Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Determination of omeprazole in human plasma by high-performance liquid chromatography

A new method for the determination of omeprazole in human plasma was developed. Omeprazole was extracted from plasma with toluene-isoamylalcohol (95:5, v/v), the organic phase was evaporated, dissolved in the mobile phase and injected into a reversed-phase C₁₈ column. Flunitrazepam was used as an internal standard. The mobile phase consisted of 47% methanol and 53% of 0.1 M dipotassium hydrogenphosphate, pH 7.8. The spectrophotometric detection was performed at 302 nm. Limit of quantitation was 9.7 ng/ml and the calibration curve was linear up to 1240 ng/ml.     

Determination by high-performance liquid chromatography of hydroxyurea in human plasma

An assay using reversed-phase high-performance liquid chromatography with electrochemical detection was developed to measure hydroxyurea in plasma at concentrations suitable for pharmacokinetic studies. The sample preparation is simple, the analysis rapid and assays can be batched. The between-run precision is excellent (coefficient of VARIATION = 2.8–4.5%) and the limit of detection is 0.02 mmol/l. Preliminary studies have shown that the method is suitable for pharmacokinetic studies.     

Determination of eltanolone in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of eltanolone in plasma has been developed. Plasma samples containing eltanolone were diluted with acetonitrile to precipitate plasma proteins, and derivatized with 2,4-dinitrophenylhydrazine before direct injection onto a C₁₈ column. The mobile phase was acetonitrile–water (70:30, v/v) containing 0.1% trifluoroacetic acid and detection was by UV absorbance at 367 nm. The quantitation limit was 0.020 μg/ml. The method has proven to be rapid, precise and sensitive in the range of concentrations found during and following intravenous anaesthesia.     

Determination of metformin in human plasma by high-performance liquid chromatography

A simple, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of antihyperglycemic agent metformin in human plasma using a novel sample extraction procedure. Liquid-liquid extraction of metformin and ranitidine (as internal standard) from plasma samples was performed with 1-butanol/n-hexane (50:50, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a silica column (250 mmx4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (25:75, v/v), pH 6. The limit of quantification (LOQ) was 15.6 ng/ml and the calibration curves were linear up to 2000 ng/ml. The mean absolute recoveries for metformin and internal standard using the present extraction procedure were 98 and 95%, respectively. The intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 8.3%.     

Determination of saquinavir in human plasma by high-performance liquid chromatography

We developed and characterized a high-performance liquid chromatography (HPLC) assay for the determination of saquinavir, an HIV protease inhibitor, in human plasma samples. Extraction of plasma samples with diethyl ether resulted in quantitative recovery of both saquinavir and its stereoisomer Ro 31-8533 which was used as an internal standard. The assay was performed isocratically using 5 mM H₂SO₄ (pH 3.5) and acetonitrile (75.5:24.5, v/v) containing 10 mM tetrabutylammonium hydrogen sulfate (TBA) as a mobile phase, a Nucleosil 3C8 column kept at 45°C and UV detection at 240 nm. Using this method, saquinavir and Ro 31-8533 can be separated from endogenous substances, and in the concentration range of 5–110 ng/ml the relative standard deviations for the determination of saquinavir were below 5%. The detection limit of saquinavir in human plasma was 1 ng/ml. The usefulness of the method was demonstrated by quantification of saquinavir in plasma of human subjects treated with 600 mg of saquinavir per os or 12 mg intravenously.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Determination of dimethylated arginines in human plasma by high-performance liquid chromatography

Using high-performance liquid chromatography (HPLC) with multigradient elution, (asymmetric-DMA, ADMA) and (symmetric-DMA, SDMA) can be separated from human plasma samples. The dimethylarginine compounds in plasma, after extraction with a cation-exchange column, are converted to fluorescent derivatives with o-phthaldialdehyde (OPA) in an alkaline medium and the derivatives are separated simultaneously within 50 min on a reversed-phase column (Ultracarb 3 ODS(20)). The recoveries of ADMA and SDMA are over 80% and the method permits quantitative determination of dimethylated arginines at concentrations as low as 0.1 μmol/l in human plasma.     

Determination of O-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic assay for O⁶-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were constituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O⁶-Benzylguanine peak heights were compared to peak heights of O⁶-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O⁶-benzylguanine.     

Determination of vitamin E in human plasma by high-performance liquid chromatography

The use of selective protein precipitation to enhance the recovery of vitamin E from plasma, by minimising binding with very-low-density lipoproteins, is reported. The procedure employed treatment of plasma with magnesium chloride and tungstate, followed by methanol protein precipitation. Separation of vitamin E was performed using reversed-phase high-performance liquid chromatography of the methanol extracts with subsequent UV detection of the compound. Using this technique the procedure was observed to be specific for vitamin E and linear over the range 1.0 to 40.0 μg/ml. The within-run imprecision (C.V.) at three different supplemented plasma vitamin E concentrations of 5.0, 10.0 and 20.0 μg/ml was 4.51, 3.33 and 2.58%, respectively, and the between-run imprecision (C.V.) estimated to be 5.19, 3.69 and 3.67%, respectively. With the same supplemented plasma vitamin E concentrations, the overall accuracy (bias) of the procedure, using an albumin matrix for calibration, was estimated to be 6.0, −5.0 and −3.5%, respectively, and the recovery of vitamin E from six different spiked plasma samples estimated to be 98.2±2.6%.     

Determination of free malondialdehyde in human serum by high-performance liquid chromatography

Lipid peroxidation involves the oxidative deterioration of polyunsaturated fatty acids in biomembranes and generates a variety of aldehydic products including malondialdehyde (MDA). To demonstrate the occurrence of lipid peroxidation in biological systems, the production of MDA has been shown to be a relevant indicator. Therefore, we describe a new method for measurement of free malondialdehyde in human serum. A simple, rapid but sensitive method for determination of MDA in human serum was applied to goiter patients and control groups. Patients with goiter had high levels of MDA compared to control groups. Our method is fast and practical for clinical measurements. The detection limit was found to be 1.2 x 10(-8) mol L(-1).     

Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The assay was linear over the concentration range of 0.25–18.00 ng/ml. Both of the within-day and day-to-day reproducibility and accuracy were less than 11.80% and 12.00%, respectively. The plasma profile following a single administration of 10 mg amlodipine to a healthy volunteer was presented.     

Determination of pyridinium crosslinks in plasma and serum by high-performance liquid chromatography

A chromatographic method for the determination of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in serum and plasma is described. The analytical procedure involved plasma or serum purification by ultrafiltration (20 000 relative molecular mass cut-off) under centrifugation at 2500 g for 4 h, as an innovative step. Analysis was done by isocratic high-performance liquid chromatography with fluorescence detection. The linearity of the method was tested from 0.6 to 15 pmol/ml and 0.12 to 3 pmol/ml for Pyr and Dpyr, respectively. The detection limit was 60 fmol/ml for both crosslinks. Except for Dpyr in plasma (coefficient of variation 19.9%), intra-assay variation was always below 10% in serum and plasma. The method has been applied to the quantification of crosslinks in serum and plasma of healthy volunteers and also in mouse and rat plasma. Serum proved to be the most suitable biological fluid for the systemic measurement of these compounds in humans and under the experimental conditions used, contained an average of 3.62 ± 0.65 and 0.7 ± 0.18 pmol/ml Pyr and Dpyr, respectively.