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.     

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.     

Determination of quinapril and quinaprilat by high-performance liquid chromatography with radiochemical detection,coupled to liquid scintillation counting spectrometry

Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase high-performance liquid chromatographic procedure with radiochemical detection, coupled to liquid scintillation counting spectrometry. Quinapril and quinaprilat were measured in perfusate and urine after pretreatment with acetonitrile and subsequent centrifugation. Perfusate ultrafiltrate was used as collected. Two quinapril diketopiperazine metabolites, PD 109488 and PD 113413, were separated chromatographically from quinapril, quinaprilat, and from each other. Assay performance for quinapril and quinaprilat was assessed by examining precision and accuracy of the assay over four days. Using a 100-μl sample volume, the limit of quantitation for both ³H-quinapril and ³H-quinaprilat (sp. act. ≈ 2.0 μCi/μg) was 1 ng/ml.     

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.     

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.     

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.     

A sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from Saposhnikovia root in rat plasma and urine

A sensitive and efficient liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C(18) column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0-100 ng/mL in rat plasma and 10-1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5-150 ng/mL in plasma and 15-1500 ng/mL in urine for 4'-O-D-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9% as terms of relative error (R.E.). The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC-MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.     

High throughput and sensitive determination of trazodone and its primary metabolite, m-chlorophenylpiperazine, in human plasma by liquid chromatography-tandem mass spectrometry

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine (mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm x 2.1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/z 372.2-->176.2, 197.2-->118.1 and 470.5-->274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0-3000.0 ng/mL for TRZ and 0.2-60.0 ng/mL for mCPP was evaluated with mean correlation coefficient (r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was < or =8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay to quantitate ON 01910.Na, a mitotic progression modulator, in human plasma

A reverse-phase high performance liquid chromatographic method with tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantitation of ON 01910.Na, a novel synthetic benzyl styryl sulfone, in human plasma. The assay involved a simple sample preparation with acetonitrile protein precipitation. ON 01910.Na and the internal standard temazepam were separated on a Waters X-Terra MS C(18) column with mobile phase of acetonitrile containing 0.1% formic acid /10mM ammonium acetate (55:45, v/v) using isocratic flow at 0.2 mL/min for 5 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Two calibration curves were generated over the range of 10-2000 ng/mL and 100-20000 ng/mL. The lower limit of quantitation (LLOQ) was 10 ng/mL for ON 01910.Na in human plasma. The accuracy and within- and between-day precisions were within the acceptance criteria for bioanalytical assays. ON 01910.Na was found stable in plasma at -70 degrees C for at least 1 year. The method was successfully applied to characterize the plasma concentration-time profiles of ON 01910.Na in the cancer patients in the Phase I study.     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma

A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic-lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88-93% for horse plasma and 96-103% for dog plasma. The coefficient of variation in all cases was less than 10%. This method is suitable for the analysis of clinical samples from pharmacokinetic and bioequivalence studies and drug monitoring.     

A liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of valsartan and hydrochlorothiazide in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous quantification of valsartan and hydrochlorothiazide in human plasma. After a simple protein precipitation using acetonitrile, the analytes were separated on a Zorbax SB-Aq C18 column using acetonitrile-10mM ammonium acetate (60:40, v/v, pH 4.5) as mobile phase at a flow rate of 1.2 mL/min. Valsartan and hydrochlorothiazide were eluted at 2.08 min and 1.50 min, respectively, ionized using ESI source, and then detected by multiple reaction monitoring (MRM) mode. The precursor to product ion transitions of m/z 434.2-350.2 and m/z 295.9-268.9 were used to quantify valsartan and hydrochlorothiazide, respectively. The method was linear in the concentration range of 4-3600 ng/mL for valsartan and 1-900 ng/mL for hydrochlorothiazide. The method was successfully employed in a pharmacokinetic study after an oral administration of a dispersible tablet containing 80 mg valsartan and 12.5 mg hydrochlorothiazide to each of the 20 healthy volunteers.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Simultaneous determination of doxifluridine and 5-fluorouracil in monkey serum by high performance liquid chromatography with tandem mass spectrometry

A reverse-phase high performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its active metabolite 5-fluorouracil in monkey serum. A liquid/liquid extraction with ethyl acetate (90%) and isopropyl alcohol (10%) was used to extract simultaneously doxifluridine and 5-FU which have considerable difference in the polarity. Optimum chromatographic separation was achieved on a Agilent Zorbax C(18) (100 mm x 2.1mm, 3.5 microm) column with a mobile phase of methanol-water (20:80, v/v). The flow rate was 0.2 mL/min with total cycle time of 5 min. The lower limit of quantification (LLOQ) was validated at 10.0 ng/mL of serum for both doxifluridine and 5-FU. Accuracy and precision of quality control (QC) samples for both compounds met FDA Guidance criteria of +/-15% with average QC accuracy of 95.5-105.0% and coefficients of variation of 1.1-9.5% in the 10-2000 ng/mL concentration range. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability to support the analysis of monkey serum samples.     

Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

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.     

Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS/MS) had been developed and validated to determine the concentrations of BPR0L075 in rat plasma. After a simple protein precipitation of plasma samples by acetonitrile, BPR0L075 was analyzed on a C(8) column at a flow rate of 0.5 mL/min. The mobile phase consisted of a mixture of 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile (20:80, v/v). Both BPR0L075 (analyte) and the internal standard (BPR0L092) were determined using electro-spray ionization and the MS data acquisition was via multiple reactions monitoring (MRM) in positive scanning model. The MS/MS ion transitions monitored are m/z 342.2/195.2 and 312.5/165.2 for BPR0L075 and BPR0L092, respectively. The low limit of quantitation was 0.5 ng/mL. Each plasma sample was chromatographed within 5 min. The method was validated with respect to linearity, accuracy, precision, recovery, and stability. A good linear relationship was observed over the concentration range of 0.5-1000 ng/mL (r>0.9994). Absolute recoveries ranged from 63.45 to 68.34% in plasma at the concentrations of 2, 40, 400, and 800 ng/mL. The intra- and inter-day accuracy ranged from 92.04 to 111.80%. Intra- and inter-day relative standard deviations were 1.08-3.29% and 1.96-5.46%, respectively. This developed and validated assay method had been successfully applied to a pharmacokinetic study after intravenous injection of BPR0L075 in rats at a dose of 5mg/kg.     

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.     

Quantitative determination of buagafuran in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of buagafuran in human plasma. The analyte was extracted from plasma samples with hexane after addition of isotopic internal standard and chromatographed on a RP-C(8) column. The mobile phase consisted of methanol-water (90:10, v/v) and the flow rate was 0.2 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.5-200 ng/mL. Inter- and intra-day precision (RSD%) were all within 15% and the accuracy (RE%) was equal or lower than 9.5%. The lower limit of quantitation (LLOQ) was 0.5 ng/mL. The extraction recovery was on average 38.1% and the detection was not affected by the matrix. The method was successfully applied to the pharmacokinetic study of buagafuran in healthy Chinese volunteers.     

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究(英文)

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取,汉邦C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇∶水(85∶15,v/v),水相含10 mmol/L的醋酸铵和0.1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测,草乌甲素及内标的反应离子对分别为644.4/584.4和237.2/194.2,草乌甲素血药浓度在0.010～1.0 ng/mL范围内线性关系良好,最低定量限为0.010 ng/mL可以满足口服0.4 mg草乌甲素后血药浓度的检测,日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中,20名志愿者口服0.4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0.325±0.110),(0.323±0.115)ng/mL;AUC0-16(1.627±0.489),(1.732±0.556)ng.h/mL;AUC0-∞(1.730±0.498),(1.831±0.562)ng.h/mL;t1/2(4.26±0.95),(3.80±0.90)h;Tmax(1.34±0.54),(1.83±0.99)h。