Sensitive determination of omeprazole and its two main metabolites in human plasma by column-switching high-performance liquid chromatography: application to pharmacokinetic study in relation to CYP2C19 genotypes

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether-dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 microm, 150 mm x 4.6mm i.d.) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer-acetonitrile-methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was approximately 25 min. The validated concentration ranges of this method were 3-2000 ng/ml for omeprazole, 3-50 ng/ml for 5-hydroxyomeprazole and 3-1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.     

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 urinary 2- and 3-dechloroethylated metabolites of ifosfamide by high-performance liquid chromatography

In vivo oxidation of chloroethyl side-chains on ifosfamide produces the toxin chloroacetaldehyde. Production of this labile metabolite can be indirectly quantitated by monitoring the excretion of the residual 2- and 3-dechloroethylated ifosfamide. Urinary ifosfamide and the two dechloroethylated metabolites were extracted into chloroform from alkalinized salt-saturated urine, followed by high-performance liquid chromatographic separation using an acetonitrile gradient on a reversed-phase column and ultraviolet detection at 190 nm. In five patients given 1.6 g/m² ifosfamide, 11–30% of the dose was excreted over 24 h as unchanged drug, 11–21% as 3-dechloroethylated and 3–10% as 2-dechloroethylated ifosfamide.     

Determination of lansoprazole and two of its metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography: application to measuring CYP2C19 activity

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method for the simultaneous determination of lansoprazole, a proton pump inhibitor and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human plasma. The test compounds were extracted from 1 mL of plasma using diethyl ether-dichloromethane (7:3, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 microm, 3.5 mm x 4.6 mm i.d.) for clean-up and column I (C(18) STR ODS-II analytical column, 5 microm, 150 mm x 4.6 mm i.d.) for separation. The peak was detected by a ultraviolet detector set at a wavelength of 285 nm, and the total time for a chromatographic separation was approximately 25 min. The method was validated for the concentration range from 3 to 5000 ng/mL. Mean recoveries were 74.0% for lansoprazole, 68.3% for 5-hydroxylansoprazole, and 79.4% for lansoprazole sulfone. Intra- and inter-day relative standard derivatives were less than 6.1 and 5.1% for lansoprazole, 5.8 and 5.8% for 5-hydroxylansoprazole, 4.4 and 5.9% for lansoprazole sulfone, respectively, at the different concentration ranges. This method is suitable for use in therapeutic drug monitoring and pharmacokinetic studies, and provides use tool for measuring CYP2C19 activity.     

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.     

Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography

An isocratic high-performance liquid chromatography procedure was developed for the analysis of five urinary metabolites of caffeine; caffeine or 1,3,7-trimethylxanthine (137X), paraxanthine or 1,7-dimethylxanthine (17X), 1,7-dimethylurate (17U), 1-methylxanthine (1X), 1-methylurate (1U) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU). A standardized procedure was used for oral intake of caffeine and for urine collection. Conditions for sample storage and preparation were optimized, resulting in no detectable loss of caffeine metabolites after storage of the urine samples for four months. Urine samples were extracted with chloroform–2-propanol (4:1, v/v) and separated on a reversed-phase column with acetic acid (33%)–tetrahydrofuran–acetonitrile–water (1:2.5:44:925.5, v/v) as the eluent. Peaks were monitored at 280 nm. Peak heights were measured and the five metabolites were quantified using calibration curves. Cytochrome P4501A2 (CYP1A2) activity was calculated from the molar ratio (AFMU+1X+1U)/17U, N-acetyltransferase (NAT) from the ratio AFMU/1X, XO from the ratio 1U/1X+1U and cytochrome P4502A6 (CYP2A6) from the ratio 17U/(17U+17X+1U+1X+AFMU). The inter-assay coefficients of variation ranged from 1.7% for 17U to 5.7% for 1X. The intra-individual variation in metabolite ratios determined in two people, with intervals of a few days to several weeks between measurements, ranged from 2.1% for XO to 11.0% for CYP2A6. Using this procedure, metabolic ratios were determined for four groups of subjects; healthy, non-smoking females using oral contraceptives (OC users, n=5) and non-users (n=5), healthy non-smoking males (n=9) and children (n=7). Results found in this study were comparable to results reported in the literature for subjects with similar characteristics. A significantly higher CYP1A2 ratio was found for males (4.87±0.47) compared to females (3.62±0.91; p=0.005, Mann-Whitney). For the other enzyme activities, no significant differences were found between the groups of subjects in this study.     

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.     

Simultaneous analysis of 2-methoxyphenylmetyrapone and its seven potential metabolites by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic (HPLC) assay has been developed for the quantification of 2-methoxyphenylmetyrapone (2-MPMP) and its seven potential metabolites in rat urine and whole blood. 2-MPMP, 2-hydroxyphenylmetyrapone and their N-oxides, together with 2-methoxyphenylmetyrapol, 2-hydroxyphenylmetyrapol and their N-oxides were separated on an Isco Spherisorb ODS-2 reversed-phase column (250×4.6 mm, I.D., 5 μm), with an Isco Spherisorb ODS-2 guard cartridge (10×4.6 mm I.D.). A gradient elution was employed using solvent system A (acetonitrile–water–triethylamine–acetic acid, 27.3:69.1:0.9:2.7%, v/v) and solvent system B (methanol), the gradient program being as follows: initial 0–4 min A:B=74:26; 4–10 min linear change to A:B=50:50; 10–16 min maintain A:B=50:50; 16 min return to initial conditions (A:B=74:26). Flow-rate was maintained at 1.25 ml/min, and the eluent monitored using a diode array multiple wavelength UV detector set at 260 nm. Most of the analytes were baseline resolved, and analysis of samples recovered from blood or urine (pH 12, 3×5 ml of dichloromethane, recovery 20–95%) revealed no interference from any co-extracted endogenous compounds in the biological matrices, except for 2-hydroxyphenylmetyrapol N-oxide (2-OHPMPOL-NO) at low concentrations. The calibrations (n=6) were linear (r≥0.996) for all analytes (0.5–100 μg/ml), with acceptable inter- and intra-day variability. Subsequent validation of the assay revealed acceptable precision, as measured by coefficient of variation (C.V.) at the low (0.5 mg/ml), medium (50 μg/ml) and high (100 μg/ml) concentrations. The limits of detection for 2-MPMP and their available potential metabolites, except 2-OHPMPOL-NO, in rat urine and blood were both 0.5 μg/ml, respectively.     

Analysis of purines in urinary calculi by high-performance liquid chromatography

A high-pressure liquid chromatography method has been developed for the analysis in urinary calculi of six purines: uric acid, 2, 8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol, and oxypurinol. Separation was conducted isocratically on a reversed-phase column, using 50 mM phosphate buffer (pH 5.5) / methanol (97/3, v/v) as mobile phase. Limits of detection, depending on compound, ranged from 7 to 28 microg/g stone weight. Hitherto, no reports have appeared on other purines present with uric acid in stones, due to lack of a sensitive and specific analytical method. We have now found that all calculi with more than 4% uric acid also contained 1-methyluric and 7-methyluric acids and trace amounts of hypoxanthine, xanthine, and 2,8-dihydroxyadenine. Accurate identification and quantitation of purines in urinary calculi are important for the diagnosis of rare metabolic diseases leading to urolithiasis (xanthinuria, dihydroxyadeninuria), as well as for prevention of iatrogenic complications during treatment with allopurinol of uric acid urolithiasis. The method may be used for reference purposes in clinical laboratories and for research on the pathogenesis of urolithiasis in disorders of purine metabolism.     

Determination of bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

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.     

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 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.     

Analysis of secondary metabolites from eschscholtzia californica by high-performance liquid chromatography

A rapid and precise analytical HPLC method has been developed for screening the major benzophenanthridine alkaloids produced by cell cultures of Eschscholtzia califomica, namely, sanguinarine, chelirubine, macarpine, chelerythrine and chelilutine. Separation was achieved on a C18, reversed-phase column with gradient elution using acetonitrile and 50 mM phosphoric acid. Detection was performed by both fluorescence (lambda(ex) 330 nm, lambda(em) 570 nm) and photodiode array, leading to good selectivity and precision in determining peak purity. A simple and quick sample preparation protocol was elaborated involving a methanolic extraction for the measurement of intracellular concentrations of the alkaloids and a solid phase extraction for their quantification in culture medium. Owing to the non-availability of commercially standards, a method for the purification of chelirubine, macar pine and chelilutine by semi-preparative HPLC was developed. Coupled together, the isolation method and the analytical method were highly reliable for screening the alkaloids of interest produced by E. califomica.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

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.