Enantioselective and highly sensitive determination of carvedilol in human plasma and whole blood after administration of the racemate using normal-phase high-performance liquid chromatography

A highly sensitive HPLC method for enantioselective determination of carvedilol in human whole blood and plasma was developed. Carvedilol and S-carazolol as an internal standard extracted from whole blood or plasma were separated using an enantioselective separation column (Chiralpak AD column; 2.0 diameter x 250 mm) without any chiral derivatizations. The mobile phase was hexane:isopropanol:diethylamine (78:22:1, v/v). The excitation and emission wavelengths were set at 284 and 343 nm, respectively. The limits of quantification for the S(-)- and R(+)-carvedilol enantiomers in plasma and blood were both 0.5 ng/ml. Intra- and inter-day variations were less than 5.9%. As an application of the assay, concentrations of carvedilol enantiomer in plasma and blood samples from 15 patients treated with carvedilol for congestive heart failure were determined.     

Quantification of carvedilol in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry: application to bioequivalence study

A rapid, sensitive and specific method to quantify carvedilol in human plasma using metoprolol as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a diethyl-ether solvent. After removed and dried the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile-water (50/50; v/v). The extracts were analyzed by a high performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-MS/MS). Chromatography was performed isocratically on Alltech Prevail C18 5 microm analytical column, (150 mm x 4.6 mm i.d.). The method had a chromatographic run time of 3.5 min and a linear calibration curve over the range 0.1-200 ng ml(-1) (r2>0.997992). The limit of quantification was 0.1 ng ml(-1). This HPLC-MS/MS procedure was used to assess the bioequivalence of two carvedilol 25 mg tablet formulations (carvedilol test formulation from Laboratórios Biosintética Ltda and Coreg from Roche Químicos e Farmacêuticos S.A standard reference formulation). A single 25 mg dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 2-week wash-out interval. Since the 90% CI for C(max) and AUCs ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration Agency, it was concluded that carvedilol formulation elaborated by Laboratórios Biosintética Ltda is bioequivalent to Coreg formulation for both the rate and the extent of absorption.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Liquid chromatographic method for the determination of rizatriptan in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.     

Determination of amosulalol in human plasma using solid-phase extraction combined with liquid chromatography and ultraviolet detection

Amosulalol is an antihypertensive drug with selective postsynaptic alpha 1 and non-selective beta blocking effects. A simple solid-phase extraction and high-performance liquid chromatographic (HPLC) method has been developed and validated for the quantitative determination of amosulalol in human plasma. A reversed phase C18 column was used for the separation of amosulalol and ethyl paraben (internal standard) with a mobile phase composed of 0.025 M phosphate buffer (pH 6.0).acetonitrile (73:27, v/v) at a flow rate of 1.5 mL/min. The ultraviolet detector was operated at the 272 nm wavelength. Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification of 30 ng/mL. Recovery of amosulalol from human plasma was >95.6%. Amosulalol was stable in human plasma under various storage conditions. This method was used successfully for a pharmacokinetic study in plasma after oral administration of a single 20 mg dose of amosulalol hydrochloride to 16 healthy volunteers.     

High performance liquid chromatographic method for the determination of sumatriptan with fluorescence detection in human plasma

A rapid and sensitive high performance liquid chromatography (HPLC) method with fluorescence detection has been developed for the determination of sumatriptan in human plasma. The procedure involved a liquid-liquid extraction of sumatriptan and terazosin (internal standard) from human plasma with ethyl acetate. Chromatography was performed by isocratic reverse phase separation on a C18 column. Fluorescence detection was achieved with an excitation wavelength of 225 nm and an emission wavelength of 350 nm. The standard curve was linear over a working range of 1-100 ng/ml and gave an average correlation coefficient of 0.9997 during validation. The limit of quantitation (LOQ) of this method was 1 ng/ml. The absolute recovery was 92.6% for sumatriptan and 95.6% for the internal standard. The inter-day and intra-day precision and accuracy were between 0.8-3.3 and 1.1-6.3%, respectively. This method is simple, sensitive and suitable for pharmacokinetics or bioequivalence studies.     

Simultaneous quantification of voriconazole and posaconazole in human plasma by high-performance liquid chromatography with ultra-violet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method with ultra-violet detection has been developed and validated for the simultaneous determination of posaconazole and voriconazole, two systemic anti-fungal agents. An internal standard diazepam was added to 100 microL of human plasma followed by 3 mL of hexane-methylene chloride (70:30, v/v). The organic layer was evaporated to dryness and the residue was reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The compounds were separated on a C8 column using sodium potassium phosphate buffer (0.04 M, pH 6.0): acetonitrile:ultrapure water (45:52.5:2.5, v/v/v) as mobile phase. All compounds were detected at a wavelength of 255 nm. The assay was linear and validated over the range 0.2-10.0 mg/L for voriconazole and 0.05-10.0 mg/L for posaconazole. The biases were comprised between -3 and 5% for voriconazole and -2 and 8% for posaconazole. The intra- and inter-day precisions of the method were lower than 8% for the routine quality control (QC). The mean recovery was 98% for voriconazole and 108% for posaconazole. This method provides a useful tool for therapeutic drug monitoring.     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile–n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water–acetonitrile–tetrahydrofuran–ammonium hydroxide–triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20–2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93±9.7% and 83±10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection

An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150 x 2.1 mm I.D., 5 microm particle size). The mobile phase was acetonitrile-10 mM ammonium acetate solution containing 0.3 mM EDTA-glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4-700 ng/ml of quercetin in plasma and 20-1000 ng/ml of quercetin in urine. The lower limit of quantification was approximately 7 ng/ml of quercetin in plasma and approximately 35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was approximately 0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.     

Determination of plasma testosterone using a simple liquid chromatographic method

A simple and sensitive high-performance liquid chromatographic (HPLC) method using ultraviolet detection was developed for the determination of testosterone in human plasma. Testosterone and the internal standard, griseofulvin, were extracted from 0.50 ml plasma sample using a mixture of dichloromethane-2,2,4-trimethylpentane (3:2, v/v). The mobile phase, consisted of 0.02 M sodium dihydrogenphosphate-acetonitrile-methanol (51:47:2, v/v) adjusted to pH 3.1 and delivered to a C(18) analytical column (150 x 4.6 mm I.D., 4 microm particles) at a flow-rate of 1 ml/min while the detection wavelength was set at 240 nm with a sensitivity range of 0.005 a.u.f.s. The method has a quantification limit of 1.6 ng/ml. Recoveries of testosterone were all greater than 92% over the linear concentration range of 1.6-400 ng/ml while that of griseofulvin was approximately 95%. The within- and between-day RSD values were all less than 8% while the accuracy values ranged from 96.0 to 106.0% over the concentration range studied. The method was applied to the analysis of early morning plasma testosterone levels of 12 healthy human male volunteers. The levels were found to range from 3.1 to 8.4 ng/ml, within the normal range reported in the literature.     

Simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) procedure for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma samples is described. A one-step solid-phase extraction (SPE) with C18 cartridges was coupled with a reversed-phase HPLC system. The system requires two mobile phases composed of tetrabutyl ammonium hydrogensulfate (10 mM adjusted to pH 7)-acetonitrile (62:38, v/v) for quinapril, and (25:75, v/v) for quinaprilat elution through a C18 Symmetry column and detection at a wavelength of 215 nm. Calibration curves were linear over the ranges 20 to 1,000 ng/ml for quinaprilat and 10 to 500 for quinapril. The limits of quantification were 20 and 10 ng/ml for quinaprilat and quinapril, respectively. Extraction recoveries were higher than 90% for quinapril and 80% for quinaprilat. This method has been successfully applied to a bioequivalence study of quinapril in healthy subjects.     

Liquid chromatographic method for the determination of rosiglitazone in human plasma

A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid-liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C(18), 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5-1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane—isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound α₁-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Simple liquid chromatographic method for the determination of cefotaxime in human and rat plasma

A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.