Liquid chromatographic method for the quantitative determination of Nepsilon-carboxymethyllysine in human plasma proteins

The modification of the lysine moieties of proteins to Nepsilon-carboxymethyllysine (CML) is supposed to play a major role in the development of long-term complications in patients with diabetes mellitus. This paper presents an analytical method for the quantitative determination of CML in plasma proteins, which could be used for studying the development of diabetic complications. The method is based on isolating proteins from plasma by precipitation with trichloroacetic acid and hydrolysing these under acidic conditions (6M hydrochloric acid at 110 degrees C for 20 h) to the individual amino acids. After hydrolysis, CML is derivatised along with the other amino acids to 9-fluorenylmethoxycarbonyl (FMOC) derivatives, which are subsequently separated by reversed-phase column liquid chromatography using a 150 mm x 4.6 mm C8 column and a mobile phase of 25 mM potassium phosphate buffer (pH 2.0) and acetonitrile (80:20 (v/v)) and detected using fluorescence detection (excitation at 260 nm and emission at 310 nm). Quantification of the protein-bound CML content of a plasma sample is achieved using standard addition. The impact of several aspects of the sample preparation and chromatography on method performance is discussed. Method evaluation results are reported and show that this method is capable of determining CML with good accuracy and precision (below 10%) in the relevant concentration range (1-10 microg/ml), with a limit of detection of 0.2 microg/ml.     

High-performance liquid chromatographic analysis of nalidixic acid and hydroxynalidixic acid in plasma with a dynamic anion-exchange system

A rapid high-performance liquid chromatographic method has been developed for monitoring plasma levels of patients treated intravenously with nalidixic acid. The major metabolite (in vitro also active) can be determined as well; 50-μl plasma samples are sufficient. Use is made of a dynamic anion-exchange system. Different parameters such as adsorption of the surfactant cetrimide onto the column; pH and ionic strength of the eluent, and the critical micelle concentration of the surfactant in the eluent have been studied.     

Thin-layer chromatographic method for the determination of glycosyltransferase activity

To survey glycosyltransferase activities and specificities we have developed a TLC method to separate various nucleotide sugars from both high- and low-molecular-weight sugar acceptors. Here, we report details of the procedure and its application for galactosyltransferase and fucosyltransferase detected in mouse spermatogenic cells. The assay method involves sample separation using polyethyleneimine cellulose plastic-backed thin-layer plates, developed in sodium phosphate buffer for 30 min. Nucleotide sugars, including UDP-Gal, GDP-Fuc, CMP-NeuNAc, and GDP-Man, remain at the origin, while both high- and low-molecular-weight sugar acceptors migrate within 2 cm of the solvent front. Assays for galactosyltransferase and fucosyltransferase are linear with time and yield results comparable to other methods such as gel permeation chromatography and micropartitioning filtration. The TLC protocol should be useful for determinations of many different glycosyltransferases.     

High-pressure liquid chromatographic method for the simultaneous quantitative analysis of propranolol and 4-hydroxypropranolol in plasma

A simple and rapid high-pressure liquid chromatographic procedure is reported for the simultaneous quantitative determination of propranolol and 4-hydroxypropranolol in plasma. Following an extraction the samples are chromatographed on a reversed-phase column and the components in the column effluent are detected by fluorescence monitoring. Using 1-ml plasma samples propranolol and 4-hydroxypropranolo concentrations at least as low as 1 ng/ml and 5 ng/ml, respectively, can be quantitated. The reproducibility of the method is satisfactory and no interference from endogenous plasma components or other drugs has been observed. A single plasma sample can be analyzed in approximately 20 min.     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

Liquid chromatographic method for the determination of rizatriptan in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Liquid chromatographic method for the determination of rosiglitazone in human plasma

A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid-liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C(18), 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5-1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.     

Automated high-performance liquid chromatographic method for the analysis of two novel ergoline compounds in human plasma

A rapid and sensitive high-performance liquid chromatographic method for the determination of the novel ergoline derivatives sergolexole (compound I), its acid metabolite (compound II) and cis-n-(2-hydroxycyclopentyl)-6-methyl-1-(1-methylethyl) ergoline-8-carboxamide (LY215840, compound III) in human plasma is reported. The compounds were extracted from plasma by automated solid-phase extraction and analysed on a reversed-phase C₈ column with fluorescence detection. The limit of quantification for all compounds was 10 ng/ml and the response was linear over the range 10–1000 ng/ml. Validation studies showed the method to be both repeatable and reproducible with no interference from human plasma. The method has been used to support pharmacokinetic studies and has proved to be robust and effective.     

High-performance liquid chromatographic method for the determination of gabapentin in human plasma

A sensitive high-performance liquid chromatography (HPLC) method using UV detection for the determination of gabapentin in human plasma has been developed. In this method, gabapentin was extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge followed by derivatization with phenylisothiocyanate. Analysis was achieved by using a HPLC system that was equipped with a UV detector. The quantitation limit of gabapentin in human plasma was 0.03 microg/ml. The method is sensitive with excellent selectivity and reproducibility and it has been applied to a bioequivalence clinical study with great success.     

Validated liquid chromatographic method for the determination of bexarotene in human plasma

A new liquid chromatographic method is described for the determination of the anti-tumour agent bexarotene in human plasma over the range 0.500-1500 ng/ml, using 1 ml of sample. Sample preparation consists of liberating the analyte from plasma lipids by adding acetonitrile, followed by acidification of the plasma and liquid extraction using a mixture of isoamyl alcohol and pentane or hexane. Separation and quantitation are performed by reversed-phase column liquid chromatography with fluorescence detection. Parameters affecting the performance of these steps are discussed. Validation results on linearity, selectivity, accuracy, precision, recovery and stability are shown, as well as the application of the method to samples from clinical trials.     

A gas chromatographic method for the quantitative detemination of hexuronic acids in alginic acid

A method has been developed for the quantitative determination of the relative proportions of d-mannuronic and l-guluronic acids in alginic acid. To obtain homogeneous reaction conditions the viscosity of the alginic acid sample was first decreased by limited hydrolysis with mineral acid. The carboxyl groups were then esterified by reaction with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, and reduced with sodium borohydride. The resulting hexosans were converted by acid hydrolysis to d-mannose and an equilibrium mixture of l-gulose and 1,6-anhydro-l-gulose. These were treated with sodium borohydride; the 1,6-anhydro-l-gulose was not reduced whereas d-mannose and l-gulose were converted to d-mannitol and d-glucitol. The hexitols were estimated by gas-liquid chromatography as the n-butane boronic acid esters, and the relative proportions of the uronic acids in the alginic acid were calculated by taking into account the equilibrium ratio of l-gulose and 1,6-anhydro-l-gulose. The method can be used to analyze as little as 2 mg of alginic acid.     

Thin-layer chromatographic determination of furosemide and 4-chloro-5-sulfamoyl anthranilic acid in plasma and urine

A method is described for the assay of furosemide based on thin-layer chromatography and measurement of fluorescence directly on the plates. Conditions are specified for stabilizing fluorescence over the time of measurement. As little as 10 ng can be accurately measured and fluorescence is linear up to 160 ng. The metabolite or decomposition product 4-chloro-5-sulfamoyl anthranilic acid is well separated and measured quantitatively in the procedure. Application of the method to human plasma and urine is demonstrated.