Gradient high-performance liquid chromatography for the simultaneous determination of chlorogenic acid and baicalin in plasma and its application in the study of pharmacokinetics in rats

A novel HPLC-UV method was developed for the simultaneous determination of two major active components in Yinhuang injection, chlorogenic acid and baicalin, in rat plasma. Extracted from the plasma samples with methanol-acetonitrile (3:1, v/v), the two compounds were successfully separated using a C18 column with a gradient elution composed of 15 and 54% methanol-acetonitrile (1:1, v/v) in 0.2% (v/v) phosphoric acid water solution (pH 2.0). The flow-rate was set at 1 ml min(-1) and the eluent was detected at 327 nm for chlorogenic acid, 278 nm for baicalin. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388-12.4 microg ml(-1), 0.485-124 microg ml(-1) for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7 and 9%, with the relative error ranging from -9.5 to 7.3% and from -4.2 to 1.8%. The limit of detection (LOD) and the limit of quantification (LOQ) for chlorogenic acid and baicalin in plasma were 0.194, 0.122, 0.388 and 0.485 microg ml(-1), respectively. This assay has been successfully applied in the pharmacokinetic study of chlorogenic acid and baicalin in vivo through intravenous administration of Yinhuang injection to rats.     

HPLC determination and pharmacokinetic study of homoeriodictyol-7-O-beta-D-glucopyranoside in rat plasma and tissues

Homoeriodictyol-7-O-beta-D-glucopyranoside (HEDT-Glu) was isolated from Viscum coloratum and identified by MS, 1H- and 13C-NMR. A HPLC method was developed for determination of HEDT-Glu in rat plasma and tissues. All biological samples were pretreated by protein precipitation with acetone. Vanillin was selected as internal standard. The mobile phase consisted of methanol-water-glacial acetic acid (45 : 55 : 0.5, v/v/v). Good linearity were observed over the concentration ranges of 0.1-200.0 microg.ml(-1) in rat plasma and 0.05-5.0 microg.ml(-1) in tissues. Both intra- and inter-day precisions of HEDT-Glu, expressed as the relative standard deviation, were less than 13.1%. Accuracy, expressed as the relative error, ranged from -0.8 to 5.4% in plasma and from -5.6 to 9.4% in tissues. The mean extraction recovery of HEDT-Glu was above 73.17% in biological samples. The described assay method was successfully applied to the pre-clinical pharmacokinetic study of HEDT-Glu. After intravenous administration of HEDT-Glu to rat, AUC and CL(tot) were 16.04+/-3.19 microg.h.ml(-1) and 0.85+/-0.17 l.kg(-1).h(-1), respectively. T(1/2,alpha) and t(1/2,beta) were 0.06+/-0.01 h and 1.27+/-0.31 h, respectively. HEDT-Glu was cleared from the blood and mainly distributed to the liver and small intestine.     

UPLC-MS/MS法同时测定人血浆中黄芩苷和绿原酸

建立UPLC-MS/MS法同时测定人血浆中黄芩苷和绿原酸的浓度，研究注射用银黄在健康人体的药代动力学。色谱柱为BEH C₁₈(50 mm×2.1 mm ID，1.7 μm)；流动相：A(水，0.1%甲酸)，B(甲醇，0.1%甲酸)，梯度洗脱；流速：0.35 mL·min^-1。电喷雾离子源，多反应监测。黄芩苷：［M+H］⁺，m/z 447→271；绿原酸：［M-H］^-，m/z 353→191；山柰素：［M+H］⁺，m/z 287→287。结果表明，黄芩苷和绿原酸血药浓度的线性范围分别为9.6～1 540和7.5～1 200 ng·mL^-1，日内、日间精密度均小于10.2%。志愿者静脉滴注注射用银黄后，黄芩苷和绿原酸的药时曲线分别符合二房室和三房室模型。该法简便、灵敏、特异，适用于血浆中黄芩苷和绿原酸浓度的测定。     

Bioavailability of salvianolic acid B in conscious and freely moving rats

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 microl) was vortex-mixed with 50 microl of internal standard solution (chloramphenicol 10 microg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm x 4.6 mm, 5 microm) with a mobile phase of acetonitrile-methanol-20mM NaH(2)PO(4) (adjusted to pH 3.5 with H(3)PO(4)) (20:10:70 v/v/v) containing 0.1mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration-response relationship from the present method indicated linearity over a concentration range of 0.5-200 microg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC-MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030+/-565 and 582+/-222 min microg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.     

Determination and pharmacokinetics of isoferulic acid in rat plasma by high-performance liquid chromatography after oral administration of isoferulic acid and Rhizoma Cimicifugae extract

A simple and specific high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) absorbance detection has been developed for the determination of isoferulic acid in rat plasma. The plasma samples were deproteinized with methanol after the addition of internal standard (IS) tinidazole. The analysis was performed on a Kromasil C18 column (250 mm x 4.6 mm i.d., 5 microm particle size) with acetonitrile-0.05% phosphoric acid (25:75, v/v) as mobile phase. The linear range was 0.0206-5.15 microg ml(-1) and the lower limit of quantification (LLOQ) was 0.0206 microg ml(-1). The intra- and inter-day relative standard deviations (R.S.D.s%) were less than 11.4 and 12.3%, respectively, and accuracy as relative error (R.E.%) between -6.7 and -1.1%. Mean extraction recovery was above 80%. The validated method was successfully applied to the pharmacokinetic study of isoferulic acid in rat plasma after oral administration of isoferulic acid and Rhizoma Cimicifugae extract.     

High-performance liquid chromatography method for the determination of mycophenolic acid in human plasma and application to a pharmacokinetic study of mycophenolic acid dispersible tablet

A sensitive and selective high-performance liquid chromatographic-ultraviolet (HPLC-UV) method for the determination of mycophenolic acid (MPA, CAS 24280-93-1) in human plasma has been developed. Sample treatment was based on protein precipitation with a trichloroacetic acid-water (10:90, w/v) solution. The analytical determination was carried out by HPLC with ultraviolet detection at 254 nm. Chromatographic separation was achieved on a C18 column by isocratic elution with acetonitrile-water (pH 4.4) (50:50, v/v) at a flow rate of 1.0 mL/min. The method was linear in the concentration range of 0.2-50.0 microg/mL. The lower limit of quantification (LLOQ) was 0.2 microg/ mL. The intra-day and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 7.05%. The accuracy determined at three concentrations (0.4, 5.0 and 20.0 microg/mL for MPA) was within +/- 10.0%. The method was successfully applied to the evaluation of the pharmacokinetic profile of MPA dispersible tablet in 20 healthy volunteers. The results showed that AUC, C(max) and T1/2 for the test and reference formulations were not significantly different (P > 0.05). The relative bioavailability was 96.42 +/- 15.5%.     

Determination of roxithromycin in rat lung tissue by liquid chromatography-mass spectrometry

A liquid chromatography-mass spectrometry (LC-MS) method for the determination of roxithromycin in rat lung tissue is described. Liquid-liquid extraction was adopted for sample preparation with recoveries from 72.5 to 76.9% at levels of 0.1, 5.0 and 20.0 microg/ml. Chromatographic separation was performed on a C18 column using a mixture of methanol, water and formic acid (80:20:1, v/v/v) as mobile phase delivered at a flow rate of 0.5 ml/min. Positive selected ion monitoring (SIM) mode was used for the quantification of roxithromycin at m/z 837.7 and clarithromycin (internal standard) at m/z 748.7. The linearity was obtained over the concentration range of 0.05-20.0 microg/ml and the lower limit of quantification was 0.05 microg/ml. For each QC level of roxithromycin, the intra- and inter-day precisions relative standard deviation (R.S.D.) were less than 4.1 and 7.5%, respectively, and accuracy (RE) was +/-10.0%. The proposed LC-MS method has been successfully used for the determination of roxithromycin in rat lung tissue after oral administration of roxithromycin formulations to 44 SD rats. The present study demonstrates that the concentration of roxithromycin in rat lung tissues can be significantly increased by ambroxol when they are formulated in combination.     

Validation of an HPLC method for the determination of scutellarin in rat plasma and its pharmacokinetics

A validated HPLC method was developed for the quantification of scutellarin in rat plasma using a liquid-liquid extraction and an ultraviolet detection. Chromatographic separation of scutellarin in plasma was performed on a C18 column, with a mobile phase of acetonitrile-water (23:77, v/v), adjusted to pH 2.5 with 1M phosphoric acid, and rutin was used as an internal standard. The calibration curve was linear over the range 0.1-100 microg/ml in rat plasma. The average extraction recoveries were 85.9+/-8.9, 71.0+/-4.6, 72.7+/-1.2% (n=3) at concentrations of 0.1, 2, 100 microg/ml, respectively, and the within-day and between-day precisions were less than 15%. After intravenous administration to rats over the doses range of 10-40 mg/kg, the plasma concentration-time curve of scutellarin was best conformed to three-compartment open model. The AUC of scutellarin was proportional to dose, and the systemic clearance (Cl), elimination half-life (t1/2beta) and apparent volume of distribution (Vc) were not significantly different among the three doses, suggestive of the linear pharmacokinetics characteristic of scutellarin after intravenous administration.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry

A highly selective and sensitive HPLC-ESI-MS-MS method was developed for the determination of oleanolic acid in human plasma. The oleanolic acid and glycyrrhetinic acid (internal standard) were recovered from plasma with ethyl acetate liquid-liquid extraction. The organic extracts were dried under a stream of warm nitrogen, reconstituted in mobile phase and injected into a Zorbax-Extend ODS analytical column (150 mm x 4.6 mm i.d., 5 microm), with the mobile phase consisting of methanol-ammonium acetate (32.5 mM) (85:15, v/v) pumped at a flow rate of 1.0 ml/min, and 30% of the eluent was split into a MS system with electrospray ionization tandem mass (ESI-MS-MS) detection in negative ion mode. The tandem mass detection was performed on a Finnigan Surveyor LC-TSQ Quantum Ultra AM tandem mass spectrometer operated in selected reaction monitoring mode. The parent to product ion combinations of m/z 455.4-->455.4 and 469.3-->425.2 at 38 V 1.5 mTorr Ar CID were used to quantify oleanolic acid and glycyrrhetinic acid, respectively. The assay was validated in the concentration range of 0.02-30.0 ng/ml for oleacolic acid when 0.5 ml of plasma was processed. The precision of the assay (expressed as relative standard deviation, R.S.D.%) was less than 15% at all concentrations levels within the tested range and adequate accuracy, and the limit of quantification was 0.02 ng/ml. The established method was applied for the pharmacokinetics study of oleanolic acid capsules in 18 healthy male Chinese volunteers with the mean values of C(max), T(max), AUC(0-48), AUC(0-infinity), t(1/2,) CL/F, and V/F of oleanolic acid after p.o. a single 40 mg dose obtained were 12.12 +/- 6.84 ng/ml, 5.2 +/- 2.9h, 114.34 +/- 74.87 ng h/ml, 124.29 +/- 106.77 ng h/ml, 8.73 +/- 6.11 h, 555.3 +/- 347.7 L/h, and 3371.1 +/- 1,990.1 L, respectively.     

Rapid and simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatographic method was developed for simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in human plasma. The method entailed injection of the samples after deproteination with perchloric acid and subsequent neutralizing. Primidone was used as internal standard. Chromatography was performed on a C(18) column (250 mm x 4.6 mm, 5 microm) under isocratic elution with 50 mM aqueous sodium dihydrogen phosphate-acetonitrile-triethylamine (100:25:0.5, v/v), pH 5.9. Detection was made at 240 nm and analyses were run at a flow-rate of 1.5 ml/min at a temperature of 35 degrees C. The recovery was 83.4, 88.5 and 98.2% for TMP, SMX and internal standard, respectively. The precision of the method was 2.6-9.8% over the concentration range of 0.125-2 microg/ml for TMP and 0.39-50 microg/ml for SMX. The limit of quantification (LOQ) in plasma was 0.125 and 0.39 microg/ml for TMP and SMX, respectively. The method was used for a bioequivalence study.     

Validated HPLC method for determination of PAT-5A, an insulin sensitizing agent, in rat plasma

A high performance liquid chromatographic method for the determination of PAT-5A (a potent insulin sensitizer) using DRF-2095 (a thiazolidinedione) as internal standard (I.S.) is described. A 1:1 v/v ethylacetate and dichloromethane solvent mixture was used for extraction of PAT-5A from plasma. A Kromasil KR100-5C18-250A, 5 microm, 4.6 x 250 mm SS column was used for the analysis. Mobile phase consisting of sodium dihydrogen phosphate (pH 4.0, 0.05 M) and methanol mixture (25:75, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector set at 345 nm. Ratio of peak area of analyte to I.S. was used for quantification of plasma samples. Using this method the absolute recovery of PAT-5A from rat plasma was > 90% and the limit of quantification was 0.05 microg/ml. The intra-day relative standard deviation (RSD) ranged from 2.19 to 4.98% at 1.0 microg/ml, 1.05 to 3.68% at 10.0 microg/ml and 3.14 to 5.08% at 50 microg/ml. The inter-day RSD were 1.6, 2.24 and 1.54% at 1, 10 and 50 microg/ml, respectively. The method was applied to measure the plasma concentrations of PAT-5A in pharmacokinetic and bioavailability studies in male Wistar rats.     

A simple high-performance liquid chromatographic method for the determination of acyclovir in human plasma and application to a pharmacokinetic study

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir (CAS 59277-89-3) concentrations in human plasma and its use in bioavailability studies is evaluated. The method was linear in the concentration range of 0.05-4.0 microg/ml. The lower limit of quantification (LLOQ) was 0.05 microg/ml in 0.5 ml plasma sample. The intra- and inter-day relative standard deviations across three validation runs over the entire concentration range were less than 8.2%. This method was successfully applied for the evaluation of pharmacokinetic profiles of acyclovir capsule in 19 healthy volunteers. The main pharmacokinetic parameters obtained were: AUC(o-t) 6.50 +/- 1.47 and 7.13 +/- 1.44 microg x h/ml, AUC(0-infinity) 6.77 +/- 1.48 and 7.41 +/- 1.49 microg x h/ml, C(max) 2.27 +/- 0.57 and 2.27 +/- 0.62 microg/ml, t(1/2) 2.96 +/- 0.41 and 2.88 +/- 0.33 h, t(max) 0.8 +/- 0.3 and 1.0 +/- 0.5 h for test and reference formulations, respectively. No statistical differences were observed for C(max) and the area under the plasma concentration--time curve for acyclovir. 90% confidence limits calculated for C(max) and AUC from zero to infinity (AUC(0-infinity)) of acyclovir were included in the bioequivalence range (0.8-1.25 for AUC).     

Solubilization and quantification of lycopene in aqueous media in the form of cyclodextrin binary systems

An optimized kneading method for the preparation of lycopene-cyclodextrin binary systems was developed leading to solubilization of lycopene in water and 5% (w/v) dextrose solution. Lycopene quantification in the prepared binary systems was performed by a developed spectrometric method that followed a successful single-step extraction with dichloromethane. Storage stability characteristics of the binary systems were studied at 4 degrees C in solution and at -20 degrees C in the lyophilized products. Lycopene content was monitored at lambda(max)=482 nm, the limit of detection was 0.41 microg/ml and relative standard deviation was less than 3.1%. The results obtained with the spectrometric method were confirmed by a HPLC method. In the presence of cyclodextrins, lycopene concentration in water was 8.0+/-1.0, 27.1+/-3.2 and 16.0+/-2.2 microg/ml for beta-CD, HP-beta-CD and Me-beta-CD, respectively. In 5% (w/v) aqueous dextrose solutions the corresponding values were 16.0+/-1.8, 48.0+/-5.1 and 4.0+/-0.5 microg/ml, respectively. At 4 degrees C, storage stability of lycopene-cyclodextrin binary systems in water or 5% (w/v) aqueous dextrose solutions, was limited (t(1/2)=1-4 days). Addition of the antioxidant sodium metabisulfite increased the stability of lycopene-HP-beta-CD binary system in water. At -20 degrees C, the lyophilized lycopene-cyclodextrin binary systems were stable for at least 2 weeks.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

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.     

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.     

Determination of ibuprofen in dog plasma by liquid chromatography and application in pharmacokinetic studies of an ibuprofen prodrug in dogs

A liquid chromatography (LC) method for the determination of ibuprofen in dog plasma is described. Chromatographic separation was performed on a Diamonsil C18 column with a C18 guard column using a binary mixture of acetonitrile and 0.02 mol/l phosphate buffer (pH 6.5) (35:65, v/v) delivered at a flow rate of 1.2 ml/min. The linear range for ibuprofen was from 1.0 to 40.0 microg/ml with a limit of quantitation of 1.0 microg/ml. Within-run accuracy and precision ranged from -0.1% to 4.0% and from 1.1% to 5.5% and between-run accuracy and precision ranged from -1.1% to 4.7% and from 1.3% to 7.0%, respectively. The mean extraction recoveries of ibuprofen determined over the concentrations of 1.0, 10.0, and 40.0 microg/ml were (100.5+/-1.8)%, (99.8+/-1.0)%, and (99.2+/-2.3)%. The developed LC method greatly simplified the sample preparation and adopted mild conditions to prevent the possible hydrolysis of the prodrug and was successfully applied to the pharmacokinetic studies of an ibuprofen prodrug in dogs.     

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.     

Quantification and 24-hour monitoring of mycophenolic acid by high-performance liquid chromatography in Japanese renal transplant recipients

We developed a rapid, simple, and sensitive high-performance liquid chromatography method with UV detection for the quantitative determination of mycophenolic acid (MPA) in human plasma. MPA and the internal standard (naproxen) were separated using a mobile phase of 0.04 M H(3)PO(4)-acetonitrile-methanol (3:3:4 v/v/v) over a CAPCELL PAK C18 MG column. A flow-rate of 0.5 ml/min was used at ambient temperature and sample detection was carried out at 254 nm. The assay required only 100 microl of plasma and involved liquid-liquid extraction, which gave high recovery (>94%). The lower limit of quantification for MPA was 0.05 microg/ml. Inter- and intra-day coefficients of variation were less than 9.6% and accuracies were within 9.3%. Additionally, we validated this method in 24-hour monitoring of plasma MPA concentrations after mycophenolate mofetil (MMF) morning and evening administration in 40 Japanese renal transplant recipients with 1.5 g/day MMF. The time to reach the maximum (11.7 microg/ml) and second peak (4.5 microg/ml) of MPA after morning 0.75 g MMF administration was 2.6 h and 9.0 h, and time to reach maximum (10.5 microg/ml) after evening 0.75 g administration was 4.0 h.