Quantitative liquid chromatographic determination of bromadoline and its N-demethylated metabolites in blood, plasma, serum, and urine samples

Bromadoline and its two N-demethylated metabolites were extracted into ether:butyl chloride after the addition of internal standard and basification of the various biological fluids (blood, plasma, serum, and urine). These compounds were then extracted into dilute phosphoric acid from the organic phase and separated on a reversed-phase chromatographic system using a mobile phase containing acetonitrile and a buffer of 1,4-dimethylpiperazine and perchloric acid. The overall absolute extraction recoveries of these compounds were approximately 50-80%. The background interferences from the biological fluids were negligible and allowed quantitative determination of bromadoline and the metabolites at levels as low as 2-5 ng/mL. At mobile phase flow rate of 1 mL/min, the sample components and the internal standard were eluted at the retention times within approximately 7-12 min. The drug- and metabolite-to-internal standard peak height ratios showed excellent linear relationships with their corresponding concentrations. The analytical method showed satisfactory within- and between-run assay precision and accuracy, and has been utilized in the simultaneous determination of bromadoline and its two N-demethylated metabolites in biological fluids collected from humans and from dogs after administration of bromadoline maleate.     

High-performance liquid chromatography assay for moxifloxacin: pharmacokinetics in human plasma

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of moxifloxacin in human plasma using fluorescence detection was developed. The drug and an internal standard (norfloxacin) were subjected to precolumn derivatization with 4-chloro-7-nitrobenzodioxazole (NBD-CI). The chromatographic separation was achieved by HPLC using a mixture of acetonitrile-10 mM orthophosphoric acid (pH 2.5) (80:20, v/v) as the mobile phase with isocratically system, a C18 column. The derivative is highly fluorescent at 537 nm, being excited at 464 nm. The linear and reproducible calibration curve over the range was 15-2700 ng/mL of moxifloxacin in human plasma. The limits of detection and quantitation were 6 and 15 ng/mL, respectively. This method was applied in pharmacokinetic studies moxifloxacin preparations in healthy volunteers.     

A rapid, sensitive assay for cocaine and its metabolites in biological fluids using solid-phase extraction and high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine and its metabolites, benzoylecgonine (BE), norcocaine, and ecgoninemethylester (EME), in rat plasma and urine is described. Following derivatization of EME to p-fluorococaine, chromatography was performed on two high-performance liquid chromatography (HPLC) columns in series (5-microm spheric C8 and 5-microm cyanopropyl) using a mobile phase containing acetonitrile/HPLC water/trifluoroacetic acid (28:72:0.1) with bupivacaine as an internal standard. Quantitation limits were 25 ng/mL for cocaine, BE, and norcocaine and 50 ng/mL for EME using 300-500 microL rat plasma and 500 microL of rat urine. The assay was linear from the limit of quantitation to 2000 ng/mL for cocaine and its metabolites in both plasma and urine samples. Because this method uses a small amount of sample (300 microL plasma or 500 microL of urine), it is applicable to study of the pharmacokinetics and disposition of cocaine and its major metabolites.     

Simultaneous determination of chlorpyrifos, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the separation and quantitation of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid-(3-phenoxyphenyl) methylester), and two of its metabolites, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, in rat plasma and urine. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and reversed-phase HPLC. The compounds were separated using a gradient of 1 to 80% acetonitrile in water (pH 3.2) at a flow rate ranging between 1 and 1.5 mL/min in a period of 17 min and gradient UV detection ranging between 210 and 280 nm. The retention times ranged from 9.3 to 14.5 min. The limits of detection ranged between 20 and 150 ng/mL, whereas the limits of quantitation were 150-200 ng/mL. The respective average percentage recoveries of chlorpyrifos, chlorpyrifos-oxon, TCP, permethrin, m-phenoxybenzyl alcohol, and m-phenoxybenzoic were 78.6 +/- 6.4, 72.8 +/- 6.8, 84.8 +/- 8.0, 79.2 +/- 8.4, 80.5 +/- 7.2, and 82.3 +/- 7.1 from five spiked plasma samples and 77.5 +/- 8.1, 72.8 +/- 8.3, 79.9 +/- 6.4, 79.1 +/- 8.9, 80.5 +/- 7.6, and 81.4 +/- 7.8 from urine samples. The relationship between peak areas and concentration was linear for concentrations between 200 and 2,000 ng/mL. This method was used to analyze these chemicals and metabolites following dermal administration in rats.     

Pharmacokinetic profile of glycyrrhizin in healthy volunteers by a new high-performance liquid chromatographic method.

An improved high-performance liquid chromatographic method was developed for the quantification of glycyrrhizin and its metabolites in human plasma. The improved method was selective and made it possible to determine precisely glycyrrhizin at levels as low as 500 ng/mL. The pharmacokinetic behavior of glycyrrhizin and its metabolites after oral and intravenous administration of glycyrrhizin to normal subjects was investigated. After oral administration of glycyrrhizin (100 mg) to three normal subjects, the major metabolite of glycyrrhizin (glycyrrhetic acid) appeared in plasma (less than 200 ng/mL), but glycyrrhizin was not found. On the other hand, glycyrrhizin was found in urine, and the amount excreted was 1.1-2.5% of the dose. This finding suggests that glycyrrhizin is partly absorbed in the intact form from the gastrointestinal tract. The concentration of glycyrrhizin in plasma after intravenous administration of glycyrrhizin (40, 80, and 120 mg) showed biexponential profiles during the 24-h period after administration of each dose. The glycyrrhizin metabolites, glycyrrhetic acid and glycyrrhetic acid-3-O-glucuronide, were not detected in either plasma or urine. The terminal half-life of glycyrrhizin, the apparent volume of the central compartment, the steady-state distribution volume, and the total body clearance in three dosing experiments were 2.7-4.8 h, 37-64 mL/kg, 59-98 mL/kg, and 16-25 mL/kg/h, respectively. Glycyrrhizin was not detected in plasma after oral administration of the usual therapeutic dose of glycyrrhizin, and no dose dependency of the drug was observed in the dose range of 40-120 mg.     

Extended application of an LC-MS/MS method for the analysis of vesnarinone and its metabolites in human urine and dialysate fluid

Vesnarinone, a positive inotropic drug developed for congestive heart failure, and its metabolites (OPC-8230, OPC-18136, OPC-18137) were analyzed in human dialysate and urine (plus an additional metabolite: OPC-18692 in urine) samples using a modification to a previously published LC-MS/MS assay for the analysis of human plasma and urine samples. OPC-8192, a structural analogue of vesnarinone, was used as the internal standard. The analytes of interest were extracted from human dialysate or urine by a solid phase extraction method using a pre-conditioned C-18 extraction column. The analytes were then resolved by a 7 min gradient elution on a reverse phase high performance liquid chromatographic column. Vesnarinone and metabolites were detected on a PE/Sciex API III+Biomolecular Mass analyzer in MS/MS mode using a Turbo IonSpray interface. The linear range of quantitation in dialysate was 2.00-100.00 ng/ml for vesnarinone and 0.50-25.00 ng/ml for each metabolite. In urine, the linear range was of 0.50-25.00 microg/ml for vesnarinone and 0.10-5.00 microg/ml for the metabolites. This method was used to support the analysis of urine and dialysate samples from renally impaired patients who are on vesnarinone treatment.     

Analytical methods for the determination of sulindac and metabolites in plasma, urine, bile, and gastric fluid by liquid chromatography using ultraviolet detection

A high-performance liquid chromatographic method using a linear elution gradient has been developed for the analysis of sulindac, sulindac sulfone, and sulindac sulfide in plasma, urine, bile, and gastric fluid. The methodology uses reverse-phase, radial compression chromatography with gradient elution, and UV detection. Sulindac and its metabolites in plasma can be quantitated at 0.25 microgram/mL with a mean CV of 6.0 +/- 2.9%; urine, bile, and gastric fluid (0.5 microgram/mL) yield a mean CV of 5.5 +/- 1.9%.     

An assay for methotrexate and its metabolites in serum and urine by ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic assay for methotrexate and its metabolites, 7-hydroxy-methotrexate, 4-amino-4-deoxy-N10-methyl-pteroic acid and 7-hydroxy-4-amino-4-deoxy-N10-methyl-pteroic acid in the range 10 microg/l to 50 mg/l (2.2 x 10(-8) to 1.1. x 10(-4)M) has been developed using L-tryptophyl-L-glutamic acid as internal standard. Extraction was performed using an anion exchange resin (Dowex 1-X2) with subsequent ion-pair chromatography of the appropriate eluent fraction. The method has been found to be sensitive and precise for the analysis of both serum and urine, and may also be used for the quantitation of polyglutamyl metabolites.     

Solid-phase extraction and liquid chromatography of torsemide and metabolites from plasma and urine

Torsemide is a new diuretic drug with a profile of action similar to that of furosemide. The high potency of torsemide results in low dose therapy and causes problems for the pharmacokinetic study of the drug due to low plasma levels. Described here are methods for the analysis of torsemide and two metabolites in plasma and urine using solid-phase extraction and liquid chromatography. The limits of quantitation are 10 ng/mL for plasma and 20 ng/mL for urine. The relative standard deviations for precision are less than 10% for most analytes at most concentrations in the calibration range. The recoveries from plasma were 94.3, 92.9, and 95.6%, and from urine were 77.5, 66.6, and 76.5% for torsemide and metabolites M1 and M5, respectively. The method was suitable for pharmacokinetic studies. Data from a normal volunteer are presented.     

Fluorimetric determination of norfloxacin in plasma and urine samples after thin-layer chromatographic separation

A thin-layer chromatographic (TLC) assay for the determination of norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7- (piperazinyl)-3-quinolinecarboxylic acid), a new antibacterial agent, in human plasma and urine is described. Norfloxacin was extracted from plasma samples using commercial cartridges, urine samples were simply diluted with methanol. Plasma extracts or urine samples were applied to TLC plates (silica gel). Chromatography was performed with a mobile phase consisting of methanol, ethanol and water, the atmosphere in the tank being saturated with ammonia. The fluorescence intensity of norfloxacin was enhanced by dipping the plate into a paraffin/citric acid mixture. Peaks were quantified by fluorimetric measurement (excitation 313 nm/emission 390 nm, cut-off filter). The method showed acceptable precision and linearity in the range of 0.01 to 4 micrograms/ml for plasma and 1 to 100 micrograms/ml for urine. The assay was shown to be applicable to human plasma and urine samples.     

Simultaneous high-performance liquid chromatographic determination of carbamazepine and its principal metabolites in human plasma and urine

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of carbamazepine and the 10,11-epoxide, 10,11-dihydroxy, and 2-hydroxy metabolites of carbamazepine. The chromatographic system involves the use of a 18C-microsorb, reversed-phase column with acetonitrile/water (28:72) as the mobile phase. Detection and quantitation are monitored by ultraviolet absorption at 212 nm. The compounds are extracted from 250 microliters of plasma or from 100 microliters urine with methyl-t-butyl ether and 0.1 M sodium hydroxide; 2-methylcarbamazepine is added as internal standard. If phenytoin and/or phenobarbital are present in plasma or urine samples, it is necessary to use 1.0 M sodium hydroxide. The limits of quantitation for carbamazepine and its metabolites are 10 ng/ml.     

A simple and rapid liquid chromatographic method for the determination of major metabolites of sulfasalazine in biological fluids

A simple and rapid assay for quantitation of sulfasalazine metabolites in rat urine and plasma was developed using high-performance liquid chromatography (HPLC). The method involves dilution of urine or plasma samples (0.1 mL) with methanol for protein precipitation, followed by mixing and centrifugation at 10,000 x g. Chromatography was accomplished with a reversed-phase ODS C-18 column (5 mu; 4.6 x 250 mm). The mobile phase consisted of 20% methanol in 5.0 mM phosphate buffer (pH 6.0), with 0.5 mM tetrabutylammonium chloride as an ion-pairing agent. The flow rate was 1.7 mL/min. An injection volume of 30 microL was used and the metabolites were quantitated by an ultraviolet detector at 254 nm. Benzamide was used as the internal standard. This method is linear in the range of 0.5 to 25 micrograms/mL for 5-aminosalicylic acid (5-ASA), acetylsulfapyridine (Ac-SP), and acetyl-5-aminosalicylic acid (Ac-5-ASA), and from 0.25 to 25 micrograms/mL for sulfapyridine (SP). The percent relative standard deviation ranged from 1 to 7.9% for the metabolite standard curves and precision studies. The limit of detection for 5-ASA, Ac-SP, and Ac-5-ASA is 100 ng/mL, and for SP is 50 ng/mL, in both urine and plasma. This method is rapid, precise, and accurate, and has been used to determine sulfasalazine metabolites in individual rat plasma and urine samples following an oral dose of 60 mg/kg of sulfasalazine.     

Determination of pivaloylcarnitine in human plasma and urine by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic (HPLC) method was developed for the determination of pivaloylcarnitine, one of the major metabolites of pivaloyloxymethyl (+)-(6R,7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2- pentenamido]-3-carbamoyloxymethyl-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2- ene-2-carboxylate hydrochloride hydrate (S-1108), an oral cephem antibiotic, in human plasma and urine. Fluorescence detection was done with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as the labeling reagent. Pivaloylcarnitine and cyclopropylacetylcarnitine, the internal standard, were selectively fractionated from plasma or urine on a disposable cation-exchange column. Derivatization was completed in 20 min at 40 degrees C in the presence of N,N'-diisopropylethylamine as the catalyst. A column-switching device was used to remove the excess reagent for HPLC analysis. The recovery of pivaloylcarnitine was greater than 98%, and average within-day and between-day coefficients of variation were less than 5% at concentration ranges of 0.05-2 micrograms/mL for plasma and 5-500 micrograms/mL for urine. Detection limits were 0.02 micrograms/mL for plasma and 1 micrograms/mL for urine. The urinary recovery of pivaloylcarnitine was 94% after the administration of S-1108, a result that suggested that S-1108 was almost quantitatively converted to pivalic acid and then conjugated with carnitine.     

Norfloxacin concentrations in seminal plasma measured by HPLC

Eighteen healthy volunteers aged 22 to 37 (average 30.44 +/- 4.11 years) seeking fertility evaluation at the Fertility and Sterility Centre of the Medical Clinic of the University of L'Aquila Medical School were studied for norfloxacin concentrations in serum, urine and seminal plasma 14-15 h after taking two 400 mg doses of the drug. The extraction of the biological fluids and the high pressure liquid chromatographic (HPLC) assay were performed following a method described previously (1), with seminal plasma samples processed in the same way as serum samples. The results in urine and serum substantially confirmed the figures already established for norfloxacin concentrations in such fluids. Seminal plasma levels (average 4.78 +/- 2.69 micrograms/ml) were about 5 times higher than the corresponding serum levels (average 0.89 +/- 0.59 microgram/ml). The authors believe that such concentrations, attaining a therapeutic range in the seminal plasma after only two 400 mg doses of norfloxacin, support its use as an effective chemotherapeutic agent in the management of male genital infections that frequently account for subfertility conditions in young patients.     

A sensitive analytical method for the determination of a novel lipoxygenase inhibitor, 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one, in plasma using reductive electrochemical detection

A reversed-phase high-performance liquid chromatographic assay using electrochemical detection in the reductive mode has been developed for the analysis of 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-one (1) in plasma to determine drug absorption. Free drug in plasma in concentrations as little as 0.25 ng/mL can be estimated with a mean coefficient of variation (CV) of 6.3 +/- 2.6%. Metabolites which can be converted to the parent drug by acid hydrolysis can be quantified in concentrations of 10 ng/mL or more, with a mean CV of 4.3 +/- 1.9%. To test the procedure, plasma was obtained from dogs receiving 14C-labeled 1. After acid hydrolysis of plasma, the electrochemical assay for parent drug showed good agreement with the radioactive equivalents in plasma, suggesting that parent drug and metabolites can be satisfactorily analyzed by this procedure.     

High-performance liquid chromatographic assay of the aldose reductase inhibitor spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567) in plasma and urine and its pharmacokinetics in humans

Two selective high-performance liquid chromatographic (HPLC) methods have been developed for the quantitative determination of spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567; 1) in plasma and urine, with an assay sensitivity of 0.25 micrograms/mL for plasma and 0.13 micrograms/mL for urine. The plasma assay procedure involved precipitation of proteins with acetonitrile followed by dilution with water. The diluted supernatant was analyzed on an ODS column eluting with acetonitrile:0.5% phosphoric acid (30:70) adjusted to pH 7.2 with concentrated ammonium hydroxide. The urine assay procedure involved extraction of 1 with 10% n-butanol in hexane, followed by back extraction with 0.05 M sodium hydroxide. The basic extract was neutralized and analyzed on a phenyl column eluting with acetonitrile:10 mM potassium phosphate (30:70; monobasic, pH 5.6). The pharmacokinetics of 1 was investigated in humans following single and multiple oral doses. The elimination half-life from 12 normal subjects following single 100-400-mg oral doses was independent of dose, and the overall mean half-life was 66 +/- 9 h. The overall mean oral clearance (assuming a bioavailability of 100%) was 11 +/- 3 mL/min, and the mean apparent volume of distribution was 59 +/- 13 L. The mean urinary recovery of intact drug during the first 24 h after dosing was 1.2 +/- 0.4% of the administered dose. During once daily 100-mg oral dosing of 1 to five subjects for 21 d, plasma concentrations of 1 reached apparent steady-state by 7 d.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of modafinil in plasma and urine by reversed phase high-performance liquid-chromatography

Modafinil (Provigil) is a new wake-promoting drug that is being used for the management of excessive sleepiness in patients with narcolepsy. It has pharmacological properties similar to that of amphetamine, but without some of the side effects associated with amphetamine-like stimulants. Since modafinil has the potential to be abused, accurate drug-screening methods are needed for its analysis. In this study, we developed a high-performance liquid-chromatographic procedure (HPLC) for the quantitative analysis of modafinil in plasma and urine. (Phenylthio)acetic acid was used as an internal standard for the analysis of both plasma and urine. Modafinil was extracted from urine and plasma with ethyl acetate and ethyl acetate-acetic acid (100:1, v/v), respectively, and analyzed on a C18 reverse phase column with methanol-water-acetic acid (500:500:1, v/v) as the mobile phase. Recoveries from urine and plasma were 80.0 and 98.9%, respectively and the limit of quantitation was 0.1 microg/mL at 233 nm. Forty-eight 2-h post-dose urine samples from sham controls and from individuals taking 200 or 400 mg of modafinil were analyzed without knowledge of drug administration. All 16-placebo urine samples and all 32 2-h post-dose urine samples were correctly classified. The analytical procedure is accurate and reproducible and can be used for therapeutic drug monitoring, pharmacokinetic studies, and drug abuse screening.     

Determination of 3-quinuclidinyl benzilate (QNB) and its major metabolites in urine by isotope dilution gas chromatography/mass spectrometry.

In response to the scheduled destruction of U.S. military stockpiles of the hallucinogenic agent 3-quinuclidinyl benzilate (QNB), a specific confirmatory test for human exposure to QNB was developed. The amount of the parent compound in the urine as well as the two major metabolites, 3-quinuclidinol (Q) and benzilic acid (BA), was determined because the relationship between QNB dose and levels of QNB and its metabolites in human urine is not known. QNB was determined in urine samples spiked at a target level of 0.5 ng/mL, and the metabolites BA and Q were determined at a target level of 5 ng/mL. The method uses solid-phase extraction to isolate each analyte from the urine and isotope dilution gas chromatography/mass spectrometry for quantitation. Each analyte is converted to its trimethylsilyl derivative for analysis. The analytical method was tested on eight different urine samples spiked with known amounts of the analytes near the target levels, at 10 times the target levels, and blank (unspiked) urine samples. The variabilities in the method are for the most part evenly distributed between three imprecision categories: GC/MS measurement, sample preparation, and the urine samples. The total imprecision (1 standard deviation) of a single measurement is about 15% of the value for each analyte.     

A sensitive liquid chromatographic procedure for the analysis of camphor in equine urine and plasma

A sensitive method was required to analyze low levels of camphor in equine urine and plasma. Camphorated oil (20% w/w camphor) was administered topically (6 g) and intratracheally (1 g) to standardbred mares. The drug was extracted from urine and plasma by diethyl ether and analyzed as its 2,4-dinitrophenylhydrazone derivative by reverse phase HPLC with UV detection. The UV detector was set at 368.5 nm and the samples were eluted from the C18 column by 82% acetonitrile in water. The detection limit achieved was about 10 ng/mL urine and about 20 ng/mL plasma. After topical administration, only trace amounts of camphor were found in urine, whereas in plasma detectable quantities appeared from 20 min to 12 hrs post-administration. After intratracheal administration, camphor appeared immediately in both urine and plasma and occurred in higher concentrations. The concentrations of camphor ranged from 11.4 to 21.6 ng/mL urine during 2 hrs post-administration and from 46.7 to 1650 ng/mL plasma during 1 hr. All traces of camphor disappeared from urine after 4 hrs and from plasma after 12 hrs. A GC/MS analysis of hydrolyzed urine extracts indicated two metabolites of camphor: trans-isoketopinic acid and an as yet uncharacterized hydroxycamphor.