Chiral separation of 10,11-dihydro-10,11-trans-dihydroxycarbamazepine, a metabolite of carbamazepine with two asymmetric carbons, in human serum

Chiral separation of 10,11-dihydro-10,11-trans-dihydroxycarbamazepine (CBZ-diol), a metabolite of carbamazepine (CBZ) with two asymmetric carbons, in serum taken from epileptic patients receiving CBZ alone for a long period, was performed by high-performance liquid chromatography using a polysaccharide stationary phase with n-hexane-ethanol (75:25, v/v) as the mobile phase. The enantiomeric ratio (S,S-/R,R-CBZ-diol) was 10.74 ± 1.13 (mean ± S.D.), which could demonstrate the presence of the stereospecificity in the hydrolysis of 10,11-dihydro-10,11-epoxycarbamazepine (CBZ-epoxide) to CBZ-diol and/or in the conversion of CBZ-diol to some metabolite such as 9-hydroxymethyl-10-carbamoylacridan. This is the first paper to report the determination of each enantiomer and the enantiomeric ratio of CBZ-diol in serum of epileptic patients who received CBZ.     

High-speed liquid chromatographic method for the monitoring of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma

The assays of antiepileptic drugs, which are performed by central laboratories in Phase II and III clinical trials, require both a very fast turn-around time and a suitable specificity. In order to decrease the run time and to keep the powerful specificity of the liquid chromatography (HPLC), the use of a reversed-phase 1.5 μm monosized non-porous silicon dioxide microspheres column instead of regular columns containing spherical porous C₁₈ material was studied. The determination of carbamazepine (CBZ) and its active metabolite, carbamazepine-10,11-epoxide (CBZ-E), in human plasma or serum was chosen to demonstrate the utility of these columns. As a prerequisite of this work, no modification of a regular HPLC system was allowed. The samples were prepared in autosampler vials by protein precipitation with acetonitrile, followed by a quick centrifugation. Without any change to a conventional HPLC system, CBZ and CBZ-E are well separated in less than 2.5 min using a Kovasil MS C₁₄ column. No interference was observed with endogenous compounds and with nine antiepileptic drugs commonly prescribed as co-medication, and their metabolites. Due to the very low specific surface area of the packing, the required organic modifier volume per chromatographic run was decreased by a factor of 25. The method was validated. The developed method is well suited for the determination of CBZ and CBZ-E in clinical trials. It can be easily adapted to the monitoring of other antiepileptic drugs. No modification of a regular HPLC system was required.     

High-performance liquid chromatographic determination of carbamazepine and metabolites in human hair

To study the use of hair analysis in monitoring drug compliance and historical changes in pharmacokinetics we developed a method for the quantitative determination of the anti-epileptic drug carbamazepine (CBZ) and trans-10,11-dihydro-10,11-dihydroxy-carbamazepine (CBZ-diol) in hair from carbamazepine users. Digestion by 1 M NaOH was found to be the best method for isolating CBZ and CBZ-diol from hair, followed by solid-phase extraction and reversed-phase HPLC with UV detection. Recoveries from spiked hair samples were 76–86%. Within-day precision (C.V.; n=10) for CBZ and CBZ-diol in hair of a CBZ user containing 10.9 μg/g CBZ and 3.2 μg/g CBZ-diol were 1.7 and 5.0%, respectively. Sectional hair analysis of a patient on a constant dosage of CBZ demonstrates an exponential decrease in hair concentrations of CBZ and CBZ-diol with increasing distance from the root, probably caused by shampooing. No CBZ-10,11-epoxide (CBZ-epox) could be detected. However, one component in the chromatogram is probably CBZ-β-hydroxythioether, an adduct of CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product. The concentration of this component does not decrease with increasing distance from the root.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

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.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Solid-phase extraction and high-performance liquid chromatographic determination of articaine and its metabolite articainic acid in human serum

A new method is described using solid-phase extraction (SPE) for preconcentration of articaine and the metabolite articainic acid and high-performance liquid chromatography (HPLC) for the determination of both compounds in human serum. Articaine and articainic acid were extracted in one step with SDB-RPS disk cartridges after precipitation of the serum proteins by perchloric acid. The HPLC separation was then performed on a reversed-phase C₈ column using phosphate buffer–acetonitrile (88:12, v/v). UV absorption at 274 nm was used for measuring the analytes with a low limit of quantitation of about 10 ng/ml, which is appropriate for pharmacokinetic studies of low dose submucosal injections of the local anaesthetic agent articaine hydrochloride in dentistry.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces. Both compounds are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. Separate HPLC systems have been developed for the determination of lactone and total (lactone plus hydroxycarboxylate forms) concentrations in plasma. The instability of the analytes in plasma requires immediate protein precipitation with ice-cold methanol. The lactone forms of the analytes were stable in the methanol extracts for at least 15 months when stored at −70°C. For the determination of the total levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample pretreatment procedure for urine included dilution in methanol while the faecal samples were homogenized in distilled water and then extracted twice with an acetonitrile–ammonium acetate mixture. Separation was achieved on reversed-phase columns (Zorbax SB-C18) and detection was performed fluorimetrically at 380/527 nm. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%. The methods were used in a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopotecan.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylarniodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether—acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol—acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C₁₈ column operating in combination with a radial compression separation unit and a methanol–25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay.The limit of sensitivity of the assay was 0.025 μg/ml with a precision of ± 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Simultaneous liquid chromatographic determination of doxorubicin and its major metabolite doxorubicinol in parrot plasma

A new microscale method is reported for the determination of doxorubicin and its active metabolite, doxorubicinol, in parrot plasma. Sample workup involved acetonitrile protein precipitation, ethyl acetate extraction, followed by back extraction into HCl. Separations were achieved on a phenyl-hexyl column at 30 degrees C using acetonitrile (17%, v/v) in 0.01 M orthophosphoric acid (83%, v/v) delivered via a linear flow program. Fluorometric detection wavelengths were 235 nm (excitation) and 550 nm (emission). Calibration plots were linear (r2>0.999), and recoveries were 71-87% from 20 to 400 ng/mL. Assay imprecision was 相似文献   

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.