Journal of Chromatography. B, Biomedical Sciences and Applications

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro...

A gas chromatographic-mass spectrometric method was developed for the simultaneous analysis of 15 low-dosed benzodiazepines, both parent compounds and their corresponding metabolites, in human urine. The target compounds are alprazolam, alpha-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, nitrogen-desalkylflurazepam, ketazolam, oxazepam, lormetazepam, lorazepam, triazolam and alpha-hydroxytriazolam. Nitrogen-methylclonazepam is used as the internal standard...

A highly sensitivity liquid chromatography-tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100-50.0 ng/ml, using 13C4 sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C18 solid-phase extraction cartridges. The dried residue was reconstituted with 100 microl of 0.01 N HCl. and 30 microl was injected onto the LC-MS-MS system...

A HPLC column-switching system with LiChrospher RP-8 ADS precolumn was applied for the determination of beta-blockers (atenolol, pindolol, propranolol) in human plasma. The influence of biological matrices on the changes of the chromatographic parameters such as retention time, peak symmetry, area and selectivity were investigated. After injection of 5 ml plasma a decrease of retention times of the analytes was observed of up to 25% and an increase of asymmetry factors of up to 5%. Peak areas and selectivities were not changed...

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5-500 ng/ml...

An improved isocratic high-performance liquid chromatography (HPLC) method for the analysis of L-(-)-fucose. D-(+)-galactosamine, D-(+)-glucosamine, D-(+)-galactose, obtained by hydrolysis of glycosaminoglycans (GAGs) and D-(+)-glucose and D-(+)-mannose is described. The presence in circulation of GAGs, acid polysaccharide sequences of alternate monosaccharide units, aminosugar and uronic acid (galactose in keratan sulfate), has been measured in terms of their sugar components. To evaluate concentration of these circulating sugars we considered blood samples obtained from healthy humans...

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions...

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C8 column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma...

The South African traditional remedy Impila (Callilepis laureola) contains the mitochondrial toxin atractyloside. The plant is sold widely and continues to lead to fatalities in patients. We describe, for the first time, a simple GC-MS procedure for the identification of atractyloside, which we have applied to the gastric washing from a poisoned patient and to extracts of Impila tuber.

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column...

The capacity factors of 16 anionic cholates (from six bile salts, including their glyco- and tauro-conjugates) were determined in a micellar electrokinetic chromatography (MEKC) system consisting of buffer, pH 7.5 (phosphate-boric acid; 20 mmol/l) with 50 mmol/l sodium dodecyl sulfate (SDS) as micelle former and 10% acetonitrile as organic modifier. The capacity factors of the fully dissociated, negatively charged analytes (ranging between 0.2 and 60) were calculated from their mobilities, with a reference background electrolyte (BGE) without SDS representing "free" solution...

An RP-HPLC method with photodiode array detection was established for the determination of major constituents (rutin, hyperoside, isoquercitrin, quercitrin, quercetin, pseudohypericin, hyperforin and hypericin) in St. John's Wort dietary supplements. The samples were extracted with methanol by means of sonication in low temperature. The extraction was rapid, with two steps of sonication (30 min each) recovering more than 99% of the major constituents in St. John's Wort samples. The major components were separated by RP-18 chromatography column using a 60-min water-acetonitrile-methanol-trifluoroacetic acid gradient...

We purified the activated recombinant glucicorticoid receptor (GR) overexpressed in insect cells by sequential chromatographies using Mono Q and Mono S columns. This procedure was based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH (pH 8.4). The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. The purified GR was able to bind specifically to a DNA fragment containing the glucocorticoid response element...

Fluvastatin, an inhibitor of cholesterol biosynthesis, is commercialized as a racemic mixture of the (+)-3R,5S and (-)-3S,5R stereoisomers, although inhibition of HMG-CoA reductase mainly resides in the (+)-(3R,5S)-fluvastatin isomer. The aim of the present study was to analyze fluvastatin isomers in human plasma with application to studies on kinetic disposition. Plasma samples of 1 ml were eluted into 3 ml LC-18 Supelclean (Supelco) columns equilibrated with methanol and water. The columns were washed with water and acetonitrile and then eluted with methanol containing 0...

The phase I and phase II metabolism of the anabolic steroid methandrostenolone was investigated following oral administration to a standardbred gelding. In the phase I study, metabolites were isolated from the urine by solid-phase extraction, deconjugated by acid catalysed methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. GC-MS analysis indicated the major metabolic processes to be sequential reduction of the A-ring and hydroxylation at C6 and C16. In the phase II study, unconjugated, beta-glucuronidated and sulfated metabolites were fractionated and deconjugated using a combination of liquid-liquid extraction, enzyme hydrolysis, solid-phase extraction and acid catalysed methanolysis...

A sensitive assay method was developed to determine fentanyl, an opiate agonist, in rat plasma by gas chromatography with nitrogen-phosphorus detection. For the pretreatment of plasma samples, sodium hydroxide was added to denature protein and n-butyl chloride was used to extract fentanyl. The calibration curve was linear within the concentration range 0.5 to 50 ng/ml (r=0.9997). The limit of detection was 0.1 ng/ml, and 0.5 ng/ml could be quantified with acceptable precision. Furthermore, fentanyl could be determined in only 200 microl of rat plasma...

Modern atmospheric pressure ionization (API) ion-trap mass spectrometry in connection with fast chromatographic separations using a short narrow-bore C8 column was developed to determine 5-phenyl-3-thioureido-1,2,4-thiadiazole (301029), a novel virus inhibitor in serum. Both 301029 and an internal standard (I.S.) were separated from serum samples by acetonitrile deproteinization and extraction without time-consuming reconstitution. The chromatographic separation was achieved on a C8 reversed-phase narrow-bore column using acetonitrile-water-acetic acid (90:10:0...

In this study molecular imprinting technology was employed to prepare a specific affinity sorbent for the resolution of phenylpropanolamine, a chiral drug. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting with either (-)- or (+)-phenylpropanolamine as the template. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of the template molecule. The bulk polymerization was carried out in chloroform with 2,2'-azobisisobutyronitrile as the initiator, at 4 degrees C and under UV radiation...

A sensitive, simple, and accurate method for determination of BMS-284756, a novel des-F(6)-quinolone antimicrobial agent in mouse serum was developed by HPLC with fluorescence detection. Sample preparations were carried out by protein precipitation with the addition of acetonitrile, followed by evaporation of the acetonitrile to dryness. The resultant residual was then reconstituted in 0.01 M HCl and injected onto a Nucleosil 100 10 microm, C18 25 cm x 4.6 mm analytical column. The mobile phase consisted of acetonitrile-0...

A chiral separation using carboxymethyl-beta-cyclodextrin and methyl-beta-cyclodextrin for the direct assay of tramadol in human urine by capillary electrophoresis (CE) with laser-induced native fluorescence detection was developed. Furthermore, the phase II metabolite O-demethyl tramadol glucuronide was determined from the urine samples and the ratio of the diasteromers was determined. The chiral method was validated. Correlation coefficients were higher than 0.999. Within day variation showed accuracy in the range 96...