Determination of Quinidine and its major metabolites by high-performance liquid chromatography

A specific and precise assay, capable of quantitating in human plasma simultaneously but separately quinidine, dihydroquinidine and the quinidine metabolites 2′-quinidinone, 3-OH-quinidine and a third metabolite found — tentatively identified as the product formed by rearrangement of quinidine-N-oxide — is reported. The assay uses a normal phase high-performance liquid chromatographic (HPLC) system with a variable-wavelength UV detector at 235 nm and has a limit of sensitivity at approximately 20 ng/ml. The mobile phase consists of hexanes—ethanol—ethanolamine (91.5:8.47:0.03). A 2-ml plasma sample is worked up by adding primaquine base as an internal standard and extracting with ether—dichloromethane—isopropanol (6:4:1). The organic extract is evaporated and the residue reconstituted in 100—600 μl of mobile phase and an aliquot injected onto the column.Comparison of this procedure with the Edgar and Sokolow (dichloroethane) extraction—fluorescence procedure and with the Cramer and Isaksson (benzene) double extraction—fluorescence assay indicates that both fluorescence procedures give quinidine concentrations up to 2.3 times those determined by HPLC. These discrepancies were shown to be due to carry-over of metabolites and some extraneous background fluorescence.     

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.     

Determination of propranolol and its major metabolites in plasma and urine by high-performance liquid chromatography without solvent extraction

Fast, reliable, specific and sensitive methods are reported to accurately quantitate unchanged propranolol in plasma, and its major metabolites in plasma and urine after enzymatic hydrolysis without the need for solvent extraction. These methods enable the analyst to process a large number of propranolol samples in one working day and should prove valuable to clinical laboratories demanding both speed and specificity in an assay.     

Determination of nalidixic acid and its two major metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography

This paper describes a precise and sensitive method for analysis of nalidixic acid and its two major metabolites in plasma and urine following the oral administration of a therapeutic dose in humans. After addition of an internal standard (oxolinic acid), 1-ml samples of plasma or urine are extracted at acidic pH with chloroform. The extracts are purified by re-extraction with sodium hydroxide solution and then chloroform. The final extracts are evaporated to dryness, reconstituted in mobile phase and injected into a high-performance liquid chromatograph equipped with RP-8 column and UV detector operating at 254 nm. The limit of sensitivity of the method is lower than 0.5 μg/ml of plasma or urine for each compound. The applicability of the method to pharmacokinetic studies of nalidixic acid in humans is demonstrated.     

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.     

Determination of theophylline and its metabolites in human urine and plasma by high-performance liquid chromatography

A method for the quantitation of theophylline (13DMX) and the three metabolites, 1-methyluric acid (1MU), 3-methylxanthine (3MX) and 1,3-dimethyluric acid (13DMU) in human plasma and urine has been developed. The method is based on a simple one-step liquid-liquid extraction with ethylacetate-2 propanol followed by isocratic, reversed-phase high-performance liquid chromatography with UV detection (detection wavelength: 273 nm). The overall mean recoveries ranged from 86 to 95% for the four compounds. The detection limit was 1 μm for 1MU, 3MX and 13DMU and 2 μM for 13DMX in urine, and 0.1 μM for 1MU, 3MX and 13DMU and 0.2 μM for 13DMX in plasma. The intra-day and inter-day coefficient of variation was <6% and <9%, respectively, and the accuracy was within ±10% in both urine and plasma.The simple but sensitive method is highly suitable for the development of theophylline as a probe drug for assessing CYP1A2 activity in man.     

Determination of lansoprazole and five metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of lansoprazole, a new proton-pump inhibitor, and five of its metabolites in human plasma is described. Lansoprazole, its metabolites, and internal standard (omeprazole) were extracted into diethyl ether-methylene chloride and separation was obtained using a reversed-phase column under isocratic conditions. The method features monochromatic ultraviolet detection at 285 nm, and single extraction, single evaporation sample handling. The lower limit of quantitation, based on standards with acceptable coefficients of variation, was 10 ng/ml for all compounds. No endogenous compounds were found to interfere. This method has been demonstrated to be suitable for pharmacokinetic studies in humans.     

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography

UMF-078, methyl (±)-[5-(α-amino-4-fluorobenzyl)benzimidazol-2-yl]carbamate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of ±20% at the lowest limit of quantitation, whereas at higher concentrations it was ±15%. The analytes showed stability up to two freeze–thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at −30°C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.     

Simultaneous Determination of Fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane—dichloromethane—butanol (55:40:5). The plasma extract is chromatographed on a 10-μm, C₁₈ reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration—response curve for all four compounds are linear from 0.05 μg/ml to at least 10 μg/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within ± 9% of the various amounts added with a standard deviation of ± 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.     

Determination of pharmacokinetics of 8-chloroadenosine and its two major metabolites in dogs by high-performance liquid chromatography

High-performance liquid chromatography was used to measure concentration of 8-chloroadenosine (8-Cl-A) and its two major metabolites 8-chloroadenine (8-Cl-Ad) and 8-chloroinosine (8-Cl-I), and their pharmacokinetics in dogs. 8-Cl-A and its metabolites in serum were treated by deproteinization with acetonitrile, then organic impurities were extracted with dichloromethane, followed by centrifuged and direct injection of the supernatant into the liquid chromatograph. After intravenous injection of 8-Cl-A (30 mg/kg), the parent drug and 8-Cl-I were not detected, but the other metabolite, 8-Cl-Ad, was found at a high concentration for 240 min in dog serum. The main pharmacokinetic parameters of 8-Cl-Ad, t_1/2β and AUC, were 69.30 min and 580 μg min/ml. Our finding indicates that in dogs 8-Cl-A is rapidly metabolized and forms its major metabolites, 8-Cl-Ad and 8-Cl-I. 8-Cl-Ad appeared in many tissues, but 8-Cl-A and 8-Cl-I did not. The concentration of 8-Cl-Ad in dog tissues was highest in the liver and spleen, intermediate in the kidney, intestine, and lowest in the bone marrow, heart, and lungs. However, it was not detected in some liposoluble tissues such as the testes, brain, or uterus. Our study provides useful information for clinical experiment.     

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.     

Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4′-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabas C₁₈ analytical column (5 μm particle size; 250×4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantification were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6–15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.     

Determination of nicotine and its main metabolites in urine by high-performance liquid chromatography

Nicotine and its main metabolites (cotinine, trans-3'-hydroxycotinine, trans-3'-hydroxycotinine glucuronide, nicotine-1'-N-oxide and 3-pyridylcarbinol) were analysed in urine after liquid—liquid extraction by high-performance liquid chromatography using norephedrine as internal standard, ultraviolet detection at 260 nm and scanning ultraviolet spectra with a photodiode-array detector. The conjugated trans-3'-hydroxycotinine was determined after enzymatic hydrolysis. Specific determination of 3-pyridylcarbinol was also carried out. Owing to its good selectivity, sensitivity and reproducibility, the method was applied to the analysis of urine samples from smokers and non-smokers. The results obtained suggest that the urinary markers used to assess active smoking or exposure to environmental tobacco smoke must be not only nicotine and cotinine, but also their main free and conjugated metabolites.     

Determination of tripamide and its metabolites in plasma,red blood cells and urine by high-performance liquid chromatography

A procedure for the determination of tripamide and its hydroxylated metabolites in plasma, red blood cells and urine by reversed-phase high-performance liquid chromatography is described.The concentrations in red blood cells showed a monophasic decline and the half-life was 9.5 h. The concentration in red blood cells was markedly higher than that in plasma, showing that 95–98% of the drug is present in whole blood, after a dose of tripamide (90 mg) in man. The specificity and sensitivity of this procedure appear to be satisfactory for pharmacokinetic studies.     

Determination of four carboxylic acid metabolites of felodipine in plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid—liquid and solid-phase extraction. The metabolites were applied to a C₂ cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C₁₈ column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration—time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.     

Direct determination of tamoxifen and its four major metabolites in plasma using coupled column high-performance liquid chromatography

A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 × 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 × 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.     

Determination of the concentrations of 5-fluorouracil and its metabolites in rabbit plasma and tissues by high-performance liquid chromatography

The concentrations of 5-fluorouracil, 5-fluoro-5,6-dihydrouracil, 5-fluorouridine and 5-fluoro-2′-deoxyuridine in plasma, liver, kidney, lung and heart of rabbits were determined by high-performance liquid chromatography (HPLC) after drug administration by two different routes. HPLC was carried out by using a Spherisorb 5 ODS 2 column and 0.05 M phosphate buffer as the mobile phase with UV detection at 200 nm. The pH of the mobile phase, organic modifier content and column temperature were found to have a profound influence on the results, hence it was necessary to optimize a procedure for each matrix. A comparison of the efficiency of intravenous and peritoneal administration revealed that the latter provides higher drug concentrations in the liver and minimal contents in plasma and all other tissues studied.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.