Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photodiode-array detection

A high-performance liquid chromatographic method has been developed for the determination of tolbutamide and its metabolites in human plasma and urine. The compounds examined were extracted with diethyl ether from the acidified biological fluid. Chlorpropamide was used as internal standard, and 235 nm was chosen as the wavelength for diode-array detection. A study of the relationship between the capacity factor and the mobile phase composition and pH showed that acetonitrile-2-propanol-0.1% orthophosphoric acid (17: 17: 66, v/v) was the best eluent on a C8 reversed-phase column. The method is precise, sensitive and suitable for pharmacological investigations.     

Determination of exifone in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of exifone in human plasma and urine. Exifone was extracted from acidified plasma or neutralized urine with diethyl ether and the evaporated extracts were analysed on a C18 reversed-phase column. The compound was eluted in about 8 min with acetonitrile-0.3 M orthophosphoric acid (15:85, v/v) at a flow-rate of 0.9 ml/min. This method gave accurate and reproducible results; the calibration graphs were linear (r greater than 0.99) over the range of 2.8-360 nmol/l for plasma and 0.18-36 mumol/l for urine, and concentrations as low as 1 nmol/l in plasma could be quantified. These results allowed this assay to be used for determinations in single-dose pharmacokinetic studies.     

Specific HPLC Method for the Separation of Verapamil and Four Major Metabolites After Oral Dosing

Abstract

We have developed a high performance liquid chromatographic (HPLC) method which resolves verapamil, norverapamil, D620, D617 and what we believe to be another verapamil metabolite which has been previously unreported. An alkyl-phenyl column is used with a mobile phase of 0.005% sulfuric acid in methanol. The extraction recoveries of verapamil, norverapamil and the internal standard (imipramine) from plasma ranged between 98% and 104%. The day-to-day, and within-day coefficients of variations for verapamil and norverapamil at plasma concentrations of 7.3 and 233 ng/ml ranged between 1.7 and 6.1%. The limit of sensitivity was slightly less than 1 ng for both verapamil and norverapamil. Chromatograms of extracts of serum and urine obtained from five normal subjects who took single oral verapamil doses, indicated the presence of verapamil, norverapamil, and two other known metabolites. Chromatograms of serum extracts also indicated an additional peak which is probably another verapamil metabolite.     

Simultaneous determination of rofecoxib and celecoxib in human plasma by high-performance liquid chromatography

An innovative reversed-phase high-performance liquid chromatographic method is validated for the simultaneous determination of rofecoxib and celecoxib in human plasma. The internal standard is 4-n-pentyl-phenyl-acetic acid. Good chromatographic separation is achieved using a Zorbax SB-CN (5 microm) analytical column operated at room temperature and mobile phase consisting of acetonitrile and water containing 0.1M potassium dihydrogen orthophosphate buffer adjusted to pH 2.4 with 85% orthophosphoric acid (42:58, v/v). UV detection is performed at 254 nm, and the flow rate is maintained at 1.0 mL/min. Plasma samples are extracted into an organic solvent (1-chlorobutane) and evaporated under an air flow. The calibration curve for rofecoxib is linear over the range of 10 to 500 microg/L, and the celecoxib calibration curve is linear over the range of 20 to 2000 microg/L. The lower limit of quantitation for rofecoxib and celecoxib is 10 and 20 microg/L, respectively, using 1.0 mL of human plasma. The validation data show that the assay is sensitive, accurate, specific, and reproducible for the determination of rofecoxib and celecoxib. This method is therefore appropriate for pharmacokinetic studies to quantitate these therapeutic agents in patients with arthritis conditions.     

An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

High-performance liquid chromatographic determination of navelbine in MO4 mouse fibrosarcoma cells and biological fluids.

A high-performance liquid chromatographic method is described for separating and determining navelbine and possible metabolites in plasma, cell culture medium and MO4 cells. Navelbine is extracted from these fluids by ion-pair extraction with sodium octylsulphate as the counter-ion at pH 3. The system uses a cyano column as the stationary phase and a mobile phase of acetonitrile-0.12 M phosphate buffer (pH 3) (60:40, v/v). Application of the method to a study of the pharmacokinetic behaviour of navelbine in MO4 mouse fibrosarcoma cells is reported.     

High-performance liquid chromatographic determination of dimethylxanthine metabolites of caffeine in human plasma

A normal-phase high-performance liquid chromatographic assay of caffeine and its metabolites, theophylline, theobromine and paraxanthine, in human plasma is described. The two internal standards ethyltheophylline and 1,3,7-trimethyluric acid are used simultaneously and cover the range of different polarities from caffeine to the three dimethylxanthines. Plasma (0.5 ml) in the presence of ammonium sulphate is extracted with chloroform--isopropanol (1:1, v/v). The extract is chromatographed with a LiChrosorb Si 60 5-micron column and a mobile phase of dichloromethane containing 2.5% of a formate buffer in methanol. Calibration is performed with six different calibration mixtures which take into account the large plasma concentration differences between caffeine and its metabolites in man. The method is suitable for the simultaneous determination of caffeine and its dimethylxanthine metabolites in plasma of healthy and diseased persons.     

A common method for the determination of several calcium channel blockers using an HPLC system with ultraviolet detection

We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C₁₈ column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml⁻¹ for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1–1000 ng ml⁻¹. The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.     

Sensitive liquid chromatographic-tandem mass spectrometric method for the determination of fluoxetine and its primary active metabolite norfluoxetine in human plasma

A sensitive method for the simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from alkalised plasma with hexane-isoamyl alcohol (98:2, v/v) followed by back-extraction into formic acid (2%). Chromatography was performed on a Phenomenex Luna C18 (2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (340:660, v/v) at a flow-rate of 0.35 ml/min. Detection was achieved by a Perkin-Elmer Sciex API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recoveries for fluoxetine and norfluoxetine were 98 and 97%, respectively, with a lower limit of quantification set at 0.15 ng/ml for the analyte and its metabolite. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and sensitive method for the simultaneous determination of fluoxetine and norfluoxetine in human plasma than has previously been described.     

Effects of common metal ions on the determination of anions by suppressed ion chromatography

A rapid and sensitive method for the determination of domperidone in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometry detection. The samples were rendered basic with 1 M Na2CO3 and the domperidone extracted using tert.-butyl methyl ether, followed by back-extraction into formic acid (2% in water). Chromatography was performed on a Phenomenex Luna C8 (2), 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.02% formic acid (300:700, v/v), delivered at 0.2 ml/min. Detection was performed using an Applied Biosystems Sciex API 2000 mass spectrometer set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recovery of domperidone was +/- 100%, with a lower limit of quantification set at 0.189 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric detection resulting in a rapid (extraction and chromatography) and sensitive method for the determination of domperidone in human plasma, which is more sensitive than previously described methods.     

Synthesis and characterization of norverapamil and quantification of verapamil and nor-verapamil in plasma

Verapamil is a calcium channel blocking agent which has found widespread use in the management of supraventricular tachyarrhythmias, angina pectoris, hypertrophic cardiomyopathy and hypertension. It is converted to its biologically active metabolite nor-verapamil in liver by cytochrome P450. In present communication, synthesis and characterization of nor-verapamil and development of reverse phase high performance liquid chromatographic method for the quantification of nor-verapamil along with verapamil in plasma has been carried out. The characterization of nor-verapamil was carried out using GC-MS, FT-IR and NMR spectroscopy. The separation was carried out with an isocratic JASCO RP-HPLC system using 5 μm KYA TECH HiQ Sil C₁₈ HS column (250 mm × 4.6 mm internal diameter) as a stationary phase and methanol: water: 0.01 M orthophosphoric acid: triethylamine [70: 30: 2: 0.5, v/v/v/v] as mobile phase. The flow rate was maintained at 1.0 mL/min and UV detection at 222 nm. The calibration for verapamil and nor-verapamil were found to be linear over concentration range of 50–300 ng/mL with correlation coefficient (n = 6) of 0.9995 and 0.9997, respectively. This method was validated according to USFDA guidelines. The method was found to be simple, accuare, precise sensitive and selective for the determination of verapamil and nor-verapamil in plasma and thus useful in bioequivalence studies of verapamil.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

Simple determination of pirfenidone in rat plasma via high-performance liquid chromatography

A simple, rapid and reliable high-performance liquid chromatographic method was developed and validated for the determination of pirfenidone and its major metabolites in rat plasma. Plasma proteins were precipitated with perchloric acid (10%, v/v) and the supernatant after centrifugation was determined using high-performance liquid chromatography. The analysis was carried out on a Lichrospher C(18) column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water containing 0.2% acetic acid (23:77, v/v) at a flow-rate of 1 mL/min. The eluant was detected at 310 nm. The calibration curves were linear over a concentration range from 0.15 to 76.67 microg/mL. The accuracy (relative error) of the assay ranged from -2.6 to 7.9% and the precision (coefficient of variation) was less than 4.5%. The established method has been successfully applied to a pharmacokinetic study of pirfenidone following a single oral dose to rats.     

Development and validation of an RP-HPLC-UV method for the determination of BOL-303225-A, a new coumarin-based anti-inflammatory drug, in rat plasma

A simple and rapid high-performance liquid chromatographic method was developed and validated for the analysis in rat plasma of BOL-303225-A, a new coumarin-based anti-inflammatory drug. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid (35.5:64.5 v/v) as mobile phase. The UV detector was set at 324 nm. The method proved to be linear (r(2) > 0.99) and precise (RSD < 7%) over the concentration range 29-940 ng/mL, and was suitable for the support of pharmacokinetic studies in rats.     

Determination of verapamil and its primary metabolites in serum by ion-pair adsorption high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of verapamil, norverapamil, N-dealkylverapamil (D617) and N-dealkylnorverapamil (D620) concentrations in serum is developed. Analysis is performed on a microparticulate (10 microns) silica column using a counter-ion solvent system (0.6 mM NaBr in methanol). Column effluent is monitored by fluorescence detection at an excitation wavelength of 203 nm. The limit of sensitivity is less than 1 ng for all compounds in serum. No potential sources of interference are identified and a coefficient of variation of less than 10% is observed on replicate verapamil determinations. The method has the advantages of complete resolution of the metabolites of verapamil, low limits of detection, high degree of reproducibility, and short analysis time.     

Separation and determination of synthetic impurities of sildenafil (Viagra) by reversed-phase high-performance liquid chromatography.

A simple and rapid high-performance liquid chromatographic method for the separation and determination of process-related impurities of sildenafil was developed. The separation was achieved on a reversed-phase C18 column using acetonitrile-0.05 M potassium dihydrogen orthophosphate (70:30 v/v) as a mobile solvent at a flow rate of 1.0 ml/min and UV detection at 230 nm. The method was used not only for quality assurance, but also for monitoring the chemical reactions during the synthesis of sildenafil. It was found to be specific, precise and reliable for the determination of all process-related impurities of sildenafil in bulk drugs and formulations.     

Determination of nizatidine and two of its main metabolites in human serum using high-performance liquid chromatography

A high-performance liquid chromatographic assay has been developed for the determination of nizatidine, a new histamine H2-receptor antagonist, and two of its main metabolites, N-desmethylnizatidine and nizatidine sulphoxide. Drugs were extracted with chloroform-2-propanol (90:10, v/v) from alkalinized samples of serum, using ranitidine as an internal standard. After evaporation of the extraction solvent, the residue was removed and analysed on a LiChrosorb Si60 5-microns column with a mobile phase of acetonitrile-methanol-water-ammonia solution (1000:200:20:5, v/v). The compounds were detected at 320 nm. The lower detection limits were 6-18 ng/ml at a signal-to-noise ratio of 3. This method is simple and specific, and the single-step extraction makes it rapid. It is the first high-performance liquid chromatographic assay to be described for the determination of nizatidine metabolites.     

High-performance liquid chromatographic determination of proquazone and its m-hydroxy metabolite in spiked human plasma and urine

A simple and rapid high-performance liquid chromatographic method for the determination of proquazone (PQZ) and its major metabolite, m-hydroxyproquazone, in spiked human plasma and urine was developed. Plasma samples were purified using acetonitrile as a protein precipitant, while urine samples were diluted only with the mobile phase and filtered prior to injection. Samples containing the parent compounds and glafenine (internal standard) were eluted from a reversed-phase C8 column using acetonitrile-0.025 M sodium acetate (60 + 40) adjusted to pH 5 as the mobile phase and detected at 234 nm. Peak area ratios of the analytes versus internal standard were used for calibration. The mean recoveries from plasma and urine samples spiked with PQZ and its m-hydroxy metabolite ranged from 97.87 to 103.88%. The relative standard deviation for the within- and between-day analyses were < 4%. The proposed method was applied for the assay of PQZ in laboratory-made tablets.     

Highly specific and sensitive liquid chromatography-tandem mass spectrometry method for the determination of 3-desmethylthiocolchicine in human plasma as analyte for the assessment of bioequivalence after oral administration of thiocolchicoside

A sensitive method for the determination of 3-desmethylthiocolchicine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The plasma samples were extracted with ethyl acetate and separated on a Phenomenex Luna C18(2) 5 microm, 150x2 mm column with a mobile phase consisting of acetonitrile-0.005% formic acid (350:650, v/v) at a flow rate of 0.35 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode. TurbolonSpray ionisation was used for ion production. The mean recovery for 3-desmethylthiocolchicine was 70%, with a lower limit of quantification set at 0.39 ng/ml. The increased selectivity of mass spectrometric (MS-MS) detection allowed us to distinguish between thiocolchicoside and its primary metabolite 3-desmethylthiocolchicine in human plasma, thereby giving more insight about the pharmacokinetics of the drug in humans.     

Determination of paracetamol and its four major metabolites in mouse plasma by reversed-phase ion-pair high-performance liquid chromatography.

A reversed-phase ion-pair high-performance liquid chromatographic method has been used for the separation of paracetamol and its four major metabolites (glucuronide, sulphate, cysteine and mercapturate conjugates) in mouse plasma samples. An ODS column was used and the mobile phase consisted of an aqueous solution of 0.01 M tetrabutylammonium chloride and 0.01 M Tris buffered to pH 5.0 with phosphoric acid, with methanol as the organic solvent. The gradient elution started with 30% methanol. After a delay of 0.5 min the methanol concentration was increased linearly to 75% over 7.5 min. The column was returned to the initial conditions after a delay of 1 min. A methanol solution of theophylline was added to the mouse plasma sample, centrifuged and immediately injected into the chromatographic system. The advantages of this method include good and rapid separation (last metabolite detected at 6.86 min), well resolved peaks, only a small amount of sample required for assay, adequate precision (no coefficient of variation was greater than 10% for paracetamol metabolites) and a high sensitivity (particularly for unchanged paracetamol and the cysteine conjugate).