Development of a high-performance liquid chromatography method for the determination of caspofungin with amperometric detection and its application to in vitro microdialysis experiments

Microdialysis is an increasingly employed technique for the determination of tissue pharmacokinetics. A high-performance liquid chromatography method for the quantitative determination of caspofungin in human microdialysates with amperometric detection is described. Since microdialysis of caspofungin is performed with a 100,000 molecular mass cut-off membrane, microdialysates contain protein that was precipitated at pH 4 with acetonitrile. Addition of 1-propanol (33%, v/v) to the sample extract improved the analytical recovery to 81-89%. Caspofungin and the internal standard clarithromycin were separated isocratically on a cyanopropyl silica column using acetonitrile-0.05 M citrate (33:67, v/v), adjusted to an apparent pH of 6.9, at a flow rate of 1.0 ml/min, and amperometric detection at +950 mV oxidation potential. Within-day and between-day imprecision and inaccuracy were <11%. The lower limit of quantification was 0.07 microg/ml. The method was applied to in vitro microdialysis experiments. Ringer's solution containing 1% (w/v) human albumin was used for the perfusing and surrounding medium, respectively. Albumin did not entirely prevent adsorption of caspofungin to the surface of membrane and/or tubing. When the binding-sites were saturated with albumin plus caspofungin prior to the start of sampling, the percentage of drug appearing in the microdialysate ("recovery") remained stable over the concentration range tested.     

Determination of ibuprofen in serum by high-performance liquid chromatography and application to ibuprofen disposition

A high-performance liquid chromatographic method for quantitation of ibuprofen from serum and application of this method to ibuprofen disposition in the dog is described. The drug was extracted from acidified plasma with dichloromethane. The internal standard used was a methanolic solution of 4-n-butylphenylacetic acid. A μBondapak C₁ column was used for analysis; the mobile phase was methanol—water—glacial acetic acid (pH 3.4) (75:24:1, v/v). A wavelength of 272 nm was used to monitor ibuprofen and the internal standard.Method sensitivity was 0.5 μg/ml serum using either 0.5 or 1.0 ml of sample, and no interference was found from endogenous compounds or other commonly used anti-inflammatory agents. The coefficients of variation of the method were 4.2% and 6.0% for samples containing 50.0 and 6.25 μg/ml of ibuprofen, respectively, and the calibration curve was linear for the range of 0.5 to 100 μg/ml. This method was demonstrated to be suitable for pharmacokinetic and/or biopharmaceutical studies of ibuprofen in man and the dog.     

Quantitative determination of intracellular, ferritin-associated radioactive iron by high-performance liquid chromatography and immunoprecipitation

Antibodies raised against ferritin preparations of diverse origin provide an uncertain reagent for quantitation of the ferritin present in specific cell lysates. Utilizing K562 cells, a human leukemic cell line, techniques are described to resolve and to quantitate the ferritin-bound cytosolic iron. Processing the cell lysates by HPLC employing an anion-exchange or hydrophobic interaction column resulted in recovery of a single, ferritin-containing radioactive peak widely separated from the bulk of the non-ferritin-bound iron. Comparison of the yield obtained by chromatography with that by immunoprecipitation confirmed both the specificity and the quantitation of the antibody technique.     

Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection

A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g^?1 for danofloxacin, from 25 to 100 ng g^?1 for sarafloxacin and from 50 to 315 ng g^?1 for difloxacin, respectively. The method presents detection limits under 10 ng g^?1 and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques.     

Determination of acetylmethadol and metabolites by use of high-performance liquid chromatography

A method is described for the simultaneous determination of l,α-acetylmethadol (LAAM) and five active metabolites — noracetylmethadol, dinoracetylmethadol, methadol, normethadol, and dinormethadol — in biofluids by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are recovered from biofluids by a multistep liquid—liquid extraction. The mobile phase is methanol—acetonitrile (70:30, v/v) containing 0.015% ammonium hydroxide as the modifier. Retention times can be varied by adjusting the composition of the mobile phase to maximize peak height for quantitation using l-propranolol as the internal standard or peak separation for the collection of fractions. Using a UV detector the lower limit of sensitivity is 10 ng/ml of biofluid. Using fraction collection of radiolabeled drug and metabolites followed by liquid scintillation counting the lower limit of sensitivity is 1.0 ng/ml. Commonly used or abused narcotics including morphine, heroin, meperidine, methadone and propoxyphene do not interfere with the analysis. The method has been applied to plasma and urine samples from humans, sheep and rats. Extracts of urine from patients receiving maintenance treatment with LAAM contain LAAM and each of the five active metabolites.     

Determination of dicentrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetics

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of dicentrine in rat plasma after 10 mg/kg intravenous administration. After addition of an internal standard (coumarin), plasma was deproteinized by acetonitrile for sample clean-up. The drugs were separated on a reversed-phase Nucleosil C₁₈ column (250 × 4 mm I.D., particle size 5 μm) and detected by photodiode-array detection at a wavelength of 308 nm. Acetonitrile-water (35:65, v/v, pH 2.5–2.8, adjusted with orthophosphoric acid) was used as the mobile phase. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve.     

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 guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

Determination of morphine by high-performance liquid chromatography with electrochemical detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with coulometric electrochemical detection was developed for the determination of morphine in human, rabbit, pig and dog plasma. It includes a one-step extraction procedure with hexane–isoamyl alcohol (1:1, v/v) at pH 8.9 (adjusted with phosphoric acid) and reversed-phase liquid chromatography on a μPorasil column. The mobile phase was composed of 5 mM sodium acetate buffer (pH 3.75)–acetonitrile (25:75, v/v). A flow-rate of 1.2 ml/min at 20°C was used. The working potentials for the electrochemical detector were +0.20 V for detector cell 1, +0.55 V for detector cell 2 and +0.75 V for the guard cell. The limit of detection of morphine was 100 pg/ml of plasma. Repeatability, precision and accuracy were also determined concomitantly. The calibration graphs were linear in the concentration range 0.25–250 ng/ml with correlation coefficients of 0.998±0.01 and with a minimum intercept of 0.05±0.08. The precision in plasma was acceptable, with coefficients of variation less than 11%. The absolute recoveries of morphine and nalbuphine (internal standard) were between 86 and 89% and independent of morphine concentration. Pharmacokinetics after oral morphine [MST Continus™ (morphine sulphate tablets) 30 mg, Bard Pharmaceutical, Cambridge, UK] in humans revealed a one-compartment first-order absorption model with one absorption phase and one elimination phase. The absorption and elimination half-lives were 2.46 and 1.80 h, respectively. Pharmacokinetics after intravenous morphine (3 mg/kg) in rabbits showed a linear two-compartment open model with one distribution phase and one elimination phase. The distribution and elimination half-lives were 0.5 and 33.8 h, respectively.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Determination of nalbuphine by high-performance liquid chromatography with ultraviolet detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with ultraviolet detection was developed for the determination of nalbuphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of nalbuphine and ethylmorphine (internal standard) were greater than 86%. Calibration graphs were linear over the concentration range 0.75–150 ng/ml with a coefficient of variation, both within-day and between-day, of less than 10% at any level. The limit of quantitation was 0.75 ng/ml of plasma based on a signal-to-noise ratio of 3. Seven other clinically used analgesics were investigated to check for potential interferences and their analytical conditions. The specificity of this assay was checked with a metabolite of nalbuphine (noroxymorphine). Nalbuphine in plasma did not decompose significantly at −20°C for six weeks. Pharmacokinetic application in three surgical patients and four rabbits revealed that nalbuphine followed a linear three-compartment model with two distribution phases. The two distribution and one elimination half-lives and the plasma clearance of nalbuphine were 0.9, 5.8 and 157 min and 370 ml/min in human, and 3.5, 28 and 117 min and 21 166 ml/min in rabbits.     

Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid–liquid extraction. The method was linear in the 25 to 1000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean±SD): C_max, 776±400 ng/ml; AUC_0–∞, 5368±2689 ng h/ml; AUC_0–t, 5005±2605 ng h/ml; T_max, 2.6±1.6 h; elimination half-life, 5.2±2.0 h.     

An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated.     

The use of high-performance liquid chromatography for sample clean-up in mass fragmentographic assays

A novel high-performance liquid chromatography (HPLC) sample clean-up procedure for use in mass fragmentographic assays of (sub)-nanogram amounts of drugs in human plasma is described and compared with a conventional extraction sequence for sample purification. With the assay of the new antidepressant drug mianserin hydrochloride (Org GB 94) as an example, the HPLC procedure is discussed with respect to retention time, recovery, purification, column deterioration and convenience. It is demonstrated that HPLC sample clean-up is a useful and time-saving procedure for routine clinical analyses.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.