High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of a new carbapenem antibiotic assay using ultraviolet detection has been developed for a new carbapenem antibiotic L-749,345 in human plasma and urine. A plasma sample is centrifuged and then injected onto an extraction column using 25 mM phosphate buffer, pH 6.5. After 3 min, using a column-switching valve, the analyte is back-flushed with 10.5% methanol–phosphate buffer for 3 min onto a Hypersil 5 μm C₁₈ BDS 100×4.6 mm analytical column and then detected by absorbance at 300 nm. The sample preparation and HPLC conditions for the urine assay are similar, except for a longer analytical column 150×4.6 mm. The plasma assay is specific and linear from 0.125 to 50 μg/ml; the urine assay is linear from 1.25 to 100 μg/ml.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.     

Rapid determination of tetracycline and lumecycline in human plasma and urine using high-performance liquid chromatography

A rapid and accurate method for the determination of tetracycline in human plasma and urine is presented. Determination of tetracycline in plasma is based on precipitation of plasma proteins with trifluoroacetic acid, followed by injection of the centrifuged plasma sample onto a μBondapak C₁₈ column. Acetonitrile in phosphate buffer pH 2.2 is used as mobile phase. Only tetracycline, and no trace of lumecycline can be detected in plasma and urine after administration of lumecycline, indicating that lumecycline is completely degraded to tetracycline, lysine and formaldehyde in the gastrointestinal tract prior to absorption.Determination of tetracycline in urine was performed by injection of urine diluted with phosphoric acid onto a μBondapak Phenyl column. The precision of determination of tetracycline in plasma, expressed as the relative standard deviation, was < 3% at tetracycline concentrations of 0.05 and 3.7 μg/ml. Urine determinations were made with a precision of < 1.5% at tetracycline concentrations of 0.5 and 6.7 μg/ml.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025–25 and 2–150 μg/ml in plasma and urine, respectively. An aliquot of 200 μl of plasma was extracted with solid-phase extraction using Oasis^® cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 μl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C₁₈ column at 50°C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 μg/ml in plasma and 1 to 50 μg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.     

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.     

Determination of a chemoprotective agent, 2-(allylthio)pyrazine, in plasma, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemoprotective agent, 2-(allylthio)pyrazine (I), in human plasma and urine, and in rat blood and tissue homogenate using diazepam as an internal standard. The sample preparation was simple; 2.5 volumes of acetonitrile were added to the biological sample to deproteinize it. A 50–100 μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column. The mobile phase employed was acetonitrile–water (55:45, v/v), and it was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector at 330 nm. The retention times for I and the internal standard were 4.0 and 5.1 min, respectively. The detection limits of I in human plasma and urine, and in rat tissue homogenate (including blood) were 20, 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 6.1%) in a concentration range from 0.02 to 10 μg/ml for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.     

Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring

Plasma phenobarbital (PB) concentrations in rat offspring were determined using a 9 μl capillary by high-performance liquid chromatography (HPLC). Capillary plasma which was put into a Bond Elut^® cartridge column by using 1 ml of 0.01 M KH₂PO₄ was applied to the column with 50 μl of 2 μg/ml of acetanilide (internal standard, I.S.). After washing the column, PB and I.S. were eluted with methanol and injected into the HPLC system. There were excellent linear correlation between the amount of PB and length of the capillary at three different concentrations. Calibration for PB was linear in the range of 0–50 μg/ml. The coefficients of variation were 3.4–5.0% and 5.9–7.5% in the within-day and between-day assays, respectively. The extraction recovery rates were 87.5–105.4%. By this method, it was possible to measure plasma PB concentrations in rat offspring without killing. These results suggested that this method is very useful to determine the plasma PB concentration derived from mother’s milk in newborn rats.     

Automated analysis of a novel anti-epileptic compound, CGP 33 101, and its metabolite, CGP 47 292, in body fluids by high-performance liquid chromatography and liquid-solid extraction

Automated procedures for the determination of CGP 33 101 in plasma and the simultaneous determination of CGP 33 101 and its carboxylic acid metabolite, CGP 47 292, in urine are described. Plasma was diluted with water and urine with a pH 2 buffer prior to extraction. The compounds were automatically extracted on reversed-phase extraction columns and injected onto an HPLC system by the automatic sample preparation with extraction columns (ASPEC) automate. A Supelcosil LC-18 (5 μm) column was used for chromatography. The mobile phase was a mixture of an aqueous solution of potassium dihydrogen phosphate, acetonitrile and methanol for the assay in plasma, and of an aqueous solution of tetrabutylammonium hydrogen sulfate, tripotassium phosphate and phosphoric acid and of acetonitrile for the assay in urine. The compounds were detected at 230 nm. The limit of quantitation was 0.11 μml/l (25 ng/mol) for the assay of CGP 33 101 in plasma, 11 μmol/l (2.5 μg/ml) for its assay in urine and 21 μmol/l (5 μg/ml) for the assay of CGP 47 292 in urine.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

High-performance liquid chromatographic assay for a new fasciolicide agent, αBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (αBIOF10) — a new fasciolicide agent — and its sulphoxide (SOαBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a μBondapak C₁₈ (10 μm) column, using methanol–acetonitrile–water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 12 ng/ml for αBIOF10 and SOαBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 μg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both αBIOF10 and SOαBIOF10. In urine, recovery was 99.6% and 93.1% for αBIOF10 and SOαBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Determination of lamotrigine in human serum by liquid chromatography

A rapid high-performance liquid chromatographic method was developed using a short silica column (30 mm×4.6 mm) with an aqueous methanol mobile phase consisting of methanol–water–NH₄H₂PO₄ (94:5.96:0.04) adjusted to a final apparent pH of 5.0 and pumped at a flow-rate of 1 ml/min. Ultraviolet detection was carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. Within-day variation of the method was 4.46% at 0.75 μg/ml and 2.37% at 6.0 μg/ml, and day-to-day variation was 9.10% at 0.75 μg/ml and 7.28% at 6.0 μg/ml.     

Simultaneous analysis of 2-methoxyphenylmetyrapone and its seven potential metabolites by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic (HPLC) assay has been developed for the quantification of 2-methoxyphenylmetyrapone (2-MPMP) and its seven potential metabolites in rat urine and whole blood. 2-MPMP, 2-hydroxyphenylmetyrapone and their N-oxides, together with 2-methoxyphenylmetyrapol, 2-hydroxyphenylmetyrapol and their N-oxides were separated on an Isco Spherisorb ODS-2 reversed-phase column (250×4.6 mm, I.D., 5 μm), with an Isco Spherisorb ODS-2 guard cartridge (10×4.6 mm I.D.). A gradient elution was employed using solvent system A (acetonitrile–water–triethylamine–acetic acid, 27.3:69.1:0.9:2.7%, v/v) and solvent system B (methanol), the gradient program being as follows: initial 0–4 min A:B=74:26; 4–10 min linear change to A:B=50:50; 10–16 min maintain A:B=50:50; 16 min return to initial conditions (A:B=74:26). Flow-rate was maintained at 1.25 ml/min, and the eluent monitored using a diode array multiple wavelength UV detector set at 260 nm. Most of the analytes were baseline resolved, and analysis of samples recovered from blood or urine (pH 12, 3×5 ml of dichloromethane, recovery 20–95%) revealed no interference from any co-extracted endogenous compounds in the biological matrices, except for 2-hydroxyphenylmetyrapol N-oxide (2-OHPMPOL-NO) at low concentrations. The calibrations (n=6) were linear (r≥0.996) for all analytes (0.5–100 μg/ml), with acceptable inter- and intra-day variability. Subsequent validation of the assay revealed acceptable precision, as measured by coefficient of variation (C.V.) at the low (0.5 mg/ml), medium (50 μg/ml) and high (100 μg/ml) concentrations. The limits of detection for 2-MPMP and their available potential metabolites, except 2-OHPMPOL-NO, in rat urine and blood were both 0.5 μg/ml, respectively.