Development of superparamagnetic high-magnetization C18-functionalized magnetic silica nanoparticles as sorbents for enrichment and determination of methylprednisolone in rat plasma by high performance liquid chromatography

In this study, a novel extraction and enrichment technique based on superparamagnetic high-magnetization C₁₈-functionalized magnetic silica nanoparticles (C₁₈-MNPs) as sorbents was successfully developed for the determination of methylprednisolone (MP) in rat plasma by high performance liquid chromatography (HPLC). The synthesized silica-coated magnetite modified with chlorodimethyl-n-octadecylsilane was about 320 nm in diameter with strong magnetism and high surface area. It provided an efficient way for extraction and concentration of MP in the samples through hydrophobic interaction by the interior C₁₈ groups. Moreover, MP adsorbed with C₁₈-MNPs could be simply and rapidly isolated through placing a strong magnet on the bottom of container, and then easily eluted from C₁₈-MNPs by n-hexane solution. Extraction conditions such as amounts of C₁₈-MNPs added, adsorption time and desorption solvent, were investigated. Method validations including linear range, detection limit, precision, and recovery were also studied. The results showed that the proposed method based on C₁₈-MNPs was a simple, accurate and high efficient approach for the analysis of MP in the complex plasma samples.     

金纳米粒子富集-高效液相色谱同时测定人血浆中三种氨基硫醇

建立了金纳米粒子富集-高效液相色谱-紫外检测(HPLC-UVD)同时测定人血浆中3种氨基硫醇(半胱氨酸(Cys)、高半胱氨酸(Hcys)、谷胱甘肽(GSH))的新方法。以Tween 20修饰的金纳米粒子作为选择探针萃取富集氨基硫醇。经二硫苏糖醇脱附后,采用SpursilTM C18柱(250 mm×4.6 mm, 5 μm)分离氨基硫醇,以60 mmol/L磷酸盐缓冲溶液(pH 2.0)等度洗脱,检测波长为200 nm。3种氨基硫醇的浓度分别在0.025～350 μmol/L、0.02～60 μmol/L、0.01～50 μmol/L内与峰面积具有良好的线性关系,相关系数均高于0.99。方法检出限(信噪比为3)分别为5.0、6.0和2.5 nmol/L,回收率为92.8%～106.0%。该方法能显著降低血浆样品中内源性物质的干扰,提高HPLC-UVD的选择性和灵敏度。将该方法应用于心血管病人血浆中上述氨基硫醇的分离测定,结果显示: 与对照组相比,疾病组血浆中的Hcys和GSH水平存在显著性差异,Cys不存在显著性差异。     

Application of liquid phase deposited titania nanoparticles on silica spheres to phosphopeptide enrichment and high performance liquid chromatography packings

A novel core-shell composite (SiO(2)-nLPD), consisting of micrometer-sized silica spheres as a core and nanometer titania particles as a surface coating, was prepared by liquid phase deposition (LPD). Here, we show the resulting core-shell composite to have better efficient and selective enrichment for mono- and multi-phosphopeptides than commercially available TiO(2) spheres without any enhancer. The material exhibited favorable characteristics for HPLC, which include narrow pore size distribution, high surface area and pore volume. We also show that the core-shell composite can efficiently separate adenosine phosphate compounds due to the Lewis acid-base interaction between titania and phosphate group when used as HPLC packings. After coating the silica sphere with titania by LPD, the silanol of silica spheres will be shielded and that the stationary phase, C(18) bonded SiO(2)-3LPD, could be used under extreme pH condition.     

在线衍生-高效液相色谱法同时测定血浆中的色氨酸及其代谢物

建立了醋酸锌在线衍生高效液相色谱法同时测定血浆中色氨酸(Trp)、犬尿氨酸(Kyn)、5-羟吲哚乙酸(5-Hiaa)和犬尿喹啉酸(Kyna)的方法。以3-硝基酪氨酸为内标(IS),采用Hypersil C-18柱(250 mm×4.0 mm, 5 μ m),以250 mmol/L醋酸锌溶液(pH 5.5)-乙腈(95:5, v/v)为流动相,流速为0.8 mL/min,柱温30℃。荧光检测波长设定:5-Hiaa为278 nm(λ_ex)/343 nm(λ_em), Kyna为244 nm(λ_ex)/400 nm(λ_em);紫外检测波长设定:Kyn和IS为360 nm, Trp为302 nm。4种物质的回收率在91.62%~114.17%之间;线性范围分别为2.50~320.00 μ mol/L(Trp), 0.32~15.36 μ mol/L(Kyn), 3.27~104.60 nmol/L(5-Hiaa), 14.00~464.80 nmol/L(Kyna);检出限分别为0.078 μ mol/L(Trp), 0.056 μ mol/L(Kyn), 0.690 nmol/L(5-Hiaa), 1.290 nmol/L(Kyna)。利用该方法对30例正常孕妇和28例女性健康志愿者的血浆进行测定,结果表明两组间Trp, Kyn和Kyna含量有显著性差异。该方法操作简便,重复性好,灵敏度高,适合于临床检测。     

Titania-coated monolithic silica as separation medium for high performance liquid chromatography of phosphorus-containing compounds

A method of preparing titania-coated monolithic silica stationary phase has been developed to achieve liquid chromatographic separation of phosphorus-containing compounds, which have recently been attracting increasing attention in biochemical research. The titania-coated silica columns exhibited efficient separation with low pressure drop, which is a typical feature of monolithic structures, and also possessed phospho-selectivity, which is a unique property of the titania surface. The material characteristics of titania-coated monolithic silica were examined, and then resin-clad columns were applied to the HPLC analysis of phosphorylated compounds. Highly efficient separation of phosphorylated substances indicated that the novel titania-coated monolithic silica column will find applications as a useful tool in the field of biochemistry, especially in post-genomic analyses.     

高效液相色谱法测定胭脂红酸的研究

研究了以反相C18柱,V(甲醇)∶V(乙腈)∶V(pH 2.8 H3PO4)=12∶8∶80混合溶液为流动相,对胭脂红酸进行高效液相色谱分析的方法。胭脂红酸在2.5~40μg/mL范围内的吸光度线性相关系数达0.9998,方法回收率在98.2%~101.1%,相对标准偏差为0.78%~1.2%,已用于样品中胭脂红酸的测定。     

全多孔球形硅胶基质高效液相色谱填料研究进展

结合本实验室的研究工作,评述了以全多孔球形硅胶为基质的正相反相色谱填料、手性色谱填料、离子色谱填料以及核一壳型色谱填料的研究进展．     

High‐performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles

The optimization of a porous structure to ensure good separation performances is always a significant issue in high‐performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high‐performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high‐performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high‐performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36 000 m^?1. Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans‐stilbene with separation factor as 7 and theoretical plate number as 76 000 m^?1 for cis‐stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long‐ established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.     

Extraction and preconcentration of formaldehyde in water by polypyrrole‐coated magnetic nanoparticles and determination by high‐performance liquid chromatography

In this study, a simple and rapid extraction method based on the application of polypyrrole‐coated Fe₃O₄ nanoparticles as a magnetic solid‐phase extraction sorbent was successfully developed for the extraction and preconcentration of trace amounts of formaldehyde after derivatization with 2,4‐dinitrophenylhydrazine. The analyses were performed by high‐performance liquid chromatography followed by UV detection. Several variables affecting the extraction efficiency of the formaldehyde, i.e., sample pH, amount of sorbent, salt concentration, extraction time and desorption conditions were investigated and optimized. The best working conditions were as follows: sample pH, 5; amount of sorbent, 40 mg; NaCl concentration, 20% w/v; sample volume, 20 mL; extraction time, 12 min; and 100 μL of methanol for desorption of the formaldehyde within 3 min. Under the optimal conditions, the performance of the proposed method was studied in terms of linear dynamic range (10–500 μg/L), correlation coefficient (R² ≥ 0.998), precision (RSD% ≤ 5.5) and limit of detection (4 μg/L). Finally, the developed method was successfully applied for extraction and determination of formaldehyde in tap, rain and tomato water samples, and satisfactory results were obtained.     

Extraction of aflatoxins from food samples using graphene‐based magnetic nanosorbents followed by high‐performance liquid chromatography: A simple solution to overcome the problems of immunoaffinity columns

In this research, magnetic graphene nanoparticles were prepared and used as adsorbents for preconcentrating the aflatoxins in rice, wheat, and sesame samples. For this purpose, graphene was synthesized by Hummer's method. Magnetically modified graphene formed by the deposition of magnetite (Fe₃O₄) on graphene was used for the separation of aflatoxins B₁, B₂, G₁, and G₂ from the samples. The extractants were subsequently analyzed with high‐performance liquid chromatography and fluorescence detection. Parameters affecting the efficiency of the method were thoroughly investigated. The measurements were done under the optimized conditions. For aflatoxins B₁, B₂, G₁, and G₂, limits of detection were 0.025, 0.05, 0.05, and 0.075 ng/g and limits of quantification were 0.083, 0.16, 0.16, and 0.23 ng/g, respectively. Accuracy was examined by the determination of the relative recovery of the aflatoxins. The relative recovery of aflatoxins B₁, B₂, G₁, and G₂ were quite satisfactory (between 64.38 and 122.21% for food samples). Relative standard deviations for within laboratory repeatability (n = 6) were in the range from 1.3 to 3.2. The application of this sorbent for the separation and concentration of the mentioned aflatoxins from food samples was examined.     

核壳型二氧化硅色谱填料的研究进展

复杂样品的高效快速分离分析是分离科学家所面临的挑战。近年来,核壳型二氧化硅色谱填料以其高效、快速和低背压的特点被广泛用于小分子、大分子和复杂样品的快速分离分析。该文系统综述了二氧化硅核壳色谱固定相快速分离的机理,制备方法及其在小分子、多肽和生物大分子快速分离分析方面的应用,同时对核壳型色谱固定相的发展进行了展望。     

柱切换技术-高效液相色谱法快速检测大鼠血浆中的普萘洛尔对映体

建立了快速检测大鼠血浆中普萘洛尔对映体浓度的柱切换-高效液相色谱法。将自制限进填料柱作为预处理柱,通过直接进样方式,使普萘洛尔对映体在预处理柱上保留,同时除去血浆中的蛋白质等大分子;再通过柱切换技术,使普萘洛尔对映体在键合型纤维素-三(3,5-二甲基苯基氨基甲酸酯)(Chiralcel OD-RH)分析柱上得到手性拆分。通过条件优化,确定切换前预处理流动相为硼酸盐缓冲液(pH 8.5)-甲醇(95:5, v/v),流速为1.0 mL/min;切换后分析流动相为异丙醇-乙醇-0.2 mmol/L硼酸盐缓冲液(pH 8.5)(30:30:40, v/v/v),流速为0.8 mL/min;切换时间为3 min;柱温为25 ℃;检测波长为293 nm。普萘洛尔两对映体在25～500 mg/L的质量浓度范围内具有良好的线性关系(r=0.9995), 3个加标水平(50、100、250 mg/L)的平均回收率为97.89%～101.56%,日内和日间精密度均小于5%。该方法简便、快速、灵敏、准确,适于血浆样本中手性药物的药代动力学研究。     

Direct separation and quantitative determination of glimepiride isomers by high performance liquid chromatography

A simple and sensitive high‐performance liquid chromatographic procedure for the determination of the trans isomer of glimepiride is reported. Chromatography accomplished direct separation of the cis and trans isomers of glimepiride on a Dikmonsil C18 (250×4.6 mm, 5 μm) column with a mobile phase consisting of methanol‐acetonitrile‐NH₄Ac buffer solution (1.5 mol L^–1, pH = 4.5) (1.1 : 1.3 : 1.0, v/v) at a flow rate 0.5 mL min^–1. The resolution (R_S) was 1.73 with a retention time of 24.885 and 23.018 min for the cis and the trans isomer, respectively. A standard linear calibration curve was established for the trans isomer of glimepiride over the range of 4.95–198.00 μg mL^–1 with a correlation coefficient of 0.99997. This method has been successfully used to analyze four different kinds of glimepiride product.     

扑草净、乙草胺混配除草剂的HPLC 分析方法

建立了正相高效液相色谱法定量分析二元混配除草剂"昆明6号"中扑草净和乙草胺的方法。采用ShimPackCLC CN(150mm×6.0mm)5μm柱,流动相为正己烷/氯仿(87∶13,V/V),λ=264nm,线性相关系数为0 9997～0 9999。扑草净和乙草胺的加料回收率分别为99 0%和101 0%,RSD分别为0 52%和0 60%。     

Memory effects with the on-column interface for on-line coupled high performance liquid chromatography-gas chromatography: The Y-interface

When using the on-column interface for on-line high performance liquid chromatography (HPLC)-gas chromatography (GC), there is a memory effect typically equivalent to 0.5–3% of the previous transfer. The shape of peaks distorted as a result of incomplete reconcentration of the initial bands enabled mapping of the distribution of the solute material in the uncoated precolumn and deriving the mechanism which causes the memory effect. The relatively slow transfer of HPLC eluent causes liquid being sucked backwards into the narrow interspace between the transfer line and the precolumn wall. Solvent is evaporated into the passing carrier gas and is replaced by more eluent pulled into this zone, resulting in enrichment of solute material. At the end of the transfer, some of this solute material enters the transfer line and remains there up to the subsequent transfer of an HPLC fraction. This problem is avoided by replacing the on-column injector used as interface by a Y-piece in which the eluent flow from HPLC and the carrier gas are joined. The memory effect was reduced to below 0.02%.     

高效液相色谱法同时测定反应液中的巯基乙酸和巯基乙酸异辛酯

建立了同时测定反应液中的巯基乙酸(TGA)和巯基乙酸异辛酯(TGB)的高效液相色谱分析方法.实验采用Shimpack C18色谱柱,紫外检测器,检测波长210 nm,以V(乙腈)∶V(水)=70∶30作为流动相(用H3PO4调节流动相pH为3),流速1.0 mL/min,柱温30 ℃.结果表明,TGA和TGB在上述色谱条件下可实现较好分离,测定结果的最大相对标准偏差分别为0.53%和0.46%,检出限分别为2.03×10-3 g/L和6.11×10-3 g/L,加标回收率分别在99.0%～100.8%和99.1%～100.7%.     

微柱高效液相色谱法测定丹参中的几种有效成分

研究了用微柱高效液相色谱法测定丹参中的4种有效成分(丹参素,原儿茶酸,原儿茶醛,丹酚酸B)的方法。丹参样品中的几种有效成分用体积分数40%的甲醇超声振荡提取,然后以WatersXterraTMRP18(1.0×50mm,2.5μm)微柱为固定相,甲醇和体积分数1%的HAc溶液梯度洗脱为流动相分离,在该色谱条件下,丹参中的4种有效成分在4.0min内可达到基线分离;用紫外二极管矩阵检测器检测。方法标准回收率为97%~102%,相对标准偏差为1.6%~2.3%。用此方法可测定几种丹参样品中的有效成分。     

Determination of phenazopyridine in human plasma by high performance liquid chromatography

Summary A simple, low-cost, sensitive and selective HPLC method was developed for the determination of phenazopyridine in human plasma. The method employs UV detection of phenazopyridine and of the internal Standard at 2 different wavelengths. Calibration curves were linear over a large dynamic range, i.e., within 0.05–10.0 μg mL⁻¹ with limit of quantification of 0.05 μg mL⁻¹, and a limit of detection of 0.01 μg mL⁻¹.     

Simple determination of pirfenidone in rat plasma via high-performance liquid chromatography

A simple, rapid and reliable high-performance liquid chromatographic method was developed and validated for the determination of pirfenidone and its major metabolites in rat plasma. Plasma proteins were precipitated with perchloric acid (10%, v/v) and the supernatant after centrifugation was determined using high-performance liquid chromatography. The analysis was carried out on a Lichrospher C(18) column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water containing 0.2% acetic acid (23:77, v/v) at a flow-rate of 1 mL/min. The eluant was detected at 310 nm. The calibration curves were linear over a concentration range from 0.15 to 76.67 microg/mL. The accuracy (relative error) of the assay ranged from -2.6 to 7.9% and the precision (coefficient of variation) was less than 4.5%. The established method has been successfully applied to a pharmacokinetic study of pirfenidone following a single oral dose to rats.