Simultaneous determination of lidocaine and its metabolites in plasma and myocardium

No validated method exists for measuring lidocaine and its metabolites in myocardial tissue. We modified a previously described high-performance liquid chromatographic assay and applied it to plasma and to homogenized myocardial samples obtained from dogs that had received lidocaine by a double-infusion technique. Recovery of lidocaine, monoethylglycylxylidide and glycylxylidide after homogenization and extraction is reported. Assay variability, sensitivity and linearity over a wide range of sample sizes are also described. The results obtained with high-performance liquid chromatographic analysis are compared to quantitation of 14C-labeled lidocaine plus metabolites measured by an oxidation-scintillation technique. Myocardium to plasma partition coefficients for lidocaine, monoethylglycylxylidide and glycylxylidide were 2.16, 4.27, and 2.91, respectively.     

Liquid chromatography/tandem mass spectrometric method for the simultaneous determination of tobacco-specific nitrosamine NNK and its five metabolites

A sensitive and robust high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method to analyze 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its five metabolites in one passage was developed and validated. The method achieved excellent reproducibility and accuracy. Linearity was observed for all six compounds (R² = 0.999) with detection limits (S/N ≥ 3) ranging from 0.2 to 2.4 pg on column and 0.01-0.12 ng ml⁻¹ in samples injected. Average intra-day and inter-day variations (% R.S.D.) were 1.2 and 3.5%, respectively. A sample preparation method involving C8 and C18 solid phase extraction provided satisfactory recovery of the analytes in mouse urine. Each NNK metabolite was identified by its chromatographic retention time and specific fragmentation pattern. Since the carcinogenicity of NNK is related to its metabolism, the method described in this report should facilitate toxicological investigations into the carcinogenesis due to NNK exposure in the environment.     

Simultaneous determination of monoamine transmitters, precursors and metabolites in a single mouse brain

A simple and sensitive procedure for simultaneous determination of monoamine transmitters and related substances including precursors and metabolites has been developed for a single mouse brain. The proposed procedure involves (1) primary butanol extraction, (2) separation of the substances into either acid or alkaline aqueous layers according to their physicochemical properties, and (3) determination by means of high-performance liquid chromatography with electrochemical detection. Three transmitters (noradrenaline, dopamine and 5-hydroxytryptamine) and their precursors (tyrosine, 3,4-dihydroxyphenylalanine and tryptophan) and major metabolites (normethanephrine, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid and 5-hydroxyindoleacetic acid) were selectively separated and sensitively detected in mouse whole brain sample. Although 3-methoxy-4-hydroxymandelic acid was also separated from other substances by authentic chromatography, the substance was not detected in mouse brain. Changes in levels of these substances were examined for drugs whose effects had been previously confirmed. These changes reflected putative effects of the drugs and confirmed that the procedure is effective for neurochemical research into the transmitter system.     

Simultaneous determination of pantoprazole and its two metabolites in dog plasma by HPLC

A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 microg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025-10.0 microg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between -6.4% and 12%.     

HPLC determination of serotonin and its metabolites from human platelet-rich plasma; shift to 5-hydroxytryptophol formation following alcohol consumption

A sensitive, simple, and reliable high-performance liquid chromatographic method with electrochemical detection is developed for the measurement of four natural products, the serotonin-related indols from human platelet-rich plasma (PRP) using N-methylserotonin as internal standard. Separation of serotonin (5HT), 5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL), and 5-hydroxyindole-acetic acid (5HIAA) is carried out on Supelcosil LC-18DB stationary phase. A mixture of 48mM citric acid, 28mM sodium phosphate dibasic, 0.027mM Na(2)EDTA, and 3% methanol (pH 3.18) serves as the mobile phase. Measurements are carried out at 25 degrees C at E(ox) = 0.65 V. The calibration curves are linear through the range of 10-200 pg/mL. Method validation is performed according to internationally accepted criteria. Blood is collected from healthy controls and schizophrenic subjects. Significantly higher PRP serotonin is measured in schizophrenics; patients with recent alcohol consumption could be characterized with significantly elevated 5HTOL/5HIAA ratio.     

Simultaneous determination of trimipramine and its major metabolites by high-performance liquid chromatography

Trimipramine is a tricyclic antidepressant drug often assayed by gas chromatographic or gas chromatographic-mass spectrometry techniques. A high-performance liquid chromatographic method with electrochemical detection is described for the assay of trimipramine and its major metabolites, monodesmethyltrimipramine and 2-hydroxytrimipramine, in plasma. The method is sensitive, accurate and robust and thus suitable for routinely assaying samples following single doses of trimipramine to man. The assay was applied to plasma samples obtained following a single 50-mg dose of trimipramine to healthy volunteers.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous HPLC determination of 5-fluorouracil and its metabolites in plasma of cancer patients

5-Fluorouracil (5-Fu) is a commonly used anticancer agent for treatment of solid tumours. Certain studies have reported conflicting results between individual plasma concentration levels and toxicity or therapeutic effects. For this reasons some authors proposed to evaluate the plasma levels of 5-Fu metabolites 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-5,6-dihydro-uracil. The aim of the present work is to develop and validate a new HPLC method simultaneously determining 5-fluorouracil and its three metabolites, to be used to study the plasma levels, therapeutic effects and toxicity in cancer patients. The analytes were separated on a 4.6 x 250 mm ODS1 (5 micro m) not end-capped column, operating at room temperature. Elution was performed under isocratic conditions, employing a 1.5 mM K(3)PO(4) mobile phase (pH 5). 5-Bromo-5,6-dihydro-uracil was used as internal standard. The limits of quantitation were 0.5 micro g/mL for 5-fluorouracil, 1 micro g/mL for 5-fluoro-5,6-dihydro-uracil, 3 micro g/mL for 5-fluoro-2'-deoxyuridine and 5-fluorouridine; the stability, recovery, linearity, accuracy and specificity of the compounds were evaluated according to the criteria widely accepted. Using this method we measured plasma samples of 18 cancer patients treated with folinic acid (100 mg/m(2)) by intravenous administration, followed by an i.v. bolus of 5-Fu (400 mg/m(2)). The concentration levels of 5-fluorouracil and for 5-fluoro-5,6-dihydro-uracil were detectable in all the subjects while 5-fluorouridine and 5-fluoro-2'-deoxyuridine were present only in eight patients.     

Simultaneous determination of metapramine and its demethylated metabolites in plasma by gas chromatography-mass spectrometry

A capillary column gas chromatography--mass fragmentographic method for metapramine and its three major demethylated metabolites is described. Compounds are extracted from plasma using a double-extraction procedure and transformed into N-trifluoroacetyl derivatives. The detection is performed by monitoring specific ions for metapramine and for its metabolites with a mass detector. In spite of extensive metabolism in the liver and rapid elimination of metapramine, plasma concentrations of both metapramine and its metabolites can be simultaneously followed over 24 h after a single 150-mg oral dose, because of the sensitivity and selectivity of the method. This method has been successfully applied to the analysis of samples obtained from patients who were at steady state with metapramine and to a pharmacokinetic study in a healthy volunteer.     

Simultaneous chiral determination of methamphetamine and its metabolites in urine by capillary electrophoresis-mass spectrometry

A capillary electrophoresis-mass spectrometry method for the simultaneous chiral determination of enantiomers of methamphetamine (MA), amphetamine (AP), dimethylamphetamine (DMA) and p-hydroxymethamphetamine (pOHMA), in urine has been developed. The internal standards used were 2-phenylethylamine and 1-amino4-phenylbutane. The electrolyte was 1 M formic acid (pH 2.2). The chiral selector, which was added to the electrolyte, was a mixture of 3 mM beta-cyclodextrin and 10 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin. The detection limits were 0.03 microg ml(-1) for the enantiomers of MA and AP and 0.05 microg ml(-1) for the enantiomers of pOHMA using selected ion monitoring. In the analysis of healthy adult urine samples spiked with MA, AP and pOHMA, the precision of within-run assays (n = 4) for the migration time after correction with two internal standards were under 0.04%, and the detection yields utilizing solid phase extraction were 95-105%. This method was applicable to the analysis of urine samples of MA addicts and DMA addicts.     

Simultaneous determination of albendazole and its metabolites in fish muscle tissue by stable isotope dilution ultra-performance liquid chromatography tandem mass spectrometry

A rapid, specific, and sensitive method utilizing ultra-performance liquid chromatography tandem mass spectrometry was developed and validated to determine albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole 2-aminosulfone in fish muscle tissue. The fish samples were extracted with ethyl acetate, then the organic phase was evaporated to dryness, and the residue was reconstituted in methanol–water solution and cleaned up by n-hexane. Reversed-phase separation of target compounds was achieved using a BEH C18 column and a gradient consisting of 0.2% (v/v) formic acid and methanol. Tandem mass spectrometry analyses were performed on a triple–quadrupole tandem mass spectrometer. In the whole procedure, the isotope-labeled internal standards were used to correct the matrix effect and variations associated with the analysis. The method was validated with respect to linearity, specificity, accuracy, and precision. The method exhibited a linear response from 0.1 to 20 ng mL^-1 (r ² > 0.9985). The limit of quantitation for albendazole (ABZ), albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO₂), and albendazole 2-aminosulfone (ABZ-2-NH₂SO₂) was 0.1, 0.1, 0.1, and 0.2 ng g^-1, respectively. The mean recoveries of ABZ, ABZSO, ABZSO₂, and ABZ-2-NH₂SO₂ spiked at a level of 0.2–5.0 ng g^-1 were 95.3–113.7%, and the relative standard deviations of intra- and inter-day measurements were less than 6.38%. The method was later successfully applied to the determination of albendazole and its three metabolites in 60 fish samples collected from local markets.     

Simultaneous determination of perhexiline and its monohydroxy metabolites in biological fluids by gas chromatography-electron-capture detection

A rapid and sensitive method for the simultaneous determination of perhexiline and its cis-4-axial and trans-4-equatorial monohydroxy metabolites (M1 and M3, respectively) in human plasma, urine and bile is described. The assay utilises a single diethyl ether extraction, heptafluorobutyric acid anhydride derivatisation and separation and detection by gas chromatography-electron-capture detection. The limits of detection are 0.1 microgram/ml for perhexiline and 0.025 microgram/ml for the M1 and M3 metabolites. This method has been used in a five-day kinetic study of three healthy adult males who ingested a single 300-mg dose of perhexiline maleate. One of these volunteer subjects exhibited elevated plasma perhexiline and markedly reduced plasma and urinary M1 concentrations together with profoundly prolonged plasma and urinary M1 elimination times when compared with the other two subjects. These differences are thought to be of genetic origin. There were also obvious differences in urinary M3 concentrations which were discussed.     

Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R ² 0.998–0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.     

液相色谱-串联质谱法测定黄瓜和苹果中氟啶虫酰胺及其代谢产物残留量

建立了黄瓜和苹果中氟啶虫酰胺及其3种代谢产物［N-(4-trifluoromethylnicotinoyl)glycine(TFNG)、4-tri-fluoromethylnicotinic acid(TFNA)和4-trifluoromethylnicotinamide(TFNA-AM)］同时测定的液相色谱-串联质谱分析方法。样品用磷酸盐缓冲液提取两次,调节pH值至1.5～2.0后,再用乙酸乙酯提取,液相色谱-串联质谱分析。采用Acquity BEH C18色谱柱分离,0.1%甲酸水-甲醇作为流动相进行梯度洗脱,电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式检测,外标法定量。氟啶虫酰胺、TFNG、TFNA和TFNA-AM的检出限分别为0.17、0.20、0.35和0.60 μg/kg。在黄瓜和苹果样品中添加5.0～2000 μg/kg水平的氟啶虫酰胺、TFNG、TFNA和TFNA-AM,其平均添加回收率在82.9%～104.1%范围内,批内分析相对标准偏差(RSD)在3.6%～6.9%之间。4种物质的峰面积与其浓度在0.50～200 μg/L范围内均呈良好的线性关系,线性回归系数均大于0.998。前处理步骤仅用有机溶剂6 mL。整个方法具有高灵敏度、准确、稳定的特点。     

Simultaneous determination of thioridazine and its S-oxidized and N-demethylated metabolites using high-performance liquid-chromatography on radially compressed silica

A method for simultaneously quantifying thioridazine, northioridazine, thioridazine-2-sulfoxide, thioridazine-2-sulfone and thioridazine-5-oxide in serum and plasma is described. Following solvent extraction these compounds were separated by high-performance liquid chromatography on radially compressed silica gel and detected by UV absorbance at 254 nm. Chromatography time is less than 7 min. The relative retention of these compounds as a function of the methanol and methylamine content of the mobile phase is discussed. Practical limits of detection, based upon on assayed plasma or serum volume of 1 ml, were 20 ng/ml for thioridazine-5-oxide and 10 ng/ml for the other compounds. The coefficient of variation for all compounds was less than 13%. The method is compared with more conventional high-performance liqiud chromatographic and gas chromatographic methodology.     

Simultaneous determination of fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane--dichloromethane--butanol (55:40:5). The plasma extract is chromatographed on a 10-micron, C18 reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration--response curves for all four compounds are linear from 0.05 micrograms/ml to at least 10 micrograms/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within +/- 9% of the various amounts added with a standard deviation of +/- 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.