. It was then increase to 80˚C at a rate of 2˚C per minute, followed by a further increase to 225˚C at a rate of 30˚C per minute, with a final hold at 225˚C for 10 minutes. Nitrogen was used as a carrier gas at a flow rate of 1.20 mL per minute. Dimethyl sulfoxide (DMSO) was selected as sample solvent. Results: The developed method is precise and specific. The percent RSD for the areas of six replicate injections of this gas chromatographic method was within 10.0 and the recovery result found within 80.0% to 120.0%.

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 14 Organophosphorus Pesticide Residues in Mutton by Gel Permeation Chromatography-Gas Chromatography-Mass Spectrometry(GPC-GC-MS)

[Objectives]This study was conducted to purify mutton samples by gel permeation chromatography(GPC).[Methods]Fourteen organophosphorus pesticide residues in samples were qualitatively and quantitatively analyzed by gas chromatography-mass spectrometry(GC-MS)in selective ion scanning mode(SIM).[Results]The organophosphorus pesticide standard solutions showed good linearity in the mass concentration range of 0.1-10.0μg/ml with correlation coefficients(r)not lower than 0.999,and the detection limits(S=3 N)ranged from 0.01 to 0.05 mg/kg.The average recovery values were in the range of 80.2%-99.7%,with relative standard deviations(RSDs,n=3)in the range of 1.8%-6.3%,at the addition levels of 0.5,1.0 and 2.0 mg/kg.[Conclusions]The method is simple,sensitive and accurate,and can be used for the determination of organophosphorus pesticide residues in mutton.

Comprehensive analysis of phenolic composition and antioxidant mechanisms in Gymnema sylvestre extracts using LC-MS and column chromatography

Recent studies have highlighted the potential of plant extracts as therapeutic agents for managing oxidative stress and related disorders.This study aims to elucidate the phenolic composition and antioxidant properties of Gymnema sylvestre extracts.Ethanolic reflux extraction followed by column chromatography was employed to isolate phenolic compounds.The total phenolic and flavonoid contents were quantified using the Folin–Ciocalteu and aluminum chloride colorimetric methods,respectively.Antioxidant activities were assessed by DPPH,ABTS scavenging assays and the ferric reducing antioxidant power(FRAP)assay.High-Performance Liquid Chromatography(HPLC)with a C18 column and Thermo TSQ Quantum Access Max(LC-MS)were used to determine the levels of gymnemic acid and identify other potential phenolic compounds.The analysis revealed significant antioxidant activities in the fractions.Fraction A showed the highest DPPH and ABTS scavenging activities,and Fraction C demonstrated the highest ferric reducing power.LC-MS analysis identified several phenolic compounds,indicating that these are major contributors to the antioxidant efficacy of the extract.This study provides a detailed phenolic profile and confirms the strong antioxidant potential of Gymnema sylvestre leaf extract,supporting its therapeutic use and further investigation.

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.

Development of Quantitative Analysis Method of Carbendazim in Tomatoes by High-Performance Liquid Chromatography with Fluorescence Detection(HPLC-FLD)

Carbendazim belongs to the benzimidazole fungicides,which can be used for control lots of fungi pathogens.High-performance liquid chromatography is frequently used for the analysis of carbendazim in all kinds of samples.In most occasions,the developed methods were applied for the simultaneous detection of a huge number of pesticides.Thus,an analytical method via UPLC-FLD was developed,and the sample preparation process was optimized by studying the effect of extraction solvent,approach,time and purification absorbent on the recovery rate of carbendazim.The results showed the optimized method for analysis was ultrasonication-assisted extraction with acetonitrile for 1 min,and subsequent purification by C18.In this occasion,the established analytical method of carbendazim in tomato samples displayed good linearity,accuracy and precision.

基于顶空-固相微萃取-气相色谱-质谱和顶空-气相色谱-离子迁移谱技术结合化学计量法分析芜菁冻干片挥发性成分

为探究不同品种芜菁冻干片中挥发性有机物(volatile organic compounds,VOCs)的差异,采用顶空-固相微萃取-气相色谱-质谱(headspace solid phase microextraction gas chromatography-mass spectrometry,HS-SPME-GC-MS)和顶空-气相色谱-离子迁移谱(headspace gas chromatography-ion mobility spectrometry,HS-GC-IMS)对紫色、黄色和白色3种芜菁冻干片的VOCs进行分析,并结合主成分分析(principal component analysis,PCA)和偏最小二乘判别法(partial least squares-discriminant analysis,PLS-DA)等化学计量法探究不同品种芜菁冻干片挥发性成分的差异。结果表明,通过HS-SPME-GC-MS共解析出96种VOCs,包括醛类、醇类、酮类、含硫化合物、酯类、酸类等化合物,其中含硫化合物和酯类为芜菁冻干片中相对含量最高的化合物种类;HS-GC-IMS共解析出94种VOCs,包括醛类、酯类、酮类及含硫化合物等挥发性成分。HS-SPME-GC-MS和HS-GC-IMS检出的挥发性物质种类和含量存在差异,共有VOCs有15种,二者结果互为补充,结合使用可以较全面系统地表征芜菁冻干片的挥发性成分。PCA和PLS-DA结果表明,2种方法均能够有效区分3种芜菁冻干片。通过变量投影重要度分别筛选了59种和23种差异VOCs,该结果可为芜菁冻干片VOCs的差异分析提供参考方法。

活性炭净化-离子色谱法测定茶水及茶饮料中氟含量及其健康风险评估

目的建立分析纯活性炭净化-离子色谱法检测茶水及茶饮料中氟离子含量的方法,进而研究茶水及茶饮料对人体健康风险。方法将6种茶叶分别以两种方式冲泡进行实验,方式一:按时间梯度冲泡茶叶;方式二:一次投茶,多次加水冲泡茶叶,收集茶水。茶水及茶饮料,经活性炭净化,0.45μm微孔滤膜过滤,以4.5 mmol/L碳酸钠和0.8 mmol/L碳酸氢钠溶液为淋洗液,经AS23离子色谱柱(4.0 mm×250 mm)分离,离子色谱进行检测。以冲泡方式一获得的最高茶氟溶出量,冲泡方式二获得的总茶氟溶出量以及茶饮料中氟含量分别进行健康风险评估。结果在最佳分析条件下,本法氟离子的检出限为0.016 mg/L;在0.1~5.0 mg/L质量浓度范围内的线性关系良好,相关系数(r^(2))为0.9996;加标回收率94.93%~105.32%;相对标准偏差1.02%~2.13%(n=6)。本方法检测茶叶中氟离子的溶出量,发现茶叶中氟离子的溶出量均随冲泡时间的延长不断增加达到最高值;随冲泡次数的增加,茶叶中氟离子的溶出量先增加,之后下降的趋势逐渐变缓。前2次冲泡氟离子溶出率可达茶氟总溶出量的65%以上。结论该方法灵敏度高,准确性好,操作简单,具有实用价值。以该法检测6种茶叶在两种冲泡方式下获得的茶氟日摄入量以及茶饮料中茶氟日摄入量均符合我国和世界卫生组织推荐的氟日摄入量限值要求;目标危险因素(target hazard quotient,THQ)均小于1,对人体没有显著健康风险。建议减少茶叶浸泡时间,洗茶,选用优质茶叶来科学健康饮茶。

基于非靶代谢组学探究食管鳞状细胞癌津血亏虚证患者的代谢特征

背景津血亏虚是中晚期食管癌患者的主要临床表现,但目前关于食管鳞状细胞癌津血亏虚证代谢组学特征的研究报道较少见。目的探究食管鳞状细胞癌津血亏虚证患者的代谢组学特征。方法选择2022年4—12月就诊于河北医科大学第四医院中医科、胸外科的食管鳞状细胞癌津血亏虚证患者35例作为病例组,招募同时期健康志愿者35例作为对照组。收集两组受试者血清标本,通过超高效液相色谱质谱技术分析其代谢组学特征,筛选差异代谢物及差异代谢通路。结果筛选出具有物质二级匹配名称的差异代谢物88种,具有显著性差异的关键代谢通路5条(β-丙氨酸代谢通路,鞘脂代谢通路,亚油酸代谢通路,甘氨酸、丝氨酸、苏氨酸代谢通路,花生四烯酸代谢通路)及其代谢物11种(表达下调代谢物:β-丙氨酸,磷脂酰胆碱,L-高丝氨酸,L-别苏氨酸;表达升高代谢物:肌肽,亚油酸,9,10-二羟基八氢-1-氧杂-4-氮杂苯并环庚烯,羟基丙酮酸,花生四烯酸,肝素B3,血栓素B2)。结论食管鳞状细胞癌津血亏虚证患者存在具有显著性差异的代谢通路及其代谢物,主要涉及氨基酸、脂肪酸等能量代谢异常。

在线凝胶渗透色谱-气质联用法测定腌制鱼中7种N-亚硝胺类化合物的一测多评研究

目的:建立气质联用法结合QuEChERS法和在线凝胶渗透色谱技术检测腌制鱼中7种N-亚硝胺类化合物,并对采用的一测多评法进行研究。方法:样品经优化后的QuEChERS法提取和净化后,采用在线凝胶渗透色谱技术进行二次净化。试验对QuEChERS方法进行了优化,对基质效应、样品溶液的稳定性和方法耐受性进行了试验,并进行了方法学验证;试验以NDMA为参照对象,建立了其余6种N-亚硝胺类化合物一测多评校正方法。结果:经凝胶渗透色谱技术净化后,可有效除去大部分杂质,降低基质效应,提高灵敏度。校正因子分别为f_(NMEA/NDMA)=6.7369、f_(NDEA/NDMA)=0.6679、f_(NDPA/NDMA)=0.2399、f_(NDBA/NDMA)=1.1532、f_(NPIP/NDMA)=0.7289、f_(NPYR/NDMA)=0.7103,且稳定性较好。方法学验证表明,7种N-亚硝胺类化合物的检出限为0.02~0.29μg/kg;加标回收率和精密度分别为NDMA 101.0%~107.5%,2.5%~3.9%;NMEA 102.0%~108.4%,2.8%~4.0%;NDEA 101.6%~109.0%,2.6%~3.9%;NDPA 103.1%~107.4%,1.9%~4.0%;NDBA 103.7%~107.7%,1.7%~3.8%;NPIP 104.7%~107.5%,2.3%~3.7%;NPYR 101.4%~106.4%,2.2%~3.8%。结论:该方法具有灵敏度高、操作简便、方法稳定等特点,适用于大批量腌制鱼中N-亚硝胺类化合物的测定。

新型色谱柱技术对液相色谱基础实验的提升

在本科生仪器分析实验教学中,受限于仪器效率与教学时长,许多教学内容无法展开,合理安排并丰富教学内容是教学领域的难点。例如每个单样的色谱分离时间长,是本科生液相色谱基础实验的瓶颈。通过自研的快速传质色谱柱,一个样品在3 min内可完全分离。实践不明:新型色谱柱技术的采用,显著提升了仪器效率,在教学时长不变的情况下,教学内容获得了极大的拓展:30 min即可快速完成实验样品的定性分析和色谱柱的基本性能评价;进而把优化色谱条件从验证型改为研究型实验;最后用外标工作曲线完成样品的定量分析。改进后实验教学内容充实深入,知识点覆盖面广,增强了基本操作实践,培养了科研思维,为液相色谱的实际应用打下良好基础。相关实验的设计为提升仪器分析实验教学效果提供了示范。

Study on the Chromatographic Fingerprint of Volatile Constituents from Acacia Honey

[Objective] The experiment aimed to study chromatographic fingerprint in volatile components of acacia honey and provide scientific evaluation and effective control on quality of acacia honey.[Method] Using solid-phase microextraction method to separate and detect volatile components and construct chromatographic fingerprint.[Result] The honey was preheated for 15 min in water bath at 40 ℃ and solid-phase microextraction 85 μmPA was used to extract in overhead air about 30 min,then put it into the injector and desorpted 3 min,which is in 230 ℃.The Supelco WaxTM10 30 m×0.25 mm×0.25 μm column and gradient heating program was the best method to separate volatile components from honey.83 fingerprint peaks were constructed,among which 17 common fingerprint peaks were comprised of chromatographic fingerprint of volatile components of acacia honey.[Conclusion] The chromatographic fingerprint could provide reference for quality control of acacia honey.

水力空化对玉米淀粉糊相对分子量及黏度的影响

为揭示水力空化对淀粉相对分子量及黏度的影响,以玉米淀粉糊为研究对象,采用凝胶渗透色谱法(gel permeation chromatography,GPC)检测相对分子量、乌氏黏度计检测特性黏度,考察不同水力空化条件及淀粉糊质量浓度对玉米淀粉糊相对分子量及其分布和黏度的影响。结果表明:在水力空化作用下,玉米淀粉糊的相对分子量、特性黏度均有一定程度的降低;在相同条件下,随着玉米淀粉糊的质量浓度的减小和空化作用时间的延长,空化后淀粉糊的特性黏度和相对分子量均有所减小,当pH=6.0时相对分子量降至最低值,可见水力空化对玉米淀粉糊有较好的降解作用。

Simultaneous Determination of Tetramethylpyrazine and Aspirin in a New Compound Formulation by Liquid Chromatography

Aim To establish a reversed-phase liquid chromatographic (LC) method forsimultaneous determination of tetramethylpyrazine (TMP) and aspirin in a new compound formulation.Methods Chromatographic separation of the two drugs was achieved on a Diamonsil C_(18) column, usinga binary mixture of methanol-1.5% acetic acid (35:65, V/V, pH = 3.1) as mobile phase at a flow rateof 1.0 mL·min^(-1). Results Separation was completed in less than 12 min. Benzoic acid was used asthe internal standard. Recoveries at levels corresponding to 80 % to 120 % of the label claim ofthe formulation ranged from 99.6 to 100.3 % for aspirin and from 99.9 to 101.3% for TMP. The linearrange was 12.6 - 150.9 μg·mL^(-1)(r= 0.9997, n = 5) for aspirin and 25.0- 300.0 μg·mL^(-1) (r =0.9999, n = 5) for TMP. Conclusion The method developed can be used for the simultaneousdetermination of TMP and aspirin in pharmaceutical preparations.

液液萃取-超高效液相色谱-串联质谱法同时测定尿液中乌头碱和新乌头碱

建立液液萃取-超高效液相色谱-串联质谱法同时测定尿液中乌头碱和新乌头碱的含量。向尿液样品中加入一定量的萃取溶剂,涡旋,离心,取上层有机相,重复萃取两次,合并萃取液,氮吹至干,再复溶于乙腈中。以乙腈和含0.1%甲酸的2 mmol/L甲酸铵溶液作为流动相,梯度洗脱,采用Acquity BEH C_(18)色谱柱分离,电喷雾离子源正离子方式扫描,多反应监测模式监测,以保留时间和特征离子定性,外标法定量。乌头碱和新乌头碱的质量浓度在1.0~100.0μg/L范围内与其色谱峰面积呈良好的线性关系,相关系数均不小于0.998,方法检出限为0.01μg/L。样品加标平均回收率为99.2%~105.4%,测定结果的相对标准偏差为1.7%~4.1%(n=5)。该方法灵敏度高,准确可靠,适用于食物中毒患者尿液中乌头碱和新乌头碱的检测。

Gel Filtration Chromatography Combined with Bradford Method for Determination of Total Residual Protein in Ferment Antibiotics

Aim A novel method has been developed for evaluation of the levels of total residual protein in antibiotics produced by fermentation using gel filtration chromatography (GFC) combined with Bradford assay based on determination of residual protein in lincomycin hydrochloride. Methods The chromatographic conditions were SuperdexTM peptide column, 0.01 mol*L-1 phosphate buffer solution as mobile phase, and flow rate of 1 mL·min-1. Five hundred microliters of lincomycin hydrochloride solution (3 g of lincomycin hydrochloride dissolved in 10 mL of mobile phase) was injected into the chromatograph and the eluted solution was collected between 6 min and 14.5 min (protein eluted from column within this period), and the residual content of total protein in the eluted solution was assayed using Bradford assay method. Results The average recovery was more than 90% for bovine serum albumin, the calibration equation for the range of 0-12 μg·mL-1 of protein was y=-0.002 4x2+0.064 2x+0.002 9, r2=0.999 9, RSD=0.1%-0.9%, and the LOD and LOQ were 3 and 10 ng·mL-1 of protein, respectively. Conclusion The novel method for determining the residual protein in ferment antibio-tics is simple, rapid, and precise.

Analysis of Sugar Components of Fermented Rice Wine by Ion Chromatography

[Objective] The aim was to analyze sugar components in fermented rice wine by ion chromatography. [Method] The optimal condition for chromatography system of sugar analysis was selected by measuring sugars in fermented rice wine with ion chromatography and pulsed amperometric detection. [Result] The optimal measurement conditions were as follows： Leacheate （Leachate）, consisting of NaOH and CH3COONa, was eluted by gradient concentrations, with column temperature at 35 ℃ and flow rate at 0.4 ml/min. In the condition, sugars in rice wine were ana- lyzed and the results showed that the method is featured by low detection limit, good repetition and high recovery rate. [Conclusion] The research establishes and determines the approaches and optimum conditions for sugar analysis in rice wine by ion chromatography and pulsed amperometric detection, providing references for advancement of research on quality improvement of fermented rice wine.

Determination of Dichlofluanid Residue in Soybean by Capillary Collumns Gas Chromatography

A method for the determination of dichlofluanid residue by Capillary Collumns Gas Chromatography was established in this study.Samples were extracted with acetone and subjected to through liquid-liquid extraction with N-hexane,and then they were cleanup with column that filled with florisil,subsequently separated by Capillary Column Gas Chromatography,and the electron capture detector （ECD） was used for detection.The detection limit of this method was 0.005 mg/kg （S/N=3）,and this method was rapid,sensitive and accurate,and was applied in the determination of dichlofluanid residue in soybean.