Estimation of amniotic fluid phospholipids by high-performance liquid chromatography

We have developed a high-performance liquid chromatographic (HPLC) method for the analyses of surface-active amniotic fluid phospholipids, lecithin (L), sphingomyelin (S), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and phosphatidyl serine (PS), which are important in the prediction of fetal lung maturity. The method incorporates an internal standard in the amniotic fluid extract, and utilizes a 10-microliter aliquot of a 2:1 chloroform-methanol extract of amniotic fluid injected onto a 5-micron DIOL or CN HPLC column, and a variable-wavelength detector set at 203 nm. Amniotic fluid phospholipid estimations were determined on 40 amniotic fluid samples by the HPLC method and by the routine thin-layer chromatographic (TLC) method. Good agreement was observed between the two methods for the L/S ratio, PG, and PI (rPG 0.94, rPI 0.95, rL/S 0.97). The advantages of the HPLC procedure include: Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. The internal standard allows individual concentration of phospholipids to be estimated. The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

High pressure liquid chromatography of standard and amniotic fluid phospholipids

A method for the HPLC separation of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), and sphingomyelin (SPH) was achieved using five in-series columns packed with LiChrosorb, Partisil, and μ-Porasil adsorbents, a solvent mixture of chloroform/methanol/ammonium hydroxide (50 : 36 : 6.7, by volume), and a Pye LCM2 Moving Wire (FID) detector. The same phospholipid mixture was also separated using four μ-Porasil columns with the same eluent and detector. The latter conditions were found to be suitable for the analysis of phospholipids obtained after centrifuging, extraction, and precipitation of surface-active lipid components of patient amniotic fluid collected at amniocentesis section. The lecithin/sphingomyelin (L/S) ratios, determined by the HPLC method, correlated well with those determined by the TLC technique in four normal pregnancies, whereas results of shake tests did not correlate too well with L/S ratios determined by the above two chromatographic methods. Besides the lecithin/sphingomyelin ratio, the present method was able to supply additional information: the concentrations of phosphatidylglycerol and phosphatidylinositol, for prediction of fetal lung maturity, and the palmitic acid content of amniotic fluid phosphatidylcholine.     

Amniotic fluid uric acid levels determined by reversed-phase liquid chromatography with spectrophotometric and electrochemical detection

A rapid and precise, reversed-phase high-performance liquid chromatography method for amniotic fluid uric acid is described. Detection of uric acid and other naturally occurring constitutents is based on UV absorption at a wavelength of 280 nm and direct electrochemical oxidation at a potential of +0.800 V. The total analysis time is short (20 min) and the assay requires only filtration of the samples. Uric acid levels were determined in 14 samples of amniotic fluid obtained during the 15th to 24th week of gestation. Results ranged from 0.897 to 4.39 mg per 100 ml of amniotic fluid.     

Determination of trans-resveratrol in grapes by pressurised liquid extraction and fast high-performance liquid chromatography

A study has been made of the extraction of trans-resveratrol from grapes using pressurised liquids (PLE); for this, the first stage was to determine the stability of this compound during extractions at different temperatures (50, 100, 150 degrees C), with quantitative recoveries being obtained up to 150 degrees C. By employing solid-phase extraction (SPE) it was possible to retain this compound and separate it from other interfering substances present in the grape. The method developed comprises a sequential extraction of the sample adsorbed (0.5g) on a polystyrene-divinylbenzene based sorbent in the extraction chamber, first with water at 40 degrees C and 40atm of pressure (three cycles of 5min), and then with methanol at 150 degrees C and 40atm (three cycles of 5min). The trans-resveratrol content of the methanolic extract is determined by means of liquid chromatography. A rapid (5min) chromatographic method employing a monolithic column, with fluorescence detection, has been developed; for this, the conditions for detection of the compound were optimised (excitation at 310nm and emission at 403nm). The analytical parameters of the method of chromatographic analysis developed have been calculated: linear range (0.11-2.75mg/L), detection limit (0.003mg/L), quantification limit (0.004mg/L). Using this method, three varieties of grape have been analysed and the concentration of trans-resveratrol in these has been determined.     

Capillary electrophoretic determination of oxalate in amniotic fluid

A capillary electrophoretic (CE) assay for oxalate has been applied to the quantitative determination of free oxalate in amniotic fluid. Indirect absorbance detection of oxalate is accomplished with a chromate-based background electrolyte modified with ethylenediaminetetraacetic acid (EDTA). Detection interference due to the presence of high levels (≈4 mg/ml) of inorganic chloride is eliminated through a direct sample clean-up procedure based on cation (Ag⁺-form) resins. Separation interference from amniotic fluid proteins is prevented through the use of a simple aqueous-based dilution procedure. This method for the determination of oxalate in amniotic fluid provides precision of ≈5% relative standard deviation (RSD). Within-day precisions for the oxalate response and migration time are better than 3% RSD and 1% RSD, respectively. Between-day precisions for the oxalate response and migration time are better than 6% RSD and 3% RSD, respectively. The analytical recovery of oxalate (1000 ng/ml) spiked into amniotic fluid was better than 96%. The limit of detection (LOD) for the method is ≈100 ng/ml oxalate. This method also shows promising results for the determination of oxalate in human blood plasma samples.     

反相高效液相色谱法测定实验动物血液和脑组织中川芎嗪

 用反相高效液相色谱法测定了川芎药材、实验动 物血液和脑匀浆中的川芎嗪,方法简便快速。在270 nm检测波长下,不经浓缩可检测动物体 内1 mg/L的川芎嗪,川芎嗪在5～500 mg/L范围内具有良好的线性关系,线性相关系数为0.99 9。川芎嗪的加标回收率为98%～103％。川芎提取物样品、处理后的动物血清和脑匀浆样品具 有较好的稳定性,室温下可放置1星期。     

Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.     

高效液相色谱法同时测定血清和尿中厚朴酚与和厚朴酚

 建立了大鼠服用厚朴提取物后的血清中及尿中厚朴酚与和厚朴酚的高效液相色谱测定法。色谱柱填料为SpherisorbC18,流动相为甲醇-水-冰醋酸(体积比为70∶30∶1),UV检测波长为294nm,灵敏度0.005AUFS。样品用甲醇沉淀蛋白,上清液酸化后用乙酸乙酯-乙醚萃取,然后测定其中的药物浓度。血清和尿中的药物浓度与峰面积的线性关系良好,线性范围分别为0.05～2mg/L(厚朴酚)、0.025～1mg/L(和厚朴酚);精密度和重现性良好。血清中厚朴酚与和厚朴酚的平均加样回收率分别为95.6%(RSD=3.85%)和93.8%(RSD=3.95%),尿中分别为96.0%(RSD=3.83%)和94.9%(RSD=3.54%)。     

Determination of phosphatidylcholine in amniotic fluid

A relatively rapid method for measuring phosphatidylcholine (lecithin) quantitatively in amniotic fluid has been described that requires 1 ml or less of sample for fluids having a total phospholipid concentration greater than 25 mg/liter. Following extraction with chloroform-methanol, the solvent is passed through a calcium hydroxyphosphate column which removes the acidic phospholipids and allows passage of the phosphatidylcholine and sphingomyelin. Hydrolysis with periodate-sulfuric acid selectively releases inorganic phosphate from the phosphatidylcholine that is measured by reduction of the formed phosphomolybdate complex to the usual blue color. Various mixtures of phospholipids were carried through the entire procedure with excellent recoveries. Phosphatidylcholine added to an amniotic fluid pool was also quantitatively recovered, so that the method appeared completely suitable for routine clinical laboratory use.     

Determination of didanosine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography-tandem mass spectrometry

A rapid and efficient high-performance liquid chromatography (HPLC)-tandem mass spectrometry method for the determination of didanosine concentrations in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in optima water and centrifuged. The supernatant was subjected to solid-phase extraction (SPE) prior to analysis. Plasma and amniotic fluid samples were extracted without pretreatment. An Agilent 1100 Series HPLC coupled with a Micromass Quattro II triple quadrupole mass spectrometer was used for all analyses. Chromatographic resolution was achieved on a Nova-Pak phenyl analytical column (2.0 x 150 mm, 4 microm particle size) equipped with a Phenomenex Security-guard phenyl guard cartridge (2.0 x 4.0 mm) using 60% methanol in 10 mm ammonium acetate buffer mobile phase for all matrices at a flow rate of 0.15 mL/min. The method yields retention times of 2.9 min for didanosine and 3.0 min for the internal standard, stavudine. Limits of detection were 1 ng/mL for all matrices. Recoveries were 70% or greater for both compounds in the different matrices. Within- and between-run precision (%RSD) and accuracy (%error) was less than 15% for all matrices.     

免疫吸附治疗用ProteinA切流膜色谱柱的研究

切流膜色谱柱是免疫吸附治疗中实现全血灌流的一种新的模式。实验研究表明,切流膜色谱柱的结构和血液流动形式对血细胞的损害很小。分别考察了水、血浆、血液等不同流体流速与切流膜色谱柱柱压降之间的关系,柱压降随着流体流速和粘度的升高而增高,血液流速为120mL／min时,柱压降达到93kPa;考察了ProteinA切流膜色谱柱对人血浆中免疫球蛋白IgG的吸附能力,切流膜色谱柱ProteinA健合量为139mg（6mgProteinA／g干介质）,血浆在柱中循环1h可吸附IgG553mg（23．8mgIgG／g平介质）,而血液在柱中循环1h,可吸附IgG499.4mm（21.5msIgG／g卡介质）。     

A Simple Liquid Chromatographic Method for Quantitative Extraction of Hydrophobic Compounds from Aqueous Solutions

Abstract

We are reporting a rapid, high-capacity liquid chromatographic method for quantitative extraction and concentration of hydrophobic compounds from biological fluids and aqueous solutions. Samples are injected into commerically-available cartridges (Sep-Pak C^18R) containing a microparticulate, reversed phase packing which retains hydrophobic compounds. Inorganic salts and organic hydrophilic contaminants are removed with a water wash. Hydrophobic compounds are eluted quantitatively with minimal volumes (～5 ml) of organic solvents. As demonstrated with radiolabeled taurocholate, thin-láyer chromatography, enzymatic fluorimetry and capillary gas chromatography, complete recovery of bile salts from large volumes of urine, serum, amniotic fluid and hydrolysis reaction mixtures was achieved at flow rates up to 20 ml/min. A single cartridge concentrated approximately 50 mg of either taurocholate or the more polar bile salt, taurolithocholate sulfate. The technique is simple and applicable to the isolation of a wide range of hydrophobic compounds from aqueous solutions.     

Simultaneous Separation and Determination of Seven Quinolones Using HPLC: Analysis of Levofloxacin and Moxifloxacin in Plasma and Amniotic Fluid

A reversed-phase high-performance liquid chromatographic method is described for the determination of seven quinolones in plasma and amniotic fluid. Experimental designs have been applied for the optimization of the method in order to determine the experimental conditions for maximized resolution and minimized retention time. A total desirability function D that weights the responses together was used to optimize the two different responses simultaneously. The experimental responses were fitted into a second order polynomia. The optimum assay conditions were: 15 mM citrate buffer, pH 3.2, 9% MeCN, 5% MeOH, 5 mM TMAB, 1.5 mL min⁻¹ flow rate and 40 °C column temperature. A simple and efficient solid phase extraction has been applied for preparation of samples. The recovery of quinolones is >95%. The optimized assay condition was validated according to Federal Drug Administration (FDA) guidelines to confirm specificity, linearity, accuracy, precision and robustness. The method developed has been applied to quantification of levofloxacin and moxifloxacin in amniotic fluid and plasma.

     

Isolation of tomato pectin methylesterase and polygalacturonase on monolithic columns

An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 microg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 microg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

高效液相色谱法测定新雪颗粒中栀子苷的含量

建立了新雪颗粒中栀子苷含量的高效液相色谱测定方法。色谱柱为Diamonsil C18(200 mm×4.6 mm i.d., 5 μm), 流动相为乙腈-水(体积比为15∶85),流速1.0 mL/min,检测波长238 nm,进样量20 μL。栀子苷在25~400 mg/L时其浓度与峰面积呈良好的线性关系,方法平均回收率为101.2%,相对标准偏差(RSD)为1.6％。     

柱前衍生高效液相色谱法测定鱼罐头中的组胺

建立了一种测定鱼罐头中组胺含量的柱前衍生高效液相色谱(HPLC)方法。样品匀浆后采用高氯酸水溶液超声提取,提取液经丹酰氯衍生后,采用HPLC分离,紫外检测器检测,外标法定量。采用粒径为1.8 μm固定相填料的C18色谱柱,在0.3 mL/min的流速下,样品的分析时间小于5 min,并可有效地减少流动相消耗,节约成本。组胺在0.08～8.00 mg/L内线性关系良好,相关系数为0.99998;酱煮鲐鱼罐头中组胺在不同浓度水平的平均加标回收率均大于96%,相对标准偏差(RSD)小于2.5%;鱼罐头中组胺的定量限可达5.00 mg/kg。所建立的HPLC方法快速、灵敏度高、重复性好,前处理方法简单,可用于鱼罐头中组胺的测定。     

Characterization of C18 chemically modified silica-gel and porous glass phases by high resolution solid state NMR spectroscopy and other physico-chemical methods

Summary Two types of packing materials, porous glass (PG) and silica gel (SG) have been modified by mono- and difunctional octadecylchlorosilane. The packing surfaces before and after chemical modification have been characterized by CP/MAS NMR, SIMS, porosimetrical, elemental and chromatographic methods. On the basis of the physico-chemical and chromatographic data the PG and SG (of similar porosity) used as supports of chemically bonded phases for RP HPLC, have been compared. Presented at 13th Symposium on Column Liquid Chromatography, Stockholm, June 25–30, 1989.     

用反相高效液相色谱法分析测定Cd,Hg,Pb和Cu

用直接进样（Ｃ１８柱）反相高效液相色谱法研究了Ｍｅｎ＋－Ｄｚ（二硫腙）体系的色谱行为,建立了同时测定Ｃｄ,Ｈｇ,Ｐｂ和Ｃｕ的分析方法。方法的线性范围为０．０１～２．０ｍｇ／Ｌ,最低检出质量浓度为２．４～５．０μｇ／Ｌ,相对标准偏差为１．８％～９．７％,回收率为９４％～１０３％（Ｈｇ除外）。直接进样反相高效液相色谱法比萃取进样正相液相色谱法更快速,更简便,更容易操作,已用于人发测定。     

Rapid determination of minority organic acids in honey by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the determination of organic acids in honey is reported. Malic, maleic, citric, succinic and fumaric acids were identified and quantified in 15 min. First time repeatibility, reproducibility and recoveries were determined out for these acids in honey samples. Maleic acid was also quantified for first time by a chromatographic method. The organic acids were removed from honey by using a solid-phase extraction procedure with anion-exchange cartridges. Previously, the solution of honey was adjusted to pH 10.50 with 0.1 M NaOH and stirred for 15 min at room temperature. Then, this solution was adjusted to pH 5.00 with 0.1 M H2SO4. This procedure was carried out to avoid interferences in the baseline. The chromatographic separation was achieved with only one Spherisorb ODS-2 S5 column thermostated at 25 degrees C. Metaphosphoric acid (pH 2.20) was used as mobile phase at a flow-rate of 0.7 ml/min. Organic acids were detected with a UV-vis detector (215 nm). The precision results showed that the relative standard deviations of the repeatability and reproducibility were < or =3.20% and < or =4.86%, respectively. The recoveries of the organic acids ranged from 62.9 to 99.4%. Under optimum conditions the detection limits ranged from 0.0064 to 7.57 mg/kg and the quantification limits ranged from 0.025 to 10.93 mg/kg.