Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

A new high-performance liquid chromatography assay for glutamine synthetase and glutamate synthase in plant tissues

Glutamic acid and glutamine, formed in plant tissue extracts by glutamate synthase and glutamine synthetase, respectively, were separated by derivatization with o-phthaldialdehyde followed by reverse-phase high-performance liquid chromatography on a μBondapak C₁₈ column. The derivatives were eluted by isocratic elution, and the mobile phase was a 20 mm sodium phosphate buffer (pH 6.8) with 36% methanol. The procedure is rapid, sensitive, and requires a minimum sample.     

Liquid Chromatographic Procedure for Fermentation Product Analysis in the Identification of Anaerobic Bacteria

High-performance liquid chromatography with a cation-exchange resin-packed column was used to determine fermentation products of several known and unknown Clostridium species. The column was operated at 30°C, and isocratic elution was done with 0.013 N H₂SO₄. Sample preparation for high-performance liquid chromatographic analysis required only membrane filtration. Glucose and formate were readily determined. Quantitative results were easily obtained. Chromatograms of eight unknown strains could be matched with chromatograms of at least one of the type culture strain chromatograms. In some cases, additional testing was necessary before identification could be made. The same conclusions were reached by parallel testing with gas chromatography to determine fermentation products. High-performance liquid chromatography is simple to apply and, under some conditions, is faster than gas chromatography for fermentation product analysis.     

Assay of L-aromatic amino acid decarboxylase by high performance liquid chromatography

A method is presented for the assay, by high-performance liquid chromatography, of l-aromatic amino acid decarboxylase. The K_m value of the enzyme for 3,4-dihydroxyphenylalanine (dopa) was, by this procedure, 0.16 mm, in good agreement with previous reports. When α-methyldopa was used as substrate, evidence was obtained indicating the formation of, besides α-methyldopamine, 3,4-dihydroxyphenylacetone, presumably through an internal transamination process.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C₁₄ reversed-phase column (Zorbax Bonus-RP column) and a liquid–liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid–liquid extraction. The mobile phase consists of acetonitrile–0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Resolution of RS-abscisic acid and the separation of abscisic acid metabolites from plant tissue by high-performance liquid chromatography

Attempts to resolve the enantiomers of racemic abscisic acid (ABA) by high-performance liquid chromatograpy on a chiral stationary-phase column were unsuccessful. However, reduction of RS-methyl ABA (RS-Me-ABA) with sodium borohydride generates a new chiral centre and one of the two isomeric products, the RS-Me-1′,4′-cis-diol of ABA, was separated into its enantiomers by high-performance liquid chromatography on an optically active Pirkle column.High-performance liquid chromatography on a μBondapak C₁₈ column separated the metabolites and conjugates of [2-¹⁴C]ABA fed to tomato shoots. The resolution method was used to measure the relative proportions of R and S enantiomers in the free acid liberated from conjugates of ABA.     

Application of high-performance liquid chromatographic peptide purification to protein microsequencing by solid-phase Edman degradation

Peptide purification via high-performance liquid chromatography (HPLC) and solid-phase sequencing were integrated to form a system allowing the determination of complete sequence information on a microscale without the use of radiolabels or modified phenylisothiocyanate. Mixtures of peptides (500 pmol to 10 nmol) resulting from proteolytic digestion or chemical cleavage were applied directly to reverse-phase columns. The columns, equilibrated in either 10 mm KP_i or 0.05% trifluoroacetic acid, were then developed using acetonitrile gradients. Eluates were monitored nondestructively by direct ultraviolet detection at both 214 and 254 nm. Each peak was collected as a discrete fraction, and purity was assessed by amino acid analysis prior to covalent attachment to a solid support for sequence analysis. Activation of the peptide carboxyl terminus via a water soluble carbonyldiimide was the solid-phase coupling method used 90% of the time. Coupling yields averaged 52% of starting material. Sequence analysis was performed in the range 100 pmol to 4 nmol of coupled peptide. Phenylthiohydantoin-amino acids were identified by reverse-phase HPLC using ultraviolet detection.     

Separation of Chlorophylls c(1) and c(2) from Pigment Extracts of Pavlova gyrans by Reversed-Phase High Performance Liquid Chromatography

Chlorophylls c₁ and c₂ have been separated from total pigment extracts of the alga Pavlova gyrans Butcher using a reversed-phase high-performance liquid chromatography system. Pigments were separated on a 5 micrometer C₁₈ column (25 centimeters × 4.6 millimeters) using a gradient of methanol-acetonitrile-water. Other photosynthetic pigments were also well resolved by the system used. The separation system described may replace current thin layer chromatography methods for qualitative and quantitative determination of chlorophyll c species.     

Rapid high-performance liquid chromatographic assay for atovaquone

A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Taxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C₆ column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 μg/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 μg/ml and 10.3% at 1 μg/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay.     

Determination of quercetin in human plasma using reversed-phase high-performance liquid chromatography

A method is reported for the measurement of quercetin in human plasma using reversed-phase high-performance liquid chromatography (HPLC). Quercetin and kaempferol (as internal standard) were spiked into plasma samples and extracted using C₁₈ Sep-Pak Light cartridges (efficiency > 85%). Flavonoids were eluted with aqueous acetone (50% v/v, pH 3.5), dried down and redissolved in aqueous acetone (45% v/v, pH 3.5). The increased osmolarity promoted a phase separation and the water-saturated acetone layer, containing the flavonoids, was analysed by HPLC with aqueous acetone mobile phase (45% v/v acetone in 250 mM sodium dihydrogen sulphate. The mixture was adjusted to pH 3.5 with phosphoric acid and used at a flow-rate of 1.0 ml/min) and μBondapak C₁₈ column (150 × 3.9 mm I.D., 10 μm particle size). The detection limit (A_{375 nm}) for quercetin in plasma was 0.1 μg/ml (300 nM). The method also detects metabolites of quercetin, although these are not yet identified.     

High-performance liquid chromatographic determination of α-keto acids

A high-performance liquid chromatographic method has been developed for the determination of eight kinds of α-keto acids. These acids were derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into chloroform. The quinoxalinol derivatives were separated by reversed-phase high-performance liquid chromatography using a 250 mm × 2.1 mm I.D. column packed with LiChrosorb RP-8 (5 μm). This method could be satisfactorily applied to urine samples without any prepurification.     

Determination of rifampin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifampin in human plasma. Rifampin and sulindac (internal standard) are extracted from human plasma using a C₂ Bond Elut extraction column. A 100-μl volume of 0.1 M HCl is added to the plasma before extraction to increase the retenction of the compounds on the extraction column. Methanol (1 ml) is used to elute the compounds and 0.5 ml of 3 mg/ml ascorbic acid in water is added to the final eluate to reduce the oxidation of rifampin. Separation is achieved by reversed-phase chromatography on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate-acetonitrile (55:45, v/v). Detection is by ultraviolet absorbance at 340 nm. The retention times of rifampin and internal standard are approximately 4.4 and 7.8 min, respectively. The assay is linear in concentration ranges of 50 to 35 000 ng/ml. The quantitation limit is 50 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.     

Assay of catechol O-methyltransferase by determination of the m-and p-O-methylated products using high-performance liquid chromatography.

A new assay of catechol O-methyltransferase (EC 2.1.1.6) is described by determination of the m- and p-O-methylated products of 3,4-dihydroxybenzoic acid. The method involves DEAE-Sephadex A-25 chromatography and reversed-phase high-performance liquid chromatography on a LiChrosorb 5 RP 18 column. The liquid chromatographic solvent system consisted of 0.05 m acetic acid in methanol:water (1:4, $\text{v}\text{v}$), pH 3,2. meta/para ratios of O-methylation are easily obtained by this method. Dimethylation has not been observed with a partially purified enzyme preparation from rat liver.     

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.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C₁₈ reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.     

High-performance liquid chromatographic method for the rapid and simultaneous determination of sulfamonomethoxine,miloxacin and oxolinic acid in serum and muscle of cultured fish

A rapid method for the simultaneous determination of sulfamonomethoxine (SMM), miloxacin (MLX) and oxolinic acid (OA) in serum and muscle of cultured fish by high-performance liquid chromatography has been developed. A Hisep shielded hydrophobic phase column (15 cm×4.6 mm I.D.) and a mobile phase of 0.05 M citric acid-0.2 M disodium hydrogenphosphate buffer, pH 2.5 in 10 mM tetra-n-butyl ammonium bromide-acetonitrile (85:15) with ultraviolet detection at 265 nm were used. The recoveries of SMM, MLX and OA from serum and muscle samples were 72–101%. The detection limits of the three drugs were 0.05–0.1 μg/ml or g of sample.     

Separation of basic,hydrophilic peptides by reversed-phase ion-pair chromatography: II. Analytical applications with particular reference to phosphoserine peptides

Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C₁₈ column, using phosphate buffers with pH 3.2–4.5, n-hexane sulfonic acid as counter ion, and ethanol as organic modifier.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.