Achiral and chiral high-performance liquid chromatographic determination of tramadol and its major metabolites in urine after oral administration of racemic tramadol

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of tramadol and its major metabolites O-demethyltramadol and N-demethyltramadol in urine has been developed. The determination of the enantiomeric ratios of the three compounds was achieved using a Chiralpak AD column and a Chiralcel OD column, respectively. After oral administration of racemic tramadol to five healthy volunteers, inter-individual differences of the excreted amounts and the enantiomeric ratios of the compounds were observed.     

Enantiomeric separation of tramadol and its active metabolite in human plasma by chiral high-performance liquid chromatography: application to pharmacokinetic studies

A sensitive and stereoselective high-performance liquid chromatographic assay for the quantitative determination of the analgesic tramadol and O-demethyltramadol, an active metabolite, is described in this work. Ketamine was used as internal standard. The assay involved a single tert-butymethylether extraction and liquid chromatography analysis with fluorescence detection. Chromatography was performed at 20 degrees C on a Chiracel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as stationary phase, preceded by an achiral end-capped C18 column. The mobile phase was a mixture of phosphate buffer (containing sodium perchlorate (0.2 M) and triethylamine (0.09 M) adjusted to pH 6) and acetonitrile (80:20). The method developed was validated. The limit of quantitation of each enantiomer of tramadol and its active metabolite by this method was 0.5 ng/mL; only 0.5 mL of the plasma sample was required for the determination. The calibration curve was linear from 0.5 to 750 ng/mL for tramadol enantiomers, and from 0.5 to 500 ng/mL for O-demethyltramadol enantiomers. Intra and interday precision [coefficient of variation (CV)] did not exceed 10%. Mean recoveries of 95.95 and 97.87% for (+)R,R- and (-)S,S-tramadol and 97.70 and 98.79% for (+)R,R- and (-)S,S-O-demethyltramadol with CVs < 2.15% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study in normal volunteers who received 100 mg of tramadol by the intravenous route.     

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 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.     

Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry

A quantitative liquid chromatography positive ion electrospray tandem mass spectrometric method for the simultaneous determination of sulforaphane, iberin and their metabolites in human urine and plasma is described. The stability of the metabolites was determined in aqueous solution and in human plasma. Gradient liquid chromatographic separation was performed on a Zorbax SB-Aq 3.5 microm (100 x 2.1mm) column, using a mobile phase (flow rate 0.25 mL/min) consisting of ammonium acetate buffer at pH 4 and acetonitrile. Butyl thiocarbamoyl l-cysteine was used as internal standard. The assay was linear (r(2)>0.99) over the range of 0.03-300 microM in urine and 0.03-15 microM in plasma with intra- and inter-day assay precision (<10% CV) and accuracy (<20%). The lower limits of quantitation were in the range of 10-150 nmol/L. The method has been used to report, for the first time, individual quantitative measurement of each of the mercapturic acid pathway metabolites of sulforaphane and iberin in both human plasma and urine following a dietary study of broccoli consumption.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching : I. Determination of isotretinoin and tretinoin and their 4-oxo metabolites in plasma

A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17x4.6 mm I.D.), filled with C₁₈ Corasil 37–53 μm. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-1000 ng/ml was between 1.0 and 4.7% for all compounds.     

Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction-liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol-glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol-water (75:25, v/v) containing 0.025% NH(4)OH. Chromatography of glucuroconjugate metabolites and phenyl-beta-d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10-1500 ng mL(-1) for propofol and 1-QG, 20-3000 ng mL(-1) for PG and 25-3750 ng mL(-1) for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias < or =6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.     

Detection and determination of the major metabolites of [3H]cytosine arabinoside by high-performance liquid chromatography

An ion-pair high-performance liquid chromatographic assay involving solid-phase scintillation detection was established for the rapid identification and determination of all major metabolites of tritium-labelled cytosine arabinoside (Ara-C) in an in vitro system. In a single run of 50 min, Ara-C, Ara-CMP, Ara-CDP-choline, Ara-CDP, Ara-U, Ara-UMP, Ara-CTP, Ara-UDP can be measured. The method is fast, sensitive, with limits of detection ranging from 40 to 200 pg (absolute), and highly reproducible.     

Optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma

The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid–liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 °C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ = 4.078 ng/ml for tramadol, respectively LLOQ = 3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV% = 5.147% and bias% = − 7.273% in the intra-days and CV% = 4.894% and bias% = 0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV% = 11.517% and bias% = 0.337% in the intra-days and CV% = 6.41% and bias% = 3.259% in the between-days assay.

In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at − 20 °C, but also for 48 h at 15 °C in the re-eluted solution after liquid–liquid extraction.     

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.     

Evaluation of the stereoselective metabolism of the chiral analgesic drug etodolac by high-performance liquid chromatography

The enantiomers of the racemic analgesic drug etodolac have been resolved by fractional crystallization of the diastereomeric salts with optically active 1-phenylethylamine. A high-performance liquid chromatographic method to determine racemic etodolac (assay I) and its major metabolites (assay II) in urine using a conventional reversed-phase column is described. The determination of the enantiomeric ratios of etodolac and the two metabolites 7-hydroxyetodolac and 8-(1′-hydroxyethyl)etodolac was achieved using different protein-bonded chiral stationary phases. The urinary data for five volunteers are presented and show a marked stereoselectivity of the metabolism of etodolac in humans.     

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.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

Analysis of hydroxylated and N-dealkylated metabolites of terfenadine in microsomal incubates by liquid chromatography–mass spectrometry

This report describes an assay for the H₁-receptor antagonist, terfenadine, and its two primary metabolites, terfenadine alcohol (TOH) and azacyclonol (AZ), using positive-ion, electrospray ionization–liquid chromatography–mass spectrometry. The assay was developed in support of kinetic studies of terfenadine oxidative metabolism in human liver and intestinal microsomes, which required quantification of incubate metabolites at low nanomolar concentrations. Terfenadine metabolites were extracted from basified microsomal incubates into methylene chloride. Reconstituted extracts were subject to liquid chromatographic separation on a cyano-reverse phase column. The [M+H]⁺ ions of terfenadine, terfenadine metabolites, and internal standard were monitored in the effluent by quadrupole mass spectrometry. The assay demonstrated linearity over an incubate concentration range of 5–250 and 12.5–1250 ng/ml for the metabolites and the parent drug, respectively. The respective limits of detection and quantitation for all three analytes were 1.5 and 5 ng/ml of microsomal incubate. Replicate analysis of quality control samples exhibited intra-day coefficients of variation ranging from 3.3% to 7.8% for the three analytes. The corresponding inter-day coefficients of variation ranged from 4.2% to 8.6%. The reproducibility and sensitivity of the assay, combined with the selectivity of mass spectrometric detection, should allow an accurate kinetic characterization of terfenadine oxidation mediated by the high affinity CYP3A enzymes in human liver and intestinal microsomes.     

Simultaneous automatic determination of catecholamines and their 3-O-methyl metabolites in rat plasma by high-performance liquid chromatography using peroxyoxalate chemiluminescence reaction

A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automated precolumn ion-exchange extraction of diluted plasma is coupled with HPLC separation of CAs and their 3-O-methyl metabolites on an ODS column, postcolumn coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally peroxyoxalate chemiluminescence reaction detection. The detection limits were about 3 fmol for norepinephrine, epinephrine, and dopamine, 5 fmol for normetanephrine, and 10 fmol for metanephrine and 3-methoxytyramine (signal-to-noise ratio of 3). Fifty microliters of rat plasma was used and 4-methoxytyramine was employed as an internal standard. The relative standard deviations for the method (n = 5) were 2.5-7.6% for the intraday assay and 6.3-9.1% for the interday assay. The method was applicable to the determination of normetanephrine and metanephrine in 50 microl of rat plasma.     

Rapid chiral high-performance liquid chromatographic assay for salmeterol and α-hydroxysalmeterol: Application to in vitro metabolism studies

A rapid and sensitive chiral high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of salmeterol and its principal human metabolite α-hydroxysalmeterol is described. The two pairs of enantiomers were resolved on a chiral-cellobiohydrolase column and detected by electrochemical detection at +700 mV. Standard curves were linear over the concentration range 0.1 to 4.0 μM for α-hydroxysalmeterol enantiomers and 2.5 to 40.0 μM for salmeterol enantiomers. Intra- and inter-day coefficients of variation were <10%. The method was applied to a study of human hepatic metabolism in vitro which showed that microsomal metabolism of salmeterol to α-hydroxysalmeterol is not stereoselective.     

Detection of NAD:arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. and Romig, W.R. (1979) J. Biol. Chem. 254, 5849-5854) was utilized to identify NAD:arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and 125I-labeled guanyltyramine as ADPribose acceptor, formation of ADPribosyl 125I-guanyltyramine was linear with time and enzyme concentration. The product was indistinguishable on both thin-layer and high-performance liquid chromatography from that formed by choleragen. Using 125I-guanyltyramine, ADPribosyltransferase activity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase product by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free 125I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl 125I-guanyltyramine and other metabolites of 125I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl 125I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, 125I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH₂ bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 μg/ml for plasma, 3 μg/ml for urine and 0.03 μg/ml for saliva using UV detection.     

Quantitative determination of 1-hexylcarbamoyl-5-fluorouracil and its metabolites in man

A high-performance liquid chromatographic (HPLC) method for the quantitative determination of 1-hexylcarbamoyl-5-fluororacil (HCFU) and its metabolites using μBondapak C₁₈ and μPorasil has been developed. Two mobile phases containing PIC-B7 (consisting of acetic acid and 1-heptanesulphonic acid) were used for the separation, and good separations were obtained. With methanol-water (56:44) as the mobile phase, the separation of HCFU and its three metabolites was achieved within 4 min. With methanol-water (32:68) a new metabolite, 1-υ-carboxymethylcarbamoyl-5-fluororacil, was revealed in human plasma. The recovery of each substance was 80% or greater and the sensitivity was at the nanograms per millilitre level. The coefficient of variation was less than 3.6% for each component.     

Liquid chromatographic method with ultraviolet absorbance detection for measurement of levamisole in chicken tissues, eggs and plasma

The development and validation of a high-performance liquid chromatographic and UV detection method was accomplished for quantitative determination of levamisole in chicken tissues, eggs and plasma. The chromatographic separation was achieved on Luna 5 microm C(18) column using a mobile phase of 0.2% acetic acid in water:methanol (50:50 (v/v)) and Pic B-7 low UV reagent and the pH was adjusted to 7.31 with ammonium hydroxide and UV wavelength was 225 nm. Limits of quantification were 0.025 microg/g for all tissues and 0.003 microg/ml for plasma. Limit of detection was 0.001 microg/g for tissues and plasma.