Determination of the enantiomers of chlorpheniramine and its main monodesmethyl metabolite in urine using achiral–chiral liquid chromatography

The enantiomers of chlorpheniramine and its monodesmethyl metabolite were determined separately in urine by using a coupled achiral–chiral chromatographic system. The two enantiomers of the studied compound and the internal standard were separated from the biological matrix on a cyanopropyl column and reinjected into a chiral amylose AD column where the two enantiomers were separated and quantified by UV detection. The method was validated for chlorpheniramine and for the metabolite within the range 0–1000 ng/ml. It was also applied in a pilot pharmacokinetic study to samples from a volunteer given 8 mg of racemic chlorpheniramine by mouth.     

Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography

A coupled achiral-chiral liquid chromatographic assay has been developed to determine the concentrations of metyrapone and the enantiomers of its chiral metabolite metyrapol in plasma and urine. The chromatographic system consisted of a silica precolumn (75 × 4.6 mm I.D.) coupled in-line to a 250 × 4.6 mm I.D. column containing cellulose tris(4-methylbenzoate) coated on silica gel (Chiralcel OJ-CSP). When plasma samples were analyzed, the mobile phase was hexane-ethanol (92:8, v/v) modified with 0.1% diethylamine and when urine samples were analyzed the mobile phase was hexane-ethanol (94:6, v/v) modified with 0.2% diethylamine. Under these chromatographic conditions the chromatographic retentions [expressed as capacity factors (k′)] for metyrapone were k′ = 2.35 (plasma) and 2.52 (urine); for (−)-metyrapol k′ = 4.22 (plasma) and 4.62 (urine); for (+)-metyrapone k′ = 5.16 (plasma) and 5.86 (urine); enantioselectivities (α) were 1.09 (plasma) and 1.13 (urine). The assay has been validated for use in metabolic studies. The analyses of plasma and urine samples from one subject following oral administration of 750 mg of metyrapone indicated that the enzymatic reduction of myterapone by aldo-keto reductase was enantiospecific.     

Determination of zopiclone enantiomers in plasma by liquid chromatography using a chiral cellulose carbamate column

The enantiomers of zopiclone were determined in human plasma using a sequential achiral—chiral liquid chromatographic method. Zopiclone was separated from the biological matrix and quantified on an achiral silica column. The limit of detection was 5 ng/ml. The eluent fraction containing zopiclone was collected, evaporated, reconstituted with the mobile phase and injected onto a chiral cellulose carbamate column where the enantiomeric ratio was calculated. This validated method, applied to a pilot study, suggests that pharmacokinetics of zopiclone is stereoselective.     

Analysis of the enantiomers of citalopram and its demethylated metabolites using chiral liquid chromatography

A procedure using a chirobiotic V column is presented which allows separation of the enantiomers of citalopram and its two N-demethylated metabolites, and of the internal standard, alprenolol, in human plasma. Citalopram, demethylcitalopram and didemethylcitalopram, as well as the internal standard, were recovered from plasma by liquid–liquid extraction. The limits of quantification were found to be 5 ng/ml for each enantiomer of citalopram and demethylcitalopram, and 7.5 ng/ml for each enantiomer of didemethylcitalopram. Inter- and intra-day coefficients of variation varied from 2.4% to 8.6% for S- and R-citalopram, from 2.9% to 7.4% for S- and R-demethylcitalopram, and from 5.6% to 12.4% for S- and R-didemethylcitalopram. No interference was observed from endogenous compounds following the extraction of plasma samples from 10 different patients treated with citalopram. This method allows accurate quantification for each enantiomer and is, therefore, well suited for pharmacokinetic and drug interaction investigations. The presented method replaces a previously described highly sensitive and selective high-performance liquid chromatography procedure using an acetylated β-cyclobond column which, because of manufactural problems, is no longer usable for the separation of the enantiomers of citalopram and its demethylated metabolites.     

Enantioselective determination of zopiclone and its metabolites in urine by capillary electrophoresis

A method has been developed for the stereoselective determination of zopiclone and its main metabolites in urine. After the addition of the internal standard zolpidem the urine samples were extracted at pH 8 with chloroform-isopropanol (9:1). Analyses were carried out using capillary electrophoresis (CE) with β-cyclodextrin as the chiral selector. The analytes were detected using UV laser-induced fluorescence detection with a He-Cd laser operated at 325 nm. Urine samples of two volunteers after oral administration of 7.5 mg zopiclone were investigated. The S-(+)-enantiomers of zopiclone and its metabolites were always excreted in higher amounts than the R-(−)-enantiomers. With the same method the zopiclone enantiomers were quantified in saliva. Compared to high-performance liquid chromatography, the CE method is very fast and simple.     

Determination of the enantiomers of bunolol in human urine by high-performance liquid chromatography on a chiral AGP stationary phase and identification of their metabolites by gas chromatography—mass spectrometry

A high-performance liquid chromatographic method using a chiral AGP column was developed to screen and determine the enantiomers of bunolol in human urine. The recovery of (+)- and (−)-bunolol from urine was 91.79–95.23% at different concentrations. The coefficients of variation (C.V.) were less than 2.1 and 2.3% for intra- and inter-assays, respectively. Urinary metabolites were detected using GC—MS after derivatization with N-methyl(trimethylsilyl)trifluroacetamide. The influences of pH and modifier on a chiral AGP column were studied.     

Determination of the enantiomers of citalopram,its demethylated and propionic acid metabolites in human plasma by chiral HPLC

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5, and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and steroselectively metabolized to CIT-PROP. © 1995 Wiley-Liss, Inc.     

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic—mass spectrometric assay

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.     

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction-liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific automated SPE-LC-MS-MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C(18) solid-phase extraction cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile-water (50:50, v/v) containing 10 mM 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118, m/z 329-->196 and m/z 343-->196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1-200 ng/ml for I and II and 2-200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.     

Determination of salbutamol enantiomers in human plasma and urine by chiral high-performance liquid chromatography

Enantiomers of salbutamol were directly separated (R_s=1.16) and quantitated at therapeutic concentrations after solid-phase extraction from human plasma and urine by normal-phase high-performance liquid chromatography on a chiral column with fluorescence detection. The assay was linear for each enantiomer between 1.25 and 500 ng ml⁻¹ and had a minimum limit of detection of 250 pg ml⁻¹. A 3-ml plasma or 1-ml urine sample was required for quantitation at therapeutic doses. Inter-day variation was 50% for S-(+)- and 6.5% for R-(−)-salbutamol. The assay was used to compare enantioselective disposition after single doses of racemate by the intravenous, oral and rectal routes.     

Determination of amphetamine and methamphetamine enantiomers by chiral derivatization and gas chromatography–mass spectrometry as a test case for an automated sample preparation system

An automated sample preparation system has been applied to the chiral analysis of amphetamine and methamphetamine using derivatization with trifluoracetyl-L -prolyl chloride (L -TPC) and subsequent separation on a gas chromatography–mass spectrometry (GC-MS) system. Tasks automated were the dilution of standards and the off-line preparation of the diastereoisomer derivatives. Chromatographic performance, sensitivity, and reproducibility of the automated procedure were compared to the equivalent values obtained with two existing assays methods which employ manual derivatiation, either on-column or off-line. Chromatographic performance was unaffected by the derivatization procedure and sensitivity was better for both automated and manual off-line derivatization. Qualitative reproducibility as based on enantiomeric composition was equivalent for all three approaches, while quantitative reproducibility as based on peak areas was best for the automated procedure. Considering the fact that the diastereoisomer derivatives are unstable over time, automated sample preparation with “just-in-time” derivatization can increase the overall precision of the analytical method. The procedures described here are general enough in nature that they could be applied to other chiral or even achiral analytes. © 1994 Wiley-Liss, Inc.     

Liquid chromatographic determination of the enantiomers of ibuprofen in plasma using a chiral AGP column

A reversed-phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen. The enantiomers and the internal standard 4-pentylphenylacetic acid are extracted from plasma, separated and quantified on a Chiral-AGP column using ultraviolet detection. The simplicity, sensitivity and precision of the method makes it convenient for use in pharmacokinetic studies.     

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.     

Chromatographic analysis of the enantiomers of ifosfamide and some of its metabolites in plasma and urine

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil- -val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.     

Assay of moguisteine metabolites in human plasma and urine: conventional and chiral high-performance liquid chromatographic methods

Moguisteine is a novel peripheral non-narcotic antitussive agent. Pharmacokinetic studies in animal and in man showed that no unchanged drug is present in plasma, urine and faeces after oral administration. The main active metabolite, M1, is the free carboxylic acid of moguisteine, which maintains a stereogenic centre and consists of R(+)-M1 and S(−)-M1 enantiomers. M1 is partly metabolized to M2, its sulfoxidation derivative. A conventional HPLC method is described for the simultaneous determination of M1 and M2 in human plasma and urine after administration of therapeutic moguisteine doses. Plasma samples, previously acidified with phosphoric acid, are extracted with dichloromethane; urine samples are analyzed after appropriate dilution with methanol. Chromatography is performed using a Lichrosorb RP2 column and a linear gradient. M1 enantiomers can be determined in plasma extracts and urine samples by a chiral HPLC method using a β-cyclodextrin column. The analytical characteristics of both HPLC procedures proved to be adequate to analyze samples of subjects treated with therapeutic doses of moguisteine during clinical pharmacokinetic studies.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

Comparison of two high-performance liquid chromatographic methods for the determination of oxmetidine and its metabolites in plasma, bile and urine samples

Two high-performance liquid chromatographic methods for the assay of oxmetidine are described: both utilize the same liquid extraction from plasma, urine and bile samples. A normal-phase technique is considered most suitable for the analysis of plasma extracts and a reversed-phase method is preferred for the assay of excretory fluids such as urine and bile which will contain polar metabolites in detectable quantity as well as unchanged oxmetidine.The methods are sensitive enough to follow the kinetic changes in concentration for up to 8 h after the administration of recommended therapeutic doses. Both methods can be automated in respect of the high-performance liquid chromatograph and the samples can be stored for several weeks at −20°C without prejudicing the accuracy of the analysis.