High-performance liquid chromatographic methods for the analysis of haloperidol and chlorpromazine metabolism in vitro by purified cytochrome P450 isoforms

Sensitive HPLC methods for the resolution and quantitation of metabolites of both haloperidol and chlorpromazine metabolism have been developed for use in in vitro reconstitution assays with purified P450 isoforms. Separation of haloperidol metabolites was accomplished using a Hypersil CPS column with a mobile phase of 67% acetonitrile and 10 mM ammonium acetate, pH 5.4. Separation of chlorpromazine metabolites was achieved using an Ultrasphere cyano column with a mobile phase of 87.5% acetonitrile, 5% methanol, 3% 0.12 M sodium acetate, 3% 0.12 M ammonium acetate, 0.01% diethylamine and 0.01% triethylamine, pH 9.5. Sharp resolution was observed for haloperidol and three of its major metabolites and for chlorpromazine and five of its major metabolites. Varying levels and combinations of these metabolites are formed during in vitro incubations of parent compound with purified P450 isoforms 1A1 and 2B1 in a reconstituted system.     

High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP⁺). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Analysis of perillic acid in plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO₃. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C₁₈ column and the effluent monitored for UV absorption at the analytes' respective UV_max. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 μg/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.     

Reversed-phase high-performance liquid chromatography analysis of 6-thioguanine applicable to pharmacologic studies in humans

6-Thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic method. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50–100% recovery efficiency. The neutralized extracts were chromatographed on a μBondapak C₁₈ column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5–10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate—25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.     

A preliminary pharmacokinetic study of the enantiomers of the terfenadine acid metabolite in humans

A stereoselective and sensitive achiral/chiral method for the determination of terfenadine acid metabolite in human plasma was developed. The metabolite was separated and quantitated using an achiral chromatographic procedure with a cyano column. The mobile phase was 1 mM sodium acetate buffer (pH 4.0) and acetonitrile (25:75% v/v) at a flow rate of 2 ml/min, at ambient temperature. The stereospecific resolution was accomplished using a chiral-AGP column and a mobile phase consisting of sodium acetate (0.01 M): methanol (98.7:1.3% v/v), and 20 mM di-n-butylamine at a flow rate of 1.2 ml/min. The column temperature was maintained at 32°C. The eluent was monitored at 230 nm (excitation) and 300 nm (emission) with a cut-off filter at 270 nm. This assay was used for a pharmacokinetic study in five subjects after administration of a single dose of 60 mg of terfenadine. The t_½ values of the two enantiomers were similar, but the AUC values of the (+)-enantiomer were 2.05–2.35 times higher than those of (?)-enantiomer. © 1994 Wiley-Liss, Inc.     

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

High-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil in cattle muscle tissue with on-line anion-exchange clean-up

A high-performance liquid chromatographic method for the determination of the anthelmintic nitroxynil has been developed. The drug was extracted from cattle muscle tissue with 1% triethylamine in acetonitrile. The extract was evaporated to dryness and taken up in 0.1 M ammonium acetate—acetonitrile (50:50, v/v). The extract was then injected onto a polymeric anion-exchange precolumn. After clean-up with 0.1 M ammonium acetate—acetonitrile (50:50, v/v) for 5 min, the precolumn was eluted with 1% aqueous trifluoroacetic acid—acetonitrile (50:50, v/v) onto a PLRP-S polymer column and chromatographed with a mobile phase of 0.01 M phosphate pH 7—acetonitrile (80:20, v/v). Detection was by ultraviolet at 273 nm. Average recoveries at four levels from 0.005 to 1.000 mg kg⁻¹ were > 88%. The limit of determination was 0.005 mg kg⁻¹.     

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.     

Sensitive high-performance liquid chromatographic assay for motexafin gadolinium and motexafin lutetium in human plasma

We present new HPLC methods for the quantitation in human plasma of two investigative metallotexaphyrin agents, motexafin gadolinium (Gd-Tex) and motexafin lutetium (Lu-Tex). Each assay uses: the other texaphyrin analogue as an internal standard; protein precipitation with acetonitrile:methanol (50:50, v/v); an ODS reversed-phase column; an isocratic mobile phase of 100 mM ammonium acetate, pH 4.3:acetonitrile:methanol (59:21:20, v/v/v); and absorbance detection at 470 nm. The Gd-Tex assay has a lower limit of quantitation (LLOQ) of 0.01 μM and is linear between 0.01and 30 μM. The Lu-Tex assay has an LLOQ of 0.1 μM and is linear between 0.1 and 30 μM. The assays are suited for in vivo preclinical studies and clinical trials because they require minimal amounts of plasma, are sensitive, and involve a 30-min run time. These assays are important tools for evaluating the potential of Gd-Tex and Lu-Tex as a radiation enhancer and photosensitizer, respectively.     

Elimination of caffeine interference in high-performance liquid chromatographic determination of cotinine in human plasma

A high-performance liquid chromatographic method with ultraviolet photometric detection for the determination of cotinine in human plasma was described. The use of a 30-cm reversed-phase column and of a mobile phase consisting of water—methanol—0.1 M sodium acetate—acetonitrile (72:21:5.6:1.4, v/v), pH 4.1, eliminated caffeine interference. A simplified solid-phase extraction procedure was also performed for plasma samples.     

Simple method for the simultaneous analysis of pipecolic acid and lysine by high-performance liquid chromatography and its application to rumen liquor and plasma of ruminants

A high-performance liquid chromatography method for the simultaneous determination of pipecolic acid (Pip) and lysine (Lys), a precursor of Pip, in the rumen liquor and plasma of ruminant animals was established. Samples of rumen liquor and plasma were deproteinized with 50% acetonitrile and derivatized with a fluorescent agent 9-fluorenylmethyloxy carbonyl chloride (Fmoc-Cl). Chromatographic separation was achieved on a TSK gel ODS-80TM column using a reversed-phase gradient elution system. For the gradient elution, two mobile phases, A and B, were needed, both commonly consisted of: 5 mM l-proline, 2.5 mM cupric sulfate and 6.5 mM ammonium acetate. Mobile phase B additionally contains 50% (v/v) acetonitrile. The pH of both mobile phases was adjusted to 7.0. Derivatized Pip and Lys were detected on a fluorescent detector at excitation and emission wavelengths of 260 and 313 nm, respectively. The calibration curves were linear within the range 0 to 1 mM (r>0.999). The average recoveries for Pip and Lys were 95.9±1.8 and 93.2±2.5% in rumen liquor and 98.3±1.4 and 97.5±1.3% in plasma, respectively. The limits of detection for Pip and Lys were 0.6 and 0.7 μM in rumen liquor and 0.01 and 0.05 μM in plasma. The assay has acceptable precision, relative standard deviation (RSD) for reproducibility (within-day and day-to-day variation) were less than 5.2% for aqueous (5.0 μM Pip and Lys), MB9 (5.0 μM Pip and Lys), plasma (7.1 μM Pip and 85.6 μM Lys) and rumen liquor (28.4 μM Pip and 10.2 μM Lys) samples. The levels of Pip and Lys in faunated goats, determined from three animals over a period of two days sampling, were found to be 36.8±18.1 and 14.6±2.8 μM in rumen liquor, and 7.3±2.5 and 137.3±38.0 μM in plasma at 1 h after feeding. This is the first report on the normal levels of Pip in the rumen liquor and plasma of faunated goat.     

High-performance liquid chromatographic method for the analysis of imipramine metabolism in vitro by liver and brain microsomes

A new sensitive method for resolution and quantitation metabolites of in vitro imipramine metabolism has been developed for use in liver and brain microsomes. Separation of metabolites was done using a Supelcosil PCN column with a mobile phase of acetonitrile-methanol-potassium phosphate dibasic (40:35:25, v/v/v), pH 7. Resolution is achieved for 2- and 10-hydroxyimipramine, didesmethylimipramine, and desipramine. Varying levels of these metabolites formed during in vitro incubations of rat liver and brain microsomes following treatments.     

Use of electrospray ionization liquid chromatography-mass spectrometry to study the role of CYP2D^ in the in vitro metabolism of 5-hydroxytryptamine receptor antagonists

An electrospray ionization liquid chromatographic-mass spectrometric (ESI-LC-MS) method has been developed to study the involvement of the cytochrome P450 isoenzyme CYP2D6 in the in vitro metabolism of the indole containing 5-hydroxytryptamine (5-HT₃) receptor antagonists tropisetron, ondansetron and dolasetron in human liver microsomes. Compounds were eluted using linear gradients of acetonitrile-20 mM ammonium acetate, solvent A, (10:90, v/v) (ph 6.0) and solvent B, (60:40, v/v) (pH 6.0) and a Nucleosil C₄ column. Microsomal incubations were analysed using selected ion monitoring of the molecular ion of parent drug and the molecular ion of hydroxylated metabolites. The involvement of CYP2D6 in drug metabolism was assessed by inhibition studies using quinidine (5 μM), a specific inhibitor of human CYP2D6, as well as by incubating compounds with microsomes prepared from celss transfected with cDNA encoding human CYP2D6. Results showed that the oxidation of all three compounds involved CYP2D6, but only that of tropisetron was inhibited by over 90% in the presence of quinidine. The present method can be applied to pre-clinical compounds, at an early stage of drug discovery, to assess the involvement of CYP2D6 in their metabolism and to screen for those compounds where CYP2D6 is the only isoenzyme implicated in the formation of major metabolites.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Routine and sensitive method for determination of nicorandil in human plasma developed for liquid chromatography with ultraviolet and m0ass spectrometric detection

A rapid and sensitive method using HPLC has been developed for the quantification of nicorandil (SG-75) in human plasma samples for routine bioequivalence studies. The sample preparation needs two liquid–liquid extractions, first with CH₃Cl and HClO₄ as denaturation reagent and second with addition of ethyl acetate and Na₂CO_3(aq). Detection wavelength was 256 nm. The obtained correlation coefficient for weighted linear curve in the range from 5.0 to 300 ng/ml was higher than 0.9950. The limit of quantitation (LOQ) was established at 5.0 ng/ml. The HPLC separation was accomplished on Nucleosil Phenyl (5 μm) stainless steel column within 7 min. The mixture of 0.01 M ammonium acetate buffer (pH 6.2) and acetonitrile 10:3 (v/v) was used as the mobile phase. The same separation method was examined on HPLC–MS system. Using this system, the LOQ was established at 1.0 ng/ml and the linearity was obtained in the range from 1.0 to 150 ng/ml.     

Determination of the covalent adducts of the novel anti-cancer agent 5,6-dimethylxanthenone-4-acetic acid in biological samples by high-performance liquid chromatography

The reversed-phase HPLC methods were developed to determinate the covalently bound protein adducts of the novel anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) via its glucuronides after releasing aglycone by alkaline hydrolysis in human plasma and human serum albumin (HSA). An aliquot of 75 μl of the mixture was injected onto a Spherex C₁₈ column (150×4.6 mm; 5 μm) at a flow-rate of 2.5 ml/min. The mobile phase comprising of acetonitrile:10 mM ammonium acetate buffer (24:76, v/v, pH 5.8) was used in an isocratic condition, and DMXAA was detected by fluorescence. The method was validated with respect to recovery, selectivity, linearity, precision, and accuracy. Calibration curves for DMXAA were constructed in the concentration range of 0.5–40 μM in washed blank human plasma or HSA prior to alkaline hydrolysis. The difference between the theoretical and calculated concentration and the relative standard deviation were less than 10% at all quality control (QC) concentrations. The limit of detection for the covalent adduct in human plasma or HSA is 0.20 μM. The methods presented good accuracy, precision and sensitivity for use in the preclinical and clinical studies.     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.