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.     

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.     

Supercritical fluid extraction of the fluoroquinolones norfloxacin and ofloxacin from orally treated-chicken breast muscles

Supercritical fluid extraction (SFE) of the fluoroquinolones norfloxacin and ofloxacin from chicken breast muscles was examined. A liquid chromatography with fluorescence detection was used for the determination of the fluoroquinolones. Extraction conditions of the SFE were optimized by determining the extraction parameters to achieve a sufficiently high recovery of each fluoroquinolone in fortified-muscle samples. Recovery values for the extraction of the fluoroquinolones using the SFE ranged from 70 to 87%. Chickens were treated orally with each fluoroquinolone and their muscles were extracted at set time intervals for time-course determination of the fluoroquinolones in chickens. The SFE combined with liquid chromatographic analysis showed that the concentrations of the fluoroquinolones decreased gradually with time in the chicken muscles after oral treatment, giving a concentration less than 5 ng/ml in 120 h. No further sample cleanup procedures were required after the SFE. These results suggest that SFE method is an extraction method for the determination of norfloxacin and ofloxacin in chicken muscle.     

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (μLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 μl injection volumes with on-column focusing. A 350×0.32 mm capillary column packed with Kromasil 100-C₁₈ of 5 μm particle size was used with a mobile phase of acetonitrile–10 mM ammonium acetate (85:15, v/v) at a flow of 5 μl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90°C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile–water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 μm poly(vinylidene difluoride) syringe filter before μLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 μg/kg. The within-day and between-day repeatabilities were in the range 4–18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.     

Determination of mefloquine in blood, filter paper-absorbed blood and urine by 9-fluorenylmethyl chloroformate derivatization followed by liquid chromatography with fluorescence detection

We describe a method for determination of mefloquine (MQ) in 100-microliters samples of urine, whole blood, and capillary blood collected on filter paper; quantification is by liquid chromatography with fluorescence detection at 475 nm of the 9-fluorenylmethyleneoxycarbonyl derivative. Whole blood and urine samples were prepared by extraction of MQ and internal standard from aqueous base with methyl tert.-butyl ether (MTBE), separation and evaporation of the MTBE layer, and derivatization using a solution of 9-fluorenylmethyl chloroformate in acetonitrile. Filter paper spots were immersed for 16 h in 0.1 M hydrochloric acid, followed by extraction with MTBE from aqueous sodium carbonate. The separated and evaporated organic layer was treated with the derivatizing solution. An aliquot was injected onto a high-performance liquid chromatography system using a C18 reversed-phase column and acetonitrile-water (72:28) mobile phase for filter paper spot extracts as for whole blood and urine extracts. The method has a limit of determination in blood, blood spots, and urine of 50 ng/ml with 100 microliters sample size (coefficient of variation = 16%). Linearity and precision (within-day and between-day) for the method are good. The MQ derivative was isolated and characterized spectroscopically. Values for MQ concentrations in filter paper blood spots compared favorably with values found in corresponding whole blood samples analyzed by a published method.     

Liquid Chromatographic Determination of 4-Amino-N-(2,6-dimethylphenyl)-benzamide in Rat Serum and Urine

Abstract

The compound 4-amino-N-(2,6-dimethylphenyl)-benzamide has shown potential as a new anticonvulsant. A method for the liquid chromatographic determination of serum and urine concentrations of the compound and its N-acetylated metabolite was developed for pharmacokinetic studies. Quantitation was achieved via UV detection at 275 nm following isocratic reversed phase (C₁₈) separation using a ternary solvent system of water:acetonitrile:acetic acid (60:39:1) at a flow rate of 1.5 mL/min. The compounds were isolated from a 50 μL sample of serum using solid phase extraction with prior protein precipitation. The compounds and internal standard were eluted from the extraction column with acetonitrile. Isolation from urine was achieved similarly with the exclusion of protein precipitation. The assay procedure is useful for the determination of concentrations of parent compound from 0.68 to 204.6 μg/mL.     

The determination of 2-hydroxynicotinic acid and its major metabolite in blood and urine by spectro-photofluorimetry and differential pulse polarography

A sensitive and specific spectrofluorimetric assay was developed for the determination of 2-hydroxynicotinic acid (1) and its major metabolite, the N-1-riboside (11) in blood and urine. Both compounds exhibit strong fluorescence in acidic media. Thin-layer chromatography is employed to separate the drug from its metabolite; the compounds are eluted from the silica gel into methanol—0.1 M hydrochloric acid (80+20). The sensitivity is 0.04–0.05 μg of I and 0.10–0.12 μg of II per ml of blood or urine. The two compounds can also be determined by differential pulse polarography, which is especially suitable for urine. The fluorimetric method was applied to the determination of blood levels and the urinary excretion of the drug in man after single oral 500-mg doses.     

Supercritical fluid extraction combined with solid phase extraction as sample preparation technique for the analysis of β-blockers in serum and urine

A method is developed for the determination of β-blockers in serum and urine at levels of 0.5 μg/mL. The technique uses a combination of solid phase extraction (SPE) with in situ derivatization and supercritical fluid extraction (SFE) with subsequent gas chromatography mass spectrometry. The optimization of the SFE step shows that a static extraction period can be eliminated. The method gives good linearity (r = 0.991–0.999) and repeatability in the concentration range of 0.5 to 5 μg/mL. Relative standard deviations for oxprenolol, propanolol and metoprolol were less than 5% in serum and 5–11% in urine.     

Simultaneous determination of phenolphthalein and phenolphthalein glucuronide from dog serum, urine and bile by high-performance liquid chromatography.

A procedure is described to simultaneously quantitate phenolphthalein and its glucuronide metabolite from dog serum, urine and bile using high-performance liquid chromatography. The major advantages of this over pre-existing methods include direct analysis of the parent compound and glucuronide metabolite without enzymatic hydrolysis, increased sensitivity and the potential for automation of a large number of samples. Analytes were extracted from serum and urine using a combination of liquid- and solid-phase extraction methodology. Bile samples were analyzed directly after a twenty-fold dilution with mobile phase. The components plus internal standard were separated by reversed-phase high-performance liquid chromatography using step gradient elution and quantitated by the absorbance of ultraviolet light at 230 nm. Limits of detection from 1 ml of serum, 0.1 ml of urine and 0.05 ml of bile were 0.1, 0.5 and 10 microgram/ml for phenolphthalein and 0.1, 10 and 50 microgram/ml for phenolphthalein glucuronide, respectively.     

A high-throughput liquid chromatography/tandem mass spectrometry method for simultaneous quantification of a hydrophobic drug candidate and its hydrophilic metabolite in human urine with a fully automated liquid/liquid extraction

ABT-869 (A-741439) is an investigational new drug candidate under development by Abbott Laboratories. ABT-869 is hydrophobic, but is oxidized in the body to A-849529, a hydrophilic metabolite that includes both carboxyl and amino groups. Poor solubility of ABT-869 in aqueous matrix causes simultaneous analysis of both ABT-869 and its metabolite within the same extraction and injection to be extremely difficult in human urine. In this paper, a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method has been developed and validated for high-speed simultaneous quantitation of the hydrophobic ABT-869 and its hydrophilic metabolite, A-849529, in human urine. The deuterated internal standards, A-741439D(4) and A-849529D(4), were used in this method. The disparate properties of the two analytes were mediated by treating samples with acetonitrile, adjusting pH with an extraction buffer, and optimizing the extraction solvent and mobile phase composition. For a 100 microL urine sample volume, the lower limit of quantitation was approximately 1 ng/mL for both ABT-869 and A-849529. The calibration curve was linear from 1.09 to 595.13 ng/mL for ABT-869, and 1.10 to 600.48 ng/mL for A-849529 (r2 > 0.9975 for both ABT-869 and A-849529). Because the method employs simultaneous quantification, high throughput is achieved despite the presence of both a hydrophobic analyte and its hydrophilic metabolite in human urine.     

Molecularly imprinted polymer for selective extraction of 3-methylflavone-8-carboxylic acid from human urine followed by its determination using zwitterionic hydrophilic interaction liquid chromatography

A novel water-compatible molecularly imprinted SPE combined with zwitterionic hydrophilic interaction liquid chromatography method for selective extraction and determination of 3-methylflavone-8-carboxylic acid (MFA), the main active metabolite of flavoxate in human urine, was developed and validated. The effects of progenic solvents, pH, cross linker and amount of monomer were studied to optimize the efficiency and selectivity. The molecularly imprinted polymer showed good specific adsorption capacity with an optimum of 200 μmol/g at pH 7.5 and selective extraction of MFA from human urine. The recovery of MFA from human urine was >98%. The lower limit of quantification was 1.20 μg/mL. The proposed method overcomes the matrix effects of endogenous substances generally encountered during direct analysis of urine sample.     

Screening of Benzoylecgonine in Urine by Cyclobond Solid Phase Extraction and High Performance TLC

Abstract

A method is described for screening of the cocaine metabolite benzoylecgonine in urine at 0.2 μ/ml using a Cyclobond cyclodex^?trin solid phase extraction cartridge and high performance thin layer chromatography. Results are compared with those obtained using liquid-liquid and diatomaceous earth column extraction and C-18 solid phase extraction.     

Determination of some β‐Blockers by Electrochemical Detection on Polycrstalline Gold Electrode after Solid Phase Extraction (SPE)

The electroanalytical characterization and determination of three selected β‐blocker agents, namely propranolol, atenolol and nadolol using cyclic voltammetry and differential pulse voltammetry (DPV) in phosphate buffer solution (pH 2.5) plus 22 % acetonitrile (ACN), was described. The analytes were characterized through their electrooxidation processes on polycrystalline gold electrodes. The analytical determination of the selected molecules was performed using the differential pulse voltammetry (DPV) at pH 2.5. Under DPV conditions, the detection limits (LODs) ranged between 5 μM and 20 μM for propranolol and atenolol, respectively. For all investigated molecules, two well‐defined ranges of linearity Ip vs analyte concentration have been identified which correspond to specific calibration parameters. Calibration graphs (Ip vs concentration) considered in the first interval of linearity, shown correlation coefficients >0.99. A solid phase extraction (SPE) procedure using a polymeric mixed‐mode cationic sorbent (Strata‐X‐C), was studied and optimized. The proposed DPV‐SPE method was successfully applied for the determination of propranolol in several pharmaceutical formulations and urine sample, with results in close agreement with those obtained using traditional liquid chromatography technique coupled with spectrophotometric detection.     

Developing LC–MS/MS methods to quantify rivaroxabanin human plasma and urine: Application to therapeutic drug monitoring

Rivaroxaban is an oral anticoagulant directly inhibiting the activity of Factor Xa, which is widely used for the prophylaxis of thromboembolic disorders. Therapeutic drug monitoring (TDM) is required during therapy for individual dosage adjustment. This study aimed at developing a liquid chromatography/tandem mass spectrometry method that was suitable for rivaroxaban TDM in human plasma and urine and exploring the feasibility of urine drug monitoring in medical care. A 3 min run time of the LC–MS/MS methods was established by employing an Acquity UPLC BEH C₁₈ (2.1 × 50 mm, 1.7 μm) column using gradient elution of 10 mmol/L ammonium acetate containing 0.1% formic acid–0.1% formic acid acetonitrile as a mobile phase at a flow rate of 0.4 ml/min with calibration ranges of 0.5–400 and 10–10,000 ng/ml for human plasma and urine, respectively. Rivaroxaban was detected on a triple quadrupole tandem mass spectrometer with an electrospray ionization source in positive ion mode. The methods showed good linearity within the calibration range. The precision and accuracy, matrix effect, extraction recovery and stability in both human matrices were all validated and meet the international guideline requirements. These validated methods were successfully applied to support the TDM of an aged patient receiving rivaroxaban for therapy.     

Determination of pyrethrin and pyrethroid pesticides in urine and water matrixes by liquid chromatography with diode array detection

The following pyrethrin and pyrethroid pesticides were determined in urine and water matrixes by liquid chromatography with diode array detection (LC-DAD): pyrethrin I, pyrethrin II, tetramethrin, baythroid, bifenthrin, fenvalerate, phenothrin, allethrin, resmethrin, cis-permethrin, and trans-permethrin. In addition, 3-phenoxybenzyl alcohol, a metabolite of various pyrethroids, was also successfully determined by the analytical method. The matrix extraction was simple, inexpensive, and fast, using only sodium chloride and acetonitrile. The acetonitrile extract was filtered and analyzed by LC-DAD. The method detection limits for the pyrethrin pesticides in 5 mL urine were determined to range from 0.002 to 0.04 microg/mL, depending on the individual pyrethrin. Recoveries from spiked tap water ranged from 77 to 96%; recoveries from urine ranged from 80 to 117%. This method is especially well-suited to clinical investigations, in which rapid analysis of forensic samples is often required.     

Rapid and Simple Method to Determine Tamoxifen and Its Major Metabolites in Human Liver Microsomes by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

Sensitive and reproducible analyses were developed for assaying tamoxifen, desmethyltamoxifen, didesmethyltamoxifen and hydroxytamoxifen in human liver microsomes by high performance liquid chromatography (H.P.L.C.) following protein precipitation. Detection was by fluorimetry of phenanthrene derivatives formed by on-stream U.V. irradiation with a photochemical reactor enhanced detection (P.H.R.E.D.) for post-column irradiation of the H.P.L.C. stream. The method is highly sensitive (1 ng/ml). A C8,5 μm column (4.6 x 150 mm) was used, with a mobile phase of acetonitrile / 100 mM ammonium acetate (1:1) pH 5. Precipitation of the microsomal protein was carried out by adding methanol and concentrated chloric acid to the reaction mixture, and extraction efficiencies were approximately 100 % for tamoxifen and its metabolites. The assay has been optimised for the identification and quantitation of the 4-hydroxylated metabolite, as well as the two metabolites formed by N-oxidative demethylation of the side chain. The latter catalytic activity involves cytochrome P450 3A enzymes.     

Comparison of supercritical fluid and Soxhlet extractions for the isolation of nitro compounds from soils

Supercritical fluid extraction (SFE) with CO(2), a clean and rapid alternative to conventional Soxhlet extraction, was investigated for the extraction of nitro compounds from soil samples. Quantitative extraction by SFE was accomplished at a pressure of 25 MPa and an extraction temperature of 60 degrees C, for 30 min in dynamic mode and using acetonitrile as modifier, and the results were comparable with those obtained by acetonitrile Soxhlet extraction (3 h) for all soil samples. Extracts from these two procedures were analyzed by gas chromatography coupled with mass spectrometry. Quantitative reproducibility for SFE extracts was acceptable (RSD 2-10%), and the quantity of solvent was reduced from 160 mL for Soxhlet extraction to 5 mL in the case of SFE.     

Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection.

Several procedures using high-performance liquid chromatography with photodiode-array detection have been developed to create phytochemical and toxicological profiles of phenylalkylamine derivatives in biological samples (e.g. plant materials and urine). Mescaline-containing cactus samples were extracted with basic methanol, using methoxamine as internal standard; the extraction and clean-up of urine samples were performed on cation-exchange solid-phase extraction columns. The extracts were separated on a 3-micron ODS column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was performed at 198 or 205 nm; peak identity and homogeneity were ascertained by on-line scanning of the UV spectra from 190 to 300 nm. The detection limit of phenylalkylamine derivatives in urine and cactus material was 0.026-0.056 micrograms/ml and 0.04 micrograms/mg, respectively. Following a single oral dose of 1.7 mg/kg methylenedioxymethylamphetamine (MDMA) the concentrations found in urine ranged from 1.48 to 5.05 micrograms/ml MDMA and 0.07-0.90 micrograms/ml methylenedioxyamphetamine (a metabolite of MDMA). The mescaline content of the cactus Trichocereus pachanoi varied between 1.09 and 23.75 micrograms/mg.     

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.     

Determination of a novel nonfluorinated quinolone,nemonoxacin, in human feces and its glucuronide conjugate in human urine and feces by high‐performance liquid chromatography–triple quadrupole mass spectrometry

Three methods were developed and validated for determination of nemonoxacin in human feces and its major metabolite, nemonoxacin acyl‐β‐ d ‐glucuronide, in human urine and feces. Nemonoxacin was extracted by liquid–liquid extraction in feces homogenate samples and nemonoxacin acyl‐β‐ d ‐glucuronide by a solid‐phase extraction procedure for pretreatment of both urine and feces homogenate sample. Separation was performed on a C₁₈ reversed‐phase column under isocratic elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. Both analytes were determined by liquid chromatography–tandem mass spectrometry with positive electrospray ionization in selected reaction monitoring mode and gatifloxacin as the internal standard. The lower limit of quantitation (LLOQ) of nemonoxacin in feces was 0.12 µg/g and the calibration curve was linear in the concentration range of 0.12–48.00 µg/g. The LLOQ of the metabolite was 0.0010 µg/mL and 0.03 µg/g in urine and feces matrices, while the linear range was 0.0010–0.2000 µg/mL and 0.03–3.00 µg/g, respectively. Validation included selectivity, accuracy, precision, linearity, recovery, matrix effect, carryover, dilution integrity and stability, indicating that the methods can quantify the corresponding analytes with excellent reliability. The validated methods were successfully applied to an absolute bioavailability clinical study of nemonoxacin malate capsule. Copyright © 2014 John Wiley & Sons, Ltd.