蜂王浆中抗生素残留物质氯霉素的含量测定

本文对蜂王浆中抗生素残留物质氯霉素(Chloramphenicol)的检测方法进行了探讨,建立了氯霉素残留的HPLC定量方法。样品采用液液分配和固相萃取法(SPE)进行纯化。氯霉素在0.1～10μg/mL间呈良好的线形关系,加样回收率为80.6～90.3%,RSD为3.48～5.63%。该方法稳定可靠,为蜂王浆产品中氯霉素残留的检测提供了依据。     

固相小柱对沙苑子总黄酮萃取方法的研究

目的:以沙苑子总黄酮为研究对象,考察固相萃取小柱的洗脱条件,确定固相萃取沙苑子总黄酮的实验方法。方法:以不同浓度的甲醇对不同填料的固相小柱进行洗脱,将洗脱液在岛津VP-ODS(150 mm×4.6 mm,5μm)柱上进行高效液相色谱分析。通过比较不同浓度及体积甲醇的萃取效率,确定沙苑子苷在固相萃取小柱上的洗脱条件。结果:用正相氨基小柱以50%甲醇洗脱效果好,回收率可达100%。结论:经试验,正相氨基小柱对沙苑子苷的萃取回收率约为100%,适用于沙苑子总黄酮中沙苑子苷的分析。     

反相C18固相萃取小柱用于蛋白质组分析中少量样品预处理

针对少量且复杂蛋白质组样品,开发一种耗时短、操作简便的分离方法。以人的脑海马组织蛋白样品为研究对象,采用反相C18固相萃取小柱,采用不同乙腈浓度对酶切后样品进行梯度洗脱,与反相高效液相色谱法对比分离效果。通过比较不同乙腈梯度洗脱方案所鉴定到的谱图数、非冗余多肽数、蛋白数和各洗脱组分间重复率分析,确定一种样品量少、简单易行、分离效果好的实验方案。虽然反相C18固相萃取小柱法鉴定蛋白总数占高效液相色谱法的85.5%,但其操作简单,耗时少。通过4种不同乙腈浓度方案比较,确定乙腈洗脱浓度优化为5%、15%、20%和90%时,分离30μg人的海马多肽混合物可以得到较优的分离效果。其蛋白鉴定数为反相液相色谱法的67.0%,且重复性良好。该结果证实反相C18固相萃取小柱分离效果比反相高效液相色谱法稍差,但此方法可以分离少量复杂蛋白质组样品。该方法分离样品充分,操作易行,耗时短,是进行蛋白质组学分析中少量样品的一个简便预处理方法。     

甜叶菊渣中总黄酮的纯化工艺研究

以甜叶菊渣为原料,采用大孔树脂吸附和溶剂萃取法相结合的方法,得到90％以上纯度的总黄酮.通过对大孔树脂及溶剂萃取法的各影响因素进行研究,确定纯化甜叶菊渣中总黄酮的最佳工艺条件:AB-8型大孔树脂吸附流速为2 mL/min、上样液质量浓度1.5 mg/mL、上样液pH值为3.5、上样量4 BV,解吸液为50％乙醇溶液、解吸量5 BV、解吸流速为1.5 mL/min.优化后的甜叶菊总黄酮平均纯度为50.11％.后经乙酸乙酯在常温条件下萃取5次,得到甜叶菊渣中总黄酮纯度为91.8％.结果表明:通过AB-8型大孔吸附树脂和乙酸乙酯萃取相结合的方法,可以很好地纯化甜叶菊总黄酮.     

固相萃取材料在蛋白质组学研究中的应用进展

随着蛋白质组学的发展,蛋白质的分离将发挥越来越重要的作用。固相萃取是近年发展起来的一种样品预处理技术,在对某些目标蛋白质的分离纯化中作用明显。与传统的液液萃取法相比,固相萃取可以提高分析物的回收率,操作简便、省时省力。萃取材料的选择对固相萃取效果有着极为重要的影响,硅胶和磁性纳米材料是常用的萃取材料。石墨烯的出现为萃取技术的发展提供了新思路。我们简要综述了近年来固相萃取材料在蛋白质组学研究中的应用进展。     

绿色高效液相色谱法分析四种虫草药材的游离和总甾醇含量

虫草类药材采用85%乙醇超声提取游离甾醇、采用0.5 mol/L氢氧化钾-乙醇溶液回流提取总甾醇,并用绿色溶剂(乙醇和水)固相萃取法纯化分析溶液。用HPLC-ELSD法测定麦角甾醇、胆甾醇和谷甾醇的含量,色谱柱为Poroshell 120 EC-C₁₈ (100 mm×4.6 mm, 2.7 μm),绿色流动相乙醇-水(89:11),流速为0.5 mL/min,结果表明所建立的分析方法3种甾醇成分在考察的浓度范围内,线性关系好、精密度和准确度高。20批虫草类样品均含结合型甾醇和游离型甾醇;冬虫夏草繁育品与野生品均含3种甾醇,两者含量相当;凉山虫草(野生品)、蛹虫草(培植品)、蝉花(野生品)可检测到麦角甾醇。该方法可有效鉴别冬虫夏草和其他3种虫草产品,为虫草类产品质量评价提升提供了依据。     

一种用高效液相色谱-电喷雾/串联质谱(HPLC-ESI/MSn)定量检测植物组织中微量1-氨基环丙烷-1-羧酸(ACC)含量的方法

介绍一种用高效液相色谱碳18柱(HPLC-C18)分离.电喷雾串联质谱(ESI-MSn)鉴定和定量检测植物组织中微量1-氨基环丙烷-1-羧酸(ACC)含量的方法,其最低检测限达0.7 pmol,标准曲线线性符合系数为0.9992.建立了从20～100 mg微量植物样品中提取ACC和固相萃取(SPE)预纯化的方法,加样回收率为95.1%±4.2%.此种提取方法结合HPLC-ESI/MSn分析与定性定量检测苹果'2001富士'中ACC含量为306.6 ng·g-1(FW),表明此法适合于定性定量检测植物样品中的微量ACC.     

黄芪甲苷在大鼠体内的药代动力学和组织分布研究

建立了固相萃取-HPLC-MS测定大鼠血浆中黄芪甲苷含量的方法,并对其在大鼠体内的药代动力学和组织分布进行了研究。分别以1,2,4 mg/kg的剂量对大鼠静脉给药,给药后2,10,20,30,60 min和1.5,2,3,4,6,8 h采集血样,同时以2 mg/kg的剂量对大鼠静脉给药,给药后20,60,240 min采集各组织,测定血浆样品和组织样品中的黄芪甲苷浓度。血药浓度-时间曲线按二室模型拟合最佳,t1/2(α)分别为12.36,7.05,15.98 min,t1/2(β)分别为69.14,73.28,95.24 min,AUC分别为277.36,415.36,623.15μg.min/mL,AUC与剂量的线性方程为y=113.64x 173.47(r=0.997),表明黄芪甲苷在大鼠体内呈线性消除。组织分布研究表明黄芪甲苷在体内分布较广。     

微藻胞间植物激素甄别方法与研究进展

植物激素是一类微量的内源化合物,可能是微藻生命活动过程中的通用"语言"。植物激素甄别是认知微藻群体感应和胞间通讯机制的关键。然而,由于植物激素具有超微量、性质复杂和干扰物质共存的特点,选择适宜的提取浓缩方法、提高检测灵敏度、减小基质效应和区分同分异构体仍是甄别植物激素的难点问题。本文综述了植物激素提取与检测的研究现状,关注了各类样品制备植物激素的方法和案例,重点阐释了固相萃取法、液液萃取法、磁性固相萃取法、液液微萃取法等提取方法以及液相色谱、液相色谱串联质谱、毛细管电泳等检测方法的优缺点,展望了利用串联固相萃取法从藻液提取痕量植物激素的前景,以及采用超高效液相色谱-线性离子阱-静电场轨道阱组合式高分辨质谱检测植物激素的方法,以期为藻际微生物生态相关研究提供方法参考。     

羊耳菊活性部位提取物在大鼠胆汁中代谢产物的研究

采用超高效液相色谱-四极杆串联飞行时间质谱(UHPLC/Q-TOF MS/MS)技术,分析大鼠灌胃羊耳菊活性部位提取物后,主要成分在大鼠胆汁中的排泄方式。SD大鼠胆管插管手术后,灌胃给予羊耳菊活性部位提取物(100 g/kg生药量),收集给药后12 h的胆汁样品。样品经正丁醇液液萃取法处理后,采用UHPLC/Q-TOF MS/MS对胆汁中的主要代谢产物的结构进行分析推测。实验结果显示,胆汁中共检测到27个代谢产物,样品中检测到大量的咖啡酰基奎宁酸还原,甲基化,葡萄糖醛酸化和甲基葡萄糖醛酸化代谢产物,以咖啡酰基奎宁酸的甲基葡萄糖醛酸化为主。说明大鼠口服羊耳菊提取物后,成分可通过胆汁进行消除,并且甲基葡萄糖醛酸结合物是主要的物质形式。     

Bazhen Decoction Protects against Acetaminophen Induced Acute Liver Injury by Inhibiting Oxidative Stress,Inflammation and Apoptosis in Mice

Bazhen decoction is a widely used traditional Chinese medicinal decoction, but the scientific validation of its therapeutic potential is lacking. The objective of this study was to investigate corresponding anti-oxidative, anti-inflammatory and anti-apoptosis activities of Bazhen decoction, using acetaminophen-treated mice as a model system. A total of 48 mice were divided into four groups. Group I, negative control, treated with vehicle only. Group II, fed with 500 mg/kg/day Bazhen decoction for 10 continuous days. Group III, received a single dose of 900 mg/kg acetaminophen. Group IV, fed with 500 mg/kg/day Bazhen decoction for 10 continuous days and a single dose of 900 mg/kg acetaminophen 30 min before last Bazhen decoction administration. Bazhen decoction administration significantly decrease acetaminophen-induced serum ALT, AST, ALP, LDH, TNF-α, IL-1β, ROS, TBARS and protein carbonyl group levels, as well as GSH depletion and loss of MMP. Bazhen decoction restore SOD, CAT, GR and GPx activities and depress the expression of pro-inflammatory factors, such as iNOS, COX-2, TNF-α, NF-κB, IL-1β and IL-6, respectively. Moreover, Bazhen decoction down-regulate acetaminophen-induced Bax/Bcl-2 ratio, caspase 3, caspase 8 and caspase 9. These results suggest the anti-oxidative, anti-inflammatory and anti-apoptosis properties of Bazhen decoction towards acetaminophen-induced liver injury in mice.     

Detection of β-blockers in urine by solid-phase extraction-supercritical fluid extraction and gas chromatography-mass spectrometry

The most convenient way to perform supercritical fluid extraction (SPE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). β-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SPE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the β-blockers were not significantly soluble in CO₂. SFE temperatures of 70°C and 150°C together with 200 μl of acetic anhydride and 400 μl pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, β-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.     

Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography-electrochemical detection (LC-EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC-EC, CE-UV, and CE-MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC-EC analysis than in CE-UV or CE-MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC-EC method was the most sensitive, but CE-UV and CE-MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Measurement of human urinary organophosphate pesticide metabolites by automated solid-phase extraction derivation and gas chromatography-tandem mass spectromy

Organophosphorus (OP) pesticides are among the most widely used pesticides in the United States. Human exposure to these pesticides may occur from their use on crops in agriculture and for pest control in residential settings. Most of the OP pesticides used in the United States are metabolized to up to three of six common urinary dialkyl phosphate metabolites. Quantification of these metabolites provides information on cumulative exposure to most OP pesticides. To accurately quantify OP pesticide metabolites in human urine, we developed a simple, highly sensitive, analytic method involving automated solid-phase extraction (SPE) of human urine, followed by post-extraction derivatization of the organophosphorus metabolites with 1-chloro-3-iodopropane, and analysis by isotope dilution gas-chromatography-tandem mass spectrometry. The styrene-divinyl benzene polymer-based SPE cartridges yielded good SPE recoveries of the metabolites because of their enhanced non-polar interactions. This method is less labor-intensive, more time-efficient, and reproducible than previously reported methods. Automation of the SPE allowed unattended extraction of urine samples, and hence, increased the sample throughput and reduced the inter- and intra-day variations. The method limits of detection were excellent for all analytes ranging from 50 pg/ml to 170 pg/ml. Relative standard deviations ranged from 2% to 12%.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Inclusion complex-based solid-phase extraction of steroidal compounds with entrapped beta-cyclodextrin polymer

Although the hydrophobic interaction-based solid-phase extraction (SPE) has been widely used, the extraction yields of steroids including androgens, estrogens, and corticoids were slightly different along with the physical and chemical properties of each molecule. A new SPE technique based on the formation of an inclusion complex with beta-cyclodextrin (betaCD) has been achieved for comprehensive sample purification in mass spectrometric analysis of 45 endogenous or synthetic androgens, 11 endogenous estrogens, and 21 corticoids. A copolymer of betaCD with epichlorohydrin was prepared by a cross-linking reaction followed by entrapment with 0.3M CaCl(2) to yield an improved SPE sorbent and the hydrolyzed urine samples were applied for purification. Steroidal compounds tested on the entrapped betaCD polymer were extracted with tetrahydrofuran and the overall recoveries ranged from 82% to 112% for 77 steroids in urine. Especially, the hydroxylated estrogens showed an excellent binding capacity (96-116% recovery) to betaCD through hydrogen bonding between their phenolic hydroxyl and exterior hydroxyl groups. A comparison between SPE methods with betaCD and Oasis HLB as a conventional cartridge showed that the extraction efficiency of polar steroids was significantly increased in the betaCD experiment, which has no connection with different polarity of steroid molecules. Due to its multi-functional mechanism derived from molecular inclusion and chemical interactions, this new SPE sorbent resulted in better selectivity and extraction efficiency than that obtained using the conventionally used hydrophobicity-based SPE method.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Urinary 5-HIAA measurement using automated on-line solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry

Quantification of 5-hydroxyindole-3-acetic acid (5-HIAA) in urine is useful for diagnosis and follow-up of patients with carcinoid tumors and for monitoring serotonin (5-hydroxytryptamine) metabolism in various disorders. We describe an automated method (XLC-MS/MS) that incorporates on-line solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and tandem mass spectrometric (MS/MS) detection to measure urinary 5-HIAA. Automated pre-purification of urine was carried out with HySphere-Resin GP SPE cartridges containing strong hydrophobic polystyrene resin. The analyte (5-HIAA) and internal standard (isotope-labelled 5-HIAA-d(2)) were, after elution from the cartridge, separated by reversed-phase HPLC and detected with tandem MS. Total cycle time was 5 min. 5-HIAA and its deuterated internal standard (5-HIAA-d(2)) were retained on and eluted from the SPE cartridges in high yields (81.5-98.0%). Absolute recovery was 96.5-99.6%. Intra-assay (n=20) and inter-assay (n=20) CVs for the measurement of 5-HIAA in urine in three concentration levels ranged from 0.8 to 1.4% and 1.7 to 4.2%, respectively. For urine samples from patients (n=78) with known or suspected metastatic carcinoid tumors, results obtained by XLC-MS/MS were highly correlated (R(2)=0.99) with the routinely used fluorometric method. This XLC-MS/MS method demonstrated lower imprecision and time per analysis (high-throughput) than manual solvent extraction methods and higher sensitivity and specificity than non-mass spectrometric detection techniques.     

Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.