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.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

High-performance liquid chromatographic determination of cefonicid in human plasma and urine

A high-performance liquid chromatographic assay for determination of cefonicid concentrations in human plasma and urine samples has been developed using cefazolin as an internal standard. For the analysis of plasma samples two calibration curves were utilized covering the cefonicid concentration ranges of 0.05-1.0 microgram/ml and 1.0-50.0 micrograms/ml, respectively. Coefficients of variation of 7.4% or less were obtained for cefonicid concentrations of 0.05-50.0 micrograms/ml. Mean bias was +6.0% at 0.05 micrograms/ml cefonicid and between -2.1% and +1.6% for 1.0-50.0 micrograms/ml cefonicid. Plasma samples containing 30 ng/ml cefonicid could be well distinguished from blank plasma samples. Urine samples were analysed by using a calibration curve for cefonicid concentrations between 1.0 and 50.0 micrograms/ml. ranged from 8.6% at a cefonicid concentration of 1.0 microgram/ml to 0.5% at 50.0 micrograms/ml with a mean bias between -3.0% and +0.3%.     

Analysis of a new H2 receptor antagonist, 3-amino-5-[3-[4-(1-piperidinoindanyloxy)]propylamino]-1-methyl-1 H-1,2,4-triazole, in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of a new H2 receptor antagonist, 3-amino-5-[3-[4-(piperidinoindanyloxy)]propylamino] -1-methyl-1H-1,2,4-triazole (I), in human plasma and urine was developed. The method employs liquid-liquid extraction of the analyte and an internal standard and chromatographic separation using an alkylphenyl-bonded HPLC column. The total time of chromatography was less than 10 min. Sensitivity was 10 ng/ml for the plasma analysis and 1 microgram/ml for the analysis of I from urine. The coefficients of variation, based on interpolated concentrations, were less than 10%. The method was used for more than 5000 samples during clinical pharmacokinetic studies.     

A rapid gas chromatographic method for the determination of chlorophenoxyisobutyric acid in plasma and urine.

A rapid gas chromatographic method is described for the determination of chlorophenoxyisobutyric acid (the active metabolite of clofibrate) in plasma and urine. The assay involves an extraction into toluene and back-extraction of the chlorophenoxyisobutyric acid and the internal standard (2-naphthoic acid) into the methylating reagent (trimethylanilinium hydroxide). Concentrations of 1 mug/ml in plasma and urine can easily be measured; the precision of the method is 3.3 +/- 0.7% for plasma and 2.7 +/- 0.4% for urine. There is no interference from endogenous compounds or from drugs commonly prescribed together with clofibrate.     

High-performance liquid chromatographic assay for dilevalol in human plasma and urine using a PRP-1 column and fluorimetric detection

A single high-performance liquid chromatographic (HPLC) assay for the quantitative determination of dilevalol, the R,R isomer of labetalol, was developed for both plasma and urine. A significantly improved limit of detection for dilevalol in plasma was accomplished by extensive modification of an HPLC assay originally developed in our laboratory for labetalol. This simplified method is readily adaptable to urine and represents the first reported HPLC assay for the quantitative determination of dilevalol in this biofluid. Drug was recovered from plasma or urine by partition into diethyl ether under mildly alkaline conditions and back-extraction into dilute acid. Reversed-phase separation of dilevalol and the internal standard was accomplished on a 150 X 4.1 mm column commercially packed with a spherical (5 micron) macroporous copolymer (PRP-1). No interferences were observed in extracts obtained from drug-free plasma or urine. Selectivity for dilevalol in the presence of other beta-blockers was established. This method demonstrated a linear detector response to concentrations of unchanged drug typically observed in urine and plasma following once-a-day treatment with dilevalol hydrochloride (100-800 mg). The lowest limit of reliable quantitation was established at 1 ng/ml in plasma. The intra-assay precision (coefficient of variation) remained less than 6% at all concentrations evaluated from 1 to 800 ng/ml. In urine, the lowest limit of quantitation was validated to 20 ng/ml where the intra-assay precision (coefficient of variation) for unchanged drug was less than 4% at all concentrations evaluated up to 400 ng/ml. This method is suitable for routine quantitation of unchanged drug in human plasma and urine following the administration of therapeutically effective doses of dilevalol hydrochloride.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Determination of an irreversible inhibitor of monoamine oxidase B (MDL 72974A) in human plasma and urine by gas chromatography-positive-ion chemical ionization mass spectrometry.

A sensitive and specific assay has been developed for the quantitative measurement in human plasma and urine of the irreversible inhibitor of monoamine oxidase B [(E)-4-fluoro-beta-fluoromethylenebenzene-butanamine HCl salt] (MDL 72974A) (I). This assay is based on gas chromatography-mass spectrometry with ammonia as the chemical ionization reagent gas. After addition of 1-fluoro-2-(4-chlorobenzene)-ethanamine HCl salt (MDL 71946A) as the internal standard, plasma (1 ml) and urine (100 microliter) samples were extracted using an automated solid-liquid extraction procedure on CN columns. The eluent was dried with a stream of nitrogen, and the residue was derivatized with pentafluoropropionic anhydride. Selected-ion monitoring of the [MNH4]+ ions m/z 361 (I) and 351 (internal standard) was used for quantification. The method yielded a linear response over the concentration range 0.25-100 pmol/ml in plasma with a limit of quantitation of 0.25 pmol/ml. The within-day reproducibility at a concentration of 5 pmol/ml was 4.6% and at a concentration of 50 pmol/ml was 1.3%. The day-to-day reproducibility was 5.2 and 7.0% at concentrations of 10 and 30 pmol/ml, respectively. The method was applied to the quantification of I in plasma and urine after the administration of 12-mg doses of I to a healthy male volunteer.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

Simultaneous quantitation of phenobarbitone and p-hydroxyphenobarbitone and their perdeuteroethyl analogues in biological fluids by combined gas chromatography-mass spectrometry

In order to develop 5-pentadeuteroethyl-5-phenyl barbituric acid as an alternative tracer in pharmacokinetic and metabolic studies of phenobarbitone, and to search for possible isotope effects associated with such labelling, we propose a gas chromatographic-mass spectrometric assay for simultaneous measurement of phenobarbitone, p-hydroxyphenobarbitone and their perdeuteroethyl analogues, using [1,3-15N2,2-13C] phenobarbitone as internal standard. These compounds were extracted from plasma (50 microliter) or urine (500 microliter) and pentylated according to Greeley's method. Linear calibration curves were obtained in the concentration range from 0.5 to 3 micrograms/ml. The interday precision, mean accuracy and detection limit were 0.77-5.28%, 99.99-100.80% and 0.03-0.05 microgram/ml, respectively. Results for plasma and urine concentrations, and pharmacokinetic parameters in humans, are presented to illustrate this method.     

Determination of the anti-allergenic agent, 2-methoxy-11-oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, in biological fluids by high-performance liquid chromatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) assay was developed for the determination of 2-methoxy-11-oxo-11H-pyrido-[2,1-b]quinazoline-8-carboxylic acid (I) from biological fluids. The overall recovery from blood and plasma is 69 +/- 10% (S.D.) and 84 +/- 6% (S.D.), respectively, and the sensitivity limit of quantitation is 100 ng/ml by UV absorption and 5 ng/ml by fluorescence detection using a 1 ml specimen. The assay was used in the determination of blood levels of compound in the Rhesus monkey following intravenous administration of a 10 mg/kg dose, and of blood and urine levels of compound I in a dog following intravenous and oral administration of a 1 mg/kg dose.     

High-performance liquid chromatographic determination of a new beta-lactamase inhibitor and its metabolite in combination therapy with piperacillin in biological materials

[2S-(2 alpha,3 beta,5 alpha)]-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-yl- methyl)-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid 4,4-dioxide (YTR-830H) is a new beta-lactamase inhibitor and the combination therapy of this compound with piperacillin is now under study. For the determination of the beta-lactamase inhibitor and piperacillin in biological materials, plasma and visceral tissue homogenates were deproteinized, whereas diluted urine and filtered faeces homogenates were treated with a Sep-Pak C18 cartridge. In order to assay the inactive metabolite of beta-lactamase inhibitor, each sample was treated with a Sep-Pak C18 cartridge. Aliquots of each preparation were chromatographed using ion-pair and reversed-phase chromatographic techniques on a high-performance liquid chromatograph equipped with a UV detector, set at 220 nm. The detection limits of beta-lactamase inhibitor and piperacillin were 0.2 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 0.2-0.5 microgram/g in visceral tissue and faeces. Those of the metabolite were 1.0 microgram/ml in plasma, 2.5-5.0 micrograms/ml in urine and 1.0 microgram/g in visceral tissue and faeces. A precise and sensitive assay for the determination of the beta-lactamase inhibitor, its metabolite and piperacillin is described, and their stabilities in several media are reported.     

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

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

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

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.     

Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Method for the biological monitoring of hexahydrophthalic anhydride by the determination of hexahydrophthalic acid in urine using gas chromatography and selected-ion monitoring.

A method for the determination of hexahydrophthalic acid, a metabolite of hexahydrophthalic anhydride, in human urine has been developed. The urine was worked-up by liquid-solid extraction, esterified with boron trifluoride-methanol, and analysed by capillary gas chromatography and selected-ion monitoring. Hexadeuterium-labelled hexahydrophthalic acid was used as the internal standard. The precision was 4% at 0.7 microgram/ml and 5% at 0.07 microgram/ml. The recovery of the acid for the overall method was 101% at 0.07 micrograms/ml of urine (with a coefficient of variation of 4%) and 95% at 0.7 microgram/ml (coefficient of variation 2%). The limit of detection was 20 ng/ml urine.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H- pyrido [2,1-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.