Enantioselective CE method for pharmacokinetic studies on ibuprofen and its chiral metabolites with reference to genetic polymorphism

A stereospecific CE method was elaborated for the quantification of ibuprofen enantiomers and their major phase I metabolites: 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen in plasma and urine. Optimal temperature and pH of BGE were established to obtain complete separation of eight ibuprofen chiral compounds and (+)-S indobufen, applied as an internal standard, during one analytical run. After isolation from biological matrices using SPE on an octadecyl stationary phase, the analytes were separated and resolved up to 10 min in a silica capillary filled with BGE, consisting of heptakis 2,3,6-tri-O-methyl-beta-CD in triethanolamine-phosphate buffer, pH 5.0. Complete enantioseparation of the all analytes confirmed specificity of the method. The calibration curves were linear in the range of 0.1-25.0 mg/L for IBP enantiomers and their chiral metabolites in 0.5 mL of plasma and 1.0-200.0 mg/L in 0.05 mL of urine. Following SPE procedure, recovery of the chiral analytes from the two media was in the ranges of 82-87%, 90-95% and 70-76% for ibuprofen, 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen enantiomers, respectively. The validated method was successfully applied in pharmacokinetic investigations of IBP enantiomers as well as free chiral metabolites in reference to the genetic polymorphism of CYP450 2C isoenzymes.     

Simultaneous determination of the sulphonylurea glimepiride and its metabolites in human serum and urine by high-performance liquid chromatography after pre-column derivatization

A sensitive and selective high-performance liquid chromatographic method has been developed for a new sulphonylurea, glimepiride, and its metabolites. The assay involves extraction with diethyl ether, thermolysis of the sulphonylureas at 100 degrees C and trapping of the resulting amines with 2,4-dinitrofluorobenzene. The derivatives were quantitated on a reversed-phase column by absorbance at 350 nm using a step gradient for the three compounds in serum and an isocratic run for the metabolites in urine. Analogous compounds were used as internal standards. The detection limit was 5 ng/ml for glimepiride and metabolite II and 10 ng/ml for metabolite I using 1 ml of serum. The method has been applied to the analysis of serum and urine samples from pharmacokinetic studies in humans.     

Determination of clobazam, N-desmethylclobazam and their hydroxy metabolites in plasma and urine by high-performance liquid chromatography

Owing to the pharmacological and clinical importance of the determination of plasma and urine levels of the hydroxy metabolites of clobazam and N-desmethylclobazam in healthy volunteers and in epileptic patients, a high-performance liquid chromatographic (HPLC) method was developed that permits the determination of all these compounds in the same plasma or urine sample. The method involved ether extraction at pH 13 with diazepam as internal standard for the measurement of clobazam and N-desmethylcobazam, followed by ether extraction at pH 9 with nitrazepam as internal standard for the measurement of the hydroxy derivatives. The limit of detection was about 10-20 ng/ml for each of these compounds. Applications to patients were limited by chromatographic interferences between the hydroxy metabolites and some medications currently associated with clobazam in the treatment of epilepsy. The only interference in clobazam and N-desmethylclobazam analysis was from N-desmethyldiazepam. Despite these inconveniences, this HPLC procedure appears to be the only available method for the simultaneous quantification of clobazam and its three main metabolites.     

Determination of vanilmandelic acid in urine by coupled-column liquid chromatography combining affinity to boronate and separation by anion exchange

An automated liquid chromatographic method for assaying vanilmandelic acid in urine is described. Vanilmandelic acid and potential interfering substances, such as catechol compounds and their metabolites, have been tested for affinity to boronic acid-substituted silica at various pH values. Vanilmandelic acid and the internal standard, isovanilmandelic acid, were bound to the boronate matrix at an acidic pH, whereas for instance catecholamines were unretained and passed through the column. The alpha-hydroxycarboxylic acids were then desorbed by another mobile phase (pH 6.0) and transferred to an anion exchanger for chromatography and electrochemical detection. A relative standard deviation of 2.8% was obtained for the analysis of human urine samples containing 6.6 microM vanilmandelic acid.     

High-performance liquid chromatographic determination of the stereoisomeric metabolites of ibuprofen

A stereospecific reversed-phase high-performance liquid chromatographic (HPLC) method has been developed to simultaneously quantitate the stereoisomers of the two major metabolites of ibuprofen: hydroxyibuprofen and carboxyibuprofen. The metabolites were derivatized with S-(alpha)-methylbenzylamine to form diastereomeric amides which were separated and quantified on a C8 column. The validity of the stereoselective assay was confirmed by comparison with a non-stereoselective HPLC method. The stereoselective assay was applied to the quantification of all the stereoisomeric ibuprofen metabolites in urine from human volunteers dosed with racemic ibuprofen or the individual enantiomers of ibuprofen. Significant substrate and product stereo-selectivities were observed in the formation of carboxyibuprofen.     

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.     

High-performance liquid chromatographic method for the determination of eclanamine and its N-desmethyl and N,N-didesmethyl metabolites in urine

A high-performance liquid chromatographic method has been defined for the determination of eclanamine (free base of eclanamine maleate) and two of its metabolites, N-desmethyleclanamine and N,N-didesmethyleclanamine in urine. The method employs 10-ml urine samples, has a linear range from 5 to 500 ng/ml for the three compounds, and has a detection limit of 0.5 ng/ml for each compound. Sample preparation uses a cyanopropylsilane extraction column with washes of water, acetonitrile-water (30:70, v/v), and acetonitrile, and elution with 2% trifluoroacetic acid in acetonitrile. The eluate is evaporated to dryness, the residue dissolved in 1.0 ml acetonitrile-water (10:90, v/v) and 100 microliter are injected onto a Supelcosil LC-CN column. Eclanamine and its metabolites are eluted with an acetonitrile-water (35:65, v/v) eluent containing 0.01 M triethylamine and adjusted to pH 7.0 with phosphoric acid. The method has been validated by preparing and analyzing a series of fortified urines (range 2-500 ng/ml for each compound) on four separate days. Good linearity, precision, reproducibility, and specificity were obtained. Certification of the analytical method was accomplished by analyzing urine specimens collected from one volunteer administered a single oral dose of 45 mg eclanamine maleate. The data suggest that the metabolites of eclanamine have long elimination half-lives with levels still quantifiable in the 72-96 h collection interval.     

Capillary gas chromatographic method for the determination of the thromboxane A2 receptor antagonist S-1452 and its metabolites in human plasma.

A capillary gas chromatographic method for the determination of a newly developed thromboxane A2 antagonist, S-1452, and its metabolites in human plasma has been developed. This nitrogenous compound was detected as the free acid (+)-S-145 using a nitrogen-phosphorus detector. The two metabolites bisnor-(+)-S-145 and tetranor-(+)-145 were also assayed. The three compounds, which all possess carboxylic acid group, were converted into their hexafluoro-2-propyl esters with hexafluoropropan-2-ol-pentafluoropropionic anhydride as reagent. The esterification resulted in a decreased background response from the detector, and hence higher sensitivity and better precision. The glucuronides of the three compounds were also assayed after alkaline hydrolysis. The plasma concentration of the glucuronide of I was more than twice that of the free acid. This method allows determination of the three compounds at more than 2 ng/ml with a precision of 2-6% and an accuracy variation of less than +/- 5%. The analysis was performed within 2 min after injection to assay the sample rapidly from the Phase I test. The method is suitable for pharmacokinetic studies.     

High-performance liquid chromatographic determination of the 1,4-diketo and 1,4-diketo-2,3-dihydroxy metabolites of lonapalene in rat urine

Individual high-performance liquid chromatographic (HPLC) methods have been developed for the determination of two major metabolites of lonapalene in rat urine. The highly unstable and polar 1,4-diketo-2,3-dihydroxy metabolite (II) is extracted from urine by two extraction columns (phenyl followed by silica), further purified by means of HPLC with a fully end-capped C18 HPLC column and quantified by an ultraviolet detector at 280 nm. Ascorbic acid is used as an antioxidant during extraction and overnight injection of II. Urine samples for total II (free plus conjugated) determination are incubated with arylsulfatase and beta-glucuronadase prior to extraction. The 1,4-diketo metabolite (III) is extracted from urine with a C18 extraction column, further purified with a C18 HPLC column, and quantified by an ultraviolet detector at 260 nm. The detection limit for both metabolites is 100 ng/ml of urine (signal-to-noise = 2.5). The methods were used to analyze urine samples from a long-term toxicology study of lonapalene in rats and to determine the linearity of dose-concentration relationships for both metabolites.     

Versatile isocratic high-performance liquid chromatographic assay for propranolol and its basic, neutral and acidic metabolites in biological fluids

A comprehensive high-performance liquid chromatographic method was developed for quantitating propranolol and its known metabolites in serum, bile and urine. Analysis was performed before and after incubation of the samples with beta-glucuronidase-arylsulfatase to quantitate both free and conjugate forms of the oxidative metabolites. Fractionation of the basic, neutral and acidic metabolites was achieved by differential pH solvent extraction. The basic and neutral metabolites were extracted from the biological samples at pH 10.5 with 2% n-butanol in dichloromethane. Additional clean-up of the basic fraction by back-extraction into dilute acid was needed for those samples that were subjected to enzymatic hydrolysis. The original aqueous sample was titrated with acid to pH 1, followed by extraction of the remaining acidic metabolites into either n-butanol-dichloromethane (with unhydrolyzed serum) or carbon tetrachloride (with all other samples). Chromatographic separation of the metabolites in the different extracts was achieved on a reversed-phase C18 column, using a single isocratic mobile phase consisting of 0.044 M pH 2.7 phosphate buffer, tetrahydrofuran, methanol and acetonitrile, with the addition of n-butylamine as a competing base to control retention volume and peak shape. Detection and quantitation of propranolol and its metabolites in the low nanogram to sub-nanogram range was afforded by fluorescence at a low UV excitation wavelength. The coefficients of variation for replicate assay of spiked samples were uniformly less than 6% for all the analytes.     

Determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine by liquid chromatography with fluorimetric detection

A highly sensitive and rapid liquid chromatographic method for the determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine is described. After extraction of urine with diethyl ether, these acids and phenylpropionic acid (internal standard) are converted into the corresponding fluorescent derivatives by treatment with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Radial-Pak cartridge C18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limits for phenylacetic and p- and m-hydroxyphenylacetic acids are 5, 30 and 100 fmol, respectively, at a signal-to-noise ratio of 5 in a 20-microliter injection volume. This sensitivity permits precise determination of the free and total acids in 20 microliter of normal human urine.     

High-performance liquid chromatographic determination of ibuprofen, its metabolites and enantiomers in biological fluids

An isocratic high-performance liquid chromatographic method to determine racemic ibuprofen (assay I) and its major metabolites (assay II) in biological fluids (plasma, urine, bile) using a conventional reversed-phase column is described. A third assay using beta-cyclodextrin as stationary phase (Cyclobond I) for the separation of the ibuprofen enantiomers is also described. A wavelength of 220 nm was used to monitor the substances. The sensitivity of the method was 0.1 microgram/ml for all three assays. The method was demonstrated to be suitable for stereoselective pharmacokinetic studies of ibuprofen in humans and animals.     

Simultaneous determination of gemfibrozil and its metabolites in plasma and urine by a fully automated high performance liquid chromatographic system

Sensitive and specific methods for the simultaneous determination of gemfibrozil (Lopid), a lipid-lowering agent, and its metabolites in plasma and urine are described. The methods are based on a fully automated high performance liquid chromatographic (HPLC) system with fluorescence detection. Urine samples, diluted with acetonitrile, were directly analysed by HPLC using a flow and eluent programming method. In the case of plasma, gemfibrozil and its main metabolites were extracted from acidified samples and the resulting extracts injected into the chromatographic system. The sensitivity was approximately 100 ng/mL for gemfibrozil and its four metabolites using 0.5 mL plasma or urine. An acyl glucuronide of gemfibrozil excreted in human urine after oral administration of the drug was isolated and its structure and stability examined.     

Automated high-performance liquid chromatographic determination of antipyrine and its main metabolites in plasma, saliva and urine, including 4,4'-dihydroxyantipyrine

A rapid, selective and sensitive method was developed for the determination of antipyrine and its main metabolites in plasma, saliva and urine by an automated high-performance liquid chromatographic system. Using a MOS-Hypersil reversed-phase column with a phosphate buffer--acetonitrile mobile phase, baseline separation of antipyrine, its metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine, and the internal standard, phenacetin, was achieved within 6 min. Factors regarding the accuracy and precision of the method and the stability of phase I metabolites during sample preparation are discussed, taking into account certain drawbacks of previously published methods. Based on the same chromatographic system a method was developed for the assay of 4,4'-dihydroxyantipyrine in urine. This compound is an important metabolite of antipyrine in the rat, representing 12.6 +/- 1.8% of the administered dose (n = 18).     

Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

Gas chromatographic method for determination of a piperazine derivative (Trelibet) and its metabolites in human plasma and urine

A sensitive gas chromatographic method was developed for the determination of Trelibet, 1-benzyl-4-(2'-pyridinecarbonyl)piperazine, and of its major metabolites in biological fluids. The compounds were extracted as bases into dichloromethane, and the extracts were analysed by a dimethylsilicone capillary column with a nitrogen-phosphorus flame-ionization detector. The lower limit of detection was 1 ng/ml for Trelibet and 5 ng/ml for the metabolites. Peak-area ratios of the compounds and internal standard were linearly correlated to their plasma concentrations between 1 and 1000 ng/ml. The method was used for quantification of Trelibet and two of its metabolites in depressed patients after oral administration of a single dose of 200 mg of Trelibet. Concentration data measured in plasma and urine showed that the method is sensitive enough to monitor concentrations both for pharmacokinetic studies and for plasma steady-state levels daily.     

High-temperature ultra-performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry applied to ibuprofen metabolites in human urine

The application of sub-2 microm porous particle liquid chromatography (LC) operated at elevated temperatures, coupled with time-of-flight mass spectrometry (MS), to the separation and identification of metabolites of ibuprofen present in human urine following oral administrations is illustrated. The LC/MS system generated a high-resolution analytical separation that, with an analysis time of 20 min, provided a peak capacity in the order of ca. 350. Using this system a total of nine glucuronides of the drug and its metabolites were detected, including a number of isomeric acyl glucuronides of ibuprofen itself, a side-chain-oxidized carboxylic acid acyl glucuronide and a number of acyl glucuronides of various hydroxylated metabolites. The identities of the metabolites were confirmed by their accurate mass values and the presence of the common fragment ions from ibuprofen.     

Determination of alpidem and its metabolites in human plasma by high-performance liquid chromatography and fluorimetric detection

A high-performance liquid chromatographic method has been developed for the simultaneous determination of alpidem and its metabolites in human plasma. The method involved a single extraction of the parent drug and metabolites into diethyl ether from alkalinized plasma, evaporation of the organic solution and chromatography of the extracts on a C18 column coupled to a fluorimetric detector. An internal standard was used for the quantitative determination of the compounds. The method was selective for alpidem and three of its metabolites and has a limit of detection of less than 1 ng ml-1 for all the compounds. Since the chromatographic run took more than 20 min, the chromatographic process was fully automated and performed overnight.     

液液萃取-高分辨气相色谱-高分辨双聚焦磁质谱法测定尿中羟基多环芳烃代谢物

建立了灵敏、准确的液液萃取-高分辨气相色谱-高分辨双聚焦磁质谱（LLC-HRGC-HRMS）同时测定人体尿样中8种多环芳烃（PAHs）代谢物的方法。将2 mL尿液与氘或¹³C标记的同位素内标物进行混合,在抗坏血酸的存在下酶解偶联目标物,再将游离化合物用甲苯-戊烷（1：4,v/v）萃取后浓缩至近干,再用甲苯复溶,衍生后经HRGC-HRMS分离和测定。1-羟基萘在0.14~41.6 μg/L、1-羟基菲在0.05~8.33 μg/L、2-羟基菲在0.04~8.33 μg/L、其他5种代谢产物在0.02~8.33 μg/L范围内线性关系良好,相关系数＞0.99,方法的检出限为0.006~0.042 μg/L,平均回收率为81.4%~127.0%,日内和日间精密度分别为2.7%~6.9%和5.1%~10.9%（n=6）。应用该方法测定330份人群尿液样本,3-羟基䓛和6-羟基䓛未检出,其他6种PAHs代谢物检出率为100%。该方法灵敏、准确、稳定,适用于人尿中8种PAHs代谢物的定量分析。     

Rapid and simple method for the determination of urinary benzoic and phenylacetic acids and their glycine conjugates in ruminants by reversed-phase high-performance liquid chromatography.

A simple, rapid and reproducible reversed-phase high-performance liquid chromatographic method for the simultaneous determination of benzoic acid (BA), phenylacetic acid (PAA) and their respective glycine conjugates hippuric acid (HA) and phenaceturic acid (PA) in sheep urine is described. The procedure involves only direct injection of a diluted urine sample, thus obviating the need for an extraction step or an internal standard. The compounds were separated on a Nova-Pak C18 column with isocratic elution with acetate buffer (25 mM, pH 4.5)-methanol (95:5). A flow-rate of 1.0 ml/min, a column temperature of 35 degrees C and detection at 230 nm were employed. These conditions were optimized by investigating the effects of pH, molarity, methanol concentration in the mobile phase and column temperature on the resolution of the metabolites. The total analysis time was less than 15 min per sample. At a signal-to-noise ratio of 3 the detection limits for ten-fold diluted urine were 1.0 microgram/ml for BA and HA and 5.0 micrograms/ml for PAA and PA with a 20-microliters injection.