Identification of a tolfenamic acid metabolite in the horse by gas chromatography-mass spectrometry.

A tolfenamic acid metabolite, a hydroxylated product, has been identified in equine plasma and urine samples using gas chromatography-mass spectrometry in the electron-impact and chemical-ionization modes. The method also allows the qualitative monitoring of the elimination of the drug and its metabolites from plasma. The two compounds are detected up to 48 and 24 h, respectively, after a single oral administration of a 30 mg/kg dose. The simultaneous detection of the two products increases the reliability of anti-doping control analysis.     

High-performance liquid chromatographic analysis of rosoxacin and its N-oxide metabolite in plasma and urine

A high-pressure liquid chromatographic method for the analysis of rosoxacin and its pyridyl N-oxide metabolite in plasma and urine extracts is described. A statistical evaluation of the assay data has shown acceptable accuracy and precision for 0.5 to 25 microgram of rosoxacin or the metabolite per ml of plasma and for 2.5 to 60 microgram/ml of either compound in urine. The minimum quantifiable level for rosoxacin was 0.13 microgram/ml in plasma and 0.64 microgram/ml in urine; for the metabolite in plasma and urine, the corresponding values were 0.21 and 0.60 microgram/ml, respectively. The method was applied to plasma and urine from three dogs medicated orally with 5 mg/kg of rosoxacin. The pharmacokinetic parameters calculated for rosoxacin were: plasma halflife, 1.9 h; plasma clearance, 65 ml/min; volume of distribution, 11.31. The average total urinary excretion of rosoxacin as free and conjugated rosoxacin and its free N-oxide was 7.7 +/- 0.2% over the 48-h collection period.     

Stereospecific high-performance liquid chromatographic assay of pirprofen enantiomers in rat plasma and urine.

A stereospecific high-performance liquid chromatographic method was developed for the assay of pirprofen enantiomers in rat plasma and urine. Following addition of internal standard (ketoprofen) and acidifier (L-ascorbic acid) to biological fluids, pirprofen was extracted into an isopropanol-isooctane (5:95) mixture. Diastereomers of pirprofen enantiomers, which were formed using L-leucinamide, were separated on a reversed-phase column with ultraviolet detection at 275 nm using 0.06 M KH2PO4-acetonitrile-triethylamine (64:36:0.1) as mobile phase. The limit of quantitation was 0.1 microgram/ml for each enantiomer, based on 100 microliters of rat plasma. No spontaneous oxidation of pirprofen to its pyrrole metabolite occurred during sample preparation and analysis. In three female rats which were dosed with 10 mg/kg racemic pirprofen orally, plasma concentrations of the enantiomers could be followed for 24 h. Pirprofen enantiomers in plasma were virtually unconjugated, and negligible concentrations of pyrrole metabolites were observed. Less than 10% of the total dose was recovered in urine as intact drug and its glucuroconjugates. The assay was found suitable for the study of the pharmacokinetics of pirprofen enantiomers in the rat.     

Enantiomeric Composition Tests of Ketoprofen in Equine Plasma and Urine as Diastereomeric (S)-(-)-1-Phenylethylamides by Achiral GC–MS

The enantiomeric composition of ketoprofen in equine plasma and urine after administration of a commercial racemic ketoprofen product were determined as diastereomeric (S)-(-)-1-phenylethylamidation by achiral gas chromatography–mass spectrometry in selected ion monitoring mode. This method showed linearity (r ≥ 0.9986) over the range tested (5.0–5,000 ng), with acceptable precision (% RSD ≤ 9.5) and accuracy (% RE = ?3.7–7.3). The ratio of (S)-ketoprofen in plasma after 6.0 h and in urine after 71.0 h increased progressively to final values of 67.3 ± 0.1 and 91.9 ± 2.2%, respectively, attributable to the inversion of (R)-ketoprofen to (S)-ketoprofen.     

Pharmacokinetics and excretion study of bergenin and its phase II metabolite in rats by liquid chromatography tandem mass spectrometry

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method for the determination of bergenin and its phase II metabolite in rat plasma, bile and urine has been developed. Biological samples were pretreated with protein precipitation extraction procedure and enzymatic hydrolysis method was used for converting glucuronide metabolite to its free form bergenin. Detection and quantitation were performed by MS/MS using electrospray ionization and multiple reaction monitoring. Negative electrospray ionization was employed as the ionization source. Sulfamethoxazole was used as the internal standard. The separation was performed on a reverse‐phase C₁₈ (250 × 4.6 mm, 5 μm) column with gradient elution consisting of methanol and 0.5% aqueous formic acid. The concentrations of bergenin in all biological samples were in accordance with the requirements of validation of the method. After oral administration of 12 mg/kg of the prototype drug, bergenin and its glucuronide metabolite were determined in plasma, bile and urine. Bergenin in bile was completely excreted in 24 h, and the main excreted amount of bergenin was 97.67% in the first 12 h. The drug recovery in bile within 24 h was 8.97%. In urine, the main excreted amount of bergenin was 95.69% in the first 24 h, and the drug recovery within 24 h was <22.34%. Total recovery of bergenin and its glucuronide metabolite was about 52.51% (20.31% in bile within 24 h, 32.20% in urine within 48 h). The validated method was successfully applied to pharmacokinetic and excretion studies of bergenin.     

Liquid chromatography-mass spectrometry in metabolic research. I. Metabolites of benzbromarone in human plasma and urine.

Seven benzbromarone metabolites were identified in human plasma and urine by electron-impact mass spectrometry after semipreparative high-performance liquid chromatographic fractionation and/or by liquid chromatography-mass spectrometry using a thermospray interface. The major metabolite in plasma and urine was a hydroxybenzofuranoyl species; the 1-hydroxyethyl entity was identified as a minor metabolite. Five urinary metabolites occurred in trace amounts, all of them carrying OH and/or C = O groups in different positions. The hydroxybenzofuranoyl metabolite has often been mistaken for benzarone in previous studies.     

Simultaneous determination of ZLR-8 and its active metabolite diclofenac in dog plasma by high-performance liquid chromatography

ZLR-8 is a nitric oxide releasing derivative of diclofenac for the treatment of inflammation. In this paper, a sensitive and reliable high-performance liquid chromatography method for simultaneous determination of ZLR-8 and its active metabolite diclofenac in the plasma of beagle dogs has been developed and validated. After the addition of ketoprofen as the internal standard (IS), plasma samples were extracted with n-hexane-isopropanol (95:5, v/v) mixture solution and separated by HPLC on a reversed-phase C(18) column with a mobile phase of gradient procedure. Analytes were determined by the UV detector which was set at 280 nm. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of ZLR-8 and diclofenac were linear over the range 0.05-4.0 microg/mL. The within- and between-batch precisions (RSD%) were lower than 10% and accuracy ranged from 85 to 115%. The lower limit of quantification was identifiable and reproducible at 0.05 microg/mL. The proposed method has been readily implemented in preclinical pharmacokinetics studies of ZLR-8 and its active metabolite diclofeance. Representative plasma concentration vs time profiles resulting from administration of ZLR-8 to beagle dogs are presented in this communication.     

Intake and excretion of disodium monomethylarsonate in horses: a speciation study

Capillary electrophoresis coupled to inductively coupled plasma mass spectrometry was used in a speciation study on disodium monomethylarsonate (DS-MMA^V) and its metabolites in horses, to which the drug was administered by intramuscular injection on five consecutive days at a single arsenic dosage of 270 mg day⁻¹. Samples of urine, whole blood, plasma, and mane hair were analyzed before, during, and after drug administration. The data show that blood clearing and urinary excretion of MMA is a fast process following first-order kinetics with biological half-lives of about 38 h and 44 h for urine and plasma, respectively. In the time period of 9 days studied, the only metabolite detected in urine was dimethylarsinic acid (DMA^V), which 4 days after the last drug administration accounted for up to 75% of the total excreted arsenic species. This shows, for the first time, that biomethylation of MMA^V to DMA^V is the principal metabolic pathway of this drug in horses. Although DS-MMA^V was administered only during a short 5-day period, an up to six fold increase of arsenic could be measured in the newly grown mane hair.     

Quantitation of a novel antiemetic (ADR-851) in plasma and urine by reversed-phase high-performance liquid chromatography with fluorescence detection

A sensitive and specific bioanalytical method for quantitation of a novel antiemetic (ADR-851) in plasma and urine has been developed and validated. The drug and internal standard (metoclopramide) are extracted from the plasma matrix by solid-phase extraction on cyanopropyl bonded-phase columns. After extraction, samples are separated by isocratic reversed-phase high-performance liquid chromatography. The parent drug, internal standard and a yet unidentified metabolite are detected by fluorescence. The method requires 1.0 ml of plasma or 0.1 ml of urine and has a lower limit of quantitation of 2 ng/ml with 10.9% relative standard deviation (R.S.D.). Method linearity has been established over a 2-800 ng/ml range when 1.0 ml of plasma is used. The intra- and inter-day imprecisions for the method are typically better than 6% and 11% R.S.D., respectively, in both plasma and urine over the entire dynamic range. The pooled estimate of bias is less than 5% and attests to the excellent accuracy.     

Development and validation of SPMMTE HPLC method for analysis of profens from human plasma

A fast, selective and reproducible solid‐phase membrane microtip extraction (SPMMTE) HPLC method has been developed and validated for the analyses of ibuprofen, ketoprofen, and flurbiprofen from human plasma. The analysis was carried out on a C₁₈ (150 × 4.6 mm; 5 μm) column. The mobile phase used was water–acetonitrile (55:45, v/v) adjusted to pH 3.0 using trifluoroacetic acid, at a flow rate 0.5 mL/min with a detection wavelength of 225 nm. The values for the capacity factors for the profen samples ranged from 0.47 to 1.50. The values for the selectivity factor (α) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen combinations from human plasma samples were 1.99, 1.00 and 2.10, respectively. The resolution factors (Rs) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen from plasma samples were 3.00, 1.50 and 4.10, respectively. The percentage recoveries of ibuprofen, ketoprofen, and flurbiprofen from human plasma were 75–85%. All of the profens were separated within 7.0 min, indicating a relatively fast method. During the development of the SPMMTE procedure the parameters of pH, contact time, desorption and types of solvents were optimized. The final method was also found to be efficient, effective and inexpensive. Copyright © 2016 John Wiley & Sons, Ltd.     

Investigation of the metabolic fate of 2-, 3- and 4-bromobenzoic acids in bile-duct-cannulated rats by inductively coupled plasma mass spectrometry and high-performance liquid chromatography/inductively coupled plasma mass spectrometry/electrospray mass spectrometry

Inductively coupled plasma mass spectrometry (ICPMS) has been used to determine the rate and routes of excretion of bromine following the intraperitoneal administration (50 mg kg(-1)) of 2-, 3- and 4-bromobenzoic acids to male bile-duct-cannulated rats. Analysis of urine and bile for (79/81)Br using ICPMS showed that all three bromobenzoic acids were rapidly excreted (82-98%) within 48 h of dosing, primarily via the urine. High-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS) was then used to obtain metabolite profiles for bile and urine. These profiles revealed that extensive metabolism had taken place, with the unchanged bromobenzoic acids forming a minor part of the total of compound-related material detected. Concomitant MS studies, supplemented by alkaline hydrolysis, enabled the identification of the major metabolite of all three of the bromobenzoic acids as a glycine conjugate. Ester glucuronide conjugates were also identified, but formed only a small proportion of total.     

Simultaneous determination of tranilast and metabolites in plasma and urine using high-performance liquid chromatography

A method has been developed for the simultaneous determination of Tranilast, N-(3',4'-dimethoxycinnamoyl)anthranilic acid (N-5'), and metabolites in plasma and urine from humans, dogs and rodents administered N-5'. Total N-5' and metabolite N-3 conjugates were determined in human urine. Detection limits in plasma were 0.2 micrograms/ml for metabolite N-3-S and N-5' and 0.1 micrograms/ml for metabolites N-3 and N-4. In urine, detection limits were 2 micrograms/ml for metabolite N-3-S and N-5' and 1 micrograms/ml for metabolites N-3 and N-4. Metabolite N-4 was not identified in any sample assayed.     

A Validated Ion-Pairing High Performance Liquid Chromatographic Method for the Determination of Enoxacin and its Metabolite Oxo-Enoxacin in Plasma and Urine

Abstract

A rapid, sensitive, and specific determination of enoxacin and its principal metabolite, oxo-enoxacin, in plasma and urine is described. the method, which employs the structurally related compound ciprof loxac in as internal standard, involves a protein precipitation step for plasma and solid-phase extraction for urine. Liquid chromatographic analysis is carried out on a C-18 bonded silica column; the mobile phase consists of 0.1 M citric-acid/acetonitrile employing ammonium perchlorate and tetrabutyl-ammonium hydroxide as ion-pairing agents. Quantitation is performed by UV-detection at 340 nm.

The analytical method was validated by examining the performance characteristics specificity, linearity, precision, accuracy, sensitivity, and recovery. Enoxacin calibration curves were linear between 0.02 and 3.2 μg/ml of plasma and from 0.5 to 125 μg/ml of urine. Limits of quantitation in plasma and urine were 0.01 and 0.5 μg/ml, respectively. For oxo-enoxacin, linear of calibration curves were obtained i n the range 0.05 to 1.6 μg/ml (plasma) and 1 to 50 μg/ml (urine); the respective quantitation limits were approximately 0.02 and 1 μg/ml.

The present assay procedure has been applied to monitoring plasma and urine concentrations in several pharmacokinetic studies in humans and different animal species.     

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues. The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method. Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.     

Determination of risperidone and 9-hydroxyrisperidone in plasma, urine and animal tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of the new antipsychotic risperidone and its major metabolite 9-hydroxyrisperidone in plasma, urine and animal tissues. The alkalinized plasma samples were extracted with ethyl acetate and further purified prior to reversed-phase chromatography with ultraviolet detection at 280 nm. The method could also be applied to urine samples and animal tissue homogenates. Quantification limits were 2 ng/ml for plasma and urine and 10 ng/g for animal tissue. The method was applied to pharmacokinetic studies in experimental animals, human volunteers and patients.     

Pharmacokinetic study and the extraction efficiency of bulk and precipitated ibuprofen-imprinted polymer sorbents for gas chromatography and gas chromatography-mass spectrometry ibuprofen bio-analysis

Bulk and precipitation polymerization methods were used to prepare ibuprofen-molecularly imprinted polymers. Molecularly imprinted polymer-bulk and -precipitation were synthesized in acetonitrile, likewise molecularly imprinted polymer-bulk (mixture) and molecularly imprinted polymer-precipitation (mixture) in a mixture of acetonitrile/toluene (75:25 v/v). N₂ adsorption-desorption analysis data revealed that molecularly imprinted polymer-precipitation (mixture) has the highest specific surface area (200.74 m²/g). The surface chemistry and morphology of the synthesized sorbents were investigated by Fourier-transform infrared analysis and scanning electron microscope micrographs respectively. The prepared sorbents in the mixture of solvents were used in a dispersive solid-phase extraction process for selective extraction and pre-concentration of ibuprofen from urine and human plasma samples. The detection limits were 62.91 and 7.89 ng/ml using molecularly imprinted polymer-bulk (mixture) and molecularly imprinted polymer-precipitation (mixture), respectively. Also, the sorbents showed selective behavior to extract ibuprofen in the presence of naproxen, fenoprofen, and ketoprofen. Overall, the results showed that the precipitation method in the mixture of acetonitrile/toluene resulted in the preparation of a sorbent with the highest extraction efficiency. Furthermore, a pharmacokinetic study was done. The maximum plasma concentration, the time required for maximum plasma concentration, and plasma half-life were 28.95 μg/ml, 2, and 2.39 h, respectively.     

Biodetermination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan, a metabolite of vintriptol, by high-performance liquid chromatography with fluorescence detection.

The determination of N-(deacetyl-O-4-vinblastoyl-23)-L-tryptophan (vintriptol acid, VtrpA), a metabolite of the investigational semi-synthetic vinca alkaloid vintriptol [N-(deacetyl-O-4-vinblastoyl-23)-L-ethyltryptophan, VtrpE], in plasma and urine samples is described. Sample pretreatment included liquid-liquid extraction of the buffered (pH 5.0) biological samples with chloroform-2-propanol (95:5, v/v). The analyses were performed by ion-exchange high-performance liquid chromatography on normal-phase silica with fluorescence detection. The assay was applied to the analysis of samples from cancer patients who had been treated with VtrpE in a phase I clinical study. VtrpA was found to be a principal metabolite of VtrpE with up to 1.2% of the administered dose excreted in the urine.     

Determination of enalapril and its active metabolite enalaprilat in plasma and urine by gas chromatography/mass spectrometry.

The method for the simultaneous determination of angiotensin-converting enzyme (ACE) inhibitor enalapril and its active metabolite enalaprilat in plasma and urine was developed by gas chromatography/mass spectrometry. Enalapril and enalaprilat in plasma and urine were extracted and cleaned up by using Sep-Pak C18 and silica cartridges. Derivatization was carried out using diazomethane and trifluoroacetic anhydride. Detection by selected ion monitoring was selected to m/z 288 (enalaprilat) and 302 (enalapril). The detection limit of enalapril and enalaprilat was 200 pg/mL in plasma and 2 ng/mL in urine. This method was applied to the pharmacokinetic analysis of enalapril and enalaprilat in body fluids.     

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.