Journal of Chromatography B: Biomedical Sciences and Applications最新文献

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. For comparison, the capacity factors were derived for a second reference BGE where the SDS concentration (5 mmol/l) is close to the critical micellar concentration (CMC). The capacity factors are compared with the logarithm of the octanol–water partition coefficient, log P_OW, as measure for lipophilicity. Clear disagreement between these two parameters is found especially for epimeric cholates with the hydroxy group in position 7. In contrast, fair relation between the capacity factor of the analytes and their CMC is observed both depending strongly on the orientation of the OH groups, and tauro-conjugation as well. In this respect the retention behaviour of the bile salts in MEKC seems to reflect their role as detergents in living systems, and might serve as model parameter beyond lipophilicity.

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.

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. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.

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. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.

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. The observed effect could indicate changes of chromatographic performance caused by contributions of the analytical column or the ADS precolumn. The experiments with microdialysis excluded the contribution of the analytical column. A detailed investigation of experiments have been discussed in this paper.

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 μl of rat plasma. The method has been successfully applied to an intramuscular pharmacokinetic study at a dose of 10 μg/kg. Therefore, the current method is a valuable analytical tool for investigating the pharmacokinetics of fentanyl at low clinical doses.

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. The quantification was performed by using external standards. Sample preparation and stability of methanolic extract of St. John’s Wort were extensively explored. It is worth noting that the major constituents in the methanolic extract of St John’s Wort, especially hypericin and pseudohypericin, might be retained by some filter cartridges during the filtration. The current method may serve as a valuable tool for the QA/QC of St. John’s Wort dietary supplements.

A liquid chromatographic procedure with electrospray ionization tandem mass spectrometric detection has been developed and validated for LSD and iso-LSD determination. A one-step liquid–liquid extraction on 1 ml blood or urine was used. The lower limit for quantitative determination was 0.02 μg/l for LSD and iso-LSD. The analytical procedure has been applied in two positive cases (case 1: LSD=0.31 μg/l, iso-LSD=0.27 μg/l in plasma and LSD=1.30 μg/l, iso-LSD=0.82 μg/l in urine; case 2: LSD=0.24 μg/l, iso-LSD=0.6 μg/l in urine). LSD metabolism was investigated using MS–MS neutral loss monitoring for the screening of potential metabolites. The main metabolite was 2-oxo-3-hydroxy-LSD (O–H–LSD) present in urine at the concentrations of 2.5 μg/l and 6.6 μg/l, respectively, for case 1 and 2, and was not present in plasma. Nor-LSD was also found in urine at 0.15 and 0.01 μg/l levels. Nor-iso-LSD, lysergic acid ethylamide (LAE), trioxylated-LSD, lysergic acid ethyl-2-hydroxyethylamide (LEO) and 13 and 14-hydroxy-LSD and their glucuronide conjugates were detected in urine using specific MS–MS transitions.