High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Validated liquid chromatographic ultraviolet method for the quantitation of Etoricoxib in human plasma using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (284 nm) was developed and validated for quantitation of Etoricoxib in human plasma, the newest addition to the group of nonsteroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. Following a single-step liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v), the analyte and internal standard (Zaleplon) were separated using an isocratic mobile phase of water/acetonitrile (58/42, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 5 ng/mL, with a relative standard deviation of less than 20%. A linear range of 5-2500 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 4.1-5.1% and 1.1-2.4%, respectively. The between- and within-batch bias was -3.8-4.7% and -0.6-9.4%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Etoricoxib in plasma was >90%, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive and simple with between-batch precision of <6% and was used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of rabeprazole in human plasma using solid-phase extraction

A simple, sensitive and selective HPLC method with UV detection (284 nm) was developed and validated for quantitation of rabeprazole in human plasma, the newest addition to the group of proton-pump inhibitors. Following solid-phase extraction using Waters Oasistrade mark SPE cartridges, the analyte and internal standard (Pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 8%. A linear range of 20-1000 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 2.4-7.2% and 2.2-7.3%, respectively. The between- and within-batch bias was -1.7 to 2.6% and -2.6 to 2.1%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Liquid chromatographic determination of irbesartan in human plasma

A simple and sensitive method was developed for determination of irbesartan by liquid chromatography with fluorescence detection. Irbesartan and losartan (I.S.) in human plasma were extracted using diethyl ether:dichloromethane (7:3, v/v) followed by back extraction with 0.05 M sodium hydroxide. Neutralized samples were analyzed using 0.01 M potassium dihydrogen phosphate buffer (containing 0.07% triethylamine as peak modifier, pH was adjusted with orthophosphoric acid to pH 3.0) and acetonitrile (66:34, v/v). Chromatographic separation was achieved on an ODS-C-18 column (100 mm x 4.6 mm i.d., particle size 5 microm) using isocratic elution (at flow rate 1.25 ml/min). The peak was detected using a fluorescence detector set at Ex 259 nm and Em 385 nm, and the total time for a chromatographic separation was approximately 13 min. The validated quantitation ranges of this method were 15-4000 ng/ml with coefficients of variation between 0.75 and 12.53%. Mean recoveries were 73.3-77.1% with coefficients of variation of 3.7-6.3%. The between- and within-batch precision were 0.4-2.2% and 0.9-6.2%, respectively. The between- and within-batch relative errors (bias) were (-5.5) to 0.9% and (-0.6) to 6.9%, respectively. Stability of irbesartan in plasma was >89%, with no evidence of degradation during sample processing and 60 days storage in a deep freezer at -70 degrees C. This validated method is sensitive and simple with between-batch precision of <3% and can be used for pharmacokinetic studies.     

Determination of atomoxetine in human plasma by a high performance liquid chromatographic method with ultraviolet detection using liquid-liquid extraction

A HPLC method with UV detection (210 nm) was developed and validated for the quantification of atomoxetine, a new medication for the treatment of attention deficit/hyperactivity disorder, in human plasma. Following a two-step liquid-liquid extraction with diethyl ether, the analyte and internal standard (maprotiline) were separated using an isocratic mobile phase of acetonitrile/phosphate buffer (39/61, v/v, pH 6.6) on a reverse phase Inertsil C(18) column. Linearity was verified over the range of 3.12-200 ng/mL atomoxetine in plasma. The lowest limit of detection is 2.5 ng/mL (S/N=10). This HPLC method was validated with within- and between-batch precisions of 4.9-14.4% and 4.7-13.1%, respectively. The within- and between-batch biases were -1.9 to 1.4% and 0.1-13.8%, respectively. Commonly used psychotropic drugs and frequently coadministered drugs did not interfere with the drug and internal standard. This method is simple, economical and specific, and has been used successfully in a pharmacokinetic study of atomoxetine.     

A simple and rapid HPLC/UV method for the simultaneous quantification of theophylline and etofylline in human plasma

A simple, sensitive and selective high performance liquid chromatography (HPLC) method with ultraviolet detection (272 nm) was developed and validated for the simultaneous quantification of theophylline and etofylline in human plasma. Following rapid sample preparation, the analytes and internal standard (hydrochlorothiazide) were separated using an isocratic mobile phase on a reverse phase C18 column. The lower limit of quantification was 100 ng/mL for both theophylline and etofylline with a relative standard deviation of less than 6%. A linear dynamic range of 100-10,000 ng/mL for both theophylline and etofylline was established. This HPLC method was validated with between-batch precision of 2.2-6.0 and 1.4-3.7% for theophylline and etofylline, respectively. The between-batch accuracy was 94.3-98.0 and 95.4-98.2%, respectively. Stability of theophylline and etofylline in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is simple and rugged enough to be used in pharmacokinetic studies.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.     

Validated liquid chromatographic-ultraviolet method for the quantitation of tadalafil in human plasma using liquid-liquid extraction

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid-liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm x 4.6 mm, 5 microm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine+0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5-600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within -7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.     

Sensitive quantification of ranolazine in human plasma by liquid chromatography--tandem mass spectrometry with positive electrospray ionization

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with positive electrospray ionization (ESI) was developed for the quantification of ranolazine in human plasma. After liquid-liquid extraction of ranolazine and internal standard (ISTD) phenoprolamine from a 100 microl specimen of plasma, HPLC separation was achieved on a Nova-Pak C(18) column, using acetonitrile-water-formic acid-10% n-butylamine (70:30:0.5:0.08, v/v/v/v) as the mobile phase. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode using the transition m/z 428.5-->m/z 279.1 for ranolazine and m/z 344.3-->m/z 165.1 for the internal standard, respectively. Linear calibration curves were obtained in the concentration range of 5-4000 ng/ml, with a lower limit of quantitation (LLOQ) of 5 ng/ml. The intra- and inter-day precision values were below 3.7% and accuracy was within +/-3.2% at all three quality control (QC) levels. This method was found suitable for the analysis of plasma samples collected during the phase I pharmacokinetic studies of ranolazine performed in 28 healthy volunteers after single oral doses from 200 mg to 800 mg.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (-20 degrees C) for sample preparation followed by separation on a Novapack C(18) column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at -70 degrees C.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of tolterodine tartrate in human plasma

A selective and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been developed for the determination of tolterodine tartrate in human plasma. With oxybutynin as internal standard, tolterodine tartrate was extracted from plasma with n-hexane: isopropanol (95:5, v/v). The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile-water (10 mM CH3COONH4, pH 3.0)=50:50 (v/v). An aliquot of 10 microl was chromatographically analyzed on a prepacked Shimadzu Shim-pack VP-ODS C18 column (150 mmx2.0 mm I.D.) by means of selected-ion monitoring (SIM) mode mass spectrometry. Standard curves were linear (r=0.9993) over the concentration range of 0.1-30.0 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection (LOD) was 0.05 ng/ml. The validated LC-ESI-MS method has been used successfully to study tolterodine tartrate pharmacokinetic, bioavailability and bioequivalence in 20 healthy male volunteers.     

Determination of ragaglitazar,a novel dual acting peroxisome proliferator-activated receptor (PPAR) alpha and -gamma agonist,in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

A sensitive and specific LC/MS/MS method has been developed and validated for determination of ragaglitazar (NNC 61-0029 or DRF 2725) in human plasma. After solid-phase extraction (SPEC((R)) PLUS C(8)) of plasma, separation was performed on a Symmetry Shield RP8 column (mobile phase: acetonitrile: 10 mM ammonium acetate, pH 5.6 (40:60 v/v)). Two ranges were validated having LLOQs of either 0.500 or 100 ng/ml and linearity up to either 500 or 50000 ng/ml. The intra-assay precision and accuracy were 1.1% to 15.7% and 85.8% to 118.2% (range 0.500-500 ng/ml) and 2.0% to 8.8% and 92.9% to 104.8% (range 100-50000 ng/ml). The method was applied for determination of ragaglitazar in plasma from phase 1 and 2 clinical studies.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Simple high-performance liquid chromatographic determination of the protease inhibitor indinavir in human plasma

Indinavir is a member of a class of protease inhibitors that actively prevent the acquired immunodeficiency syndrome virion from maturing. A high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of indinavir in human plasma. Indinavir and the internal standard were isolated from the plasma by ether extraction. The residue after evaporation of ether was reconstituted with buffer and injected onto a C₄ reversed-phase column eluted isocratically with a mobile phase consisting of 35:65 (v/v) of acetonitrile and buffer. A wavelength of 210 nm was found to be optimum for detection. The calibration range of this assay was from 10 to 5000 ng/ml and coefficients of variation for the assay ranged from 4.6% to 11.0% for three different drug concentrations and the limit of quantitation was 10 ng/ml. During the validation, short-term stability of the drug in plasma, stability during heat deactivation and on repeated freezing and thawing of plasma was evaluated. The overall recovery of indinavir by the ether extraction method was 91.4%. This HPLC assay was found to be a simple and reproducible method for monitoring indinavir levels in human plasma obtained during clinical trials of the drug.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.