Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy

A preparative reversed-phase HPLC system utilizing an isocratic mobile phase to purify up to 10-mg quantities of phospholipids is described. The method was developed to separate oxidation products of polyunsaturated phospholipids from intact, parent lipids. The method is useful for phosphatidylcholine and phosphatidylethanolamine on a preparative scale and for phosphatidylserine and phosphatidic acid on an analytical scale. Both intact phospholipids and oxidized phospholipids were monitored by absorbance at 206 nm. The oxidation products were simultaneously monitored at 234 nm where the intact phospholipids have only a very slight end absorption. Second-derivative uv spectroscopy proved to be extremely useful to identify the presence or to verify the absence of oxidation products in phospholipid samples. For autoxidized docosahexaenoic acid containing phospholipids, the absorbance maximum of diene oxidation products is 237 nm for the trans,trans (t,t) isomer and 246 nm for the cis,trans (c,t) isomers. Similarly, five classes of triene oxidation product stereoisomers have distinct absorbance maxima detected by second-derivative spectroscopy ranging from 269 to 292 nm.     

Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography

Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-¹⁴C]ABA, of which 98.9% was recovered.     

Sensitive flow-injection method with peroxyoxalate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum

A sensitive and rapid flow-injection analysis (FIA) of total choline-containing phospholipids (PLs) and a selective FIA method for the class assay of choline-containing PLs combined with preparative HPLC were described. The FIA method is based on peroxyoxaxalate chemiluminescence (PO-CL) detection of hydrogen peroxide enzymatically formed from choline-containing PL. The linear standard curves were obtained up to 1 nmol/20-μl injection (r>0.999) with the detection limits of 1.3–1.6 pmol at a signal-to-noise ratio of 2. The total amounts of choline-containing PLs in human serum were ranged from 1.63 to 3.19 mg/ml. The HPLC separation of choline-containing PLs was achieved with an aminopropyl-modified silica gel column using a mixture of acetonitrile-methanol-10 mM ammonium phosphate buffer pH 5.8 as eluent. The eluate corresponding to each choline-containing PL was collected, evaporated, dissolved in 0.1% Triton X-100 aqueous solution, and then injected into FIA system. The FIA method combined with preparative HPLC was applied to the assay of human serum.     

Antibacterial activity of a pepsin-derived bovine hemoglobin fragment

Peptic digestion of bovine hemoglobin yields a fragment with antibacterial activity. This peptide was purified to homogeneity by a two-step procedure including anion exchange chromatography and preparative reversed-phase HPLC. Mass determination and fragmentation indicated that this peptide corresponded to the 1-23 fragment of the alpha chain of hemoglobin. The minimum inhibitory concentration and mode of action of this peptide towards Micrococcus luteus strain A270 were determined. Hemolytic assay, interaction with liposomes, and study of its structure in solution were also performed.     

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 μm). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.     

A new chromatographic method for the preparative isolation of phosphoinositides and other anionic phospholipids

A new chromatographic procedure for preparative isolation of mono-, di- and triphosphoinositides and other anionic phospholipids with the use of adsorbents containing primary amino groups is described. Sorbents with immobilized neomycin, L-lysine and aminoalkyl groups were tested. Conditions for isolation of chromatographically pure phospholipids of separate classes on the above sorbents were developed. Isolation of polyphosphoinositides on the amino sorbents represents a new type of chromatography involving bioaffinity and ion-exchange interaction.     

Enantiomerization of chiral 2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4] benzothiadiazine 5,5-dioxide by stopped-flow multidimensional HPLC

An on-column stopped-flow multidimensional HPLC (sfMDHPLC) procedure using two chiral stationary phases (CSPs) and one achiral C18 column was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)(R,S)-2,3,3a,4-tetrahydro-1H-pyrrolo[2,1-c][1,2,4]benzothiadiazine 5,5-dioxide. Moreover, a stopped-flow HPLC (sfHPLC) method previously developed was applied to the determination of kinetic parameters of enantiomerization of the above compound in the presence of a CSP. The individual enantiomers of the studied compound were isolated in parallel by preparative HPLC and the rate constants and free energy barriers of enantiomerization were determined in different solvents (off-column method). The data obtained by sfMDHPLC, sfHPLC and off-column methods were compared. The (S) enantiomer of the studied compound (S18986) was prepared by asymmetric synthesis and subsequently purified by preparative HPLC, followed by the determination of rate constants and free energy barriers of enantiomerization in different buffer solutions at pH 2-9.3.     

Effective resolution of racemic pirlindole at the preparative scale

Pirlindole, a racemic antidepressant drug, was recently resolved using the derivatization method coupled with preparative HPLC. In order to improve this technique, the use of amino acid derivatives as chiral derivatizing agents (CDA) was investigated. Among different residues, the (L)-phenylalanine methyl ester was found to be very effective to separate pirlindole enantiomers using a medium pressure liquid chromatographic (MPLC) method. This procedure is better adapted to preparative application than HPLC. Thus, several grams of the pirlindole antipodes were isolated and characterized. These two enantiomers permitted the study of the stereochemical influence at the pharmacological level. Chirality 11:261–266, 1999. © 1999 Wiley-Liss, Inc.     

The heterogeneity of brewer's yeast old yellow enzyme

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.     

Separation of phospholipid molecular species by high performance liquid chromatography: potentials for use in metabolic studies

Different molecular species of phospholipids exhibit distinctly different patterns of biologic behavior. In this minireview, the utility of HPLC for analysis of molecular species of phospholipids is illustrated in studies in which it has been demonstrated that molecular species are selectively synthesized, selectively transported, and selectively participate in enzymatic reactions. HPLC appears to be more adaptable for routine use than older procedures used to separate phospholipid molecular species. Since the metabolism of intact molecules can be characterized with HPLC, this procedure promises to provide particularly novel information with respect to changes in composition brought about by remodelling reactions during the biologic life of specific phospholipids.     

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-μl aliquot of a 2:1 chloroform—methanol extract of amniotic fluid injected onto a 5-μm 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 (r_PG 0.94, r_PI 0.95, r_L/S 0.97).The advantages of the HPLC procedure include: (i) Selective separation for PG, PI, PS, and PE, as well as L and S at the same time. (ii) The internal standard allows individual concentration of phospholipids to be estimated. (iii) The procedure is rapid: 16 min for a single assay compared with 50 min for the standard TLC procedure.     

Separation of phosphatidylethanolamine from its oxidation and hydrolysis products by high-performance liquid chromatography

1-Palmitoyl-2-linoleoyl-phosphatidylethanolamine degrades relatively quickly when subjected to common storage and handling procedures. The degradation products consist of compounds in which double bonds in the sn-2 position acyl chain are partially oxidized and of products arising from the hydrolysis of the acyl ester bonds. Thin-layer chromatography (TLC), which is widely utilized to isolate and to ascertain the purity of phospholipids, does not readily separate the oxidation products from the parent lipid class. High-performance liquid chromatography (HPLC), however, employing a normal phase column and an isocratic, UV-transparent solvent system, can be employed to produce a rapid analytical or preparative of phosphatidylethanolamine (PE) from these degradative impurities.     

Studies of enantiomerization of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds

An on-column HPLC procedure using a chiral stationary phase (CSP) was developed for the determination of rate constants and free energy barriers of enantiomerization of (+/-)IDRA21. Subsequently, the HPLC method was applied for investigation of two structurally related chiral compounds. The individual enantiomers of the studied compounds were isolated in parallel by preparative HPLC and rate constants and free energy barriers of enantiomerization were determined in different solvents. The on-column enantiomerization data revealed that CSP induces different rate constants for the two enantiomers. The results generated off-line were used to determine the influence of solvents on the racemization of (+) and (-) IDRA21 and to gain further insight into the enantiomerization mechanism of chiral 3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide type compounds.     

Synthesis of antisense oligonucleotides with minimum depurination

The removal of 4,4'-dimethoxytrityl (DMTr) groups from oligonucleotides at low pH and the acid lability of the glycosidic linkage of purine nucleotides constitute an inherent conflict in preparative oligonucleotide chemistry. The use of a mildly acidic NaOAc buffer (10 mM, pH 3.0-3.2) allows adjustment of the pH in a range where the progress of the DMTr removal reaction can be monitored conveniently by HPLC and the optimum reaction time can be calculated. As a result, oligonucleotides with minimum depurination are obtained.     

Purification of Astroprotein (Astrocyte-Specific Cerebroprotein) by Reversed-Phase C-1 HPLC

A method for purification of astroprotein (an astrocyte-specific cerebroprotein) with HPLC is described. A linear gradient from 30 to 70% acetonitrile in 0.1% trifluoroacetic acid (pH 2.2) was applied to the reversed-phase C-1 (particle size 10 micron) column. Cerebroproteins from the crude extract from human glioma were clearly separated by this procedure. Highly purified astroprotein was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has immunoreactivity to antiserum against astroprotein. Reversed-phase C-1 HPLC offers advantages over previously available preparative techniques in the higher purity and better separation time of the products.     

High-performance liquid chromatography of phospholipids: quantitation by phosphate analysis

Quantitation of individual phospholipids separated by HPLC from tissue extracts by colorimetric analysis of phosphate was investigated. Elution of inorganic phosphate and breakthrough of lecithin were determined using radioisotopes. A substance which interfered with sample phosphate determinations was found in the column eluant, and a method to minimize its effect was developed. This method allows accurate quantitation of individual phospholipids present at a minimum of 20 nmol phosphate.     

Large Scale HPLC Purification of Oligodeoxyribonucleotides for Studies of the Binding of CC-1065 to DNA by NMR Spectroscopy

Abstract

We have investigated four different HPLC columns and several conditions in search of the most efficient procedure for purification of milligram amounts of DNA. We are reporting the optimal conditions for a preparative purification using a Dupont Zorbax Bioseries Oligo column.     

Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modem procedures for identifying Bifidobacterium species.     

Preparation of deacylated phosphoglycerides

O-(sn-glycero-3-phosphoryl) ethanolamine and O-(sn-glycero-3-phosphoryl) choline can conveniently be isolated on a preparative scale from egg-yolk powder or soybean phospholipids by the following procedure:

1.

1.|Mild-alkaline methanolysis of the lipids.     

Analysis of phospholipids in bifidobacteria

Methods of preparative chromatography on silica gel columns were used for obtaining preparations of polar lipids of bifidobacteria. Studies of the preparations by one-dimensional and two-dimensional TLC demonstrated that diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were the predominant phospholipids; minor phospholipids (phosphorus-containing components present in considerably lower amounts) included phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). Parameters of qualitative composition of phospholipids and glycolipids may serve as a set of chemotaxonomic markers in modern procedures for identifying Bifidobacterium species.