Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

Simultaneous determination of haloperidol and bromperidol and their reduced metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography

This study describes a new simultaneous determination of haloperidol and bromperidol and their reduced metabolites by modification of automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1ml of plasma using chloroform-hexane (30:70 (v/v)), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C(18) analytical column for separation. The mobile phases consisted of phosphate buffer (0.02M, pH 4.6), perchloric acid (60%) and acetonitrile (54:1:45 (v/v)) and was delivered at a flow-rate of 0.6ml/min. The peak was detected using a UV detector set at 215nm. The method was validated for the concentration range 1-100ng/ml, and good linearity (r >0.999) was confirmed. Intra-day coefficient variations (CVs) for haloperidol, reduced haloperidol, bromperidol and reduced bromperidol were less than 2.5, 3.1, 2.4 and 2.5%, respectively. Inter-day CVs for corresponding compounds were 3.9, 5.1, 2.6 and 4.4%, respectively. Relative errors ranged from -5 to 10% and mean recoveries were 96-100%. The limit of quantification was 1.0ng/m for each compound. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring, particularly in patients receiving both haloperidol and bromperidol.     

Isolation of teratogenic alkaloids by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography was used for both analytical and preparative separations of several steroidal alkaloids which occur in extracts of Veratrum californicum. The inclusion of 0.1% trifluoroacetic acid in the mobile phase improved the efficiency of the chromatography and the solubility of the compounds in aqueous acetonitrile. Nuclear magnetic resonance was used to assist the identification of the isolated steroidal alkaloids. The effect of the interaction of trifluoroacetic acid with the alkaloids could be clearly seen by changes in the chemical shifts in the nuclear magnetic resonance spectra.     

Analysis of isoxazolyl penicillins and their metabolites in body fluids by high-performance liquid chromatography

A high-performance liquid chromatographic assay method to quantitate the isoxazolyl penicillins, their active metabolites, and their penicilloic acids in serum or urine is described. Separation and analysis is performed using reversed-phase chromatography. Urine samples, after the appropriate dilution, can be assayed directly. Serum samples (0.1 ml) are either extracted with methylene chloride or treated with perchloric acid—methanol. Serum levels as low as 0.4 μg/ml (extraction procedure) can be assayed accurately.     

Simultaneous determination of quinine and four metabolites in plasma and urine by high-performance liquid chromatography

The determination of quinine, (3S)-3-hydroxyquinine, 2′-quininone and (10R)- and (10S)-10,11-dihydroxydihydroquinine in plasma and urine samples is described. This is the first time the R and S configurations have been correctly assigned to the two metabolites of 10,11-dihydroxyquinine. One hundred microliter-plasma samples were protein precipitated with 200 μl cold methanol. Urine samples were 10–100× diluted and then directly injected into the HPLC. A reversed-phase liquid chromatography system with fluorescence detection and a Zorbax Eclipse XDB phenyl column and gradient elution was used. The within and between assay coefficients of variation of the method for quinine and its metabolites in plasma and urine was less than 13%. The lower limit of quantitation was in the range of 0.024–0.081 μM.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid–liquid and liquid–solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 μm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20–600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20–1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at −20°C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.     

Simultaneous determination of moricizine and its sulphoxidation metabolites in biological fluids by high-performance liquid chromatography

A simultaneous assay for moricizine, its two sulphoxidation metabolites, morizine sulphoxide and moricizine sulphone, using high-performance liquid chromatography (HPLC) is described. The drug and metabolites and clozapine (internal standard) in biological fluids were extracted using pentanesulphonic acid into diethyl ether. The ethereal extract was evaporated to dryness and the residue was redissolved in the mobile phase (methanol-water-triethylamine, 65:35:0.5, v/v). The analyses were performed on a μBondapak reversed-phase C₁₈ column housed in a Waters Z-module, linked to a C₁₈ pre-column, with a run-time of 12 min. The retention times were 2.7, 3.5, 6.2 and 9.7 min for moricizine sulphone, moricizine sulphoxide, moricizine and clozapine, respectively. The recovery of the compounds from plasma ranged from 89.9% for the sulphoxide to 98.1% for clozapine. The limits of detection of the assay for moricizine, moricizine sulphoxide and moricizine sulphone were 20, 10 and 5 ng/ml, respectively.     

Determination of heroin and its metabolites by high-performance liquid chromatography

A method is described for the simultaneous determination of heroin (3, 6-diacetylmorphine, DAM) and its two active metabolites 6-acetylmorphine and morphine in blood by high-performance liquid chromatography using a normal-phase column and a UV detector at 218 nm. The compounds are stabilized in blood by rapid freezing and recovered by a multistep liquid—liquid extraction. The mobile phase is acetonitrile—methanol (75:25, v/v) buffered to apparent pH 7 with ammonium hydroxide and acetic acid. Usingl--acetylmethadol as an internal standard, UV detection and a 1-ml biofluid sample, the lower limit of sensitivity is 12.5 ng/ml. Commonly used narcotic analgesics including codeine, propoxyphene, meperidine, methadone and levorphanol do not interfere with the analysis. The method has been applied to blood samples from humans and rats. Extracts of blood from a patient who had received an intravenous dose of 14 mg of DAM contained DAM and both of its active metabolites.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography

An isocratic liquid chromatographic method employing one extraction step and a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195–200 μg/ml for felbamate; 1.563–200 μg/ml for the p-hydroxy metabolite; 0.391–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195–200 μg/ml for felbamate; 0.781–200 μg/ml for the p-hydroxy metabolite; 0.195–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite.     

Method for determination of angiotensin-converting enzyme activity in blood and tissue by high-performance liquid chromatography

A simplified method for an angiotensin-converting enzyme activity assay in biological samples was developed. Samples were incubated with hippurylhistidylleucine, an artificial substrate of angiotensin-converting enzyme. The reaction was terminated by the addition of metaphosphoric acid and liberated hippuric acid in the supernatant was quantitated directly by reversed-phase high-performance liquid chromatography. Tissues were homogenized in the presence of Nonidet-P40, a detergent, and the resulting supernatant was used for the assay of tissue angiotensin-converting enzyme activity by high-performance liquid chromatography. The present procedure made it possible to determine angiotensin-converting enzyme activity in whole blood and the total activity in tissues. A comparative study of angiotensin-converting enzyme activity in plasma, kidney and lung of five experimental animals showed a high degree of variation from species to species.