A large-scale purification of phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylethanolamine, and phosphatidylcholine by high performance liquid chromatography: a partial resolution of molecular species

Egg yolk phospholipids, on a 10 g scale, were resolved by high-performance liquid chromatography on an 8-m silica column with elution by a stepwise chloroformmethanol gradient into homogenous phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylethanolamine fractions. Within these fractions, partial resolution on the basis of fatty acyl side chain composition was achieved.     

高效液相法测定蜂蜜中甘油的含量

建立了以混合溶剂直接提取测定蜂蜜中甘油含量的方法.采用ZORBAX Carbohydrate a-nalysis(4.6 mm×250 mm 5-Micro)色谱柱,以乙腈/水(80:20,v/v)为流动相,示差检测器,使用乙腈/甲醇/水混合作为溶剂快速检测甘油.结果表明,应用此法的甘油浓度在10.0 mg/L～250...     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Inositol phospholipid metabolism in human platelets stimulated by ADP

ADP-induced changes in inositol phospholipids, phosphatidic acid and inositol phosphates of human platelets have been studied in detail, using not only 32P labelling, but also by examining changes in amounts of the phospholipids, their labelling with [3H]glycerol and their specific radioactivities; changes in the labelling of inositol phosphates in platelets prelabelled with [3H]inositol were also measured. During the early (10 s) stage of reversible ADP-induced primary aggregation in a medium containing fibrinogen and with a concentration of Ca2+ in the physiological range (2 mM), the amounts of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and phosphatidylinositol 4-phosphate (PtdInsP) decreased (by 11.2 +/- 4.9% and 11.3 +/- 5.3%, respectively) while the labelling, but not the amount, of phosphatidic acid increased. The decreases do not appear to be attributable to the action of phospholipase C because the specific radioactivity of phosphatidic acid labelling with [3H]glycerol was not significantly increased at 10 s (although the initial specific radioactivities of the inositol phospholipids and PtdCho were more than double that of phosphatidic acid), and no increases in the labelling of inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) or inositol phosphate (InsP) were detectable at 10 s. Shifts in the interconversions between PtdInsP2 and PtdInsP, and PtdInsP and PtdIns may occur. By 30 to 60 s, when deaggregation was beginning, the amounts of PtdInsP2, PtdInsP and phosphatidic acid were not different from those in unstimulated platelets, but large increases in the 32P-labelling and [3H]glycerol labelling of phosphatidic acid were observed. Formation of [3H]inositol-labelled InsP3 was not detectable at any time in association with ADP-induced primary aggregation, indicating that degradation of PtdInsP2 by phospholipase C is not appreciably stimulated by ADP. These findings were compared with those obtained when platelets were aggregated by ADP in a medium without added of Ca2+ in which secondary aggregation associated with thromboxane A2 (TXA2) formation and release of granule contents occurs. At 10 s (during primary aggregation) the changes were similar in the two media. At 30 s and 60 s (during secondary aggregation in the low-Ca2+ medium), the increases in PtdInsP2, PtdInsP and phosphatidic acid in platelets suspended in the absence of added Ca2+ were larger than those in platelets suspended in the presence of 2 mM Ca2+. In the absence of added Ca2+, ADP-induced increases in the labelling of InsP3, InsP2 and InsP which were probably due to the effects of TXA2 since they were abolished by aspirin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of puerarin in human plasma by high performance liquid chromatography

Puerarin, an isoflavone C-glycoside, has been identified as the major active component isolated from Pueraria lobata (Kudzu) responsible for suppression of alcohol drinking. In order to conduct clinical studies of Kudzu's efficacy, a method for measuring its bioavailability and pharmacokinetic profile is needed. We have developed a gradient reversed-phase HPLC system for pharmacokinetic study of puerarin in human plasma. Solid-phase extraction was performed on an abselut Nexus cartridge (60 mg/3 ml) possessing adsorbent function with a recovery of >97% and 4-hydroxybenzoic acid was used as an internal standard. The HPLC assay was performed on a YMC ODS-A column (150 mm x 4.6mm i.d., 5 microm particle size). The HPLC mobile phase consisted of methanol/0.5% acetic acid with 20-35% methanol gradient at a flow-rate of 0.8 ml/min. The UV wavelength was set at 254 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a puerarin concentration range of 5-500 ng/ml in human plasma. The lower limit of quantification was ca. at 8 ng/ml of puerarin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 3 ng/ml. The preliminary pharmacokinetic study after oral administration of the Kudzu capsules containing 400mg of puerarin to a healthy volunteer confirmed that the present method was suitable for determining puerarin in human plasma.     

Analysis of sofalcone in human plasma by high performance liquid chromatography

A simple, rapid, sensitive and reliable high performance liquid chromatography (HPLC) method for the determination of the anti-ulcer drug sofalcone in human plasma was developed. Plasma was extracted with ethyl acetate under acidic conditions and sofalcone was determined by HPLC using a C18 column and (methanol-0.1% formic acid aqueous 80:20) mobile phase. The linear calibration curves of sofalcone in human plasma were obtained over the concentration range of 0.01-5.0 microg/ml. The lower limit of quantitation (LLOQ) was 10 ng/ml in human plasma. The precision measured for plasma was within 15%. Extraction recovery was over 85% in blood. The method was successfully applied to the identification and quantification of sofalcone in pharmacokinetic studies.     

Analysis of bacterial menaquinone mixtures by high performance liquid chromatography

Menaquinone mixtures of microbial origin were separated according to the chain length and the degree of hydrogenation of the polyisoprenyl side-chain by reversephase partition high performance liquid chromatography. Menaquinones can be measured easily and sensitively by the chromatographic system described here.     

Analysis of protein-glutathione mixed disulfides by high performance liquid chromatography

After precipitation of proteins; serum, hepatocytes, or glutathione-derivatized bovine serum albumin, by perchloric acid, dithiotheritol was used to reduce glutathione-protein mixed disulfides in the ether-washed, resuspended pellet. Following neutralization and S-carboxymethylation of free sulfhydral groups in the acid soluble fraction by iodoacetic acid, 2,4-dinitrophenyl derivatives of released compounds were produced by addition of ethanolic fluorodinitrobenzene. The 2,4-dinitrophenyl derivative of S-carboxymethylglutathione was measured by high-performance liquid chromatography. The method was found to be reproducible and limited only by the sensitivity of the glutathione analysis (about 10 pmol/sample). Quantitation of protein-bound glutathione was shown to be indepedent of the ratio of bound to soluble glutathione as well as the protein concentration in the sample. This method was found to produce glutathione values identical to those measured after borohydride reduction without the problems of foaming, sample loss, and the need of continuous pH adjustment during reduction.     

Assay of bovine fibrinopeptides by high performance liquid chromatography.

A method for separating small amounts (<10^?5 mol) of bovine fibrinopeptides A and B employing high performance liquid chromatography has been developed. The limit of detectability of this method is about 10^?10 mol of fibrinopeptide. The separation was achieved within 20 min under reversed phase conditions using isocratic elution with aqueous buffer-acetonitrile solvent systems.     

Purification of Clostridium perfringens enterotoxins by high performance liquid chromatography

Abstract Treatment of Saccharomyces cerevisiae a cells with α-factor partially inhibits mannosylation of the high M _r mannoproteins, although there is an increase in the total amount of these molecules present in the wall. They show a similar mobility in SDS-acrylamide gels to those from untreated mnn2 cells. No other significant effects on wall mannoproteins have been observed, except a decrease in the amount of the 29 kDa species.     

Fractionation of fecal neutral steroids by high performance liquid chromatography

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Complete separation by high performance liquid chromatography of metabolites of arachidonic acid from incubation with human and rabbit platelets

Separations of all major cyclooxygenase and lipoxygenase metabolites of arachidonic acid were obtained by high performance liquid chromatography (HPLC). A C₁₈ reverse-phase column was used in ion suppression mode to separate underivatized metabolites of arachidonic acid isolated from human and rabbit platelets. The metabolites were monitored by measuring radioactivity or ultraviolet light absorption at 192 nm (absorption by double bonds). Comparisons of TLC and HPLC separations demonstrated that the HPLC separation of metabolites of [1-¹⁴C]arachidonic acid was quantitative. HPLC also resolved several minor metabolites that were not detected by scanning of TLC separations.     

Measurement of muramic acid in marine sediments by high performance liquid chromatography

Bacterial biomass in marine sediments may be estimated from the amount of muramic acid present. A method for determining muramic acid by high performance liquid chromatography is described, which is simpler and faster than other methods. Muramic acid is released from sediment by acid hydrolysis, and assayed as an o-phthaldialdehyde derivative.     

Rapid separation of anionic oligosaccharide species by high performance liquid chromatography

We have developed a method for the rapid separation of anionic oligosaccharide species by high-performance liquid chromatography utilizing a MicroPak AX-10 ion-exchange column (Varian Associates) with the mobile phase consisting of 25–500 mm KH₂PO₄, pH4.0. Separation of oligosaccharides bearing zero, one, two, three or four sialic acid residues requires less than 45 min. Oligosaccharides containing mannose-6-PO₄ moieties in monoester or diester linkage can also be analyzed in this system. Preparative separations of as much as 20 mg of oligosaccharide can be accomplished in a single chromatographic analysis with quantitative yields of oligosaccharide. This method should prove useful for the rapid isolation and characterization of anionic oligosaccharide species.     

Menaquinone composition of some micrococci determined by high performance liquid chromatography

The menaquinone composition of some species of the genus Micrococcus was analysed by high performance liquid chromatography. Both unsaturated and hydrogenated menaquinones were detected. Micrococcus kristinae, M. lylae and M. nishinomiyaensis contained hydrogenated menaquinones while M. sedentarius contained unsaturated menaquinone. The predominant menaquinone isoprenologues of these species are MK- 7 (H₂), MK- 8 (H₂), MK- 8 (H₂) and MK- 8 , respectively. These observations confirm further the heterogeneity of the genus.