Use of a silicic acid microcolumn to assay acyl-CoA: lysophosphatidylcholine acyltransferase

A simple and rapid method for assaying acyl-CoA:lysophosphatidylcholine acyltransferase is described. This method is based on silicic acid microcolumn chromatography using labelled lysophosphatidylcholine (lysoPC) as substrate. The reaction was stopped by conventional Folch extraction. The chloroform extract (2 ml) was deposited on the silica gel and pushed through with air, and then elution was performed with methanol/water (50:50, v/v). Under these conditions, only the labelled phosphatidylcholine (PC) synthesized was retained on the gel, and this was then removed from the column and counted immediately. This method gave enzyme activities comparable to those obtained with the TLC method, and has proved to be reproducible. The new method, however, is both faster and safer than the classical TLC method.     

Quantitative derivatization and high-performance liquid chromatographic analysis of cyanobacterial heterocyst-type glycolipids.

Procedures are described for the rapid and quantitative analysis of cyanobacterial heterocyst-type glycolipids (HGs) by normal-phase HPLC of their per-O-benzoylated derivatives. Total lipids are obtained from 1 ml of nitrogen-fixing cyanobacterial culture by triplicate extraction with chloroform/methanol, 1/1 (v/v), and the HGs are isolated from other complex lipids by preparative silica gel TLC. A C18 solid-phase extraction cartridge is used to ensure quantitative salt-free recovery of the HGs, and the purified glycolipids are then rendered uv-absorbing by a per-O-benzoylation derivatization reaction for which optimal conditions have been established. Derivatives are analyzed within 12 min on a 3-microns silica HPLC column using a linear gradient of 2-propanol in n-hexane and uv monitoring at 230 nm. The reaction product was also used to determine the relative proportions of the glucosyl and galactosyl epimers of individual members of this class of glycolipid.     

Acyl-coenzyme A: cholesterol acyltransferase assay: silica gel column separation of reaction products

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) assays are usually performed by incubation of the enzyme with a labeled substrate followed by thin-layer chromatography separation and subsequent quantification of cholesteryl esters (CE) formed. Herein, a method is described for rapid separation of CE from other lipids, by elution from a silica gel column with a solvent mixture of petroleum ether/diethyl ether (98:2, v/v). Silica gel column chromatography is reliable and more rapid and safer than TLC. The best results were obtained when the reaction was stopped by Dole extraction followed by CE separation on a silica gel column. Assays for ACAT from rat intestinal microsomes showed that the specific activity values obtained using this method were reproducible and in good agreement with those obtained by conventional TLC method.     

Selective Solid-Phase Extraction of Urinary 2,3-Dinor-6-ketoprostaglandin F1α for Determination with Radioimmunoassay

This paper describes a method for selective two-step solid-phase extraction of urinary 2,3-dinor-6-ketoprostaglandin F_1α for reliable determination with radioimmunoassay. In the immunoreactivity profile of nonselectively extracted urine after HPLC separation, over 90% of the total 2,3-dinor-6-ketoprostaglandin F_1α immunoreactivity consisted of interfering material coeluting with 6-ketoprostaglandin F_1α and 2,3-dinor-6-ketoprostaglandin F_1α. Among the alkyl silica sorbents studied (methyl, butyl, octyl, and octadecyl), an efficient separation of 2,3-dinor-6-ketoprostaglandin F_1α from 6-ketoprostaglandin F_1α and the lowest immunoreactive concentration of analyte were achieved in extraction on the methyl silica sorbent by elution of 2,3-dinor-6-ketoprostaglandin F_1α with chloroform:hexane (85:15, v/v) from the cartridge. The proportion of specific immunoreactivity could be further increased by two-step extraction of sample on methyl silica cartridges, first at pH 3 and then at pH 10 using diethyl ether:hexane (85:15, v/v) and chloroform as eluent, respectively. After this, a high correlation was found with concentrations of samples determined by radioimmunoassay using three different antisera. A significant correlation of values was also observed between samples measured by radioimmunoassay and those measured by GC-MS. The values of 12-h excretion of 2,3-dinor-6-ketoprostaglandin F_1α in eight volunteers (268 ± 204 ng/g creatinine, mean ± SD) as well as the inhibitory effect of acetylsalicylic acid (74 ± 12%) are in accordance with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring subsequent TLC or HPLC purification.     

Isolation and comparative analysis of glycolipid fractions in bifidobacteria

A comparative TLC analysis of lipid extracts from Bifidobacterium longum B 379 M, B. bifidum 791, and B. adolescentis 94 BIM has been performed. It is demonstrated that carbohydrate-containing lipid components were present in the bacteria, which differed in their chromatographic mobility (Rf) from similar compounds isolated from actinomycetes Stomatococcus mucilaginosus PCM 2415T, Nocardiopsis dassonvillei PCM 2492, Propionibacterium propionicum PCM 2431, Saccharopolyspora hirsuta PCM 2279 (= ATCC 27875T), Rhodococcus equi PCMT 559 (= ATCC 3969), and Gordonia bronchialis PCM 2167. Polar lipids of bifidobacteria exhibited the closest similarity to their counterparts from propionic acid bacteria. Preparative chromatography (silica gel column I; elution with chloroform, acetone, and methanol) of the lipid extract of B. adolescentis 94 BIM made it possible to isolate fractions containing nonpolar lipids, glycolipids, and phospholipids. Further purification of the glycolipid fraction (column II; eluant, methanol gradient in chloroform) produced preparations of glycolipids and phospholipids. The preparations were studied by two-dimensional TLC using solvent systems chloroform-methanol-H2O MiLi Q (65 : 25 : 4, v/v/v) and n-butanol-acetic acid-H2O MiLi Q (60 : 20 : 20, v/v/v) for directions I and II, respectively. Two major glycolipids were revealed (G1 and G2), in addition to compounds characteristic of the polar lipid group and minor glycolipids (g), the latter being present in considerably lesser amounts.     

紫茎泽兰9-羰基-10,11-去氢泽兰酮分布积累动态

9-羰基-10,11-去氢泽兰酮为紫茎泽兰（Eupatorium adenophorum）的主要致肝脏毒性成分及杀虫的生物活性成分。从紫茎泽兰叶片中分离提纯得到9-羰基-10,11-去氢泽兰酮（Euptox A）标准品,建立了高效液相色谱法测定紫茎泽兰中Euptox A含量的分析方法。采用C18反相色谱柱,柱温30°C,以甲醇-水（60：40,v/v）为流动相、流速为0.8 mL.min–1、检测波长为255 nm进行测定。Euptox A在紫茎泽兰中的添加回收率为97.3%–103.7%,检测限为0.4μg.g–1。利用建立的方法测定Euptox A在紫茎泽兰体内分布与积累的动态变化规律。结果表明,Euptox A主要分布在紫茎泽兰的叶片中,且在营养生长期积累量高,生殖生长期积累量低。该方法快速、简捷,可用于紫茎泽兰原料及其产品中Euptox A成分的测定。     

Semipreparative isolation of individual cyanobacterial heterocyst-type glycolipids by reverse-phase high-performance liquid chromatography.

Procedures are described for the quantitative, semi-preparative isolation of individual cyanobacterial heterocyst-type glycolipids (HGs) by reverse-phase HPLC (RP-HPLC) and the modifications to conventional techniques necessary to prevent significant HG losses during sample preparation. Total lipids are obtained from cultures of nitrogen-fixing cyanobacteria by triplicate extraction with 200 packed-cell volumes of chloroform/methanol (1/1, v/v), filtered, and redissolved in chloroform for loading onto a short disposable column of acid-washed silica. After removal of neutral lipids, pigments, and the majority of monogalactosyldiacylglycerol with chloroform and chloroform/acetone, HGs are eluted along with other complex lipids in methanol. The complex lipids are then fractionated by TLC and the HGs isolated as two classes of differing mobilities. The individual components of each class are then resolved by isocratic elution with methanol/water (91/9, v/v) from a C18 RP-HPLC column with refractive index detection. Samples of up to approximately 1.0 mg lipid can be completely separated and the major components isolated in high purity from a single run. Structural studies on the major HG of Nostoc azollae show it to be the glycosylated hexacosane-1,3,25-triol found by others in Anabaena cylindrica.     

High-performance liquid chromatographic determination of alpha-acetyldigoxin in Digitalis lanata leaves.

An analytical method for the determination of alpha-acetyldigoxin in Digitalis lanata leaves by HPLC has been developed. The procedure consists of extraction of dry leaf powder with 50% methanol and cleanup by a Sep-Pak C18 cartridge prior to HPLC analysis. The quantitation is carried out by the incorporation of beta-methyldigoxin as an internal standard. HPLC is performed on an octylsilyl bonded silica column with acetonitrile/methanol/water (100/11/188, v/v). The effluent is monitored by uv absorption at 220 nm. The amount of alpha-acetyldigoxin per 100 mg of dry leaf powder is estimated at 5.55 +/- 0.21 micrograms (mean +/- SD). The average recovery of alpha-acetyldigoxin from added samples is 97.2%. The present method is sensitive, reliable, and relatively simple. Application of this HPLC method to the analysis of samples obtained by fermentation of the leaf powder is also demonstrated.     

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether–n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.     

Determination of cytochrome P450 2E1 activity in microsomes by thin-layer chromatography using [2-14C]chlorzoxazone

A thin-layer chromatographic assay was developed as an alternative method for the determination of cytochrome P450 2E1 (CYP2E1) in microsomes using [2-¹⁴C]chlorzoxazone. After incubation of microsomes with 0.125 μCi/mmol chlorzoxazone, chlorzoxazone and its single metabolite, 6-hydroxychlorzoxazone, were extracted using chloroform-2-propanol (85:15, v/v) and chromatographed on silica gel 60 F254 plates with acetone-hexane (45:55, v/v) as solvent. The plates were then exposed to X-ray film for 2 days to localize the radiolabelled chlorzoxazone and 6-hydroxychlorzoxazone. The metabolite and substrate regions were scraped and counted in a liquid scintillation analyzer. This method is sensitive enough to determine constitutive and induced CYP2E1 activities in liver or kidney microsomes. The precision of the method was similar to that of the HPLC method. The correlation coefficient between both methods was found to be 0.97 (n=21). Therefore, the TLC method constitutes a valuable tool for the determination of chlorzoxazone metabolism in microsomes.     

Solid-phase extraction of urinary 11-dehydrothromboxane B2 for reliable determination with radioimmunoassay.

In this paper we elaborate a one-step procedure for the selective extraction of urinary 11-dehydrothromboxane B2 on octylsilyl silica cartridges for reliable determination with radioimmunoassay. The immunoreactivity profile of nonselectively extracted urine after HPLC separation showed that as much as 70% of the total 11-dehydrothromboxane B2 immunoreactivity comigrates with polar interfering material. Its amount could be considerably decreased using acetonitrile:water (18:82, v/v) as wash solvent before elution of 11-dehydrothromboxane B2 from the cartridge. Alternatively, very high immunoreactive purity was achieved without the preceding wash step by selective elution of the analyte with dichloromethane:hexane (70:30). After both optimized steps in the extraction procedure were combined, immunoreactivity was found only in HPLC fractions corresponding to the retention volume of authentic 11-dehydrothromboxane B2. The homogeneity of this immunoreactivity was confirmed by two-step HPLC separation. A significant correlation of values was observed between samples measured after extraction and those measured after subsequent HPLC purification. A high correlation was also found with concentrations determined by radioimmunoassay using four different antisera. The values of 24 h excretion of 11-dehydrothromboxane B2 in 10 male volunteers (595 +/- 114 ng/g creatinine, mean +/- SD) as well as the inhibitory effect of acetylsalicylic acid (80 +/- 13%) closely correspond with those reported in the literature. This selective extraction procedure provides a high validity in radioimmunoassay without requiring any further purification step.     

Separation of some neutral lipids by normal-phase high-performance liquid chromatography on a cyanopropyl column: Ubiquinone, dolichol, and cholesterol levels in sheep liver

The normal-phase high-performance liquid chromatographic separation of neutral lipids into molecular classes was carried out on a cyanopropyl (CN) column eluted with isopropanol in hexane. Cholesteryl, retinyl, and dolichyl esters, triglycerides and vitamin E, ubiquinone, dolichol, phytol, and cholesterol eluted as separate peaks with 0.05% isopropanol in hexane. Cholesterol, retinol, diglyceride, and monoglyceride eluted as separate peaks with 0.75% isopropanol in hexane. These separations could not be achieved on a silica gel column. The method was used to assay sheep liver ubiquinone, dolichol, and cholesterol levels, that were determined as 77, 108 and 1864 micrograms/g wet wt, respectively.     

A Rapid Method,Combining Microwave-Assisted Extraction and Gas Chromatography-Mass Spectrometry with a Database,for Determining Organochlorine Pesticides and Polycyclic Aromatic Hydrocarbons in Soils and Sediments

A rapid method for determining organochlorine pesticides and polycyclic aromatic hydrocarbons in soils and sediments was developed to allow pollution surveys to be performed in emergencies. The method involves microwave-assisted extraction and uses an automated identification/quantification system with a gas chromatography mass spectrometry database. A sample (3 g) is extracted with a 3:2 v/v hexane:water mixture (10 mL) for 30 min using a microwave-assisted extraction system at 120°C. The hexane extract is then cleaned using silica gel, then analyzed by gas chromatography mass spectrometry. The total analysis time is approximately 4 h. The precision of the quantitative results and accuracy of the analyte identification were determined. The total analyte concentrations were generally comparable to (61%–110% of) the concentrations determined using a Soxhlet extraction method, but the concentrations of individual high-molecular-weight polycyclic aromatic hydrocarbons were unacceptably low compared with the concentrations determined using the Soxhlet method. However, these compounds (e.g., benzo(ghi)perylene and indeno(1,2,3-cd)pyrene) were subsequently efficiently extracted using a hexane:water:ethanol mixture. The accuracy of identification was evaluated using accurate masses determined by gas chromatography time-of-flight mass spectrometry, and the mass error was 2 ppm for 21 of the 22 compounds identified using the new method.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

快速鉴定格尔德霉素产生菌——吸水链霉菌17997中的洋橄榄叶素

对格尔德霉素产生菌吸水链霉菌17997的发酵液乙酸乙酯提取物进行了硅胶板TLC 初步分离和NaOH溶液喷涂显色,对显红色、具有抗革兰阳性菌活性的条带进行了HPLC分析,提示抗革兰阳性菌活性化合物可能为大环二内酯类抗生素洋橄榄叶素;以dTDP-葡萄糖-4,6-脱水酶 (Tgd) 基因保守区设计PCR引物,扩增了吸水链霉菌17997基因组DNA中的tgd并进行了序列分析,表明吸水链霉菌17997含有洋橄榄叶素生物合成基因簇中的tgd基因;对NaOH溶液喷涂显红色的化合物进行LC-(+)-ESI-MS分析,证实     

Determination of ochratoxin A by immunoaffinity column clean-up and HPLC In wheat and pig liver

Determination of ochratoxin A (OTA) by immunoaffinity column clean-up and HPLC detection was performed on wheat and pig liver. Several extraction protocols involving methanol and ethyl acetate were investigated. The optimum experimental conditions for analysis of OTA in artificially contaminated wheat (87.4% recovery) using immunoaffinity column clean-up was found to be the methanol: PBS (1∶1 v/v) protocol. These conditions, however, gave low recoveries for pig liver (40.4%).     

Extraction Efficiency and HPLC Determination of Imidacloprid in Soil

Two extraction methods were successfully applied to study the extraction of imidacloprid from soil. The first method, using a mix of acetone and hexane, was based on Soxhlet extraction, and the second method, using acetonitrile, methanol, and water, was a modified version of a liquid extraction method. Quantification was performed by reversed-phase High Performance Liquid Chromatography (HPLC) with Diode Array Detection (DAD) at 270 nm using 40:60 (v/v) acetonitrile/water as a mobile phase. The mean recoveries for imidacloprid from soil ranged from 82.6 to 109%, with a relative standard deviation between 1.9 and 5.6% for both extraction methods. The detector linearity and the reproducibility of the method proved to be very precise. The limits of detection were 0.08 and 0.06 mg kg^?1 for liquid extraction and Soxhlet extraction, respectively. Overall, the efficiency of the Soxhlet extraction at lower concentrations was better than at higher concentrations, while liquid extraction proved efficient for all spiked levels. Liquid extraction performed better at higher concentrations compared to Soxhlet extraction. Taken together, our study suggests that the analysis of imidacloprid in soil can be performed with the modified liquid extraction method with a higher recovery and a lower RSD than Soxhlet extraction.     

Determination of lamotrigine in whole blood with on line solid phase extraction

A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40microm C8 silica and separated on a C18 reversed-phase column (150x4.6mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 - acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0ml/min and a connection time of 1.0min, the complete cycle time was 10.0min. Detection was carried out at 260nm. No internal standard was necessary. The method was linear over concentration range 0.2-20.0microg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.     

Analysis of barbiturates in blood by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for the identification and quantification of barbiturates in blood at therapeutic levels has been developed. An ODS-silica column is used with an eluent of 40% methanol at pH 8.5. The barbiturates are detected at 240 nm. The sample preparation procedure involves extraction of unfractionated blood (100 μl) with hexane—diethyl ether (50:50, v/v) and is very rapid. Talbutal is used as an internal standard.The method has been applied to the determination of five barbiturates (amylobarbitone, butobarbitone, cyclobarbitone, pentobarbitone and quinalbarbitone) in blood after therapeutic doses of the drugs. An application of the HPLC assay to forensic casework is demonstrated.