Resolution of cartilage proteoglycan and its proteolytic degradation products by high-performance liquid chromatography using a gel filtration system

Cartilage proteoglycan subunits are resolved from their various-size proteolytic degradation products by a gel filtration high-performance liquid chromatography system using a Bio-Gel TSK-60 column in tandem with a Bio-Gel TSK-50 column. Molecules ranging in size from the intact proteoglycan to single chondroitin sulfate chains are eluted in the included volume. Each analysis takes less than 30 min to complete, and with purified samples as little as 20 micrograms of proteoglycan is required. The method can be applied to the measurement of proteoglycan in mixtures, such as tissue culture media, by monitoring effluent fractions using the dimethylmethylene blue dye-binding assay.     

A procedure for the separation and quantitation of tryptophan and amino sugars on the amino acid analyzer

Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 Na⁺) buffer, followed by pH 6.4 (1 Na⁺) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.     

A column procedure for the esterification of organic acids with diazomethane at the microgram level

A simple procedure is described in which micro- and submicrogram amounts of organic acids can be converted to methyl esters with diazomethane. The gas, generated in only micromole amounts, contacts the acids which have been applied to a column of Celite contained in a capillary. The resultant esters are then eluted for chromatographic analysis. The entire procedure can be done in less than 5 min.     

Gaschromatographische analyse von sauren urinmetaboliten nach trennung auf kieselgelfertigsäulen

Gas chromatographic estimation of acidic urinary metabolites after separation on prepacked silica gel columnsThe acidic ethylacetate extracts of 24-h urine specimens are evaporated and redissolved in chloroform—methanol—acetic acid. The resulting solution is transferred to a prepacked silica gel column. Elution takes 160 min using a specially designed chloroform—methanol—acetic acid gradient. The eluate is divided into fractions (16 min each) which are evaporated to dryness. The residues are silylated and determined quantitatively by gas chromatography. The capacity of the silica gel column allows analysis of 30% of a 24-h urine specimen. In consequence, metabolites can be quantitated at concentrations less than 1 mg per 24 h. The method is suitable to obtain more detailed metabolic profiles of the carboxylic acids in urine.     

High-performance liquid chromatographic analysis of galactosamine, glucosamine, glucosaminitol, and galactosaminitol

Both N-acetylgalactosamine and N-acetylglucosamine covalently link oligosaccharides to peptide in glycoproteins. In order to identify the N-acetylhexosamine involved in this linkage, the corresponding hexosaminitol generated by alkaline borohydride treatment must be determined. An HPLC method modified from the Waters PICO-TAG amino acid analysis procedure is described. Phenylisothiocarbamyl derivatives of galactosamine, glucosamine, glucosaminitol, galactosaminitol, and the internal standard, p-aminophenyl-beta-D-galactoside, are eluted from the Waters PICO-TAG column at 3.9, 4.3, 6.9, 8.1, and 10.1 min, respectively. The standard curves for the hexosamines and hexosaminitols are linear between 1 and 75 nmol. In addition to p-aminophenyl-beta-D-galactoside, several synthetic hexosamines and hexosaminitols can be employed as internal standard. These include 3-allosamine, 3-glucosamine, allosamine, 3-allosaminitol, mannosaminitol, and allosaminitol, which are eluted at 3.1, 3.4, 4.8, 6.4, 7.4, and 7.8 min, respectively. The analysis time is 15 min but can be shortened to 10 min if only hexosamines are to be analyzed and either 3-glucosamine or 3-allosamine is used as the internal standard. This rapid method is superior to previous methods for the analysis of hexosamines in glycoconjugates and hexosaminitols generated from glycoconjugates following alkaline borohydride treatment.     

Separation of substituted pteroyl monoglutamates and pteroyl oligo-gamma-L-glutamates by high pressure liquid chromatography.

Rapid analysis of substituted and unsubstituted pteroyl-oligo-γ-l-glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.     

The separation of bovine prothrombin and descarboxyprothrombin by high-performance liquid chromatography

Prothrombin contains 10 γ-carboxyglutamic acid (Gla) residues which are absent in the warfarin-induced descarboxyprothrombin; hence prothrombin has 10 more negative groups than has descarboxyprothrombin. The two proteins can be separated by HPLC with the aid of an anion-exchange column. Plasma from warfarin-treated animals could be analyzed without pretreatment of the samples and a full analysis was obtained in 30 min.     

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel

The performance of monolithic HPLC columns Chromolith (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma-Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases - for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) - was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0-5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

High-performance ion-exchange chromatographic separation of proteoglycans

Proteoglycans synthesized by cultured human muscle cells were separated by ion-exchange high-performance liquid chromatography using a Bio-gel TSK DEAE 5-PW analytical column. The procedure requires only 40 min to complete. The same analytical size column can be used for either analytical or semipreparative scale separations without significant loss of resolution. Proteoglycans elute from the TSK column with a similar recovery and at similar elution ionic strengths when compared to the established cellulose-based chromatographic gel, DEAE-Sephacel. The technique has been applied to the analysis of chondroitinase-digested samples and is particularly useful for rapid screening of large numbers of cultures for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.     

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.     

Automated analysis of urinary catecholamines by high-performance liquid chromatography and on-line sample pretreatment

A simple and automated solid-phase extraction for the selective and quantitative HPLC analysis of free catecholamines in urine is described. The urinary catecholamines react with diphenylboric acid, giving a complex at pH 8.5 which is strongly retained on a PLRP-S cartridge; elution is accomplished with the same mobile phase used for HPLC analysis. Separation is performed by ion-pair reversed-phase HPLC, with sodium heptanesulphate as counter-ion, and a totally end-capped C₁₈ analytical column. Quantitation is achieved with an electrochemical detector. A Spark Holland Prospekt system controls the on-line solid-phase extraction, preconcentration and direct elution to the LC column. Chromatography run-time is 10 min and the total time to process one urine sample is ca. 12 min.     

A high-performance liquid chromatography procedure for recovering subnanomole amounts of protein from SDS-gel electroeluates for gas-phase sequence analysis

A high-performance liquid chromatographic procedure for recovering subnanomole amounts of protein from SDS/polyacrylamide gel electroeluates in a form suitable for gas-phase sequence analysis has been developed. By a judicious choice of reversed-phase column packing, proteins can be retained at high concentrations of n-propanol (90-100%) where sodium dodecylsulfate and acrylamide gel-related contaminants are washed through the column. Retained proteins can be recovered from the column in high yield (greater than 90%) by the simultaneous adding of an ion-pairing reagent into the mobile phase and elution with a gradient of decreasing n-propanol concentration (i.e. an 'inverse or negative gradient'). Furthermore, by using a steep gradient (e.g. 50%/min) at a low flow rate (20-200 microliters/min) the proteins can be recovered in less than 100 microliters and can be used for gas-phase sequence analysis without further manipulation. This procedure is independent of sodium dodecylsulfate concentration (up to 1.2% w/v) in sample loading volumes of up to 1.5 ml. Microbore columns (2.1 mm internal diameter) have been employed for recovering small amounts of protein (1-100 micrograms from electroeluates of protein-containing gel spots while conventional columns (4.6 mm internal diameter) were used for isolating larger amounts of protein (greater than 500 micrograms) from electroeluates of preparative gel bands. The general utility of this inverse-gradient high-performance liquid chromatography procedure has been demonstrated by its successful application in recovering a wide variety of proteins from sodium dodecylsulfate gel electroeluates in a form suitable for N-terminal sequence analysis in the 10-500 pmol range.     

Automated sample introduction for an imaged capillary isoelectric focusing instrument via high-performance liquid chromatography sampling devices

Sample introduction of an imaged capillary isoelectric focusing (cIEF) instrument is fully automated by using commercially available high-performance liquid chromatography (HPLC) injection valves and autosamplers. Sample carryover can be controlled to under 1% when the valve and separation column are washed for 1 min between sample runs. The standard deviation of peak areas for 20 injections is 3.5%, which includes deviations created by the absorption imaging detector and the isoelectric focusing process inside the 75 μm I.D. column. Sample throughput is up to 10 samples per hour. The instrument has been applied to fast analysis of many proteins including monoclonal antibodies.     

Simple,rapid and micro high-pressure liquid chromatographic method for the simultaneous determination of tolbutamide and carboxy tolbutamide in plasma

A rapid high-pressure liquid chromatographic (HPLC) assay is described for the quantitative analysis of tolbutamide and its major metabolite, carboxy tolbutamide, in plasma. An aliquot (25–100 μl) of plasma was prepared for chromatography by deproteinization as follows. One volume of plasma and 2.5 volumes of acetonitrile were vortex mixed for a few seconds and then centrifuged for approx. 1 min. A 50-μl sample of the clear supernatant was injected into the chromatograph. A μBondapak C₁₂ reversed-phase column was used with a mobile phase of acetonitrile-0.05% phosphoric acid (45:55) at a flow-rate of 1.5 ml/min. The column effluent was monitored by a variable-wavelength UV detector set at 200 nm. Tolbutamide and its metabolite had retention times of 5.75 and 3.25 min, respectively. The procedure yields reproducible results with sensitivity adequate for routine clinical monitoring of plasma levels or for single-dose pharmacokinetic studies. A number of commonly used drugs do not interfere with the method. A single plasma sample can be analyzed in approx. 9 or 10 min.     

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.     

High performance liquid chromatographic method for the determination of cetirizine and ambroxol in human plasma and urine--a boxcar approach

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid-liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column Supelcosil LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile-phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C(8) micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C(18) analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile-triethylamine (0.5%) in phosphate buffer (pH 3.5; 20mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7-300 ng/ml for cetirizine and ambroxol respectively in plasma and 1-500 ng/ml and 5-400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.     

Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.     

Determination of biogenic amines using heterocyclic cation and aromatic anion elution

Heterocyclic cation and aromatic anion are used in the chromatographic buffers for the analysis of monoamines and diamines on a sulfonated cation-exchange column using an amino acid analyzer. All elution buffers employed for these analyses had a pH of 5.0 to maximize the ninhydrin color reaction. These methods have been successfully used for biological samples. Using a two-column (0.8 × 12 cm) system, with a buffer flow rate of 50 ml/h, analysis can be carried out in 330 min for monoamine and diamine mixtures on 0.5 to 10 nmol of each amine.     

Short column gas chromatography–mass spectrometry and principal component analysis for the identification of coeluted substances in doping control analysis

The identification of four doping control substances in an artificial mixture, using short column gas chromatography–mass spectrometry (GC–MS) analysis was examined. Two chromatographic peaks were recorded in the chromatogram, using a short capillary column (1.8 m) at an oven temperature of 180°C. The first peak was associated with a mixture of a solvent derivative and an artifact. The second one corresponded to the mixture of four control substances. Principal component analysis was applied on a selected GC–MS data set of the latter peak to determine clear full spectra of pure substances from mixture spectra. The time of GC–MS analysis was significantly reduced to less than 1 min from 30 min which is a typical GC–MS analysis time, using standard methods of doping control analysis.