Quantitative separation of estrogens,androgens and progestogens using reverse phase high pressure liquid chromatography

Dual UV wavelength scanning at 206 and 254 nm was used to develop a sensitive, separation and quantitation procedure for estrone, estradiol-17β, testosterone, 4-androstene-3,17,-dione, progesterone and 17-hydroxyprogesterone using reverse phase high pressure liquid chromatography. The difference in UV absorption of the estrogens from the androgens and progestogens allows for correction of the co-elution of testosterone with estradiol-17β and 4-androsten-3,17,-dione with estrone. Incorporation of an isocratic step-gradient provides improved separation while maintaining shortened elution times for the less polar steroids.     

Fractionation of gibberellins in plant extracts by reverse phase high performance liquid chromatography

In studies on endogenous plant gibberellins (GAs), reverse phase (Bondapak C₁₈) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).     

Separation of nucleoside phosphoramidate diastereoisomers by high performance liquid chromatography and capillary electrophoresis

Separations of five diastereoisomers of nucleoside phosphoramidate derivatives (pronucleotides) were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and CE method using anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using in a normal-phase methodology. Capillary electrophoresis was used as an alternative technique to HPLC for the separation of pronucleotides. The diastereoisomers were fully resolved with sulfated cyclodextrins at both BGE pH (2.5 and 6.2). Limits of detection and limits of quantification, calculated for both methods, are up to 200 times higher in CE separations than in HPLC separations. The analytical HPLC method was then applied in a preliminary study for the pronucleotide 1 quantification in cellular extract.     

Analysis of the protein subunit structure of the oligomycin sensitive ATPase proteolipid fraction by reverse phase high pressure liquid chromatography.

The proteolipid fraction which is obtained from the bovine mitochondrial oligomycin sensitive ATPase complex by extraction with chloroform:methanol is resolvable into 7 components by preparative reverse phase high pressure liquid chromatography. Each of these 7 components is present together with 4 additionally resolved components in the proteolipid fraction which is obtained by extraction of submitochondrial particles with chloroform: methanol. Of the 7 components derived from the oligomycin sensitive ATPase, 4 have been identified with known protein subunits of the membrane sector of this complex, 2 are newly documented subunits of this complex, and 1 remains uncharacterized.     

Determination of hydroxyproline by high pressure liquid chromatography.

A rapid, precise, and simple HPLC method provides an assay of hydroxyproline from tissue extracts or solutions of collagen. Samples are hydrolyzed with 6 N HCl, derivatized with phenyl isothiocyanate, and chromatographed on a small, C18 reverse-phase HPLC column. Hydroxyproline (Hyp) is separated from other amino acids and detected by absorption at 254 nm. The method detects 0.40 to 36 micrograms of Hyp with a linear response. Separation requires a total of 6 min, including column cleanup and reequilibration. All components are commercially available, making this a convenient method for routine measurement of collagen concentration.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Oxidation/reduction of methionine residues in CCK: a study by radioimmunoassay and isocratic reverse phase high pressure liquid chromatography

The study was undertaken to investigate the oxidation and reduction of cholecystokinin (CCK) both as pure standards and as endogenous porcine peptides. Furthermore an attempt was made to prevent oxidation of the endogenous porcine peptides in the extraction procedure. CCK-8 and CCK-33 standards were always oxidized in weak solutions, CCK-8 varying from 26% to 67% oxidized and CCK-33 from 18% to 70%. Similarly, tissue extracts of porcine brain and duodenum contained oxidized forms of the peptide. CCK standards were readily oxidized in the presence of hydrogen peroxide. Oxidized CCK-8 standard and CCK-8 in porcine brain was 90% reduced and oxidized CCK-33 standard and in duodenal extracts was reduced by 70% by a 40 hour incubation with 0.725 mol/l dithiothreitol at 37 degrees C. Extraction of CCK peptides in the presence of 65 mmol/l dithiothreitol resulted in almost complete prevention of oxidation with over 95% of the peptides being obtained in the reduced state. This additive is therefore recommended for all tissue quantitation studies.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Analysis of enzymatic steroid conversions by high pressure liquid chromatography.

Enzyme catalyzed introduction of the 1–2 double bond into a steroid can be monitored through spectrophotometric changes accompanying electron acceptor reduction or through paper or thin-layer chromatographic analysis of the reaction product. The spectrophotometric method is not applicable to cases in which the oxidized form of the electron acceptor is continually regenerated. In studying such cases, we have found high pressure liquid chromatography (HPLC) to be a method of direct analysis more convenient than paper chromatography or tlc. Use of a water based eluant and a reverse phase column for the HPLC analysis allows direct injection of a sample of the aqueous reaction solution after acidification, and no extraction with an organic solvent is necessary.     

III. Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides

Reversed phase HPLC has wide applications in studies on neuropeptides. It provides a fast and effective technique for assessing the purity of synthetic peptides and for purifying mg amounts of synthetic peptides (examples: angiotensins II and III and analogues; neurohypophysial hormones). Due to the very small quantities of peptides which can usually be safely recovered after HPLC, the method is also useful in the isolation, purification and sequencing of peptides from biological sources (examples: urotensins I and II), and in the identification of neuropeptides in tissues when coupled with radioligand-binding displacement assays (example: [arginine⁸]vasotocin in the anterior ganglia of $\text{Aplysia}$$\text{california}$).     

Racemization studies in peptide synthesis through the separation of protected epimeric peptides by reversed-phase high performance liquid chromatography.

Separation of protected epimeric peptides, Z-Gly-Xaa-Xbb-OMe (where Xaa and Xbb = chiral amino acid residues), by reversed-phase HPLC was utilized for studying racemization in peptide synthesis. Thus, the following factors which might affect the extent of racemization during the coupling by the carbodiimide method were investigated: the combination of amino acid residues to be coupled, coexisting tertiary amine salts, and the relative configuration of the amino acid residues. The following points were revealed: the combination of bulky residues at the coupling site results in extensive racemization in a polar solvent such as DMF, the amine hydrochlorides cause less racemization than the p-toluene-sulfonates in DMF, and the influence of relative configuration differs depending on the solvent and the individuality of the amino components. Furthermore, the racemization-suppressing effect of some additives in the carbodiimide method was reevaluated by employing the same procedure.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

Separation and quantification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography

Separation and quantification of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE2 and PGE1 were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE1 or PGE2 and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE1, made significantly more PGE1 than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE2. PGE2 synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).     

Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.     

Quantitation of malondialdehyde (MDA) in plasma, by ion-pairing reverse phase high performance liquid chromatography

An ion-pairing high performance liquid chromatography method is described for the separation and quantitation of malondialdehyde in plasma. The MDA is determined as the thiobarbiturate chromogen formed by reaction of the plasma with 2-thiobarbituric acid under acid and heating conditions. However, under these conditions other interfering chromogens can also be formed. Using DEAE-cellulose chromatography followed by ion-pairing HPLC, we have been able to separate and quantitate the levels of MDA-TBA chromogen formed in plasma from other interfering chromogens. Measurements of MDA levels in the plasma of six normal individuals by HPLC gives a mean value of 4.57 +/- 0.33 nmole/ml, whereas the spectrophotometric determined value is 8.83 +/- 1.15 nmole/ml. These data suggest that some reevaluation of the numerous papers published on MDA levels in plasma using spectrophotometric methods may be necessary.     

Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.     

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.