Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of the enantiomers of a novel anticonvulsant, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine, in rats

A selective chiral high performance liquid chromatographic (HPLC) method was developed and validated to separate and quantify the enantiomers of a novel anticonvulsant agent, N-(4-chlorophenyl)-1-(4-pyridyl)ethylamine (AAP-Cl), in rat plasma. After extraction of the plasma samples with ethyl acetate, the separation was accomplished by an HPLC system consisting of a Chirex chiral column (250 mm x 4.6 mm i.d.) and a mobile phase of hexane:ethanol:tetrahydrofuran (280:20:40 (v/v)) containing trifluroacetic acid (0.3% (v/v)) and triethylamine (0.018% (v/v)) at a flow rate of 0.8 ml/min with UV detection. Male Sprague-Dawley rats were given (+)-AAP-Cl (10 and 20 mg/kg), (-)-AAP-Cl (10 mg/kg) or the racemic mixture (20 mg/kg) by i.v. bolus injection and serial blood samples were collected at different times after drug administration. (+)-AAP-Cl and (-)-AAP-Cl were separated with a resolution factor, Rs, of at least 1.4, and a separation factor, alpha, greater than 1.09. Linear calibration curves were obtained over the concentration range of 0.5-30 microg/ml in plasma for both (+)-AAP-Cl and (-)-AAP-Cl (R2 > or = 0.996) with a limit of quantitation of 100 ng/ml and the recovery was greater than 80% for both enantiomers. The accuracy and precision for both enantiomers ranged from 96 to 102% (+/-0.2-7%) at upper and lower concentrations. The plasma concentration-time profiles of the enantiomers of AAP-Cl were best described by a two-compartment open model with a mean terminal half-life of about 5h, volume of distribution at steady state of 3 l/kg and clearance of about 0.6l/(hkg) in rats. There was no significant difference between the pharmacokinetic parameters of (+)-AAP-Cl and (-)-AAP-Cl, suggesting that the disposition of AAP-Cl in rats is not enantioselective. In addition, no chiral inversion of (+)-AAP-Cl to (-)-AAP-Cl or vice versa was observed. The results of this investigation have shed some light on the mechanism of action and disposition of AAP-Cl in rats.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.     

The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.     

Fast and reliable determination of the antifungal drug voriconazole in plasma using monolithic silica rod liquid chromatography

In the present study, we developed a fast and reliable HPLC assay for the determination of the new triazole antifungal agent voriconazole in plasma, using a Chromolith RP 18e (100 mm x 4.6 mm) monolithic silica rod HPLC column. After liquid-liquid extraction, plasma samples were separated with a mobile phase consisting of ammoniumdihydrogencarbonate buffer (pH 5.8)-acetonitrile-tetrahydrofuran (72:25:3) at a flow-rate of 3.5 mL/min and UV detection at 255 nm. The retention times for voriconazole and internal standard (UK-115794) were 2.3 and 2.7 min, respectively, and total run time was 4 min. The calibration curves were linear between 0.05 and 10 microg/mL, and within-assay and between-assay coefficients of variation were <4%. The proposed assay for voriconazole in plasma is fast, sensitive and reliable, and, thus, well suited for routine therapeutic monitoring of patients and for pharmacokinetic studies. It can be predicted that the use of monolithic silica rod chromatography will substantially shorten the turn-around time in the therapeutic drug monitoring laboratory.     

High-performance liquid chromatography spectrometric analysis of trans-resveratrol in rat plasma

A HPLC method for determination of trans-resveratrol concentrations in rat plasma was developed. Plasma samples were treated with acetonitrile to deposit proteins. The analysis used a Hypersil ODS(2) C(18) column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow-rate=1 mL/min). The UV detection wavelength was 303 nm, and chlorzoxazone was used as the internal standard. The calibration curve was linear over the range of 0.02-40 microg/mL with a correlation coefficient of 0.9997. This concentration range corresponds well with the plasma concentrations of resveratrol in pharmacokinetic studies. There was 98.7%, 91.3% and 84.4% recovery from 0.02, 0.4 and 40 microg/mL plasma samples respectively. The R.S.D. of intra- and inter-day assay variations were all less than 12%. This HPLC assay is a quick, precise and reliable method for the analysis of resveratrol in pharmacokinetic studies.     

Determination of josamycin in rat plasma by capillary electrophoresis coupled with post-column electrochemiluminescence detection

A novel determination method for josamycin (JOS) based on capillary electrophoresis-electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 microm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 microm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3(2+) and 50mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 microg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.     

Determination of ergometrine maleate by fluorescence detection.

A simple, accurate, sensitive and selective fluorescence analysis method for rapid determination of trace amounts of ergometrine maleate has been developed. The method is based on the fluorescence of ergometrine maleate. The calibration graph was linear in the range of 1 x 10(-4) to 0.2 microg/mL and the detection limit was 4 x 10(-5) microg/mL, with a relative standard deviation of 0.32% at 0.05 microg/mL (n = 11) ergometrine maleate. The influence of foreign compounds was tested. The proposed method was applied successfully to the determination of ergometrine maleate in pharmaceutical preparations and human plasma.     

Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection

A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm x 4.6 mm, 5 microm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 microg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05-50 microg/mL for all four matrices.     

Determination of glucosamine sulfate in human plasma by precolumn derivatization using high performance liquid chromatography with fluorescence detection: its application to a bioequivalence study

A simple, rapid, selective and specific high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of glucosamine sulfate in human plasma and application to a bioequivalence in healthy volunteers. Precipitation of plasma was accomplished with acetonitrile to separate interfering endogenous products from the compound of interest. After vortex mixing and centrifugation, the supernatant was transferred and derivatized with 9-fluorenylmethoxycarbonyl chloride-acetonitrile solution in borate buffer (pH=8.0) at 30 degrees C for 30 min. The chromatographic separation was performed on a Diamonsil C18 column (150.0 mmx4.6 mm, 5 microm) with a mobile phase gradient consisting of water and acetonitrile at a flow rate of 1 mL/min. The method was linear in the range of 0.1-10.0 microg/mL with a correlation coefficient (r) of 0.9996. The limit of detection was 15 ng/mL. Inter- and intra-day precisions were 相似文献   

Determination of inositol hexanicotinate in rat plasma by high performance liquid chromatography with UV detection

A HPLC method with UV detection at 262nm was developed to analyze inositol hexanicotinate in rat plasma. Plasma samples were extracted with an equal volume of acetonitrile, followed by dilution with mobile phase buffer (5mM phosphate buffer, pH 6.0) to eliminate any solvent effects. Inositol hexanicotinate and the internal standard (mebendazole) were separated isocratically using a mobile phase of acetonitrile/phosphate buffer (35:65, v/v, pH 6.0) at a flow rate of 1.0mL/min and a reverse-phase XTerra MS C(18) column (4.6mmx150mm, 3.5microm). The standard curve was linear over a concentration range of 1.5-100.0microg/mL of inositol hexanicotinate in rat plasma. The HPLC method was validated with intra- and inter-day precisions of 1.55-4.30% and 2.69-21.5%, respectively. The intra- and inter-day biases were -0.75 to 19.8% and 2.58-22.0%, respectively. At plasma concentrations of 1.5-100microg/mL, the mean recovery of inositol hexanicotinate was 99.6%. The results of a stability study indicated that inositol hexanicotinate was unstable in rat plasma samples, but was stable in acetonitrile extracts of rat plasma for up to 24h at 4 degrees C. The assay is simple, rapid, specific, sensitive, and reproducible and has been used successfully to analyze inositol hexanicotinate plasma concentrations in a pharmacokinetic study using the rat as an animal model.     

A new high-performance liquid chromatographic method for determination of warfarin enantiomers

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.     

LC-MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC-MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm x 2.0 mm, 2 microm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 microL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1+285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46-91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([(2)H(3)(15)N] sorafenib) was achieved on a Waters X-Terra C(18) (150 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3-7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).     

Application of solid phase microextraction to the determination of strychnine in blood

A simple and rapid method based on solid phase microextraction (SPME) via direct immersion followed by gas chromatography coupled with electron impact ionization/mass spectrometry (GC/EI-MS) was developed for the determination of strychnine in blood. Papaverine was used as internal standard (I.S.). Two types of fibre coating were tested, 100 microm polydimethylsiloxane and 65 microm Carbowax/Divinylbenzene, the latter giving higher recoveries of the compound. The main factors affecting the SPME process, such as sample dilution (1:10), adsorption and desorption times (20 and 10 min, respectively), carry-over effect (not observed), pH and salt addition (no modifications on pH or salt concentration) were optimized. The procedure was validated in terms of linearity (r(2)=0.9992 for concentrations ranging from 0.10 to 5.00 microg/mL), intra and interday precision (0.93 and 4.62%, respectively at 0.50 microg/mL; 3.33 and 8.06%, respectively at 2.50 microg/mL), sensitivity (6.83 and 8.91 ng/mL for LOD and LOQ, respectively) and extraction recovery (0.54 and 0.39% at 0.50 and 2.50 microg/mL, respectively). The developed procedure was found suitable for forensic investigations and was considered a good alternative to the liquid-liquid extraction methods normally used for the determination of this compound in biological media.     

Determination of lansoprazole and two of its metabolites by liquid-liquid extraction and automated column-switching high-performance liquid chromatography: application to measuring CYP2C19 activity

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method for the simultaneous determination of lansoprazole, a proton pump inhibitor and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human plasma. The test compounds were extracted from 1 mL of plasma using diethyl ether-dichloromethane (7:3, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 microm, 3.5 mm x 4.6 mm i.d.) for clean-up and column I (C(18) STR ODS-II analytical column, 5 microm, 150 mm x 4.6 mm i.d.) for separation. The peak was detected by a ultraviolet detector set at a wavelength of 285 nm, and the total time for a chromatographic separation was approximately 25 min. The method was validated for the concentration range from 3 to 5000 ng/mL. Mean recoveries were 74.0% for lansoprazole, 68.3% for 5-hydroxylansoprazole, and 79.4% for lansoprazole sulfone. Intra- and inter-day relative standard derivatives were less than 6.1 and 5.1% for lansoprazole, 5.8 and 5.8% for 5-hydroxylansoprazole, 4.4 and 5.9% for lansoprazole sulfone, respectively, at the different concentration ranges. This method is suitable for use in therapeutic drug monitoring and pharmacokinetic studies, and provides use tool for measuring CYP2C19 activity.     

Estimation of p-coumaric acid as metabolite of E-6-O-p-coumaroyl scandoside methyl ester in rat plasma by HPLC and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of p-coumaric acid in rat plasma and applied to a pharmacokinetic study in rats after administration of a prodrug, E-6-O-p-coumaroyl scandoside methyl ester, isolated from Hedyotis diffusa (Willd.). Sample preparation involved protein precipitation with acetonitrile. The supernatant was then injected onto a Diamonsil C(18) column (250 mm x 4.6mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water (21:79, v/v) with 1% glacial acetic acid. The UV detector was set at 310 nm. The lower limit of quantification of p-coumaric acid in rat plasma was 0.02 microg/mL. The calibration curves were linear over the concentration range 0.02-5 microg/mL with correlations greater than 0.999. The assay procedure was applied to the study of the metabolite pharmacokinetics of E-6-O-p-coumaroyl scandoside methyl ester in rat.