高效液相色谱法测定右旋儿茶素血浆浓度及药代动力学参数

本文建立了体液中右旋儿茶素的RP-HPLC测定方法。采用C_(18)键合相硅胶为填料的固相提取柱进行样品预处理,右旋儿茶素的提取回收率为79.8%.应用二极管阵列检测器对色谱峰纯度进行鉴定。该法精密度好,方法回收率近100%,日内、日间的变异系数为2.4～5.6%,血浓69.6～1160 ng/ml范围内呈线性关系,r=0.9993。家兔静注右旋儿茶素18mg/kg,其药代动力学过程符合二室模型,分布相半衰期为0.129 h,消除相半衰期为1.19h。     

HPLC determination of omeprazole in human plasma using a monolithic column

A rapid and sensitive HPLC method using a monolithic column has been developed for quantification of omeprazole (CAS 73590-58-6) in plasma. The method was specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/l disodium hydrogen phosphate buffer-acetonitrile (73:27 v/v) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng x ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%.     

Quantification of carvedilol in human plasma by liquid chromatography using fluorescence detection: application in pharmacokinetic studies

A simple, rapid and sensitive isocratic reversed-phase HPLC method with fluorescence detection using a monolithic column has been developed and validated for the determination of carvedilol in human plasma. The separation was performed on a Chromolith Performance (RP-18e, 100mm x 4.6mm) column with an isocratic mobile phase consisting of 0.01 M disodium hydrogen phosphate buffer-acetonitrile (40:60, v/v) adjusted to pH 3.5. The sample preparation involves protein precipitation procedure and analytical recovery was complete. Letrozole was used as internal standard. The assay enables the measurement of carvedilol for therapeutic drug monitoring with a minimum quantification limit (LOQ) of 1 ng ml(-1). The excitation and emission wavelengths were set at 240 and 340 nm, respectively. The calibration curve was linear over the concentration range 1-80 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8.0%.     

Pharmacokinetic comparison of two 40 mg tablet formulations of citalopram using a new amperometric detection technique

OBJECTIVES: To assess the bio-equivalence of two citalopram 40 mg tablet formulations (Lecital of the Jordan Sweden Medical and Sterilization Co. (JOSWE) as a test product, and Cipramil of Lundbeck (Denmark) as a reference product), and to develop a new high-performance liquid chromatography (HPLC) method using liquid-liquid extraction followed by addition of acid for the quantification of citalopram in human plasma. METHODS: A single-blind, randomized, single-dose, 2-treatment, 2-period, 2-sequence, crossover bioequivalence study with a 20-day washout period in 24 healthy volunteers. The drug was administered with 240 ml of water after 10-h overnight fasting. After dosing, serial blood samples were collected for a period of 192 h. Plasma harvested from blood was analyzed for citalopram by a novel method using HPLC coupled with an electrochemical detector. The limit of quantitation of citalopram was 1.493 ng/ml. Matrix-based calibration curves were linear over the range 1.493 - 80.640 ng/ml for citalopram. RESULTS: The average bioavailability and pharmacokinetic parameters of the two citalopram tablets were as follows: peak plasma concentration Cmax was 35.0 +/- 10.04 ng/ml and 33.4 +/-7.80 ng/ml for Lecital and Cipramil, respectively. The time to peak plasma concentrations tmax were 3.81 +/- 1.18 and 4.08 +/- 1.54 h, while the plasma half-life (t1/2) values were 54.0 +/- 7.50 and 54.7 +/- 10.6 h. The area under the plasma concentration-time profiles AUCo-t were 1,820 +/- 582 ng x h/ml and 1,660 +/- 510 ng x h/ml, whereas the AUC0-infinity were 2,010 +/- 663 ng x h/ml and 1,850 +/- 577 ng x h/ml for Lecital and Cipramil, respectively. The 90% confidence intervals for test/reference ratio were found within the acceptable limits of 80 - 125%, consequently no significant difference was found between the test and reference. CONCLUSION: Based on the pharmacokinetic and statistical results, it was concluded that Lecital 40 mg tablets of JOSWE is bioequivalent to Cipramil 40 mg tablets of Lundbeck (Denmark).     

HPLC-APCI-MS for the determination of teprenone in human plasma: method and clinical application

A sensitive high performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method for the determination of teprenone (GGA) in human plasma using menatetrenone as the internal standard (I.S.) was established. After protein precipitation with ethanol, the plasma sample was extracted by cyclohexane and separated by high performance liquid chromatography on a reversed phase C8 HPLC column with a mobile phase of water-methanol (4:96, v/v). GGA was determined with atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). HPLC-APCI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M+H]+m/z 331.3 for GGA and [M+H]+m/z 445.4 for the I.S. Calibration curve was linear over the range of 0.3-1000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 7.8% and 8.7%, respectively. The method was successfully applied in the pharmacokinetic study in which plasma concentrations of GGA in 20 healthy Chinese volunteers were determined up to 24 h after administration of capsule containing 50 mg GGA. The maximum GGA plasma concentration (Cmax) was 246.9+/-85.4 ng/ml, the elimination half-life (t1/2) was 3.38+/-1.20 h, and the time to the Cmax was 5.35+/-1.39 h.     

Simultaneous determination of aceclofenac and diclofenac in human plasma by narrowbore HPLC using column-switching

A fully automated narrowbore high performance liquid chromatography (HPLC) with column-switching was developed for the simultaneous determination of aceclofenac and diclofenac from human plasma samples. Plasma sample (100 microl) was directly introduced onto a Capcell Pak MF Ph-1 column (20 x 4 mm I.D.) where primary separation was occurred to remove proteins and concentrate target substances using acetonitrile potassium phosphate (pH 7, 0.1 M) (14:86, v/v). The drug molecules eluted from MF Ph-1 column were focused in an intermediate column (35 x 2 mm I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on the narrowbore phenyl hexyl column (100 x 2 mm I.D.) using acetonitrile-potassium phosphate (pH 7, 0.02M) (33:67, v/v) when the valve status was switched back to A position. The method showed excellent sensitivity (detection limit of 10 ng ml(-1)) with small volume of samples (100 microl), good precision and accuracy, and speed (total analysis time 17 min) without any loss in chromatographic efficiency. The response was linear (r2 > or = 0.999) over the concentration range of 50-10,000 ng ml(-1).     

Determination of ABT-089 in human plasma by high performance liquid chromatography using in situ precolumn derivatization with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

ABT-089 is a potent, selective neuronal cholinergic channel modulator with cognition enhancing activity in several animal paradigms. A simple and sensitive chromatographic method for the specific determination of ABT-089 in human plasma has been developed and validated. The method utilizes in situ precolumn fluorescence derivatization of the sample with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) prior to liquid-liquid extraction followed by reverse phase HPLC and fluorescence detection (lambda(ex) 495 nm, lambda(em) 533 nm). The described method significantly simplifies sample preparation. The derivatized product was separated from interference using a YMC ODS-AQ, 5 microm, 250x4.6 mm i.d. column using a mobile phase consisting of 30:5:65 (v:v:v) acetonitrile/methanol/aqueous buffer at a flow rate of 0.75 ml min(-1). The aqueous buffer consisted of 0.01 M tetramethylammonium perchlorate, 0.1% (v:v) trifluoroacetic acid, pH 3.0. An Alltech Absorbosphere CN, 5 microm, cartridge guard column was also used before the analytical column. Plasma samples were alkalinized with 0.1 M NaHCO3, 300 microl of a 1 mg ml(-1) ethanolic solution of NBD-F was added and the samples were heated in a water bath for 10 min at 50 degrees C. The samples were then extracted with tert-butylmethylether, evaporated to dryness and then reconstituted in mobile phase. For 1 ml of plasma, a limit of quantitation (LOQ) of 1.6 ng ml(-1) was obtained. The method was linear from 1.6 to 836 ng ml(-1). Inter and intra-day assay RSD (n = 6) were less than 9%. Accuracy determinations showed the quality control samples to range between 88-114% of the theoretical concentration.     

High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: application to a pharmacokinetic study in humans

A high-performance liquid chromatographic (HPLC) method was developed using diethylamine (DEA) solid-phase extraction (SPE), 9-fluorenylmethyl derivative (FMOC) and fluorescence detection for quantifying alendronate in human plasma. Sample preparation involved a manual protein precipitation with trichloroacetic acid, a manual coprecipitation of the bisphosphonate with calcium phosphate and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer at pH 11.9. Liquid chromatography was performed on a Capcell Pak C(18) column (4.6 mm x 150 mm, 5 microm particles), using a gradient method starting with mobile phase acetonitrile/methanol-citrate/pyrophosphate buffer (32:68, v/v). The total run time was 25 min. The fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). Pamidronate was used as the internal standard. The limit of quantification was 1 ng/ml using 3 ml of plasma. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg of alendronate sodium to volunteers, the maximum plasma concentration (C(max)) and elimination half-life were 40.94 +/- 19.60 ng/ml and 1.67 +/- 0.50 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies including bioequivalence test of alendronate sodium in humans.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.     

HPLC-紫外法测定人血浆中芬太尼浓度

目的 :建立高效液相色谱法 -紫外检测器测定人血浆中芬太尼浓度的方法。方法 :本实验采用外标法 ,以Shim -PackCLC -ODS(6 0mm×150mm ,5μm )为固定相 ,含0 015mol/LNaH2PO4 的乙腈 -水溶液 (30∶70 ,v/v)为流动相 ,流速1 5ml/min ,紫外检测波长195nm。结果 :标准曲线在2 0～100ng/ml范围内线性关系良好 (r=0 999) ,最低检测浓度为1ng/ml,方法回收率为(91 70±4 70) % ,提取回收率为 (97 38±3 69) % ,日内变异RSD (6 50±2 79) % ,日间变异RSD (6 70±3 04) %。结论 :本方法简便 ,准确 ,检测浓度低 ,能够满足血浆中低浓度芬太尼的测定及临床药代动力学研究的要求。     

Rapid high-performance liquid chromatographic method for determination of adefovir in plasma using UV detection: application to pharmacokinetic studies

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of adefovir (CAS 106941-25-7) in human plasma. The separation was achieved on a monolithic silica column (Chromolith Performance RP-18e, 100 x 4.6 mm) using acetonitrile-ammonium dihydrogen phosphate buffer (6:94, v/v), pH 5.2, as the mobile phase at a flow rate of 1.5 ml min(-1). The wavelength was set at 260 nm. The assay enables the measurement of adefovir for therapeutic drug monitoring with a minimum quantification limit of 1 ng ml(-1). The method involves a simple protein precipitation procedure. Analytical recovery was complete. The calibration curve was linear over the concentration range 1-40 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 5%. The method was applied to the determination of adefovir in plasma from 12 subjects dosed with adefovir 2 x 10 mg tablets and pharmacokinetic parameters were evaluated.     

Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for quantification of amlodipine in plasma. The assay enables the measurement of amlodipine for therapeutic drug monitoring with a minimum detectable limit of 0.2 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was about 97%. The separation was performed on an analytical 125 x 4.6 mm i.d. Nucleosil C8 column. The wavelength was set at 239 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (63:37, v/v) adjusted to pH 3.5 at a flow rate of 1.5 ml min(-1). The calibration curve was linear over the concentration range 0.5-16 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 10%.     

HPTLC method for guggulsterone. I. Quantitative determination of E- and Z-guggulsterone in herbal extract and pharmaceutical dosage form

A sensitive, selective, precise and robust high-performance thin-layer chromatographic method of analysis of E and Z stereoisomers of guggulsterone (the hypolipidemic agent in the gum-resin exudates of Commiphora mukul) both as a bulk drug and in formulations was developed and validated. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of toluene-acetone (9:1, v/v). Densitometric analysis of guggulsterone was carried out in the absorbance mode at 250 nm. This system was found to give compact spots for E- and Z-guggulsterone (Rf value of 0.38 +/- 0.02 and 0.46 +/- 0.02, respectively) following double development of chromatoplates with the same mobile phase. The linear regression analysis data for the calibration plots for E- and Z-guggulsterone showed good linear relationship with r2 = 0.9977 +/- 0.054 and 0.9975 +/- 0.068, respectively, in the concentration range of 100-6000 ng/spot. The mean value of slope and intercept were 0.11 +/- 0.006 and 0.11 +/- 0.005, 14.26 +/- 0.56 and 10.92 +/- 0.76, respectively, for E- and Z-guggulsterone. The method was validated for precision, robustness and recovery. The limit of detection and quantitation were 12, 10 and 24, 20 ng/spot, respectively, for E- and Z-guggulsterone. Statistical analysis proves that the method is repeatable and selective for the estimation of the said drug. Since the proposed mobile phase effectively resolves the E- and Z-isomers of guggulsterone, this HPTLC method can be applied for identification and quantitation of these isomers in herbal extracts and pharmaceutical dosage form.     

Determination of deoxyschizandrin in rat plasma by LC-MS

A simple, rapid and sensitive LC-MS method was developed for quantification of deoxyschizandrin in rat plasma. A 50 miccrol plasma sample was extracted by ether and performed on Elite Hypersil C(18) column (200 mm x 4.6 mm, 5 microm) with the mobile phase of methanol-water (84:16, v/v) in a run time of 6.5 min. The analyte was monitored with positive atmospheric pressure chemical ionization (APCI) by selected ion monitoring (SIM) mode. A good linear relationship was obtained over the range of 1.0-50.0 ng/ml and the validated method was successfully applied for the pharmacokinetic studies of deoxyschizandrin in rat. After oral administration of 4 mg/kg deoxyschizandrin and Schisandra extract which contained the same dose of deoxyschizandrin to male rats, the C(max) of deoxyschizandrin were 15.8+/-3.1 and 34.3+/-16.8 ng/ml, T(max) were 0.51+/-0.13 and 3.83+/-1.83 h, T(1/2) were 5.3+/-2.2 and 6.5+/-3.4h.     

A rapid reversed phase high performance liquid chromatographic method for determination of etoposide (VP-16) in human plasma

A rapid, simple and sensitive isocratic High Performance Liquid Chromatography (HPLC) method was developed to measure the concentration of etoposide in plasma samples with UV detection at 220 nm. The method uses a Bondapac C18 column at 60 degrees C. The mobile phase consists of Methanol: water (45:55 v/v) at a flow rate of 2.8 ml/min. Phenacetin was used as an internal standard. The plasma samples were extracted using ether with the organic layer evaporated under nitrogen. The residue was dissolved in 200 microl methanol with 20 microl injected into the HPLC column. The extraction method showed a recovery of 91.5+/-3% for etoposide. In this system, the retention time of phenacetin and etoposide were 3.3 and 4.4 min, respectively. The limit of detection of etoposide in plasma is 20 ng/ml and the limit of quantitation is 40 ng/ml. This analytical method has very good reproducibility (8.1% between-day variability at a concentration of 50 ng/ml). It is a fast, sensitive and economic method applicable for clinical and pharmacokinetic studies.     

Improved LC method to determine ivermectin in plasma

A simple, rapid and sensitive high-performance liquid chromatographic (HPLC) method has been developed to quantify Ivermectin (IVM) in plasma using an isocratic system with fluorescence detection. The method included a fast liquid phase extraction using cold methanol. HPLC separation was carried out by reversed phase chromatography with a mobile phase composed of methanol:acetonitrile:water with 0.2% acetic acid (45:50:5 v/v/v), pumped at flow rate of 2 ml min(-1). Fluorescence detection was performed at 365 nm (excitation) and 475 nm (emission). The calibration curve for IVM was linear from 0.25 to 100 ng ml(-1). The validation method yielded good results regarding linearity, precision, accuracy, specificity and recoveries. The values of the limit of detection (LOD) and limit of quantification (LOQ) were 0.032 and 0.167 ng ml(-1), respectively.     

Quantitative determination of spinosin in rat plasma by liquid chromatography-tandem mass spectrometry method

A sensitive method for the quantitative determination of spinosin in rat plasma was developed and validated using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by methyl tert-butyl ether (MTBE) after acidification with 1.0% acetic acid aqueous solution. Chromatographic separation was achieved on an Agilent Zorbax SB-C(18) (50 mm x 4.6 mm, 5 microm) using a isocratic mobile phase consisting of acetonitrile-water (30:70, v/v) with 1% isopropyl alcohol and 0.01% heptafluorobutyric acid. The flow rate was 0.2 ml/min. The column temperature was maintained at 25 degrees C. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear over the range of 1.00-400 ng/ml in rat plasma, with 1.00 ng/ml of the lower limit of quantification (LLOQ). The inter- and intra-day precisions and accuracy for all samples were satisfactory. The validated method was successfully applied for the pharmacokinetic study of spinosin in rat. After oral administration of 20mg/kg spinosin to rats, the main pharmacokinetic parameters of T(max), C(max), T(0.5) and AUC(0-T) were 5.33+/-0.58 h, 132.2+/-10.6 ng/ml, 4.89+/-0.37 h, 1.02+/-0.09 microg h/l, respectively.     

Rapid determination of minoxidil in human plasma using ion-pair HPLC

A rapid, simple and sensitive ion-pair high-performance liquid chromatography (HPLC) method has been developed for quantification of minoxidil in plasma. The assay enables the measurement of minoxidil for therapeutic drug monitoring with a minimum detectable limit of 0.5 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was performed on an analytical 150 x 4.6 mm i.d. microbondapak C18 column. The wavelength was set at 281 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (60:40, v/v) containing 2.5 mM sodium dodecyl sulphate adjusted to pH 3.5 at a flow rate of 1 ml/min. The column temperature was set at 50 degrees C. The calibration curve was linear over the concentration range 2-100 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.     

Simultaneous determination of ranitidine and metronidazole in human plasma using high performance liquid chromatography with diode array detection

The development and validation of a simple method for the simultaneous determination of ranitidine and metronidazole in human plasma is described. Plasma samples (250 microL) were deproteinized by precipitation with 60% perchloric acid, centrifuged and the supernatant directly injected into the HPLC. Separation was achieved in isocratic mode with a Shimpak C(18) column and a mobile phase consisting of 10mM potassium dihydrogen phosphate pH 3.5:acetonitrile (90:10, v/v) with UV detection at 315 nm. The method showed good selectivity and sensitivity. Good and consistent recovery for metronidazole and ranitidine was obtained: 96.22+/-3.52 and 95.00+/-4.50% for ranitidine (25-1000 ng/mL) and metronidazole (60-10,000 ng/mL), respectively (n=3). With this one-step sample preparation method, both ranitidine and metronidazole could be quantified simultaneously in human plasma with good precision (R.S.D.<15%) and accuracy (bias values below 15%). The limit of quantification for ranitidine and metronidazole were 20 and 40 ng/mL plasma, respectively.     

Determination of fexofenadine enantiomers in human plasma with high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatography (HPLC) method was developed as an assay for fexofenadine enantiomers in human plasma. Fexofenadine enantiomers were separated using a mobile phase of 0.5% KH(2)PO(4)-acetonitrile (65:35, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 ml/min and measurement at 220 nm. Analysis required 400 microl of plasma and involved solid-phase extraction with an Oasis HLB cartridge, which gave recoveries for both enantiomers from 67.4 to 71.8%. The lower limit of quantification was 25 ng/ml for (R)- and (S)-fexofenadine. The linear range of this assay was between 25 and 625 ng/ml (regression line r(2)>0.993). Inter- and intra-day coefficients of variation were less than 13.6% and accuracies were within 8.8% over the linear range for both analytes. This method can be applied effectively to measure fexofenadine enantiomer concentrations in clinical samples.