瑞格非尼片在健康志愿者体内的药动学研究

目的利用固相萃取–高效液相色谱,建立测定人血浆中瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼的方法,研究其人体药动力学。方法采用固相萃取–高效液相色谱法进行检测,以索拉非尼为内标,Oasis HLB型固相萃取柱(200 mg×6 m L);流动相:0.5%磷酸二氢钾溶液–乙腈(30∶70);检测波长:260 nm;体积流量:0.5 m L/min;柱温:30℃;进样量:5μL。健康志愿者空腹条件下用50 m L温水送服40、160 mg/片瑞格非尼片。利用DAS 2.1.1软件对药–时数据进行统计学分析,并得到主要药动学数据。结果瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼在10~10 000 ng/m L线性关系良好;检测限(LOD)分别为0.67、0.72、0.57 ng/m L;定量限(LOQ)分别为1.98、2.32、1.61 ng/m L;得到瑞格非尼及其代谢产物N-氧化瑞格非尼和去甲-N-氧化瑞格非尼药–时曲线和主要药动学数据。40、160mg/片规格瑞格非尼片中瑞格非尼的Cmax、AUClast、AUCmf、t1/2分别为(2 215.7±216.9)、(5 721.2±672.1)ng/m L,(13 772.9±10 231.1)、(51 182.3±43 219.2)ng·h/m L,(12 858.3±12 510.2)、(66 531.8±51 093.1)ng·h/m L,(6.58±1.31)、(13.12±4.23)h。结论采用固相萃取–高效液相色谱法考察瑞格非尼片在健康志愿者体内的药动学研究具有操作简便、可靠性高等优点,对该药的应用具有重要意义。     

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C₁₈ column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10-1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.     

Determination of L-ephedrine, pseudoephedrine, and caffeine in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid and simple liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of L-ephedrine, pseudoephedrine, and caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter concentrations for use in support of toxicology studies. Only 25 μL of plasma is required, and extraction is performed using a simple, single-step protein precipitation. The method was validated over a range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary gradient elution at 0.3 mL/min was used with a Waters XBridge Phenyl (2.1 × 150 mm, 3.5 μm) column and a Waters XBridge Phenyl 2.1- × 10-mm guard column at ambient temperature. The mobile phase consisted of 10 mM ammonium acetate in water (pH 5.0) and methanol. Caffeine trimethyl-(13)C(3) was used as the internal standard. The method was evaluated for linearity, recovery, precision, accuracy, and stability, and it was successfully applied in toxicokinetic studies of ephedrine, administered alone, in combination with caffeine, and in the herbal source Ma Huang.     

HPLC Determination of tolperisone in human plasma

A simple high performance liquid chromatographic (HPLC) method was developed for the determination of tolperisone in human plasma. Tolperisone and internal standard (chlorphenesin) were isolated from 1 mL of plasma using 8 mL of dichlormethane. The organic phase was collected and evaporated under nitrogen gas. The residue was then reconstituted with 300 mL aliquot of mobile phase and a 100 mL aliquot was injected onto the C18 reverse-phased column. The mobile phase, 45% methanol containing 1% glacial acetic acid and 0.05% 1-hexanesulfonic acid was run at a flow rate of 1 mL/min. The column effluent was monitored using UV detector at 260 nm. The retention times for tolperisone and the internal standard were approximately 7.1 and 8.4 min, respectively. The standard curve was linear with minimal intra-day and inter-day variability. The quantification limit of tolperisone in human plasma was 10 ng/ mL. The proposed method has been applied to the determination of pharmacokinetic profile of tolperisone in Koreans. The Tmax of tolperisone in Koreans (0.94 +/- 0.42 h) was not significantly differ from that reported in Europeans (0.5-1 h), but the mean half-life in Koreans (1.14 +/- 0.27 h) was shorter than that in Europeans (2.56 +/- 0.2 h). The proposed HPLC method is simple, accurate, reproducible and suitable for pharmacokinetic study of tolperisone.     

Determination of clopidogrel metabolite (SR26334) in human plasma by LC-MS

A new, sensitive, specific and reproducible method for determination of clopidogrel metabolite (SR26334) in human plasma has been developed. After liquid-liquid extraction on Chem Elut cartridges with dichloromethane, samples were quantified using reversed-phase high performance liquid chromatography with mass detection. The determination was performed on a Luna C18, 3 microm (75 mmx4.6 mm i.d.) column with an acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) as a mobile phase. The flow rate was set at 0.2 mL/min. Repaglinide was chosen as an internal standard and the time of analysis was 12 min. For SR26334 the limits of detection and quantification were 7.5 ng/mL and 20 ng/mL, respectively, and the calibration curve was linear up to 3000 ng/mL. The extraction recovery of SR26334 from plasma was within the range of 85-90%. The method has been successfully used to study clopidogrel metabolite pharmacokinetics in healthy volunteers.     

High throughput assay for the determination of piperaquine in plasma

A high throughput assay for the determination of the antimalarial piperaquine in plasma has been developed and validated. The assay utilises 96-wellplate formats throughout the whole procedure, and easily enables a throughput of 192 samples a day using a single LC system. Buffer (pH 2.0; 0.05 M) containing internal standard was added to 0.25 mL plasma in a 96-wellplate (2 mL wells). The samples were extracted on a MPC solid phase extraction deep well 96-wellplate (3M Empore). Piperaquine and internal standard were analysed by liquid chromatography with UV detection on a Chromolith Performance (100 mm x 4.6 mm) column with a mobile phase containing acetonitrile-phosphate buffer (pH 2.5; 0.1 M) (8:92, v/v) at a flow rate of 3.0 mL/min. The within-day precisions for piperaquine were 3.3 and 2.3% at 40 and 1250 ng/mL, respectively. The between-day precisions for piperaquine were 5.8 and 1.3% at 40 and 1250 ng/mL, respectively. The total assay precisions using 29 replicates over 5 days were 6.7, 4.5 and 2.7% at 40, 200 and 1250 ng/mL, respectively. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 10 and 5 ng/mL, respectively using 0.25 mL plasma. Using 1 mL of plasma, it was possible to decrease LLOQ and LOD to 2.5 and 1.25 ng/mL, respectively.     

Determination of donepezil hydrochloride in human plasma and pharmaceutical formulations by HPLC with fluorescence detection

A sensitive, isocratic reversed-phase high performance liquid chromatographic method involving fluorescence detection was developed for the determination of donepezil hydrochloride in tablets and in human plasma. Pindolol was used as an internal standard. Good chromatographic separation was achieved by using an analytical column C18. The system operated at room temperature using a mobile phase consisting of methanol, phosphate buffer (0.02 mol L?1) and triethyl amine (pH 3.5) (55 : 45 : 0.5, V/V/V) at a flow rate 0.9 mL?1 min. The analyte and internal standard were extracted from human plasma via liquid-liquid extraction. The proposed method was validated for sensitivity, selectivity, linearity, accuracy and precision. The calibration curve was linear over the range of 5-2000 ng mL?1 of donepezil with detection limit of 1.5 ng mL?1. Intra- and inter-day relative standard deviations were less than 2.5%. The method was found to be suitable for quality control of donepezil hydrochloride in bulk drug as well as in human plasma.     

Determination of olanzapine in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic method with ultraviolet absorbance detection for the determination of olanzapine in human plasma is described. Clozapine was used as internal standard. Analytes were concentrated from alkaline plasma by liquid-liquid extraction with n-hexane-isoamyl alcohol (90:10, vol/vol). The organic phase was back-extracted with 150 muL phosphate buffer (KH2PO4 with 25% H3PO4) 0.1 mol/L pH 2.2. The chromatographic separation required 6 minutes at a flow-rate of 1.0 mL/min and was carried out on a Water Spherisorb S5 C6 analytical column (250 mm x 4.6 mm ID) with a mobile phase water-acetonitrile 55:45 vol/vol containing 0.009 moL/L eptansulfonic acid sodium salt and 0.06 mol/L potassium phosphate monobasic pH 2.7. The peaks were detected at 254 nm. Mean recoveries for olanzapine and internal standard were 89.4+/-3.3% and 90.4+/-1.0%, respectively. Coefficient of variation value for intraday was 5.0% at concentrations of 10, 50, and 100 ng/mL and for interday was 4.0% at concentrations of 5 and 25 ng/mL. Accuracy, expressed as percent error, ranged from -8.00% to 1.24%. Limit of detection and limit of quantification for olanzapine were 2 and 5 ng/mL, respectively. The method is suitable for pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatographic method for the determination of trace amounts of acetaminophen in plasma

A method is described for the rapid, sensitive, and precise quantitation of acetaminophen in human plasma. The assay involved a single acetonitrile extraction and high-performance liquid chromatographic analysis using a reverse-phase column, with a mobile phase of methanol and water. The limit of quantitation of acetaminophen by this method was 8 ng/mL; only 0.1 mL of the plasma sample was required for the determination. N-Propionyl-p-aminophenol was used as the internal standard.     

Determination of bifendate in human plasma by HPLC-MS and bioequivalence on bifendate pills in healthy volunteers

A sensitive and specific method for the determination of bifendate in human plasma was developed, based on high-performance liquid chromatography (HPLC)-mass spectrometry (MS). The samples were extracted from plasma with diethyl ether, followed by separation and evaporation after addition of internal standard diazepam. The residue was reconstituted in methanol and injected into the HPLC-MS. Chromatography was performed on an Inertsil ODS column with a mobile phase consisting of methanol-distilled water (70/30, v/v) at a flow rate of 0.3 mL/min. Quantitative analysis was achieved by MS detection, using a mass spectrometer equipped with an electrospray ionization interface (ESI) and operated in selected ion monitoring (SIM) and positive-ionization mode using target ions at m/z 419 for bifendate and m/z 285 for internal standard, respectively. The linearity was confirmed in the concentration range of 2-200 ng/mL in human plasma and the precision of this assay was not more than 6.79% over the entire concentration range. The method was sensitive and repeatable enough to be used in pharmacokinetic and bioavailability studies.     

Quantification of oxcarbazeipine and its active metabolite 10-hydroxycarbazepine in human plasma by high-performance liquid chromatography

A simple, sensitive and selective high-performance liquid chromatography (HPLC) method with ultraviolet detection (230 nm) was developed and validated for the quantification of oxcarbazepine (CAS 28721-07-5), a new antiepileptic drug, and its active metabolite 10-hydroxycarbazepine (CAS 29331-92-8) in human plasma. Following solid-phase extraction, the analytes and internal standard (zaleplon, CAS 151319-34-5) were separated using an isocratic mobile phase on a reverse phase C18 column. The lower limit of quantification was 50 ng/mL for oxcarbazepine and 100 ng/mL for 10-hydroxycarbazepine with a relative standard deviation of less than 10%. A linear dynamic range of 50 to 5000 ng/mL for oxcarbazepine and of 100 to 10000 ng/mL for 10-hydroxycarbazepine was established. This HPLC method was validated with between-batch precision of 0.8 to 8.6% and 3.2 to 7.5% for oxcarbazepine and 10-hydroxycarbazepine respectively. The between-batch accuracy was 94.0 to 102.4% and 95.4 to 105.6%, respectively. Stability of oxcarbazepine and 10-hydroxycarbazepine 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.     

LC-MS determination and bioavailability study of imidapril hydrochloride after the oral administration of imidapril tablets in human volunteers

The purpose of the present study was to develop a standard protocol for imidapril hydrochloride bioequivalence testing. For this reason, a specific LC-MS method was developed and validated for the determination of imidapril in human plasma. A solid-phase extraction cartridge, Sep-pak C18, was used to extract imidapril and ramipril (an internal standard) from deproteinized plasma. The compounds were separated using a XTerra MS C18 column (3.5 microm, 2.1 x 150 mm) and acetonitrile-0.1% formic acid (67:33, v/v) adjusted to pH 2.4 by 2 mmol/L ammonium formic acid, as mobile phase at 0.3 mL/min. Imidapril was detected as m/z 406 at a retention time of ca. 2.3 min, and ramipril as m/z 417 at ca. 3.6 min. The described method showed acceptable specificity, linearity from 0.5 to 100 ng/mL, precision (expressed as a relative standard deviation of less than 15%), accuracy, and stability. The plasma concentration-versus-time curves of eight healthy male volunteers administered a single dose of imidapril (10 mg), gave an AUC12hr of imidapril of 121.48 +/- 35.81 ng mL(-1) h, and Cmax and Tmax values of 32.59 +/- 9.76 ng/mL and 1.75 +/- 0.27 h. The developed method should be useful for the determination of imidapril in plasma with sufficient sensitivity and specificity in bioequivalence study.     

Rapid liquid chromatographic assay for the determination of amiodarone and its N-deethyl metabolite in plasma, urine, and bile

A rapid high-performance liquid chromatographic assay was developed for the determination of amiodarone (1) and its N-deethyl metabolite (desethylamiodarone, 2) in plasma, urine, and bile. Analysis was performed on a C18 reversed-phase column and precolumn using a mobile phase consisting of methanol:water:58% ammonium hydroxide (94:4:2) delivered at a flow rate of 1.5 mL/min. The eluant was monitored at 244 nm. Under these conditions, 1, 2, and the internal standard eluted with retention times of 5.5, 4.6, and 6.8 min, respectively. Samples (100 microL) of plasma were prepared by precipitating the plasma proteins with acetonitrile containing the internal standard and injecting an aliquot of the supernatant directly onto the column. Samples (100 microL) of urine and bile were prepared for injection by acidifying the sample with concentrated HCl and then extracting the mixture with six volumes of 2,2-dimethoxyproprane. The recovery of 1 and 2 from plasma was virtually complete. The recovery from urine and bile was 80-90% for 1 and 60-65% for 2. The limit of sensitivity of both compounds in plasma was 100 ng/mL. For urine and bile, the detection limits were 1 and 5 micrograms/mL, respectively. Over the plasma concentration range of 0.1-10.0 micrograms/mL, the within-day CV ranged from 1 to 10% for 1 and from 1 to 8% for 2. The between-day CV ranged from 2 to 12% and from 1 to 17% for 1 and 2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of tiapride in human plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometric (HILIC-MS/MS) method for the determination of tiapride in human plasma was developed. Tiapride and internal standard, metoclopramide were extracted from human plasma with dichloromethane at basic pH and analyzed on an Atlantis HILIC silica column with the mobile phase of acetonitrile-ammonium formate (190 mM, pH 3.0) (94:6, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.999) over the concentration range of 1.00-200 ng/mL. The coefficient of variation and relative error for intra- and interassay at three QC levels were 6.4-8.8% and -2.0-3.6%, respectively. The recoveries of tiapride ranged from 96.3 to 97.4%, with that of metoclopramide (internal standard) being 94.2%. The lower limit of quantification for tiapride was 1.00 ng/mL using 100 microL of plasma sample.     

超高效液相色谱-串联质谱法测定大鼠血浆中替诺福韦的浓度

目的:建立大鼠血浆中替诺福韦浓度的测定方法。方法:采用超高效液相色谱-串联质谱法。取6只大鼠单次灌胃给予富马酸替诺福韦酯100mg.kg-1,检测给药45min后血浆中药物浓度,以恩替卡韦为内标,选用阳离子固相萃取柱除去血浆中的蛋白质。色谱柱为AcquitybenUPLC-C18,流动相为0.2%甲酸水溶液(含40mmo.lL-1醋酸铵)-乙腈=97:3,流速为0.25mL.min-1,柱温为40℃,电喷雾正离子(ESI+)源,监测离子对分别为质荷比(m/z)287.9→175.7(替诺福韦)和m/z277.9→151.7(恩替卡韦)。结果:替诺福韦检测浓度的线性范围为10～800ng.mL-1(r=0.9992),最低检测限为10ng.mL-1,回收率为(100.22±9.47)%～(102.76±4.99)%,日间、日内RSD均小于9.4%;给药45min后血浆样品中替诺福韦的平均浓度为819.2ng.mL-1。结论:该方法操作简便、专属性强、灵敏度高,可用于血浆中替诺福韦的浓度测定。     

Determination of bevantolol in human plasma by high performance liquid chromatography using solid phase extraction technique

A method was developed and fully validated for the determination of bevantolol, an adrenergic-receptor blocker, in human plasma. Bevantolol and betaxolol as internal standard (I.S) were extracted from 1 mL of human plasma by solid phase extraction technique using Sep-pak silica cartridge. Chromatographic separation was accomplished under isocratic conditions using a reverse-phase C8 analytical column and mixture of dibasic ammonium phosphate (pH 5.7; 50 mM)-acetonitrile (75:25, v/v) as mobile phase, with a detection wavelength at 220 nm. The method was proved to be specific by testing six different human plasma sources. Linearity was established for the concentration ranges of 40-1600 ng/mL with correlation coefficent of 0.9995. The lower limit of quantification 40 ng/mL with precision of 10.9% as C.V%.     

Quantification of aesculin in rabbit plasma and ocular tissues by high performance liquid chromatography using fluorescent detection: application to a pharmacokinetic study

A simple and sensitive high performance liquid chromatography method with fluorescence detection (HPLC-FD) was described for the determination of aesculin (AL) at low concentrations in rabbit plasma and ocular tissues. After deproteinization by methanol using pazufloxacin mesilate (PM) as an internal standard (I.S.), supernatants were evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was reconstituted in mobile phase and a volume of 20μL was injected into the HPLC for analysis. Analytes were separated on an Ultimate XB-C18 column (250mm × 4.6mm i.d., 5μm particle size) and protected by a ODS guard column (10mm × 4.0mm i.d., 5μm particle size), using acetonitrile-0.1% triethylamine in water (adjusted to pH 3.0 using phosphoric acid) (12:88, v/v) as mobile phase with a flow rate of 1.0mL/min. The wavelengths of fluorescence detector (FD) were set at 344nm for excitation and 466nm for emission. The lower limit of quantitation (LOQ) for AL was 0.80ng/mL for plasma and vitreous body, 1.59ng/mL for aqueous humor, and 6.55ng/g for iris and 1.66ng/g for retina. The method was used in the study of AL concentrations in plasma and ocular tissues after topical administration of AL eye drops.     

Validation of a reversed-phase high-performance liquid chromatography method with fluorescence detection for the bioequivalence study of norfloxacin in plasma samples

A robust method for the determination of norfloxacin in human plasma, using reversed-phase high-performance liquid chromatography (RP-HPLC) with fluorescence detection, has been developed. The method involves precipitation of plasma protein with acetonitrile and the use of ciprofloxacin as internal standard (IS). Chromatographic separations were performed on a Synergi MAX-RP 150 x 4.6-mm, 4-micro column with an elution system consisting of a mixture of phosphate buffer-acetonitrile (85:15, v/v). The calibration curve was linear, in the range of 30 to 3500 ng/mL. The recoveries at concentrations of 90, 1400, and 2800 ng/mL were 103.5%, 100.2%, and 100.2%, respectively. The quantification limit for norfloxacin was 30 ng/mL per 10-microL injection employing fluorescence detection with excitation and emission set at 300 and 450 nm, respectively. The method validation included examining the within-run and between-run precision and accuracy and ensuring that these were within accepted limits; in summary, the precision was <8.6% and accuracy ranged from 95.8% to 104.1% for concentration from 90 to 2800 ng/mL. The precision and accuracy for the lowest calibration standard (30 ng/mL) was well within accepted limits for lower limit of quantification. The method was then applied in a bioequivalence study in healthy volunteers given 400-mg doses of reference and test formulations of norfloxacin in random order and including a 7-day washout phase.     

Quantitative determination of azithromycin in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for determination of azithromycin in human plasma was developed and validated. Azithromycin in plasma (0.2mL) was extracted with methyl tert-butyl ether-hexane (50:50, v/v), organic phase was transferred to another clear 1.5mL Eppendorf tube and evaporated to dryness at 40 degrees C and dissolved in mobile phase, samples were separated using a Thermo Hypersil HyPURITY C18 reversed-phase column (150mmx2.1mm i.d., 5microm), together with a mobile phase containing of 20mM ammonium acetate (pH 5.2)-acetonitrile-methanol (50:40:10, v/v/v) and was isocratically eluted at a flow rate of 0.2mL/min. Azithromycin and its internal standard, clarithromycin, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated that good linearity ranged from 2 to 1000ng/mL with r=0.9977. The limit of quantification for azithromycin in plasma was 2ng/mL with good accuracy and precision. The higher mean extraction recovery, say 81.2% and 75.5% for azithromycin and internal standard (IS), respectively, was obtained in this work. The intra-day and inter-day precision ranged from 4.8% to 8.6% and 6.4% to 10.7% (R.S.D.), respectively. The established method has been successfully applied to bioequivalence study of 2 azithromycin formulations for 24 healthy volunteers.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of zofenopril and its active metabolite zofenoprilat in human plasma

A novel, sensitive and rapid liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated for the determination of zofenopril and its active metabolite zofenoprilat in human plasma. The method was based on a single extraction step using methyl tert-butyl ether and did not require chemical derivatization. The chromatographic conditions were optimized; separation was performed on a phenyl-hexyl column (5μm, 250mm×4.6mm i.d.) with a mobile phase consisting of a solution of methanol and water (95:5, v/v) that also contained 0.1% of formic acid. A flow rate of 1.0mL/min was used. Zofenopril, zofenoprilat and the internal standard (IS) fosinopril sodium were measured using an electrospray ion source in a positive reaction monitoring mode. Linear calibration curves were generated for zofenopril concentrations between 0.1052 and 1052ng/mL and for zofenoprilat concentrations between 0.2508 and 2508ng/mL. In both cases, the coefficients of determination were greater than 0.995. The extraction recovery for zofenopril was 93.5% on average. It was 92.5% for zofenoprilat. The inter- and intra-batch precision and accuracy for both zofenopril and zofenoprilat were higher than 14%. The method was applied to measure the concentrations of zofenopril and zofenoprilat in plasma samples.