Reversed-phase high-pressure liquid chromatography of arachidonic acid metabolites formed by cyclooxygenase and lipoxygenases

High-pressure liquid chromatography is required to resolve the complex mixtures of arachidonic acid metabolites synthesized by many tissues. We have investigated some of the factors which affect the retention times of these substances in reversed-phase HPLC on columns of 5-micron octadecylsilyl silica. There are considerable differences in selectivity between mobile phases based on methanol and those based on acetonitrile, the latter being much better for cyclooxygenase products. The chromatographic behavior of peptidoleukotrienes (LTC4, LTD4, and LTE4) is quite different from that of other arachidonic acid metabolites which do not contain amino acids. Addition of phosphoric acid to the mobile phase results in very long retention times for peptidoleukotrienes. Very low concentrations of trifluoroacetic acid have effects similar to that of phosphoric acid, but as its concentration is raised, the retention times of peptidoleukotrienes decrease, whereas those of other arachidonic acid metabolites are unaffected. Changing the concentration of acetonitrile in the mobile phase also affects the retention times of peptidoleukotrienes differently from those of other metabolites. This information has been used to devise simple linear gradients which separate most of the major cyclooxygenase and lipoxygenase products of arachidonic acid metabolism.     

Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor

We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC. The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column. The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances. The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation. This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination. This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts. The recovery of atrial natriuretic factor is in the range of 80-100%. This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells.     

Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological samples using column switching and flow-through radioisotope detection

For the determination of salmon calcitonin and its degradation products in biological samples, a reversed-phase HPLC method with column switching and flow-through radioisotope detection has been developed using high specific activity [¹²⁵I]salmon calcitonin. Effects of the precolumn packing material and washing solvent were examined in terms of [¹²⁵I]salmon calcitonin recovery. Spiked samples of [¹²⁵I]salmon calcitonin in plasma and kidney homogenate were injected onto a LiChroprep RP-8 precolumn after dilution with 0.1% trifluoroacetic acid. After washing the polar interfering compounds with 0.1% trifluoroacetic acid, the concentrated [¹²⁵I]salmon calcitonin and its degradation products were eluted and separated on a W-Porex C₁₈ column with a gradient of 0.1% trifluoroacetic acid in acetonitrile-water. Detection and calibration of [¹²⁵I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [¹²⁵I]salmon calcitonin by rat kidney homogenate could be traced.     

Purification of human brain metallothionein by organic and reversed-phase high-performance liquid chromatography under acidic conditions

A simplified high-performance liquid chromatographic method for the detection of metallothioneins, notably metallothionein-III, has been developed. In order to purify metallothionein, differential acetone precipitation at 50% (v/v) and at 80% (v/v) was employed on a 20% normal human brain homogenate. The reconstituted pellet was injected into a C₁₈ microbore reversed-phase HPLC column, equilibrated with 0.1% trifluoroacetic acid, and developed at a flow-rate of 800 μl/min with a linear gradient from 0% to 60% acetonitrile in 0.094% trifluoroacetic acid for 60 min. Western blots indicated that metallothioneins-I and II eluted at 16% acetonitrile and metallothionein-III eluted at 37% acetonitrile.     

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.     

Selective extraction of β-blockers from biological fluids by column-switching high-performance liquid chromatography using an internal-surface phenylboronic acid precolumn

A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of β-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 μl) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed breifly, the selectively retained β-blockers were eluted with methol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non-treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol.     

Isolation of hyperglycaemic peptides from the corpus cardiacum of the american cockroach,Periplaneta americana

Two peptides, HGHI and HGHII, have been isolated from the CC (corpus cardiacum) of the American cockroach, Periplaneta americana, both showing hyperglycaemic and phosphorylase activating potency when tested in adult cockroaches. The isolation procedure involved extraction of CC with 80% methanol and two steps of HPLC (high-performance liquid chromatography). In the first step extracted material was eluted in 0.1% TFA (trifluoroacetic acid) from a molecular size exclusion column. Estimates of molecular mass indicate that both peptides have a molecular mass of about 1000 daltons. In the second step reversed-phase HPLC utilizing a solvent system consisting of 0.1% TFA and acetonitrile was used; a gradient starting at 25% acetonitrile, with a slope 0.3% acetonitrile min⁻¹ was run over 25 min. The first major peak, HGHI, eluted after 13.7 min and caused a strong hyperglycaemic response as well as strong activation of fat body glycogen phosphorylase when the equivalent of 0.1 of a pair was tested. For HGHII eluting after 21.3 min the hyperglycaemic effect as well as phosphorylase activation were only 50% of the response obtained for HGHI when the equivalent of 0.1 of a pair was injected.     

High-performance reversed-phase chromatographic mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides.

Xyloglucan oligosaccharides from cotton cell walls and tamarind seeds were derivatized with 2-aminopyridine and subsequently separated by reversed-phase chromatography (r.p.c.) using an octadecylsilyl silica stationary phase and aqueous-organic eluents with 0.01% (v/v) trifluoroacetic acid. The chromatographic behavior of the 2-pyridylamino derivatives of xyloglucan oligosaccharides was examined under a wide range of elution conditions, including gradient steepness and shape, initial acetonitrile concentration in the eluent, and pore size of the r.p.c. packings. Relatively steep acetonitrile gradients resulted in poor resolution of the different xyloglucan fragments, which is believed to be the result of acetonitrile-induced conformational changes. Under these circumstances the elution order of the derivatized xyloglucan oligosaccharides was such that the smaller fragments eluted from the column before the larger ones. R.p.c. packing with a 70-A pore size necessitated relatively high acetonitrile concentration in the eluent when compared with 300-A stationary phase. The r.p.c. mapping of 2-pyridylamino derivatives of xyloglucan oligosaccharides was best achieved when both a wide-pore octadecyl-silyl silica stationary phase and a shallow gradient with consecutive linear segments of increasing acetonitrile concentration in the eluent were employed. This combination yielded rapid r.p.c. maps of the xyloglucan fragments from different sources with high separation efficiencies and concomitantly high resolution. The effects of the nature of the sugar residues in the xyloglucan oligomers and their degree of branching on r.p.c. retention and selectivity are also highlighted.     

High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems

This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [³H]leucine into insulin in biological systems. The first step of the procedure was size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH₂PO₄—methanol (9:1, v/v). By this step the bulk of both protein and radioactivity was separated from tritiated insulin. The second step, which employs reversed-phase chromatography on an octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simple and reproducible method was employed to test insulin synthesis in cultured human retinoblastoma Y79 cells.     

Simple determination of mycophenolic acid in human serum by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic analysis was established to monitor the serum concentration of mycophenolic acid, the active metabolite from mycophenolate mofetil administered for the prophylaxis of acute organ rejection in renal transplantation. The system consisted of two pumps for solvent delivery, a column-switching valve, a precolumn, and a reversed-phase analytical column. The present method enabled us to determine MPA by injecting serum samples directly into HPLC without any pretreatment. The mobile phases with different amounts of organic solvent were delivered to the precolumn and analytical column by separate lines, and samples were applied to the precolumn. The column switching valves were switched automatically following the processes for the elimination of protein and the drug analysis. The peak heights of MPA were linearly related to the concentrations (r=0.999) in the range of 0.1-20 micro g/ml, and the limit of quantification was 0.1 micro g/ml (S/N ratio=3). This method was accurate and reproducible on the basis of the results of recovery (94.0-98.0%) and small coefficient of variations of intra and inter-assay (less than 8.3%).     

Histone fractionation by high-performance liquid chromatography

A method for the rapid chromatography of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C₁₈ column containing a packing of octadecylsilane chemically bonded to silica and a linear elution gradient running from water to acetonitrile is described. Two conditions were found to be necessary to achieve histone fractionation: (i) silylation of the active groups of the silica solid support, and (ii) trifluoroacetic acid (TFA) in the eluting solvents. Greater than 90% of the total [³H]lysine-labeled protein applied to the column was eluted from the column. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The HPLC histone fractions (identified by their electrophoretic mobilities) were eluted from the column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A + H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less hydrophobic and more hydrophobic histone variants). Phosphorylated histone species were not resolved from their unmodified parental species. The volatile nature of the water/acetonitrile/TFA eluting solvent facilitated the recovery of salt-free histones from the eluted HPLC fractions by simple lyophilization. This system is very useful for the rapid isolation of the lysine-rich histones, H1 and H2B, and the variants of histone H3. With further development, this system is expected to extend the advantages of HPLC to the fractionation of histone H4 and the variants of histone H2A as well.     

High-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil in cattle muscle tissue with on-line anion-exchange clean-up

A high-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil has been developed. The drug was extracted from cattle muscle tissue with 1% triethylamine in acetonitrile. The extract was evaporated to dryness and taken up in 0.1 M ammonium acetate—acetonitrile (50:50, v/v). The extract was then injected onto a polymeric anion-exchange precolumn. After clean-up with 0.1 M ammonium acetate—acetonitrile (50:50, v/v) for 5 min, the precolumn was eluted with 1% aqueous trifluoroacetic acid—acetonitrile (50:50, v/v) onto a PLRP-S polymer column and chromatographed with a mobile phase of 0.01 M phosphate pH 7—acetonitrile (80:20, v/v). Detection was by ultraviolet at 273 nm. Average recoveries at four levels from 0.005 to 1.000 mg kg⁻¹ were > 88%. The limit of determination was 0.005 mg kg⁻¹.     

Separation of oligo-RNA by reverse-phase HPLC.

A rapid and highly reproducible chromatographic technique has been developed for analysis and purification of complex mixtures of oligoribonucleotides. The method utilizes a column of microparticulate porous silica beads fully derivatized with octadecylsilyl groups. The column is eluted with gradients in acetonitrile/water/ammonium acetate pumped at pressures of 1500-300 psi. Most separations are completed in 5-15 min. with usually better than 1% reproducibility of absolute retention times and about 0.1% reproducibility of relative retention times. A single column accomplishes separations of mononucleosides, mononucleotides, and larger oligomers through at least 20-mers. The absolute detection limit is approximately 1 pmole of base though most of the analytical separations described use approximately 1 nmole. In favorable circumstances it is possible to use the analytical colums to purify approximately 1 mg of an oligonucleotide in a single 10-30 min. elution.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

Increased throughput and reduced carryover of mass spectrometry-based proteomics using a high-efficiency nonsplit nanoflow parallel dual-column capillary HPLC system

We report a new design of a fully automated, high-efficiency parallel nonsplit nanoflow capillary HPLC system, coupled on-line with linear ion trap (LTQ) and high performance nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoESI LTQ-FTICR MS). The system, intended for high-throughput proteome analysis of complex protein mixtures, notably serum and plasma, consists of two reversed-phase trap columns for large volume sample injection with high speed sample loading and desalting and two reversed-phase analytical capillary columns. Through a nanoscale two-position, 10-port switching valve, the whole system is terminated by a 10 microm i.d. of nanoemitter mounted on the nanoelectrospray source in front of the sampling cone of the LTQ-FTICR MS. Gradient elution to both nanoflow-rate capillary columns is simultaneously delivered by a single HPLC system via two independent binary gradient pump systems. The parallel capillary column approach eliminates the time delays for column regeneration/equilibration since one capillary column is used for separating the sample mixtures and delivering the separated fractions to the MS, while the other capillary column is being regenerated and equilibrated. The reproducibility of retention time and peak intensity of the present automated parallel nanoflow-rate capillary HPLC system is comparable to that obtained using a single column configuration. Replicate injections of tryptic digests indicated that this system provided good reproducibility of retention time and peak area on both columns with average CV values of less than 1.08% and 7.04%, respectively. Throughput was increased to 100% for 2-h LC-MS analysis compared to the single capillary column LC-MS pipeline. Application of this system is demonstrated in a plasma proteomic study. A total of 312 868 MSMS events were acquired and 1564 proteins identified with high confidence (Protein Prophet > or = 0.9, and peptides matched > or = 2). Comparison of a series of plasma fractions run using the single-column LC-MS versus the parallel-column LC-MS demonstrated that parallel-column LC-MS system significantly reduced the sample carryover, improved MS data quality and increased the number of MS/MS sequence scan events.     

Rapid and sensitive high-performance liquid chromatographic method for busulfan assay in plasma

A reversed-phase liquid chromatographic method with ultraviolet detection has been developed to determine busulfan concentrations in plasma of children undergoing bone marrow transplantation. Plasma samples (200 μl) containing busulfan and 1,6-bis(methanesulfonyloxy)hexane as an internal standard were prepared by a simple derivatization method with diethyldithiocarbamate followed by extraction with ethyl acetate and solid-phase purification on C₈ columns conditioned with methanol and water and eluted with acetonitrile (recovery 99%). Chromatography was accomplished using a Hypersil octadecylsilyl column (10 cm×4.6 mm I.D.) and a mobile phase of acetonitrile, tetrahydrofuran and distilled water (65:5:30, v/v). The limit of detection was 25 ng/ml (signal-to-noise ratio of 5). Calibration curves were linear up to 25 000 ng/ml. Intra-day and inter-day coefficients of variation of the assay were ≤5%. This method was used to analyse busulfan plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in children.     

Alternative mobile phases for enhanced chromatographic selectivity and increased sensitivity in peptide separations.

The standard method for separating peptide mixtures is reversed-phase high-performance liquid chromatography with gradients of increasing concentrations of acetonitrile in the presence of trifluoroacetic acid. With modern instruments and columns, complex peptide mixtures can be separated, and low picomole amounts can be collected in tens of microliters. Difficult separations are addressed by modifying the gradient slope or organic eluant composition. Further improvements in resolution are often needed, requiring fundamental changes in mobile phase composition or selection of complementary chromatographic separation mechanisms. For the present study, tryptic digests of cytochrome c from various species were separated in the presence of dilute hydrochloric acid by reversed phase on a Waters Delta-PakTM C18 high-performance insert column and by strong cation exchange on a Waters Protein-PakTM SP 8HR. Different and enhanced reversed-phase selectivity was obtained by replacing trifluoroacetic acid with dilute hydrochloric acid at the same pH. The increased optical clarity of hydrochloric acid-based mobile phases in the low ultraviolet wavelengths yielded increased sensitivity. Very different selectivity was observed with the cation-exchange chromatography. These data expand the options for peptide mapping by providing additional selectivity combined with increased mass sensitivity and spectral information in the low ultraviolet.     

Determination of [d-Ala2, d-Leu5]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Differentiation of Cirsium japonicum and C. setosum by TLC and HPLC-MS

The Chinese Pharmacopoeia indicates the use of field thistle (Cirsium setosum) and Japanese field thistle (C. japonicum) in the treatment of bleeding and inflammation. In the absence of an analytical method for the differentiation and analysis of these two species, TLC and HPLC-MS methods have been developed for this purpose. Both species could be readily distinguished by their flavonoid pattern as revealed by TLC on silica gel layers eluted with ethyl acetate:formic acid:acetic acid:water. The quantitative determination of four flavonoids, namely hispidulin-7-neohesperidoside, linarin, pectolinarin and luteolin, was possible using HPLC. Their optimum separation was achieved on a C12 column eluted with water and 0.025% trifluoroacetic acid in acetonitrile. HPLC-MS experiments were performed to confirm peak identity. In samples of C. japonicum, pectolinarin was the major flavonoid (0.32-2.00%), followed by linarin, hispidulin-7-neohesperidoside and luteolin; the total flavonoid content varied from 0.81 to 3.67%. In C. setosum only one flavonoid (linarin; 1.36-2.83%) was assignable. The HPLC method was validated for linearity, limit of detection (< or = 1.7 ng on-column), peak purity, repeatability (< or = 2.3%) and accuracy (recovery rates of spiked samples were between 99.2 and 101.6%).