Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10–2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from −2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Sensitive and specific method for the determination of josamycin in human plasma by liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of josamycin in human plasma by LC–MS was developed and validated. Josamycin was extracted from human plasma by a single-step liquid–liquid extraction and analyzed by LC–MS via an electrospray ionization interface. Selected ion monitoring was used to detect josamycin and its internal standard. The intra-day precision and accuracy, expressed as C.V. and R.E., ranged from 2.8% to 13.5% and −10.3% to 7.6%, respectively. The lower limit of detection was 0.1 ng/ml and the lower limit of quantitation was set at 1 ng/ml when 0.5 ml of plasma was used. No endogenous interference was observed in human plasma obtained from drug-free volunteers.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.     

Quantitation of promethazine and metabolites in urine samples using on-line solid-phase extraction and column-switching

A chromatographic method for the quantitation of promethazine (PMZ) and its three metabolites in urine employing on-line solid-phase extraction and column-switching has been developed. The column-switching system described here uses an extraction column for the purification of PMZ and its metabolites from a urine matrix. The extraneous matrix interference was removed by flushing the extraction column with a gradient elution. The analytes of interest were then eluted onto an analytical column for further chromatographic separation using a mobile phase of greater solvent strength. This method is specific and sensitive with a range of 3.75–1400 ng/ml for PMZ and 2.5–1400 ng/ml for the metabolites promethazine sulfoxide, monodesmethyl promethazine sulfoxide and monodesmethyl promethazine. The lower limits of quantitation (LLOQ) were 3.75 ng/ml with less than 6.2% C.V. for PMZ and 2.50 ng/ml with less than 11.5% C.V. for metabolites based on a signal-to-noise ratio of 10:1 or greater. The accuracy and precision were within ±11.8% in bias and not greater than 5.5% C.V. in intra- and inter-assay precision for PMZ and metabolites. Method robustness was investigated using a Plackett–Burman experimental design. The applicability of the analytical method for pharmacokinetic studies in humans is illustrated.     

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.     

Gas chromatographic–mass spectrometric analysis of perillyl alcohol and metabolites in plasma

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1′-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC–MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC–MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH₂Cl₂ extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC–MS using NH₃ chemical ionization. POH-d₃ was used as the internal standard for POH while ¹³C-PA-d₂ was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1–2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m² p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.     

Quantitation of the anticonvulsant cinromide (3-bromo-n-ethylcinnamamide) and its major plasma metabolites by thin-layer chromatography

A quantitative thin-layer chromatography (TLC) procedure is described for the analysis of cinromide (3-bromo-N-ethylcinnamamide) and its two major metabolites, 3-bromocinnamamide and 3-bromocinnamic acid in plasma of the dog. These compounds were recovered from acidified plasma by extraction into benzene with a recovery of 95 ± 5%. All three compounds were quantitated directly on a TLC plate by ultraviolet absorbance densitometry at 270 nm. The linear dynamic range for the quantitation of the compounds on a TLC plate ranged between 10 and 1000 ng. The complete procedure is useful in the working range of 50 ng/ml to 100 μg/ml of plasma with a coefficient of variability of about 10%. Specificity of the method for parent drug and each of its plasma metabolites was confirmed by high-performance liquid chromatography. The method was used to determine the pharmacokinetics of cinromide and its two major plasma metabolites in dogs following a single oral dose of the drug.     

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity     

Method development and validation of a high-performance liquid chromatographic method for tramadol in human plasma using liquid-liquid extraction

An HPLC system using a simple liquid-liquid extraction and HPLC with UV detection has been validated to determine tramadol concentration in human plasma. The method developed was selective and linear for concentrations ranging from 10 to 2000 ng/ml with average recovery of 98.63%. The limit of quantitation (LOQ) was 10 ng/ml and the percentage recovery of the internal standard phenacetin was 76.51%. The intra-day accuracy ranged from 87.55 to 105.99% and the inter-day accuracy, 93.44 to 98.43% for tramadol. Good precision (5.32 and 6.67% for intra- and inter-day, respectively) was obtained at LOQ. The method has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.     

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36±12.53% and 93.52±7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma by liquid chromatography

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <+/-20%). The method was validated for human plasma in the concentration range 10-2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.     

Application of liquid chromatography–turbo ion spray tandem mass spectrometry for quantitative analysis of a potent motilin receptor agonist,EM574, and its metabolites in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the simultaneous determination of a new potent motilin receptor agonist as erythromycin derivative, EM574 (erythromycin derivative), and its three metabolites, M-IV, M-V and M-VI, in human plasma was developed. The internal standards (I.S.s) used were deuterated EM574, M-IV and M-V. For the quantitation of M-VI, deuterated M-V was used. The analytes and I.S. were extracted from plasma samples with diethyl ether at neutral pH. A turbo ion spray interface was used as the ion source of LC–MS–MS, and the analysis was performed in the selected reaction monitoring mode. The lower quantitation limits for all the analytes were 0.05 ng/ml when 0.2 ml of plasma was used, and the standard curves were linear in the range 0.05 to 20 ng/ml. The method was precise; the intra- and inter-day precisions of the method were not more than 19.8% for all the analytes. The accuracy of the method was good with the deviations between added and calculated concentrations of each analyte being typically within ±11.2%.