家蝇滞育幼虫血淋巴中抗病毒肽甲醇萃取方法的优化

为了优化甲醇萃取滞育家蝇Musca domestica幼虫血淋巴中抗病毒肽的方法, 本实验分别比较甲醇及乙腈萃取血淋巴中抗病毒组分效果及最佳使用梯度、上样蛋白浓度、洗脱次数、洗脱流速及操作环境温度, 用Tricine-SDS-PAGE分析甲醇及乙腈的洗脱组分的构成。结果表明：甲醇萃取的活性组分多于乙腈萃取的; 用连续梯度（10％～100％）甲醇萃取的抗病毒活性组分, 比分别用10％, 30％, 50％, 70％, 90％和20％, 40％, 60％, 80％, 100％间断梯度洗脱的回收率高, 同时获得活性组分的重现性好。在0～4℃冰浴的环境下, 于250 mg/6 mL C18萃取柱内加入1.5±0.4 mg/mL蛋白浓度的血淋巴粗提液, 用1 mL/min流速4次洗脱, 抗病毒活性组分可以得到较好的分离效果。     

A simplified purification method for the analysis of abscisic acid

A fast and convenient method is given for the purification of abscisic acid (ABA) in plant extracts. Using a dual pH elution system on Sep-Pak C18 reverse phase prepacked cartridges, abscisic acid appears in the third eluent (32% methanol pH 8.0). The cartridges can be regenerated for multiple reuse.     

The quantitative analysis of endogenous folate catabolites in human urine.

In man folates are catabolized and excreted as inactive cleaved degradation products, a mixture of pteridines and p-aminobenzoylglutamate (pABGlu) or its acetamido derivative (apABGlu). The daily rate of excretion represents the inescapable use of the vitamin in metabolic activity and thus has implications for determining the recommended dietary allowance for the vitamin. Furthermore, the rate of catabolism has been suggested to rise during pregnancy and in certain disease states. A method is described for the quantitative extraction and assay of the folate catabolites pABGlu and apABGlu in human urine. Aliquots of 24-h urine collections are acidified and applied to columns of Dowex 50W cation-exchange resin. The catabolites are selectively batch-eluted with increasing concentrations of HCl. The fraction containing pABGlu is diazotized and then applied to a C18 Sep Pak column for further purification and concentration. The fraction containing apABGlu was deacetylated and reapplied to the Dowex column and then treated identically to the pABGlu fraction. The methanolic concentrates of both extracts were evaporated to dryness and reconstituted with water and pABGlu was regenerated by reductive cleavage of the diazotized material with Zn/HCl. The extracts of the two catabolites were separated by reverse-phase HPLC using a Radial Pak C18 column. Recovery of isolated material was monitored by the addition of high specific activity tritiated labels of both compounds added as internal standards to all urine aliquots prior to purification and analysis.     

Extractions of pyrroloquinoline quinone from crude biological samples

The best conditions for extractions of free pyrroloquinoline quinone (PQQ) from crude biological samples were investigated with various organic solvents and Sep-Pak C18 cartridges. PQQ was measured with use of its native fluorescence in aqueous solution. PQQ was well extracted into n-butanol under acid conditions, and addition of NaCl did not improve the solvent extraction. PQQ, which had been extracted into n-butanol, could be re-extracted into an aqueous phase by addition of either n-heptane or pyridine, or combination of them. PQQ, which had been adsorbed to Sep-Pak C18 cartridges, could be eluted with a mixture of pyridine and water with very excellent recovery. The recovery of 1 micrograms PQQ, which had been added to 1 g human liver, brain and 1 ml plasma and had undergone the n-butanol and the Sep-Pak extractions, was 50, 75 and 105%, respectively. From the blank fluorescence, endogenous levels of free PQQ in human liver, brain and plasma were found not greater than 0.41, 0.08 and 0.13 micrograms/g or ml, respectively, if present.     

A method combining solvent and gel extraction for isolation and preliminary purification of steroids in tissues

A method for the combined extraction and purification of steroids from testicular tissue is described. The tissue is homogenized and extracted with n-hexane/isopropyl alcohol, and the column to which a Sep-Pak C18 cartridge is attached. Following a wash of the Lipidex/Sep-Pak beds with water to remove inorganic and polar organic substances, steroids are eluted with 85% aqueous methanol. Most of the nonpolar lipids and phospholipids remain on the Lipidex/Sep-Pak. The steroid fraction is acidified with acetic acid, diluted to 70% methanol, and passed through a small bed of Lipidex 5000 to remove cholesterol. Recoveries of testosterone and progesterone are about 90%.     

Rapid determination of sixteen sulfonylurea herbicides in surface water by solid phase extraction cleanup and ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry

A sensitive and very fast analytical method has been developed for the simultaneous quantification of sixteen sulfonylurea herbicides in surface water. An ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry method with solid phase extraction for sample cleanup has been developed for screening sixteen sulfonylurea herbicides (oxasulfuron, thifensulfuron-methyl, cinosulfuron, metsulfuron methyl, sulfometuron methyl, triasulfuron, rimsulfuron, ethametsulfuron methyl, sulfosulfuron, tribenuron methyl, bensulfuron methyl, iodosulfuron methyl, pyrazosulfuron ethyl, prosulfuron, chlorimuron ethyl, ethoxysulfuron) in water samples simultaneously within 12 min. Water samples were acidified, and the target herbicides were extracted by passing through ProElut C18 extraction cartridges. After drying by nitrogen flow, the cartridges were eluted with elution solvents, and the eluate was then evaporated to dryness, redissolved and analyzed. The mobile phase composed of 0.02% formic acid and acetonitrile using gradient elution. A triple quadrupole mass spectrometer equipped with an electrospray ionization source operated in the positive ion with selective reaction monitoring mode. Each of the analytes in all the samples was monitored using protonated molecule and its two characteristic fragment ions for confirmation. The limits of detection for all analytes were below 1.0 ng/mL, except for sulfosulfuron and prosulfuron, and limits of quantitation were between 1 and 8 ng/mL for this method. Three water types were used for the validation of the method.     

Isolation and identification of androstanediol glucuronide from human plasma

[3H]Dihydrotestosterone (50 microCi) was infused into normal men and women for 8 h. It was previously shown that this was sufficient time for this material to reach a steady state. Venous plasma was obtained at 6 and 8 h, pooled, and the unconjugated steroids removed by ether extraction. The remaining plasma was adjusted to pH 4.9 and the steroid conjugate was extracted first with ethyl acetate and then with an ether-ethanol mixture. The extracts were combined and taken to dryness. Steroid sulfates were solvolyzed using dioxane, and the mixture partitioned between ether and 1% NaOH. The aqueous phase was acidified and added to an XAD-2 column, washed with water, and the glucuronide fraction eluted with methanol. The solvent was concentrated and the methanol extract was passed through a C18 Sep-Pak, filtered through an Acrodisc CR and then subjected to gradient high performance liquid chromatography [HPLC] (Nova-Pak C18, KH2PO4, pH 3, and methanol). The fractions containing steroid glucuronides were collected and esterified with diazomethane and then acetylated with acetic anhydride in pyridine. The glucuronide triacetyl methyl ester (GAME) derivatives were then run in a second HPLC system (3 Lichrosorb 5 mu columns, 4 mm x 25 cm) using a gradient of ethanol-heptane and heptane. We clearly established that this system separates 3 alpha-diol GAME conjugated at the 17 and 3 positions (44 vs 50 min) with authentic samples previously synthesized in our laboratory. We concluded that the pooled plasma contained only the 17-GAME conjugate. No significant activity of the 3-glucuronide was detected. The natural compound in circulation, therefore, is 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.     

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.     

Determination of amoxicillin in plasma samples by capillary electrophoresis

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C₁₈ cartridges followed by elution with water–methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 μg l⁻¹ by the FASI technique.     

SPE-GC-MS for the sampling and determination of unmetabolized styrene in urine

The urinary excretion of unmetabolized styrene can be a very good indicator for biomonitoring styrene in occupationally exposed people. The use of a new urine sampling system, involving a solid-phase extraction cartridge, offers several advantages for determining styrene. The advantages are especially related to the pre-analytical phase of styrene determination, which may be influenced by many variables. The effect on styrene recovery of sorbent type, eluting solvent, elution volume, elution flow-rate, and the addition of methanol to the washing solvent, was evaluated by experimental design methodology. As a result, Oasis HLB cartridges were selected for urine sampling, as well as 1.5 mL of ethyl acetate at 0.5 mL/min for eluting the retained styrene. These conditions were then applied to the validation of the solid-phase extraction combined with GC-MS method for the sampling and analysis of unmetabolized styrene in urine. The overall uncertainty was in the 12-22% range and the limit of detection was 2.2 microg/L for a 4 mL urine sample. The stability of styrene has been studied both in cartridges and in vials under different storage periods. After 1 month period the styrene stored on cartridges at room temperature remained stable, whereas this is not the case for styrene recovery from vials. The results obtained indicate that on-site solid-phase extraction of urine can provide a simple, accurate and reproducible sampling and analytical method for the biomonitoring of styrene in urine.     

Microanalytical GCMS method for the assay of the food additive 2-t-butyl-4-methoxyphenol (BHA) in plasma

A GC/Ms method is described for the determination of the antioxidant 2-t-butyl-4-methoxyphenol (BHA) in plasma using Sep-Pak C18 cartridges for extraction. Picogram amounts of BHA could be detected in rat plasma with a high degree of specificity, accuracy and significant time and material saving.     

A convenient, unified scheme for the differential extraction of conjugated and unconjugated serum C19 steroids on Sep-Pak C18-cartridges

In order to conveniently and rapidly isolate by group both conjugated and unconjugated serum androgens, a scheme has been devised for their differential extraction from commercially available, disposable octadecylsilane cartridges (Sep-Pak C18). Using added radioactive steroid standards and detection of endogenous serum steroids by group-specific enzymatic assays, the quantitative recovery of steroid glucuronides and sulfates in the 47% methanol fraction and of unconjugated steroids in the 100% methanol fraction was observed. Maximum recovery of serum protein-bound steroids (e.g. testosterone) was achieved with serum denatured by urea and heat. In order to separate glucuronides from sulfates, sequential hydrolysis of the conjugated fraction (47% methanol) by enzymatic hydrolysis and then organic solvolysis as well as an additional Sep-Pak cartridge extraction step was required. Groups of extracted steroids may be further separated and assayed by any appropriate method(s). An application is given which employs HPLC and an enzymatic assay for 17 beta-hydroxy- and 17-oxo-steroids to provide separate profiles of unconjugated, glucuronidated, and sulfated androgens in human, male serum.     

Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole-3-acetic acid in plants

Introduction  – Jasmonic acid (JA), abscisic acid (ABA) and indole‐3‐acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection. Objective  – To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species. Methodology  – Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C₁₈ cartridges. The final extracts were derivatised with diazomethane and then measured by GC‐MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure. Results  – Sequential elution of the assimilates from the C₁₈ cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones. Conclusion  – A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue. Copyright © 2008 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic assay for a new fasciolicide agent, αBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (αBIOF10) — a new fasciolicide agent — and its sulphoxide (SOαBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a μBondapak C₁₈ (10 μm) column, using methanol–acetonitrile–water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 12 ng/ml for αBIOF10 and SOαBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 μg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both αBIOF10 and SOαBIOF10. In urine, recovery was 99.6% and 93.1% for αBIOF10 and SOαBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.     

Mutagenicity in salmonella of hazardous wastes and urine from rats fed these wastes

15 hazardous industrial waste samples were evaluated for mutagenicity in the Salmonella plate-incorporation assay using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat liver S9. Dichloromethane/methanol extracts of the crude wastes were also evaluated. 7 of the crude wastes were mutagenic, but only 2 of the extracts of these 7 wastes were mutagenic; extracts of 2 additional wastes also were mutagenic. In addition, 10 of the crude wastes were administered by gavage to F-344 rats, and 24-h urine samples were collected. Of the 10 raw urines evaluated, 3 were mutagenic in strain TA98 in the presence of S9 and beta-glucuronidase. The 3 crude wastes that produced these 3 mutagenic urines were, themselves, mutagenic. Adequate volumes of 6 of the 10 raw urines were available for extraction/concentration. These 6 urines were incubated with beta-glucuronidase and eluted through Sep-Pak C18 columns; the methanol eluates of 3 of the urines were mutagenic, and these were the same 3 whose raw urines also were mutagenic. In general, the C18/methanol extraction procedure reduced the cytotoxicity and increased the mutagenic potency of the urines. To our knowledge, this is the first report of the mutagenicity of urine from rodents exposed to hazardous wastes. Based on the present results, the use of only strain TA98 in the presence of S9 might be adequate for general screening of hazardous wastes or waste extracts for genotoxicity. The urinary mutagenesis assay does not appear to be a useful adjunct to the Salmonella assay for screening hazardous wastes. The problems associated with chemically fractionating diverse types of hazardous wastes for bioassay are also discussed.     

Rapid purification of proteolipids from rat brain subcellular fractions by chromatography on a lipophilic dextran gel

Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform—methanol mixture.The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral—acidified chloroform—methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95–100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles.When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB2 by HPLC on silica columns is reported.     

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB₂ by HPLC on silica columns is reported.