Direct capillary electrophoretic determination of three chemotherapeutic drugs in human urine

Summary Capillary zone electrophoresis (CZE) has been used for direct determination of 6-thioguanine, methotrexate, and 5-fluorouracil in human urine, by use of a fused-silica capillary (60.2 cm×75 μm i.d.). Separation was performed after hydrodynamic injection for 7 s; the separation potential and capillary temperature were 25 kV and 35°C, respectively. A 45mm borate buffer solution (pH 9.2) was used as separation electrolyte. Under these conditions the analysis takes approximately 10 min and interday precision of migration times and corrected peak areas is satisfactory. A linear response over the concentration range 3.0–20.0 mg L¹ was observed for the three chemotherapeutic drugs in diluted human urine. Detection limits (s/n=3) for 6-thioguanine and methotrexate were approximately 1.60 mg L¹ in diluted human urine; that for fluorouracil was 2.60 mg L¹. A 2-ml volume of human urine was diluted with 2-mL of water and introduced directly into the electrophoresis system. CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity. This method resulted in especially excellent recoveries for determination of methotrexate in all the different urine samples analysed (n=10).     

Determination of norfloxacin in pharmaceuticals,human serum,and urine using a luminol—dissolved oxygen chemiluminescence system

A sensitive chemiluminescence method, based on the enhancive effect of norfloxacin on the reaction between luminol and dissolved oxygen in a flow injection system, was proposed for the determination of norfloxacin. The increment of the chemiluminiscence intensity was proportional to the concentration of norfloxacin, giving a calibration graph linear over the concentration from 0.4 ng mL⁻¹ to 400.0 ng mL⁻¹ (r ² = 0.9988) with the detection limit of 0.1 ng mL⁻¹ (3 × σ _noise). At the flow rate of 2.0 mL min⁻¹, a complete determination of norfloxacin, including sampling and washing, could be accomplished in 30 s with the relative standard deviation lower than 3.0 %. The proposed method was applied successfully to determine norfloxacin in pharmaceuticals, human urine, and serum. Possible mechanism of the reaction was also discussed.     

Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometric method for the analysis of paroxetine in human plasma

A sensitive, simple, fast and rugged hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of paroxetine was developed and validated over curve range 0.050-50 ng/mL using only 0.4 mL plasma. This is the first published LC-MS/MS method and the low limit of quantitation of this method is 10-fold lower than previously published methods. A simple liquid-liquid extraction method using methyl-tert butyl ether (MTBE) as the extraction solvent was used to extract paroxetine and the internal standard (IS) fentanyl-d(5) from plasma. The extract was evaporated to dryness, reconstituted and injected onto a silica column using a low aqueous-high organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.1 and 1.2 min for paroxetine and IS, respectively. The detection was by monitoring paroxetine at m/z 330 --> 192 and IS at m/z 342 --> 188, respectively. The inter-day precision and accuracy of the quality control (QC) samples were <5.0% relative standard deviation (RSD) and <2.9% relative error (RE). This method can be used for supporting therapeutical drug monitoring and pharmacokinetic or drug-drug interaction studies.     

High performance liquid chromatographic determination of metoclopramide in plasma and urine and its application to biopharmaceutical investigations

An optimized HPLC method for the quantification of metoclopramide (MCP) in human plasma and urine is described. MCP and internal standard are extracted from alkalinized substrate into diethyl ether and back-extracted into dilute acid. The analytes are separated with a ternary mobile phase at cyanopropyl-silica and detected at 312 nm (UV detection). The lower limit of quantification is 0.5 ng/ml in plasma and 50 ng/ml in urine. Optimization of extraction, chromatography, and detection is discussed. The method is selective to numerous common drug substances with excellent accuracy and precision data. After validation, the method is applied to the samples of a pharmacokinetic study. Pharmacokinetic parameters indicate the need for a sophisticated method as tool for optimization of metoclopramide formulations.     

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

Summary An analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 g L^–1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.     

Determination of physiological noble metals in human urine using liquid-liquid extraction and Zeeman electrothermal atomic absorption spectrometry

An extremely sensitive, reliable and simple procedure is described for the determination of physiological palladium, platinum and gold in human urine. The urine samples were adjusted to pH 4 (Pd, Au) or pH 5 (Pt), followed by conversion of the analytes to their pyrrolidinedithiocarbamate complexes. These complexes were separated from the matrix by liquid-liquid extraction into 4-methyl-2-pentanone resulting in a 25-fold enrichment. Determination was by electrothermal atomic absorption spectrometry (ET-AAS) using longitudinal inverse alternating current Zeeman-effect background correction. The limits of detection calculated from three standard deviations of the blank values were 20 ng l⁻¹ for Pd and Au and 70 ng l⁻¹ Pt. Within-day precision (n = 10, 5 μg l⁻¹) ranged 5.2%–7.7%. The procedure is successfully applied to determine urinary palladium, platinum and gold in nine unexposed persons. Palladium levels in urine ranged < 20–80 ng l⁻¹ (arithmetical MEAN=38.7 ng l⁻¹), while gold levels ranged < 20–130 ng l⁻¹ (36.0 ng l⁻¹). Physiological platinum levels in urine were all < 70 ng l⁻¹. The accuracy of the procedure was checked by analyzing a series of urine samples by a second independent method (magnetic sector field inductively-coupled plasma-mass spectrometry) in combination with UV photolysis.     

HPLC Analysis of Cotinine in Urine After SPE with a Cholesterol-Modified Adsorbent

A one-step solid-phase extraction procedure, based on a new silica gel adsorbent modified with cholesterol groups, has been investigated for measurement of cotinine in urine. Cotinine is the main metabolite of nicotine in the human body and is analyzed as a biomarker for assessment of direct or passive exposure to tobacco smoke. New cholesterol-modified adsorbents have been obtained by chemical modification of silica gel of different porosity with cholesterol ligands. Although recovery by this extraction procedure were optimum over a relatively broad range of sample pH (3.1–8.0), analytical conditions such as sample loading, washing and elution conditions, concentration of cotinine to be extracted, and the type of adsorbent used for extraction were found to affect the efficiency of the procedure and had to be controlled for optimum recovery. When these conditions were controlled, recovery of cotinine from spiked human urine was reproducible and depended on compound ionization. Quantitative analysis of cotinine was performed by reversed-phase high-performance liquid chromatography with UV detection. Presented at: Conference of the Hyphenation of Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy, Liquid Chromatography–Mass Spectrometry and Related Topics, Tuebingen, Germany, March 25–29, 2006.     

Determination of Picogram-Levels of Acetylspiramycin in Human Urine and Serum by Flow Injection Chemiluminescence

A sensitive chemiluminescence method for the determination of acetylspiramycin is presented. It is based on the greatly enhancive effect of acetylspiramycin on the chemiluminescence reaction between luminol and hydrogen peroxide in the flow system. The increase in chemiluminescence intensity was linearly proportional to the acetylspiramycin concentration in the range from 10pg·mL^–1 to 2.0ng·mL^–1 (r²=0.9979). The detection limit was 3pg·mL^–1 (3). At a flow rate of 2.0mL·min^–1, the process of determination, including sampling and washing, could be performed in 0.5 min, and the relative standard deviations of seven replicates are less than 5.0%. The proposed method was applied successfully to the determination of acetylspiramycin in pharmaceutical preparations, human urine and serum without pre-treatment. It was found that the excretive ratio of acetylspiramycin reached its maximum 2.0 hours after having been administered orally, and the excretive ratio in 12.0 hours was 8.4.     

一种转基因猪血中重组人血清白蛋白分离纯化新方法

基因工程技术已经成为研究和生产重组人血清白蛋白(rHSA)替代人血清白蛋白(HSA)的重点技术,而白蛋白的纯化则是该技术的关键。本文主要介绍了从转基因猪血中纯化rHSA的一种新方法,即热乙醇沉淀与多级色谱分离相结合的rHSA纯化方法。热乙醇沉淀法可从猪血浆中获得rHSA粗提取液,此时rHSA的纯度可达69.5%,回收率达51.3%。进一步采用多级色谱分离法,即阴离子交换色谱和反相色谱法进一步纯化,得到rHSA的最终纯度约为100.0%,总回收率为41.1%。该方法为从转基因猪血浆中大规模纯化用于临床和生化研究的高纯度rHSA提供可能,同时也为rHSA替代HSA奠定了基础。     

Sensitive determination of probenecid in urine samples by reversed-phase liquid chromatography and UV-visible detection using solid-phase extraction techniques for sample clean-up

Summary This study describes a rapid method for the determination of probenecid in human urine by liquid chromatography with UV detection at 254 nm, after clean-up through a C8 solid-phase extraction column. Liquid chromatography was carried out on a C18-bonded phase using an acetonitrile-acetate buffer (pH=4) gradient elution. Ethacrynic acid was used as internal standard. The system has been applied to the determination of probenecid in the 0.10–100.0 g/ml concentration range; the limit of detection was 5 ng/mL.