Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

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.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Improved assay method for the determination of pyronaridine in plasma and whole blood by high-performance liquid chromatography for application to clinical pharmacokinetic studies

An improved high-performance liquid chromatography method using a diisopropyl-C₁₄ reversed-phase column (Zorbax Bonus-RP column) and a liquid–liquid extraction technique with UV detection is presented for the analysis of pyronaridine in human whole blood and plasma. Tribasic phosphate buffer (50 mM, pH 10.3) and diethyl ether were used for liquid–liquid extraction. The mobile phase consists of acetonitrile–0.08 M potassium dihydrogen phosphate buffer (13:87, v/v) with the pH 2.8 adjusted by orthophosphoric acid. Amodiaquine was found to be a suitable internal standard for the method. The quantification limit with UV detection at 275 nm was 3 ng on-column for both plasma and blood samples. The method was applied to plasma and blood specimens from a rabbit after a single intramuscular dose of pyronaridine tetraphosphate (20 mg/kg as base). From this in vivo study, evidence was found that pyronaridine is concentrated in blood cells, with a blood:plasma ratio ranging from 4.9 to 17.8. We conclude that blood is the preferred matrix for clinical pharmacokinetic studies.     

Extraction and quantification of nicardipine in human plasma

A novel simple method of extraction, separation, identification and quantification of nicardipine in human plasma samples was completely studied. The human plasma samples were initially purified by solid-phase extraction (SPE) using a C₁₈ cartridge. The extracted samples were separated and nicardipine present in the samples was quantified by high-performance liquid chromatography (HPLC) on a reversed-phase C₁₈ column employing a mobile phase consisting of 60% (v/v) acetonitrile in 0.02 M NaH₂PO₄ with pH of 6.3 and a variable wavelength UV detector set at 254 nm. The recovery of nicardipine from plasma samples using selective SPE was 91±6.0% and had less interfering compounds in the HPLC analysis compared to the use of liquid–liquid (L/L) extraction. In the HPLC analysis, examining the effect of pH values of the mobile phase on the capacity factor (k′) of nicardipine revealed a method for selecting a critical k′ value of nicardipine to eliminate interfering peaks near the peak specific to the analyte. This method for quantification of nicardipine in human plasma samples was suitable for studying the pharmacokinetic profile of nicardipine administered as an intravenous bolus to cardiac surgical patients.     

Determination of naringenin and its glucuronide conjugate in rat plasma and brain tissue by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method with ultraviolet detection was utilized for the investigation of the pharmacokinetics of naringenin and its glucuronide conjugate in rat plasma and brain tissue. Plasma and brain tissue were deproteinized by acetonitrile, then centrifuged for sample clean-up. The drugs were separated by a reversed-phase C₁₈ column with a mobile phase consisting of acetonitrile–orthophosphoric acid solution (pH 2.5–2.8) (36:64, v/v). The detection limits of naringenin in rat plasma and brain tissue were 50 ng/ml and 0.4 μg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme β-glucuronidase. The naringenin conjugation ratios in rat plasma and brain tissue were 0.86 and 0.22, respectively, 10 min after naringenin (20 mg/kg, i.v.) administration. The mean naringenin conjugation ratio in plasma was approximately four fold that in brain tissue.     

Simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine by high-performance liquid chromatography using electrochemical detection

A rapid, selective and sensitive method for the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine has been developed using high-performance liquid chromatography with electrochemical detection.The unchanged drugs and internal standard extracted from plasma and urine were separated by reversed-phase high-performance liquid chromatography. The influence of acetonitrile concentration and of the pH of the mobile phase were investigated. The detection limits were 100 pg for chlorpromazine and for levomepromazine. In comparison with three other detection systems this was found to be the most sensitive method.This method was successfully applied to the simultaneous determination of chlorpromazine and levomepromazine in human plasma and urine for pharmacokinetic studies.     

High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue distribution studies

A reliable reversed-phase high-performance liquid chromatographic method was developed for the determination of liposomal nystatin in plasma. Nystatin is extracted by 1:2 (v/v) liquid–liquid extraction with methanol. Separation is achieved by HPLC after direct injection on a μBondapak™ C₁₈ analytical column with a mobile phase composed of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is based on the sum of the peak area concentration of the two major isomers of nystatin, which elute at 7.5–8.5 and 9.5–10.5 min. The assay was linear over the concentration range of 0.05 to 50 μg/ml. The lower limit of quantitation was 0.05 μg/ml, sufficient for investigating the plasma pharmacokinetics of liposomal nystatin in preclinical studies. Accuracies and intra- and inter-day precision showed good reproducibility. With minor modifications, this method also was used for assaying nystatin in various non-plasma body fluids and tissues.     

Bioanalysis and preliminary pharmacokinetics of the acridonecarboxamide derivative GF120918 in plasma of mice and humans by ion-pairing reversed-phase high-performance liquid chromatography with fluorescence detection

We have developed and validated a sensitive and selective method for the determination of the P-glycoprotein modulator GF120918 in murine and human plasma. Chlorpromazine is used as internal standard. Sample pretreatment involves liquid–liquid extraction with tert-butyl methyl ether. Chromatographic separation is achieved by reversed-phase high-performance liquid chromatography using a Symmetry C₁₈ column and detection was accomplished with a fluorescence detector set at excitation and emission wavelengths of 260 and 460 nm, respectively. The mobile phase consists of acetonitrile–50 mM ammonium acetate buffer, pH 4.2 (35:65, v/v). To achieve good separation from endogenous compounds and to improve the peak shape the counter-ion 1-octane sulfonic acid (final concentration 0.005 M) was added to the mobile phase. The lower limit of quantitation was 5.7 ng/ml using 200 μl of human plasma and 23 ng/ml using 50 μl of murine plasma. Within the dynamic range of the calibration curve (5.7–571 ng/ml) the accuracy was close to 100% and within-day and between-day precision were within the generally accepted 15% range. The stability of GF120918 was tested in plasma and blood from mice and humans incubated at 4°C, room temperature, and 37°C for up to 4 h. No losses were observed under these conditions. This method was applied to study the pharmacokinetics of orally administered GF120918 in humans and mice. The sensitivity of the assay was sufficient to determine the concentration in plasma samples obtained up to 24 h after drug administration.     

Simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar)

A rapid clean-up and high-performance liquid chromatographic method for the simultaneous determination of ormethoprim and sulphadimethoxine in plasma and muscle of Atlantic salmon (Salmo salar) has been developed. Sample preparation is based on protein precipitation using trichloroacetic acid or methanol for plasma and muscle, respectively. The drugs are separated using a reversed-phase C₁₈ analytical column and phosphate buffer—acetonitrile (80:20, v/v) containing 1-heptanesodiumsulphonate and triethylamine, as mobile phase. Detection was performed at 270 nm. The average recovery of ormethoprim was 97.2% in muscle and 95.7% in plasma, whereas the average recovery of sulphadimethoxine was 86.5% in muscle and 90.2% in plasma. The limit of detection at a signal-to-noise ratio of 3 was 50 ng/g and 30 ng/ml for ormethoprim in muscle and plasma respectively and 30 ng/g and 15 ng/ml in muscle and plasma respectively for sulphadimethoxine.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of sildenafil in human plasma

A method to determine sildenafil in human plasma involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Sildenafil and the internal standard (I.S.), diazepam, are extracted from human plasma with ether-dichloromethane (3:2, v/v) at basic pH and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol-10mM ammonium acetate pH 7.0 (85:15, v/v) as the mobile phase. Detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was linear over the concentration range 0.125-40.0 ng/ml. Intra- and inter-day precision of the assay at four concentrations within this range were 2.5-8.0%. The method was used to evaluate plasma concentration-time profiles in healthy volunteers given an oral dose of 20mg sildenafil as a combination tablet also containing apomorphine.     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Determination of the new podophyllotoxin derivative NK 611 in plasma by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method has been developed for the determination of the new podophyllotoxin derivative NK 611 in plasma samples. A solid—liquid extraction procedure with C₁₈ extraction columns was used for extraction of plasma samples containing NK 611. The adsorbed NK 611 was eluted from the extraction columns with methanol—acetonitrile (50:50, v/v). The elution liquid was injected into a reversed-phase system consisting of a Chrompack C₁₈ column. The mobile phase was acetonitrile—20 mM phosphate buffer, pH 7 (30:70, v/v). The UV detection mode allows sensitive determination of NK 611 in plasma within phase I trials. The limit of detection was 10 ng/ml, the limit of quantitation 35 ng/ml (for 1 ml of extracted plasma and 20-μl injection volume). The calibration curve is linear within the concentration range 100–1000 ng/ml. The recovery of NK 611 from spiked plasma samples was approximately 80%.     

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 μl of mobile phase (0.01 M phosphate buffer—acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C₁₈ reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drug's pharmacological effects.     

Monitoring of benzodiazepines (clobazam, diazepam and their main active metabolites) in human plasma by column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic method for the simultaneous determination of clobazam, diazepam and their main metabolites in human plasma is described. A 200-μl plasma sample was directly injected into a precolumn filled with TSK-gel PW. After a washing step with potassium phosphate buffer, the retained substances were backflushed into a reversed-phase column with a mobile phase of acetonitrile—phosphate buffer—diethylamine. Various drugs frequently co-administered with clobazam or diazepam do not interfere with the determination.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.     

Rapid simple high-performance liquid chromatographic determination of paroxetine in human plasma

A rapid, simple method for the measurement of paroxetine in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described. This method includes only one-step extraction of paroxetine and dibucaine, an internal standard, with chloroform. Their recoveries were around 90%. The mobile phase, 10 mM phosphate buffer–acetonitrile (40:60, v/v) was eluted isocratically. Between- and within-day coefficients of variation were in the range of 1.9–9.4% and 2.3–13.3%, respectively. The detection limit was 0.2 ng/ml. The method we describe can be easily applied to the measurement of plasma paroxetine concentration for pharmacokinetic studies as well as for therapeutic drug monitoring in patients taking paroxetine.     

Determination of midazolam and its metabolites 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 midazolam and its two hydroxy metabolites in rat serum microsamples (50 μl). The separation used a 2 mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol-acetonitrile-14.9 mM sodium acetate in water at pH 3.0 (10:23:67, v/v). The detection limit was 10 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of midazolam after an intravenous bolus dose (0.75 mg/kg).     

Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography

A simple, selective, sensitive and precise high-performance liquid chromatographic plasma assay for the antihypertensive drugs, irbesartan and hydrochlorothiazide is described. Good chromatographic separation was achieved using a Supelcocil C(18) (5 micrometer 15 cmx4.6 mm) column and a mobile phase consisting of 10 mM potassium dihydrogen phosphate:methanol:acetonitrile (5:80:15 v/v/v) (pH:2.5) while at a flow-rate of 1.0 ml min(-1). Irbesartan and hydrochlorothiazide were detected at 275 nm and were eluted 5.8 and 7.8 min, respectively, after injection. No endogenous substances were found to interfere. The method utilizes protein precipitation with acetonitrile as the only sample preparation involved prior to reversed-phase high-performance liquid chromatography. No internal standard was required. Linearity range for irbesartan and hydrochlorothiazide was 10.0-60.0 microgram ml(-1) and 4.0-20.0 microgram ml(-1), respectively. The determination of intra- and inter-day precision (RSD) was less than 2.5 and 3.5%, at all concentration levels, while the inter- and intra-day accuracy (% difference) was less than 4.9-6.2%. This method is being used in a therapeutic drug monitoring service to quantitate these therapeutic agents in patients for pharmacokinetic studies.     

Determination of hydroxyurea in plasma and peritoneal fluid by high-performance liquid chromatography using electrochemical detection

A sensitive method has been developed for the determination of hydroxyurea in plasma and peritoneal fluid using reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. Plasma or peritoneal fluid samples were treated with acetonitrile to precipitate proteins then injected to the HPLC. A C₁₈ analytical column was used to separate hydroxyurea from interfering substances in the biological matrix. The mobile phase, consisting of 0.2 M sodium perchlorate–methanol (95:5, v/v) adjusted to pH 5.0, was delivered isocratically at a flow-rate of 1 ml/min and hydroxyurea was detected using a glassy-carbon electrode operating at an applied potential of +800 mV. Hydroxyurea eluted with a retention time of 3 min. The cycle time for analysis is short and the assay precision is acceptable (C.V. plasma=1.4–3.9%, C.V. peritoneal fluid=2.1–9.7%). The method has been validated and is linear from 25 to 400 ng/ml in plasma and 5 to 30 ng/ml in peritoneal fluid. The method has been shown to be applicable for pharmacokinetic studies.