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-μm μBondapak 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-(-)-α-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 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/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 μg/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 puerarin in human plasma by high performance liquid chromatography

Puerarin, an isoflavone C-glycoside, has been identified as the major active component isolated from Pueraria lobata (Kudzu) responsible for suppression of alcohol drinking. In order to conduct clinical studies of Kudzu's efficacy, a method for measuring its bioavailability and pharmacokinetic profile is needed. We have developed a gradient reversed-phase HPLC system for pharmacokinetic study of puerarin in human plasma. Solid-phase extraction was performed on an abselut Nexus cartridge (60 mg/3 ml) possessing adsorbent function with a recovery of >97% and 4-hydroxybenzoic acid was used as an internal standard. The HPLC assay was performed on a YMC ODS-A column (150 mm x 4.6mm i.d., 5 microm particle size). The HPLC mobile phase consisted of methanol/0.5% acetic acid with 20-35% methanol gradient at a flow-rate of 0.8 ml/min. The UV wavelength was set at 254 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a puerarin concentration range of 5-500 ng/ml in human plasma. The lower limit of quantification was ca. at 8 ng/ml of puerarin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 3 ng/ml. The preliminary pharmacokinetic study after oral administration of the Kudzu capsules containing 400mg of puerarin to a healthy volunteer confirmed that the present method was suitable for determining puerarin in human plasma.     

Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Rapid determination of valsartan in human plasma by protein precipitation and high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of valsartan in human plasma is reported. The assay is based on protein precipitation with methanol and reversed-phase chromatography with fluorimetric detection. The preparation of a batch of 24 samples takes 20 min. The liquid chromatography was performed on an octadecylsilica column (50 mm x 4 mm, 5 microm particles), the mobile phase consisted of acetonitrile -15 mM dihydrogenpotassium phosphate, pH 2.0 (45:55, v/v). The run time was 2.8 min. The fluorimetric detector was operated at 234/374 nm (excitation/emission wavelength). The limit of quantitation was 98 ng/ml using 0.2 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

High-performance liquid chromatographic determination of pyrazoloacridine, a nitro-9-methoxyacridine anticancer agent, in human plasma

Pyrazoloacridine (PZA) is a 9-methoxy substituted acridine with a reducible nitro group. PZA has shown selective solid tumor cytotoxicity with activity against hypoxic cells, non-cycling cells and cells expressing the multidrug resistant phenotype. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of PZA in human plasma to support phase II clinical trials. PZA and ethyl orange, the internal standard, were isolated from human plasma by precipitating plasma proteins with methanol, and centrifuging to pellet the proteins. The resulting supernatant was injected onto a cyanopropyl HPLC column eluted isocratically with a mobile phase consisting of 125 mM ammonium acetate buffer pH 4.75-acetonitrile (76:24, v/v). A single wavelength at 460 nm was used for detection. Relative standard deviations for the assay ranged from 5.0% to 12.2% for four different drug concentrations and the limit of quantitation was 100 ng/ml. During the validation short term stability of the drug in plasma and stability of PZA on repeated freezing and thawing of plasma was evaluated. Overall recovery of PZA was 88%. This simple assay was found suitable for studying the clinical pharmacokinetics of PZA.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

A rapid and sensitive microscale HPLC method for the determination of indomethacin in plasma of premature neonates with patent ductus arteriousus

Indomethacin (IND) is the drug of choice for the closure of a patent ductus arteriosus (PDA) in neonates. This paper describes a simple, sensitive, accurate and precise microscale HPLC method suitable for the analysis of IND in plasma of premature neonates. Samples were prepared by plasma protein precipitation with acetonitrile containing the methyl ester of IND as the internal standard (IS). Chromatography was performed on a Hypersil C(18) column. The mobile phase of methanol, water and orthophosphoric acid (70:29.5:0.5, v/v, respectively), was delivered at 1.5 mL/min and monitored at 270 nm. IND and the IS were eluted at 2.9 and 4.3 min, respectively. Calibrations were linear (r>0.999) from 25 to 2500 microg/L. The inter- and intra-day assay imprecision was less than 4.3 % at 400-2000 microg/L, and less than 22.1% at 35 microg/L. Inaccuracy ranged from -6.0% to +1.0% from 35 to 2000 microg/L. The absolute recovery of IND over this range was 93.0-113.3%. The IS was stable for at least 36 h when added to plasma at ambient temperature. This method is suitable for pharmacokinetic studies of IND and has potential for monitoring therapy in infants with PDA when a target therapeutic range for IND has been validated.     

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.     

Quantitative analysis of rifampicin and 25-desacetylrifampicin in the plasma using high performance liquid chromatography

A procedure for determination of rifampicin and 25-desacetylrifampicin in plasma by HPLC was developed. The plasma proteins are precipitated by acetonitrile and the supernatant layer (50 microliters) is used for the assay under isocratic conditions on an analytical column 250 x 4.6 mm in size containing the reversed phase sorbent (C18). The size of the precolumn is 50 x 4.6 mm. An UV detector (at lambda 335 nm) is used. For preparing the mobile phase 630 ml of methanol and 370 ml of 0.058 M sodium nitrite solution are mixed. The flow rate of the mobile phase is 40.7 ml/min. The assay duration is about 10 min. The retention time is 9.6 min for rifampicin and 6.5 min for 25-desacetylrifampicin. The minimum detectable amount of the antibiotic and its metabolite is 0.10 micrograms/ml. The standard curves of rifampicin and 25-desacetylrifampicin are linear within the concentration ranges of 0.5-100 and 0.5-10 micrograms/ml respectively. The procedure is useful in studies on pharmacokinetics of rifampicin and 25-desacetylrifampicin.     

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10 000 pg/ml. Sample extraction (efficiencies >86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantited using post-column UV-photochemical cyclization coupled with fluoremetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.     

Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of metformin in human plasma using phenformin as internal standard has been developed and validated. Sample preparation of plasma involved acidification with acetic acid, deproteination with acetonitrile and washing with dichloromethane. Samples were then analyzed by HPLC on a short Nucleosil C18 column (5 microm, 50 mm x 4.6 mm i.d.) using a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate pH 7.0 (20:20:60, v/v/v) delivered at 0.65 ml/min. Detection was performed using an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring (MRM) mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The assay was linear over the range 1-2000 ng/ml with intra- and inter-day precision of <8.6% and accuracy in the range 91-110%. The limit of detection was 250 pg/ml in plasma. The method was successfully applied to a clinical pharmacokinetic study of an extended-release tablet of metformin hydrochloride (500 mg) administered as a single oral dose.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

Determination of scopoletin in rat plasma by high performance liquid chromatographic method with UV detection and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for determination of scopoletin in rat plasma using psoralen as internal standard. Chromatographic separation was achieved on a C(18) column using methanol and distilled water (49:51, v/v) containing 0.05% (v/v) phosphoric acid as mobile phase. The UV detector was set at 345 nm. The calibration curve was linear over the range of 0.165-9.90 microg/ml with a correlation coefficient of 0.9994. The recovery for plasma samples of 0.165, 1.32 and 6.60 microg/ml was 93.2%, 95.9% and 95.5%, respectively. The RSD of intra- and inter-day assay variations was less than 6.7%. This HPLC assay is a precise and reliable method for the analysis of scopoletin in pharmacokinetic studies.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

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 aceto-nitrile—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-carbonxamide (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 10mg/kg oral dose of I · 2HCl and of metabolite I-A.     

Quantitation of ibogaine and 12-hydroxyibogamine in human plasma by liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic (HPLC) method with fluorimetric detection was developed for the simultaneous determination of ibogaine and noribogaine in human plasma using fluorescein as internal standard. This method involved a solid phase extraction of the compounds from plasma using N-vinylpyrrolidone-divinybenzene copolymer cartridges. Separation of the three analytes was performed on a reversed-phase Supelcosil C18 analytical column (75 mm x 4.6mm i.d., 3 microm particle size). The excitation wavelength was set at 230 nm for the first 15.8min and then at 440 nm for the following 14.2 min; the emission wavelength was set at 336 nm for the first 15.8 min and then at 514 nm for the following 14.2 min. Obtained from the method validation, inter-assay precision was 6.0-12.5% and accuracy was 95.4-104%. The extraction efficiencies of the assay were higher than 94% and were constant across the calibration range. The lower limits of quantitation were 0.89 ng/ml for ibogaine and 1 ng/ml for noribogaine; at these levels, precision was < or =17% and accuracy was 95-105%. In this paper, extensive stability testing was undertaken using a wide range of storage conditions. Special attention must be paid to sample handling to avoid light degradation of the compounds.     

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (-20 degrees C) for sample preparation followed by separation on a Novapack C(18) column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at -70 degrees C.     

High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm x 150 mm, 4 microm) protected by a Nova-Pak C18 guard column (3.9 mm x 20 mm, 4 microm), where 10 mM KH(2)PO(4)-methanol-triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 microl mouse plasma. The assay was shown to be linear over a concentration range of 1-500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT.