Selective quantification of arachidonic acid hydroperoxides and their hydroxy derivatives in reverse-phase high performance liquid chromatography

For the quantification of lipid hydroperoxides by high performance liquid chromatography (HPLC), it has been necessary to improve the detection system specific to the hydroperoxy group. We first developed a technique which combined detection by uv absorption due to conjugated diene and detection based on electrochemical (EC) reduction in reverse-phase HPLC for the selective determination of arachidonic acid hydroperoxides (hydroperoxyeicosatetraenoic acid, HPETE) and its reduced derivative, hydroxyeicosatetraenoic acid (HETE). 15-HPETE was quantified selectively by EC detection, although both 15-HPETE and 15-HETE were detected by uv absorption and were hardly resolved in the chromatogram. Isomers in HPETE obtained from autoxidized arachidonic acid were partially separated in the chromatogram and seem to have been quantified similarly to 15-HPETE. The application of this analytical system to the analysis of 15-HPETE added in human plasma has demonstrated that the recovery of HPETE extracted from human plasma is much lower than that from normal saline and that HPETE is reduced to HETE by incubation at 37 degrees C. The fact that a high concentration of glutathione accelerated this reduction may indicate that human plasma possesses a glutathione-dependent HPETE-reducing ability as a defense system against excess accumulation of lipid hydroperoxides. Blood plasma effectively suppressed the decomposition of HPETE induced by ferrous ion indicating the presence of factors which prevent the action of ferrous ion on HPETE.     

Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography

Following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. Upon development with a 60 to 100% (v/v) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. Only the lipids and very hydrophobic proteins were retained and resolved. Most noticeable among retained proteins was the Mr 100,000 catalytic polypeptide of each species' primary plasma membrane cation pump, the Na+,K+-ATPase of pig kidney and the H+-ATPase of Neurospora crassa hyphae. This simple 60-min procedure yielded nearly pure ATPase starting from crude membranes and in a completely volatile solvent, without detergent. When fungal plasma membranes were phosphorylated in vitro with [gamma-32P]ATP prior to injection, protein kinase activity was observed and this resulted in the phosphorylation of the H+-ATPase as well as of several other less-abundant hydrophobic membrane proteins. This procedure is useful as an alternative method for the rapid characterization of those membrane-associated polypeptides that contain several hydrophobic, transmembrane sequences.     

Analytical peptide mapping by high performance liquid chromatography. Application to intestinal calcium-binding proteins.

Peptide mapping of underivatized tryptic digests of bovine and chick intestinal calcium-binding proteins has been accomplished by high performance liquid chromatography (HPLC). High precision analysis of nanomolar quantities of peptides were achieved in less than 1 h (recycle time). Peak resolution and definition are superior compared to conventional techniques and recoveries of both small (4-residue) hydrophilic and large (30-residue) hydrophobic peptides are excellent. The total amino acid composition of the bovine intestinal calcium-binding protein has been accounted for on the basis of two tryptic maps of 20 microgram of protein each.     

The analysis of acyl-coenzyme A derivatives by reverse-phase high-performance liquid chromatography

A method for determining tissue levels of Coenzyme A and various short-chain-length acyl-CoA derivatives using high-performance liquid chromatography is presented. Separation of the various compounds was accomplished using a reverse-phase Spherisorb ODS II, 5-microns C18 column. Mobile-phase solvents were (a) potassium phosphate, 220 mM; thiodiglycol (2,2-thiodiethanol), 0.05% (v/v), pH 4.0 and (b) methanol, 98%; chloroform; 2% (v/v). The various acyl-CoA derivatives were detected by monitoring the column effluent at 254 nm. Nearly baseline separation was obtained for a standard mixture of free CoASH, methylmalonyl-CoA, beta-hydroxy-beta-methylglutaryl-CoA, succinyl-CoA, acetoacetyl-CoA, acetyl-CoA, propionyl-CoA, isobutyryl-CoA, beta-methyl-crotonyl-CoA, and isovaleryl-CoA. CoA derivative profiles were determined in neutralized perchloric acid extracts of perfused rat hearts and livers and of isolated rat liver mitochondria to demonstrate the utility of this method for assessing the levels of CoA derivatives in biological samples.     

Ion-pair reverse-phase high performance liquid chromatography method for determination of Huperzine-A in beagle dog serum

Huperzine-A (Hup-A), a biologically potent, reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (AD) in China, has very low blood concentration. In order to study the pharmacokinetics of newly developed Hup-A transdermal patches in animal, a rapid and sensitive ion-pair reverse-phase high performance liquid chromatography (RP-HPLC) method for the determination of Hup-A in beagle dog serum using mebendazole as internal standard has been developed and validated. The analyte and internal standard were extracted from serum using chloroform-isopropanol (95:5, v/v), analyzed on a C (18) column (5 microm, 150 mm x 4.6 mm i.d.) with a mobile phase consisting of methanol-water-glacial acetic acid (50:48.5:1.5, v/v/v), using sodium dodecylsulfonate as an ion-pair reagent, and detected with UV detector at 313 nm. The chromatographic run time was within 15 min. The assay was linear over the concentration range of 1-12 ng/ml and intra- and inter-day precision over this range was not more than 12.8%. The limit of quantification in serum was 1 ng/ml. The method was successfully applied to characterize the Hup-A concentration-time profiles and study the single and multiple doses phamacokinetics of Hup-A transdermal patches in beagle dogs. The pharmacokinetic study results showed that Hup-A patches has the characteristic of sustained or controlled drug release in vivo.     

Separation of fuchsin homologs using high performance liquid chromatography

Commercial samples of basic fuchsin contain variable proportions of four homologs, pararosaniline, rosaniline, magenta II, and new fuchsin. That different samples of dyes give variable staining is documented in the literature. Three commercial samples of basic fuchsin were investigated using high performance liquid chromatography. Separation of homologs was achieved using a C18 adsorbant and a solvent system of methanol:water:glacial acetic acid (66:24:10).     

Identification of N-myristoylated proteins by reverse-phase high performance liquid chromatography of an azlactone derivative of N-myristoylglycine.

A method is presented for the identification of N-myristoylated proteins. N-Myristoyl transferases have an absolute requirement for a free N-terminal glycine. N-Myristoylglycine is released upon mild acid hydrolysis of myristoylated peptides and proteins and its derivitization to a p-nitrobenzylazlactone with subsequent analysis by reverse phase h.p.l.c. enabled its detection to pmol levels. This facilitated the identification of N-terminal myristate in nmol quantities of purified proteins. Using this method we demonstrate that the alpha-subunit of the GTP-binding protein G0 is N-terminally myristoylated.     

Separation of tocopherol and tocotrienol isomers using normal- and reverse-phase liquid chromatography

This optimization study for tocopherols and tocotrienols involved both normal- and reverse-phase liquid chromatography using various columns and mobile phases. Normal-phase systems showed elution of the homologs in order of increasing polarity with separation based on methyl substituents on the chromanol moiety. Reverse-phase systems showed class separation based on the saturation of the phytyl side chain; the more saturated tocopherols were retained on the column longer. When the Zorbax ODS was used with an isocratic ternary acetonitrile:methanol:methylene chloride (60:35:5) mixture, the optimized resolution was greater than 2.0 and separation was achieved in less than 13 min, but there was no separation of beta- and gamma-tocopherols. The normal-phase silica and amino columns provided separation of all available isomers with resolution greater than 1.1 and separation times of less than 5.5 and less than 10 min, respectively. Optimized isocratic binary solvent mixtures of hexane:2-propanol were used for silica (99:1) and amino (98:2) columns. Derivative spectra showed differences depending on substituents in the chromanol moiety but not the phytyl side chain. Second- and fourth-derivative spectra gave the best differentiation of the vitamin E isomers.     

Microheterogeneity of the mammalian high mobility group (HMG) proteins 1 and 2 investigated by reverse-phase high performance liquid chromatography

Microheterogeneity within the high mobility group (HMG)-1 and HMG-2 groups of nonhistone chromatin proteins has been investigated using reverse-phase high-performance liquid chromatography (RP-HPLC) under conditions (acetonitrile elution with 0.1% trifluoroacetic acid (TFA) as the counter ion) which separate proteins primarily on the basis of differences in their overall hydrophobicity. RP-HPLC proved to be a fast and efficient means for separating multiple subspecies of both the HMG-1 and HMG-2 proteins from both crude nuclear extracts and from ion-exchange column "purified" protein samples obtained from different types of mammalian cell nuclei. In crude nuclear extracts at least eight different HMG-2 protein species (two major and six minor), but only one major HMG-1 species, could be resolved by RP-HPLC. Three of the minor HMG-2 protein species could be isolated in "pure" form from crude extracts in one RP-HPLC step whereas under the same conditions the two major HMG-2 peaks (as well as the other minor species) were contaminated with either HMG-1 or HMG-3 (a degradation product of HMG-1). In crude extracts the major HMG-1 fraction always seems to be contaminated with one of the HMG-2 subfractions. RP-HPLC analysis of apparently "pure" protein preparations isolated by ion-exchange chromatography techniques revealed that "pure" HMG-1 can be resolved into at least three different protein species and "pure" HMG-2 into at least four different species. Amino acid analyses of different resolvable forms of the HMG proteins were not inconsistent with the suggestion that at least some of these may be primary sequence variants of the individual proteins, but other possibilities also exist.     

A rapid method for the preparation of 125I-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography

Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies.     

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.     

Monosaccharide determination of glycoconjugates by reverse-phase high-performance liquid chromatography of their phenylthiocarbamyl derivatives.

A method for the determination of neutral sugars and hexosamines present in glycoconjugates by reverse-phase high-performance liquid chromatography (HPLC) of their phenylthiocarbamyl (PTC) derivatives has been developed. After acid hydrolysis, neutral sugars are converted to glycamines by reaction with ammonium acetate in the presence of sodium cyanoborohydride and are subsequently derivatized with phenylisothiocyanate, while the hexosamines present in the same hydrolysate, after separation on Dowex 50, are treated directly with this reagent. HPLC of the PTC-glycamines of the neutral sugars is performed on Microsorb C18 in an isocratic manner while chromatography of the PTC-hexosamines employs a Pico-Tag column with gradient elution to achieve separation from the PTC-amino acids. The procedure has proven to be highly sensitive, requiring as little as picomole amounts for the chromatographic step; monosaccharide compositions determined on glycoproteins and glycopeptides by this method were found to compare favorably to those previously obtained by other techniques.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Determination of firocoxib in equine plasma using high performance liquid chromatography

A new method of analysis has been developed and validated for the determination of firocoxib, a new nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses and dogs to control pain and inflammation associated with osteoarthritis. Following a liquid extraction using ethyl acetate:hexane (40:60), samples were separated by isocratic reversed-phase HPLC on a Sunfire C(18) column and quantified using UV detection at 290 nm. The mobile phase was a mixture of water with 0.025% trifluoroacetic acid and acetonitrile, with a flow-rate of 1.1 ml/min. The procedure produced a linear curve over the concentration range 5-1500 ng/ml with a lower limit of quantification of 5 ng/ml. Intra- and inter-assay variability was less than 7%. The average recovery was 98%. The method is suitable for the analysis of clinical samples from pharmacokinetic studies and can also be used for small volume sample sizes.     

Desalting of nucleotides by reverse-phase high-performance liquid chromatography

Chromatography of AMP, NAD⁺, or NADH on a reverse-phase C₁₈ Porasil B column rapidly removes ammonium formate or potassium phosphate from 90% of the nucleotide. Earlier reports showed these salts could not be separated from nucleotides by conventional desalting using gel filtration.     

Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives

This report describes a convenient, highly sensitive, and reproducible HPLC procedure for the quantitative analysis of gangliosides from brain tissues. The procedure involves the conversion of gangliosides to their perbenzoyl derivatives, isolation of the derivatives on a C18-reversed-phase cartridge, separation of the derivatives on a column (3-micron silica) maintained at an elevated temperature, and UV detection of the derivatives at 230 nm. The convenience of the procedure, its sensitivity, reproducibility, and application to the analysis of gangliosides from tissue sources make it the method of choice for ganglioside quantification in our laboratories. Three aspects of the procedure contribute to its convenience: reaction conditions that lead to single products, a convenient isolation procedure for the derivatives, and chromatographic conditions that provide resolution of the derivatives.     

Monitoring bacterial consumption of low molecular weight lignin derivatives by high performance liquid chromatography

The lignolytic capacity of some natural bacterial isolates was examined. Strains were selected from samples of decaying wood by growth in a minimal medium containing aromatic compounds with a structural relationship to lignin as the sole carbon sources. These included derivatives of benzoic and phenylpropanoic acids, as well as a mixture of low molecular weight compounds obtained by fractionation of kraft lignin. Reversed-phase high-performance liquid chromatography analyses before and after cell growth in the latter revealed a degradation pattern of the different compounds present in the culture which was characteristic for each of the strains studied.     

Development of structural analysis of sulfated N-glycans by multidimensional high performance liquid chromatography mapping methods

Although the biological importance of sulfated oligosaccharides has been widely recognized, there are only a few reports that describe detailed structures of sulfated N-glycans. This is largely due to the lack of a convenient method to identify structures of sulfated glycans found in low incidence. Here we develop multidimensional high performance liquid chromatography (HPLC) mapping methods for rapid and convenient identification of sulfated N-glycans. By using adequate quantities of sulfated N-glycans derived from LS12 cells, which are transfected with sulfotransferase cDNA, 40 different sulfated glycans have been successfully mapped. Furthermore, we have applied the HPLC data to identification of isomeric products resulting from an enzymatic reaction of N-acetylglucosamine 6-O-sulfotransferase-1 in vitro and revealed that this enzyme preferentially catalyzes sulfation of the GlcNAcbeta1-->2Manalpha1-->3Man branch in a biantennary acceptor.