Trace Determination of Tamoxifen in Biological Fluids Using Hollow Fiber Liquid-Phase Microextraction Followed by High-Performance Liquid Chromatography-Ultraviolet Detection

The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a 15 mL aqueous sample (source phase;SP) into an organic phase impregnated in the pores of the hollow fiber (membrane phase;MP) followed by the back-extraction into a second aqueous solution (receiving phase;RP) located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent, compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction efficiency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 - 500 ng?mL–1 and the limit of detection was found to be 0.5 ng?mL–1 in aqueous medium. A reasonable relative recovery (≥89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3) precision illustrated good performance of the analytical procedure in spiked human urine and plasma samples.

Rapid Determination of Dopamine and Its Metabolites During in vivo Cerebral Microdialysis by Routine High Performance Liquid Chromatography With Electrochemical Detection

To determine dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection. Methods Microdialysis probes were placed into the right striatum of Wistar rat brains and perfused with Ringer＇s solution at a rate of 1.5 pL/min. A reverse phase HPLC with electrochemistry was used to assay DA, DOPAC, and HVA after cerebral microdialysates were collected every 20 minutes from awake and freely moving rats. In order to identify the reliability of this method, its selectivity, linear range, precision and accuracy were tested and the contents of DA, DOPAC, and HVA in rat microdialysates were determined. Results The standard curve was in good linear at the concentration ranging from 74 nmol/L to 1.5 pmol/L for DOPAC （r^2= 0.9996）, from 66 nmol/L to 1.3 gmol/L for DA （r^2=l.0000） and from 69 nmol/L to 1.4 pmol/L for HVA （r^2=0.9992）. The recovery of DOPAC （0.30, 0.77, 1.49 gmol/L）, DA （0,26, 0.69, 1.32 gmol/L）, and HVA （0.27, 0.71, 1.37 gmol/L） was 82.00±1.70%, 104.00±4.00%, 98.70±3.10%; 92.30± 1.50%, 105.30±2.30%, 108.00±2.00%; 80.00±7.80%, 107.69±8.00%, and 108.66±3.10%, respectively at each concentration. Their intra-day RSD was 3.3%, 3.4%, and 2.5%, and inter-day RSD was 4.2%, 2.3%, and 5.6%, respectively. The mean extracellular concentrations of DOPAC, DA, and HVA in rat brain microdialysates were 10.7, 2.4, and 9.2 gmol/L （n=6）, respectively. Conclusion The findings of our study suggested that the simple, accurate and stable method can be applied to basic researches of diseases related to monoamines neurotransmitters by cerebral microdialysis in rats.

Advancements in the preparation of high-performance liquid chromatographic organic polymer monoliths for the separation of small-molecule drugs

The various advantages of organic polymer monoliths, including relatively simple preparation processes,abundant monomer availability, and a wide application range of pH, have attracted the attention of chromatographers. Organic polymer monoliths prepared by traditional methods only have macropores and mesopores, and micropores of less than 50 nm are not commonly available. These typical monoliths are suitable for the separation of biological macromolecules such as proteins and nucleic acids, but their ability to separate small molecular compounds is poor. In recent years, researchers have successfully modified polymer monoliths to achieve uniform compact pore structures. In particular, microporous materials with pores of 50 nm or less that can provide a large enough surface area are the key to the separation of small molecules. In this review, preparation methods of polymer monoliths for high-performance liquid chromatography, including ultra-high cross-linking technology, post-surface modification, and the addition of nanomaterials, are discussed. Modified monolithic columns have been used successfully to separate small molecules with obvious improvements in column efficiency.

Determination of Fructose and Glucose Contents in Honey by Liquid Chromatography-refractive Index Detection Method and Direct Titration Method

In this study, fructose and glucose contents in honey were determined by liquid chromatography-refractive index detection method and alkaline copper tartrate solution-direct titration method. According to the results, there were great differences between determination results of reducing sugar contents by liquid chromatography-refractive index detection method and alkaline copper tartrate solution-direct titration method. Specifically, average determination results of reducing sugar contents by liquid chromatography-refractive index detection method were reduced by about 9.5% compared with alkaline copper tartrate solution-direct titra- tion method. Subsequently, determination results of reducing sugar contents by two methods were compared and analyzed. Liquid chromatography-refractive index detection method was used to determine fructose and glucose monomers in honey, but alkaline copper tartrate solution-direct titration method was used to determine reducing sugar composition in honey, which might lead to significantly different results. Due to small sample size and limited conditions, the determination results were not necessarily representative. Bee product acquisition and processing enterprises and relevant departments should pay much attention on these issues and fully consider the current situation of grass-roots units to develop scientific, rigorous, simple, universal, convenient, low-cost and practicable standards, thus ensuring the safety and reliability of food quality.

Novel method for the determination of five carbamate pesticides in water samples by dispersive liquid-liquid microextraction combined with high performance liquid chromatography

A novel method for the determination of five carbamate pesticides （metolcarb, carbofuran, carbaryl, isoprocard and diethofencard） in water samples was developed by dispersive liquid-liquid microextraction （DLLME） coupled with high performance liquid chromatography-diode array detector （HPLC-DAD）. Some experimental parameters that influence the extraction efficiency were studied and optimized to obtain the best extraction results. Under the optimum conditions for the method, the calibration curve was linear in the concentration range from 5 to 1000 ng mL^-1 for all the five carbamate pesticides, with the correlation coefficients （r^2） varying from 0.9984 to 0.9994. Good enrichment factors were achieved ranging from 80 to 177- fold, depending on the compound. The limits of detection （LODs） （S/N = 3） were ranged from 0.1 to 0.5 ng mL^-1. The method has been successfully applied to the analysis of the pesticide residues in environmental water samples.

A high-performance liquid chromatography with fluorescence detection method for the simultaneous quantitation of monoamine neurotransmitters and their metabolites in subregions of rat brain

Abstract： In the presem study, we simultaneously quantified the levels of monoamine neurotransmitters （MANTs） and their metabolites （levodopa, norepinephrine, epinephrine, dopamine, 5-HT, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid） in different brain subregions of rats using a newly developed simple, sensitive and selective high-performance liquid chromatography with fluorescence detection （HPLC-FLD） method. In this new HPLC-FLD method, analytes were directly extracted and separated without deriveatization step within 20 min. The FLD wavelength was set at 280 nm and 330 nm for excitation and emission, respectively. The analytes were separated on an Agilent Eclipse Plus Cls column （4.6 mm×150 mm, 5.0 μm） equipped with an Agilent XDB-C18 security guard column （4.6 mm×12.5 mm, 5.0 lam）, and the column temperature was maintained at 35 ℃. The mobile phase for elution was isocratic. The mobile phase consisted of citric acid buffer （50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L octane sulfonic acid sodium salt, 0.5 mmol/L Na2EDTA and 5 mmol/L triethylamine, pH 3.8） and methanol （90：10, v/v） at a flow rate of 1.0 mL/min. The detection limit （DL） was 0.9-23 nM for all the MANTs and their metabolites with a sample volume of 50 μL. The method was shown to be highly reproducible in terms of peak area （intraday, 0.08%-1.85% RSD, n = 5）. The simultaneous measurement of these MANTs and their metabolites improved our understanding of the neurochemistry in the central nervous system （CNS） in relation to different addictive drugs （methamphetamine, heroin and their mixture） in drug-addicted rat models.

Comparison of protocatechuic aldchyde in Radix Salvia miitiorrhiza and corresponding pharmacological sera from normal and fibrotic rats by high performance liquid chromatography

AIM： To observe the effect of protocatechuic aldchyde on the proliferation of hepatic stellate cells （HSCs）. METHODS： Liver fibrosis was induced in rats by carbon tetrachloride （CCh）. Then normal and fibrotic drug sera were extracted from rats. The effects of protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza on HSC growth were determined by CCKoS. The protocatechuic aldchyde was separated by high performance liquid chromatography （HPLC） in a AIItima C18 column （250 mm × 4.6 mm, 5 μm） with a mobile phase of acetonitrile-4% glacial acetic acid solution （gradient elution） at the wavelength of 281 nm. RESULTS： Protocatechuic aldchyde, raw Radix Salvia miltiorrhiza and drug sera of Salvia miltiorrhiza were found to have inhibitory effects on proliferation of rat HSCs. Raw Radix Salvia miltiorrhiza had a stronger inhibitory effect than the drug sera. The fibrotic drug sera showed a higher suppressive effect than the normal drug sera （P 〈 0.05）. Protocatechuic aldchyde was found in crude materials of both Radix Salvia miltiorrhiza and its corresponding drug sera. The average recovery （n = 6） was 110.5% for raw Salvia miltiorrhiza Bge, 102% for normal drug sera and 105.2% for fibrotic drug sera. The relative standard devitation （RSD） was 0.37%, 1.96% and 1.51%, respectively （n=6）. The contents of protocatechuic aldchyde were 0.22%, 0.15% and 0.19%, respectively （n = 6） （P〈 0.05）. The RSD was 0.33%, 0.75% and 1.24% （n=6） for raw material of Radix Salvia miltiorrhiza, normal drug sera and fibrotic drug sera, respectively. The samples were stable for 6 d. CONCLUSION： Protocatechuic aldchyde can inhibit the growth of HSCs. HPLC is suitable for the determination of virtual bioactive components of Chinese herbal medicines in vitro.

Development of a Fast and Facile Analytical Approach to Quantify Radiometabolites in Human Plasma Samples Using Ultra High Performance Liquid Chromatography

Introduction: Conventional metabolite analyses often require manual sample preparation, generating variability of measurements. This study describes a new method to quantify radiometabolites in blood, combining ultra high performance liquid chromatography (UHPLC) and turbulent flow chromatography, an alternative fully automated process allowing analyte’s extraction. Methods: A new radiotracer for dopamine transporter imaging, namely LBT-999, was used to demonstrate the method’s robustness. Matrix effect, Turboflow column loading, linearity, specificity and precision were evaluated with in vitro samples of LBT-999 in human plasma. Radiodetector sensitivity and preliminary evaluation were respectively determined by analysis of calibrated samples of [18F]LBT-999 and blood samples from 4 healthy subjects injected with [18F]LBT-999, withdrawn at 5, 15, 30 and 45 min pi. Results: With three sequential loadings (3 × 100 μL) of the Turboflow column, mean coefficients of variation were 1%, below 2%, 2% and 30.9% for matrix effect, specificity, repeatability and intermediate precision, respectively. Correlation coefficients for linearity were superior to 0.97. Limits of detection and quantification of the radiodetector were fixed at 3 and 9 c/s. Retention times for [18F]LBT-999 and the two radiometabolites detected by radio-UHPLC were 6.5, 4.8 and 9.6 min. Forty-five min after the injection, parent fraction was still predominant with 57.8% ± 25% of the total radioactivity. Conclusions: An innovative approach, allying UHPLC and Turboflow column, was developed and its sensitivity, linearity, specificity and repeatability validated. Preliminary results of the clinical trial are in accordance with literature data, demonstrating its efficiency in radiometabolites quantification.

Separation and identification of moxifloxacin impurities in drug substance by high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform ion cyclotron resonance mass spectrometry

In this paper, a high-performance liquid chromatography coupled with ultraviolet detection and Fourier transform-ion cyclotron resonance mass spectrometry （HPLC-UV/FrICRMS） method was described for the investigation of impurity profile in moxifloxacin （MOX） drug substance and chemical reference substance. Ten impurities were detected by HPLC-UV, while eight impurities were identified by using the high accurate molecular mass combined with multiple-stage mass spectrometric data and fragmentation rules. In addition, to our knowledge, five impurities were founded for the first time in MOX drug substance.

Optimization of multicomponent solvent selection in high-performance liquid chromatography using a statistical method

A computer-assisted method is presented for optimization of multicomponent solvent mobile phase selection for separation of O-ethyl-N-isopropyl phosphoro(thioureido)thioates in reversed-phase HPLC and four geometric isomers of pesticides Decis in normal-phase HPLC.The method is based on Snyder's solvent selection triangle concept using a statistical method.The optimization of the separation over the experimental region is based on a special polynomial esti- mation from seven experimental runs,and resolution(R_s)is used as the selection criterion.Excellent agreement was obtained between predicted data and experimental results.

改进和优化黑果枸杞及其制品中花青素测定的pH示差法

建立一种基于美国官方分析化学师协会(Association of Official Analytical Chemists,AOAC)方法检测黑果枸杞及其制品中花青素含量的改进pH示差法。考察了黑果枸杞及其制品中花青素的最佳提取和检测条件,通过液相色谱-三重四级杆串联质谱法鉴别出黑果枸杞中花青素的具体化学结构,并计算出混合花青素的平均摩尔质量。通过分光光度法测得混合花青素的平均摩尔消光系数,对改进后的pH示差法进行方法学验证和花青素的含量测定。结果显示,最佳提取和检测条件如下:黑果枸杞花青素提取溶剂为盐酸-80%(体积分数)乙醇(3∶97,体积比),料液比为1∶100(g∶mL),提取温度为50℃,提取时间为30 min,缓冲溶液稀释5倍后静置平衡20 min。液相色谱-三重四级杆串联质谱法鉴别黑果枸杞中主要以矮牵牛素类花青素为主(占97.96%),黑果枸杞特有的混合花青素平均摩尔质量为912.7 g/mol,平均摩尔消光系数为29591 L/(mol·cm)。pH示差法改进后能够满足方法学验证要求,固体样品和液体样品最低检出限分别为28.2 mg/100 g、0.282 mg/100 mL。方法改进后花青素提取增长率均大于20%,静置平衡20 min后单次检测结果精密度小于0.3%。以矮牵牛素类花青素代替矢车菊素-3-O-葡萄糖苷计算花青素含量平均提高了2.41倍,能真实地反映黑果枸杞及其制品中花青素的含量。

高效液相色谱及其串联质谱技术在牙膏风险物质分析中的应用

牙膏是日常生活中必不可少的口腔清洁护理用品,牙膏的基料较为复杂,牙膏中潜在的风险物质对人体存在一定危害,因此牙膏产品的安全性已成为当今社会关注的焦点之一。利用现代先进的检验检测技术对牙膏中潜在的风险物质进行分析、研究,将有利于实现对牙膏产品的安全监管,本文综述了近几年来高效液相色谱及其串联质谱技术在牙膏产品检验标准和检验方法开发等方面的研究以及应用进展,一定程度上能为牙膏产品的质量控制和科学监管提供参考。

不同产地杜仲叶活性成分的定量分析

通过杜仲叶中主要活性成分绿原酸的提取率,优化杜仲叶活性成分的提取工艺,比较不同产地杜仲叶组成及含量的差异.以绿原酸为目标产物,通过单因素实验和Box-Behnken模型,并采用高效液相色谱法测定秦仲叶和华仲叶中芦丁、松脂醇二葡萄糖苷、金丝桃苷、绿原酸及没食子酸五种活性成分含量.单因素条件对两者叶的提取效果影响均为:提取温度>提取时间>乙醇体积分数>料液比.在最佳提取条件下,秦仲叶绿原酸提取率为62.94%,华仲叶绿原酸提取率为47.84%.两者叶中绿原酸含量最高,秦仲叶中芦丁、松脂醇二葡萄糖苷含量比华仲叶高出1.97倍和6.59倍,但华仲叶中金丝桃苷和绿原酸含量比秦仲叶高出1.41倍和1.16倍.本研究旨在为不同地域杜仲树种的选育以及临床上应用标准提供理论依据,帮助更加全面地评价不同产地杜仲药材质量.

高效液相色谱-串联四极杆质谱法测定人参组培不定根中11种皂苷成分

建立高效液相色谱-串联四极杆质谱(HPLC-MS/MS)法定量分析人参组培不定根中11种皂苷成分。样品经粉碎研磨,以70%甲醇水溶液为溶剂进行超声提取,离心过滤后测定。采用C_(18)色谱柱(100 mm×2.1 mm,1.8μm),用水和乙腈进行梯度洗脱,流量为0.3 mL/min,柱温为35℃。质谱采用电喷雾电离源,负离子扫描,多反应监测模式进行检测。11种皂苷的质量浓度在0.1~10μg/mL(Rb1为0.2~10μg/mL)范围内和响应强度线性相关,相关系数均大于0.995,各皂苷的定量限为0.001~0.010 g/kg,加标回收率为90.43%~97.82%,相对标准偏差为1.93%~6.33%(n=6)。该方法操作简单,分析时间短,灵敏度高,准确可靠,适用于人参组培不定根中多种皂苷类成分的同时测定。

高效液相色谱-串联质谱法检测牛组织和奶中咪多卡残留的研究

建立了一种检测牛组织和牛奶中咪多卡残留检测的高效液相色谱-串联质谱法。牛组织(肌肉、肝脏、肾脏、脂肪)和奶在NaAc缓冲体系中酶解,经HCl溶液提取,WCX固相萃取柱净化,以0.3%甲酸水溶液(含20 mM甲酸铵)和0.3%甲酸乙腈为流动相进行梯度洗脱,在HILIC色谱柱上分离,在电喷雾正离子(ESI^(+))模式下,用多反应监测(MRM)模式检测,同位素内标法定量。结果表明:咪多卡在2.5~1000 ng/mL的浓度范围内呈现良好线性关系,相关系数(R^(2))大于0.99;咪多卡在牛组织和奶中的检测限均为10μg/kg,定量限均为20μg/kg;咪多卡在牛组织和奶中20~4000μg/kg添加浓度水平上的回收率在70.9%~109%范围内;批内RSD在0.55%~9.59%之间,批间RSD在2.21%~12.1%之间。该方法具有灵敏度高、定量准确,重复性好等特点,可以满足牛组织和奶中咪多卡残留检测的要求。

高效液相荧光色谱法同时测定六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

目的建立一种用免疫亲和柱净化-柱后光化学衍生-高效液相色谱同时检测六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A的方法。方法样品采用60%乙腈超声提取,免疫亲和柱净化,采用XBridge^(®)Phenyl苯基色谱柱(4.6 mm×250 mm,5μm),以乙腈和0.1%磷酸溶液为流动相,梯度洗脱,光化学衍生仪衍生,通过切换荧光波长检测。结果黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A标准曲线的线性范围分别为0.006~0.108、0.500~2.500和0.202~1.009 ng,6种毒素的线性关系在0.9994以上,检出限分别为1.2、100.0和40.3 ng/ml,平均加标回收率为73.2%~92.3%,相对偏差为2.1%~5.4%。结论该方法具有专属性强、操作方便等特点,能够有效用于六神曲中3种毒素的同时检测和安全质量控制。

高效液相色谱内标法测定海水叶绿素a浓度

叶绿素a浓度是海水水质监测和海洋赤潮预警的重要指标,高效液相色谱法作为叶绿素a浓度最准确的测量方法之一被广泛应用。本文选用β-阿朴-8’-胡萝卜素醛(β-Apocarotena-8’-carotenal,Apocarotenal)为内标物,建立了一种基于液相色谱内标法的海水叶绿素a浓度分析方法,在实验检测范围内,方法相关性良好,相关系数为0.9998,检出限为0.05μg/L,回收率为97.0%~100%,相对标准偏差为0.38%~0.55%,精密度良好。同时,利用实验室培养的小球藻藻液和现场海水样品对内标法和外标法进行了显著性差异评估。结果表明:两种方法不存在显著性差异,12组现场海水样品内标法和外标法的测定结果相关性良好,相关系数为0.92,平均相对误差为6.21%。液相色谱内标法测定海水叶绿素a浓度分离效果好、准确度高,能对实验室叶绿素a浓度的测量进行较为准确的质量控制。

UPLC-MS/MS法检测3种食品中松仁过敏原

基于食品基质中松仁过敏原Pin k 2建立了一种超高效液相色谱-串联质谱法。将松仁经过研磨、脱脂、浸提、酶解后经Easy-nLC 1000-QExactive高分辨质谱仪进行分离分析,结合Uniprot蛋白数据库以及ProteinPilotTM软件对质谱图进行数据处理,经BLAST验证特异性,最终筛选3条松仁特异性肽段。方法学验证结果表明,方法在0.001~50mg/mL范围内线性关系良好,定量限为1mg/kg;在饼干、巧克力和饮料3种空白基质中的平均回收率为88.50%~107.57%,相对标准偏差不高于6.08%,基质效应为89.77%~96.13%。该方法具有灵敏度高、特异性好的优势,可应用于饼干、巧克力、饮料等食品样品中松仁过敏原的检测,为我国食品标签真实性检验及食品中隐性过敏原的检测提供技术支持。

配合饲料中64种药物超高效液相色谱-三重四极杆串联质谱检测方法的研究

[目的]建立同时检测配合饲料中64种药物的超高效液相色谱-三重四极杆串联质谱(ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry,UHPLC-MS/MS)法,提高非法添加物的检测效率。[方法]采用Waters HSS T3型色谱柱(2.1 mm×100 mm,1.8μm)进行分离,流动相A为0.1%甲酸水溶液,流动相B为含0.1%甲酸的乙腈溶液,梯度洗脱,流速为0.40 mL/min,进样量为2μL;采用电喷雾离子源正离子扫描模式进行检测,多反应监测模式进行信号采集。比较4种样品提取溶剂以及2种固相萃取柱处理对目标药物的回收率,确定样品前处理的最佳方法。利用建立的UHPLC-MS/MS法对宁夏回族自治区不同来源的100批次配合饲料样品进行64种药物检测。[结果]配合饲料样品均质后,用含0.2%甲酸的乙腈水溶液(乙腈∶水=8∶2,V/V)提取,利用Oasis PRiME HLB型固相萃取柱对样品净化,多数目标药物的回收率在60%以上。64种药物在浓度为5.0~200.0μg/L的范围内线性关系良好,相关系数(R)均大于0.99;不同药物的定量限在5.0~10.0μg/kg;阳性添加5.0、20.0、50.0μg/kg 3个浓度的平均回收率在41.00%~120.49%,批内相对标准偏差(RSD)在0.54%~15.94%,批间RSD在1.25%~13.64%。在100个批次的配合饲料样品中均未检出目标药物。[结论]建立的UHPLC-MS/MS法线性关系良好、回收率高、精密度好,具有较高的重现性和较好的可操作性,可用于配合饲料中非法添加64种药物的筛查。

超高效液相色谱-串联质谱法同时测定当归9种真菌毒素

通过优化液相色谱条件和质谱条件等,建立超高效液相色谱-串联质谱法(UPLC-MS/MS)测定当归中药材中9种真菌毒素的检测方法。样品经70%甲醇溶液超声提取,经固相萃取柱净化;采用流动相A:2 mmol/L甲酸铵0.1%甲酸水溶液;流动相B:甲醇,经C18色谱柱分离后注入质谱仪,电喷雾电离源(ESI)和多反应监测模式(MRM)进行检测,基质外标法定量。经方法学验证,9种真菌毒素标准曲线线性关系良好(R^(2)>0.9950),方法的检出限0.04~1.43μg/kg,定量限0.12~4.75μg/kg,高、中、低3个浓度加标回收率为77.8%~113.6%,测定结果的相对标准偏差为0.2%~17.7%。该检测方法应用于72批当归样品9种真菌毒素同时检测,2批当归样品检出真菌毒素,检出率为2.8%,样品中主要的污染真菌毒素为黄曲霉毒素B_(1)(AFTB_(1))、黄曲霉毒素B2(AFTB_(2))、黄曲霉毒素G_(1)(AFTG_(1))。本研究建立的方法具有预处理简便、经济等优点,适用于当归样品9种真菌毒素的快速检测。