Gas chromatographic—mass spectrometric procedure for the quantitation of chlorpromazine in plasma and its comparison with a new high-performance liquid chromatographic assay with electrochemical detection

A gas chromatographic—mass spectrometric assay using selected ion monitoring is compared with a high-performance liquid chromatographic assay using an electrochemical detector for single-dose studies of the psychotherapeutic phenothiazine drug chlorpromazine. Measurements were made after extraction of chlorpromazine and the internal standard, prochlorperazine, from basified plasma with an isopropanol—pentane solvent mixture. Following evaporation of the organic solvents the residue was reconstituted in a small volume of methanol and subjected to gas chromatographic—mass spectrometric selected ion detection. The residual sample was then evaporated and made up in a larger volume of acetonitrile and analyzed by high-performance liquid chromatography using an electrochemical detector. These specific methods display excellent correlation for plasma concentration determinations in the range of 0.25–10 ng ml⁻¹ and will allow for the study of the pharmacokinetics of chlorpromazine following single low doses of the drug.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 μg/ml for 2-chloroprocaine and 0.5 μg/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.     

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection

A rapid high-performance liquid chromatographic method for the quantitation of pseudoephedrine in human plasma is presented. The sample preparation involved liquid-liquid extraction of pseudoephedrine from alkalised plasma with hexane-isoamylalcohol (9:1, v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on an octadecylsilica column (50 x 4 mm, 5 microm particles); the mobile phase consisted of acetonitrile-phosphate buffer containing 0.1% of triethylamine, pH 2.4 (5:95, v/v). The run time was 4 min. The spectrophotometric detector was operated at 195 nm. Codeine was used as the internal standard. The limit of quantitation was 5.8 ng/ml using 0.5 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

High-performance liquid chromatographic assay for laudanosine in biological fluids and tissue for neurotoxic studies in rats

A procedure for the determination of laudanosine, the central nervous system active metabolite of the neuromuscular blocking drug atracurium, in serum, cerebrospinal fluid and brain is described. The method uses a readily available internal standard, ethavrine, and a single-step protein precipitation with acetonitrile followed by high-performance liquid chromatographic separation with ultraviolet detection. Norlaudanosine, the major metabolite of laudanosine, can also be quantified. Linearity of detector response was obtained between 1 and 25 μg/ml or μg/g and the method is suitable for determining neurotoxic concentrations of laudanosine in experimental animals.     

High-performance liquid chromatography determination of dipotassium clorazepate and its major metabolite nordiazepam in plasma

A rapid and sensitive high-performance liquid chromatographic method is described for the quantitative analysis of dipotassium clorazepate (CZP) and its major metabolite nordiazepam (ND) in fresh human and dog plasma. The method consists of two separate selective ND extractions from a plasma sample without and with conversion of all the CZP to ND. For quantitation, diazepam (DZP) is used as the internal standard. The chromatographic phase utilized in a reversed-phase Hibar® EC-RT analytical column prepacked with LiChrosolv RP-18 with a solvent system consisting of acetonitrile-0.05 M sodium acetate buffer, pH 5.0 (45:55). The UV absorbance is monitored at 225 nm using a variable-wave-length detector. The mean assay coefficient of variation over a concentration range of 20–400 ng per ml of plasma is less than 3% for the within-day precision. Recoveries of ND, DZP and CZP (as ND) are essentially quantitative at all levels investigated. The calibration curves of ND are rectilinear (r² = 0.99) from the lower limit of sensitivity (2 ng/ml) to at least 2000 ng/ml in plasma. Applicability of the method to CZP and ND disposition studies in the anaesthetized mongrel dog is illustrated. When the two separate selective nordiazepam extractions from plasma cannot be performed immediately after blood sampling, an extrapolation kinetic method is suggested for the estimation of CZP concentration. In all previous in vivo studies, CZP has been determined only with gas-liquid chromatographic methods.     

Determination of the major metabolite of phentolamine in human plasma and urine by high-performance liquid chromatography

A reversed-phase, high-performance liquid chromatographic method using UV detection is described for the assay of the major metabolite of phentolamine in plasma and urine before or after enzymatic hydrolysis. Plasma is deproteinized with methanol. The sensitivity limit is 200 ng/ml using 150-μl samples. Urine is either diluted with water or purified after enzymatic hydrolysis. Concentrations down to 2–3 μg/ml could be quantified with acceptable precision. This method was applied to plasma and urine samples from subjects given phentolamine.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

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

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

Simple and rapid high-performance liquid chromatography method for the determination of ofloxacin concentrations in plasma and urine

A high-performance liquid chromatographic method for the determination of ofloxacin in human plasma and urine was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was linear from 0.5 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 5%. The average recovery of ofloxacin from plasma was 93%. The method was evaluated in samples from healthy subjects whose drug levels were already measured by microbiological assay.     

Sensitive high-performance liquid chromatographic method for a dopamine receptor agonist,CI-1007, and its metabolite PD 147693 in monkey plasma

A sensitive gradient high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of a dopamine autoreceptor agonist CI-1007 (I) and its metabolite PD 147693 (II) is described. Monkey plasma samples were purified by liquid-liquid extraction using hexane. Liquid chromatographic separation was achieved on two C₁₈ analytical columns (installed in series) using gradient elution. Column effluent was monitored using a fluorescence detector programmed to change wavelengths at specified times. Minimum quantitation limits of I and II were 3.0 and 5.0 ng/ml, respectively, for a plasma sample volume of 0.100 ml. Linearity was demonstrated up to 300 ng/ml. The assay has been applied to the analysis of I and II in plasma from monkeys following intravenous and oral doses of I.     

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%.     

High-performance liquid chromatographic determination of nemonapride and desmethylnemonapride in human plasma using an electrochemical detection

A high-performance liquid chromatographic method using an electrochemical detector (HPLC–ED) was developed for the determination of nemonapride and its active metabolite, desmethylnemonapride in human plasma. Nemonapride, desmethylnemonapride and moperone chloride, which was used as the internal standard (I.S.) in plasma, were extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction, and were separated by C₈ reversed-phase HPLC column. Nemonapride and desmethylnemonapride were detected by high conversion efficiency amperometric detection at +0.84 V. Determination of both nemonapride and desmethylnemonapride were possible in the concentration range at 0.25–5.0 ng/ml, and the limit of detection for each was 0.1 ng/ml. The recoveries of nemonapride and desmethylnemonapride added to plasma were 97.0–98.2% and 96.7–98.8%, respectively, with coefficients of variation of less than 7.2% and 10.3%, respectively. This method is applicable to drug level monitoring in the plasma of schizophrenia patients treated with nemonapride and to the study of pharmacokinetics.     

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.     

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 propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

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%.