High-performance liquid chromatographic analysis of amiloride in plasma and urine

A high-performance liquid chromatographic method has been developed for amiloride in rabbit plasma and urine which uses a reversed-phase C18 column, a mobile phase (flow-rate 2 ml/min) consisting of 32% acetonitrile in 0.15 M perchloric acid, pH 2.2, and spectrofluorometric detection via excitation at 286 nm. A simple extraction step with ethyl acetate eliminates interfering peaks. Short retention times of about 2.3 and 3.8 min are observed for amiloride and the internal standard, triamterene, respectively. The method can measure 4 ng/ml amiloride in plasma. This assay has been used to explore the pharmacokinetics of amiloride in rabbits. The plasma disposition profile is biexponential after a 50-mg intravenous bolus dose and there is no evidence for saturable elimination at zero-order infusion rates of 1.8, 3.6 and 7.2 mg/h.     

High-performance liquid chromatographic method for the determination of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL-39123) in human plasma and urine

A rapid, sensitive and reliable reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection has been developed for the assay of a novel anti-herpes agent, 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL-39123), in human plasma and urine. The drug and the internal standard, the structural analogue BRL-42377, were extracted from the biological matrix by adsorption on a cation-exchange column and were subsequently eluted under alkaline conditions prior to HPLC. The method is reproducible, with coefficients of variation of ca. 5%, and linear from 0.1 to at least 30 micrograms ml-1 in plasma and from 50 to at least 2000 micrograms ml-1 in urine. The method has been used extensively to measure BRL-39123 in plasma and urine samples generated during clinical studies and is adequate for defining pharmacokinetics at projected therapeutic doses.     

Determination of neomycin in plasma and urine by high-performance liquid chromatography. Application to a preliminary pharmacokinetic study.

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the determination of neomycin in plasma and urine. The plasma was deproteinated with trichloroacetic acid and centrifuged. The supernatant was mixed with ion-pair concentrate and centrifuged again. The resultant supernatant was analyzed by HPLC. Urine was centrifuged to remove debris, if any, mixed with ion-pair concentrate and analyzed directly by HPLC. The HPLC conditions consisted of an ion-pairing mobile phase, a reversed-phase column, post-column derivatization with o-phthalaldehyde (OPA) reagent and fluorescence detection. The overall average recovery of neomycin was 97 and 113% from plasma spiked at 0.25-1.0 micrograms/ml, using standard curves prepared in plasma extract and in water, respectively, and 94% for urine spiked at 1-10 micrograms/ml using a standard curve prepared in water. The method was used to detect neomycin in plasma and urine obtained from animals injected intramuscularly with neomycin. Various pharmacokinetic parameters of neomycin were also determined from its profile of plasma concentration versus time.     

Determination of imipenem (N-formimidoyl thienamycin) in human plasma and urine by high-performance liquid chromatography, comparison with microbiological methodology and stability

High-performance liquid chromatographic (HPLC) methods using ultraviolet (UV) detection have been developed for the assay of the antibiotic imipenem (N-formimidoyl thienamycin) in human plasma and urine. A reversed-phase analytical column is employed in the plasma assay method and a cation-exchange column is used in the urine assay method. Both methods use borate buffer in the mobile phase. The method of preparation of human fluid samples for HPLC injection has been optimized with respect to the stability of imipenem in aqueous buffers, in morpholine buffer--ethylene glycol stabilizer, and in urine and plasma. Preparation of the samples before injection into the HPLC systems involves deproteination/filtration of the plasma/urine samples. The open lactam metabolite and the coadministered dehydropeptidase inhibitor, cilastatin sodium, do not interfere with the 313-nm detection of imipenem in either the plasma or the urine assay. Thienamycin, the precursor of imipenem and an impurity in imipenem formulations, is separated from the drug using both of these methods. Concentrations generated from the HPLC analysis of plasma and urine samples from two healthy volunteers compare favorably with results using a microbiological assay method. Correlation of the two methods gives r greater than or equal to 0.990 for both fluids.     

High-performance liquid chromatographic assay of sulbactam using pre-column reaction with 1,2,4-triazole

A high-performance liquid chromatographic (HPLC) method for the determination of sulbactam in human and rat plasma and urine has been developed. Sulbactam was reacted with 1,2,4-triazole to yield a product having an ultraviolet absorption maximum at 326 nm. The product was separated using reversed-phase HPLC from the regular components of plasma and urine with an ion-pair buffer at 50 degrees C and detected at the ultraviolet maximum. The limits of accurate determination were 0.2 and 1.0 micrograms/ml in plasma and urine, respectively. The coefficients of variation of inter- and intra-assays in human plasma spiked at 4.0 micrograms/ml (n = 5) were 1.02 and 3.05%, respectively. Coexisting cefoperazone, penicillins, or the alkaline degradation product(s) of sulbactam did not interfere in the sulbactam assay. The pharmacokinetic behaviour of sulbactam and cefoperazone coadministered to rats was estimated by moment analysis.     

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.     

High-performance liquid chromatographic assay for amiloride in plasma and urine

A sensitive and simplified high-performance liquid chromatographic procedure has been developed for quantification of amiloride in rabbit plasma, as well as human plasma and urine. Following protein precipitation with perchloric acid, the supernatant was directly injected into a C18 Nucleosil column. The mobile phase consisted of methanol-water (45:55) containing 0.1 M perchloric acid, and the compound was quantitated using a fluorescence detector at excitation and emission wavelengths of 286 and 418 nm, respectively. The average recovery was 97.6%. The calibration curve was linear over the range 2.0-20.0 ng/ml. The limit of detection was 0.5 ng/ml.     

HPLC Assay for Trimethoprim in Plasma and Urine

Abstract

A sensitive HPLC method for the quantitation of trimethoprim in plasma and urine was developed using fluorescence detection. Plasma (or urine) samples were made basic by the addition of 3.8N sodium hydroxide and extracted with chloroform:2-propanol (95:5). After evaporation of the organic layer, a portion of the residue was analyzed by HPLC with fluorescence detection. The minimum detectable quantity is 0.1 μg/ml for this method. This method has been applied to the analysis of plasma and urine obtained from subjects after a single 160 mg dose of trimethoprim.     

Determination of a potential antiasthmatic compound, tibenelast, and its application to phase I clinical trials

A sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of tibenelast, 5,6-diethoxybenzo[b]thiophene-2- carboxylic acid, in plasma and urine. The plasma assay involves protein precipitation with 4% trichloroacetic acid, while the urine assay is an automated solid-phase extraction procedure that utilizes the Waters Millilab workstation. The analysis was achieved by reversed-phase HPLC with ultraviolet detection at 313 nm. The quantitation limit of the assay was 50 ng/ml in plasma and 100 ng/ml in urine. The intra-day coefficient of variation for the plasma analysis was between 2.2 and 8.4%, while the overall inter-day coefficient of variation was 5.5 and 6.0% for the high and low calibration curves, respectively. The intra-day coefficient of variation for the urine analysis was between 0.3 and 3.0%, while the inter-day coefficient of variation was 2.1% for both the low and high validation samples. The assay methodology has been used in the evaluation of samples from pharmacokinetic and clinical safety studies.     

Quantitation of R-836, a New Oral Bronchodilator,by High Performance Liquid Chromatography using Manual or Robotic Sample Preparation

Abstract

A simple and selective high performance liquid chromatography (HPLC) method has been developed for the quantitation of R-836 (an investigational oral bronchodilator) in human plasma and urine, dog plasma and urine, and rat plasma. The method consists of reversed-phase HPLC with ultraviolet detection. Sample preparation involved a one-step protein precipitation procedure and was performed both manually and by a Zymate robotic system. Precision and accuracy results showed excellent reproducibility; results using the robotic procedure were slightly better than the manual procedure. The robotic procedure was capable of preparing the samples with minimal operator handling.     

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.     

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.     

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

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

A new method for determination of buformin in plasma and urine by ion-paired reversed-phase HPLC with ultraviolet detection

Buformin is a widely used as an antidiabetic agent but its renal excretion is still controversial. A new HPLC method with ultraviolet (UV) detection for the determination of buformin in plasma and urine has been developed. After protein precipitation or dilution, buformin and internal standard phenformin were resolved on an octadecyl silica column and detected by UV detection at 233 nm. Intra- and inter-day coefficients of variation were <9%. The limit of quantification was around 0.05 micro g/ml for plasma and 2.5 micro g/ml for urine.     

High-Performance Liquid Chromatographic Determination of SAZ-VII-23, A Novel Antiarrhythmic Agent,in Dog Plasma and Urine

Abstract

A HPLC method has been developed to determine the concentrations of SAZ-VII-23 (3-benzoyl-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane HClO₄), a novel antiarrhythmic agent, in dog plasma and urine. Plasma treated with acetonitrile and alkalinized urine were extracted with chloroform- propanol (9:1). An aliquot was injected on to HPLC system using a C₆ reversed-phase column and acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8 (28.5:28.5:43 v/v) containing 4.0 mM triethylamine as mobile phase. Detection wavelength was 255 nm. The linear range were 0.04–8 μg/ml, and the lower limit of quantitation was 0.04 μg/ml in plasma and urine, respectively. The method was applied to determine plasma and urine concentrations and preliminary pharmacokinetic profiles of SAZ-VII-23 in a dog.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

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.     

Determination and identification of amiloride in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A simple and sensitive high-performance liquid chromatographic method was developed to screen and determine amiloride (I) in human urine. The detection limit of the method is 0.12 micrograms/ml and the recovery of amiloride from urine was 80.4-85.5% at different concentrations. The coefficients of variation were less than 2.8 and 4.4% for intra- and inter-assays, respectively. Total urinary excretion of I in 24 h after oral administration of 5 mg or 15 mg of I ranged from 22.0 to 33.3% of the total dose for three different subjects. I could be detected in urine up to at least 44 h after a 5-mg dose and 72 h after a 15-mg dose. A gas chromatographic-mass spectrometric (GC-MS) confirmatory method was established based on the methanolysis of I to methyl 3,5-diamino-6-chloropyrazine-carboxylate (II). The di-N-trimethylsilyl derivative of II showed very good GC-MS properties and provided reliable structure information for confirmation analysis of I. This is the first time that a reliable GC-MS method has been reported for the detection of urinary I.     

HPLC‐MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute‐and‐shoot sample preparation methods,respectively

A high‐performance liquid chromatography tandem–mass spectrometry (HPLC‐MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP‐Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute‐and‐shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin‐3‐galactoside, cyanidin‐3‐glucoside, cyanidin‐3‐arabinoside, cyanidin‐3‐xyloside and quercetin‐3‐galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.     

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

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.