A high-performance liquid chromatographic method for determination of mitragynine in serum and its application to a pharmacokinetic study in rats

A simple HPLC technique for determining mitragynine levels in serum was developed. The separation system consisted of a C18 column heated to 35 degrees C, a methanol-water (80:20, v/v) mobile phase, a flow rate of 0.8 mL/min and detection in the ultraviolet at 225 nm. Mitragynine, with a retention time of 10.09 min, was well resolved from any interferences in human serum and the internal standard peak. The calibration curve was linear from 0.1 to 10 microg/mL (r = 0.9995). Extraction of mitragy-nine from alkalinized serum using diethyl ether gave a high recovery (>or=85%). The intra- and inter-day precisions of the method were 4.29-5.88%RSD and 7.06-8.45%RSD, respectively. The accuracy ranged from -9.54 to +0.67%DEV. The limit of detection was 0.03 microg/mL and the lower limit of quantification was 0.1 microg/mL. Mitragynine in the stock solution was stable during 30 days of storage at 4 degrees C. This method was successfully applied to determine the pharmacokinetic characteristics of mitragynine levels in the serum of rats after it was administered orally.     

Determination of cocaethylene, cocaine and their metabolites in rat serum microsamples by high-performance liquid chromatography, and its application to pharmacokinetic studies in rodents.

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating cocaethylene in rat serum microsamples (50 microliters), a substance formed in vivo when cocaine and ethanol are present concurrently. The separation used a 2 mm I.D. reversed-phase Nova-Pak C18 column with a mobile phase of acetonitrile-phosphate buffer containing an ion-pairing reagent. With an ultraviolet detector operated at 230 nm, a linear response was observed from 0.05 to 2.0 micrograms/ml with a detection limit of 5 ng/ml for cocaethylene, cocaine and norcocaine. The method showed a longer half-life for cocaethylene than for cocaine in rat.     

Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

A simple high-performance liquid chromatography (HPLC) method is described for the determination of matrine in rat plasma. The plasma was deproteinized with acetonitrile that contained an internal standard (phenacetin) and was separated from the aqueous layer by adding sodium chloride. Matrine was extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: YMC-pack ODS-A, 5 μm, 150×4.6 mm, I.D.; eluent: acetonitrile-0.02 mol ammonium acetate buffer-triethylamine (35:65:0.035, v/v/v) and ultraviolet detection (220 nm). The limit of quantitation for matrine was 200 ng ml⁻¹ in plasma, and the recovery was greater than 89%. The assay was linear from 0.5 to 50.0 μg ml⁻¹. Variation over the range of the standard curve was less than 6%. The method was used to determine the concentration-time profiles of matrine in the plasma following oral administration of matrine aqueous solution or bolus injection from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method for the first time.     

Determination of forsythiaside in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

A simple high-performance liquid chromatographic method was developed to study the pharmacokinetics of forsythiaside in rat plasma after intravenous administration. Hesperidin was used as the internal standard. The drugs were separated on a reversed-phase C(18) column and detected at 332 nm. Good linearity was achieved in the range of 0.067-26.667 microg/mL. The intra- and inter-assay variation coefficients for this analysis were no more than 10.94 and 14.56%, respectively. The average recovery for forsythiaside was 87.42% from plasma. The analytical sensitivity and accuracy of this assay were adequate for characterization of the pharmacokinetics of intravenous administration of forsythiaside to rats and the assay has been successfully applied to provide pharmacokinetic data. The mean t(1/2Z) was 20.36, 19.40 and 23.62 min for 2, 5 and 20 mg/kg for forsythiaside after i.v. administration, respectively. The AUC(0-t) increased linearly from 40.64 to 624.14 microg min/mL after administration of the three doses.     

Determination of tanshinone I in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

This paper describes a rapid and sensitive high-performance liquid chromatographic (HPLC) method for the determination of the concentration of tanshinone I in rat plasma, and applies the method to pharmacokinetic study. The plasma is deproteinized with acetonitrile containing an internal standard (estradiolbenzoate). The HPLC assay is carried out using a Cosmosil C18 column. The mobile phase is acetonitrile, 0.05 mol/L(-1) ammonium acetate buffer with 1% acetic acid (66:34, v/v). The flow rate is 1.0 mL/min. The detection wavelength is set at 263 nm. The assay accuracy is better than 92%, and the precision of tanshinone I at low to high concentrations is better than 9% and 11% for intra-day and inter-day assays, respectively. The recovery of the method exceeds 88.3% for tanshinone I. The assay shows good linearity (r = 0.9998) over a relatively wide concentration range from 0.05 to 10.0 microg/mL. The method is used to determine the concentration-time profiles of tanshinone I in plasma following an intravenous injection of tanshinone I solution, and the pharmacokinetic parameters of tanshinone I are calculated for the first time by the Drug and Statistics 1.0 program. This assay is successfully applied to the determination of tanshinone I in rat plasma, and the developed method is applied to pharmacokinetic studies for the first time.     

A high-performance liquid chromatographic method for determination of scopolin in rat plasma: application to pharmacokinetic studies

An analytical method based on high-performance liquid chromatographic (HPLC) with ultraviolet (UV) detection was developed for determination of scopolin in rat plasma using aesculin as internal standard (IS). After protein precipitation of plasma sample with methanol, the supernatant was directly injected and analyzed. Chromatographic separation was achieved on a C18 column using methanol and distilled water (22:78, v/v) containing 0.2% (v/v) glacial acetic acid as mobile phase with a column temperature of 30 degrees C. The UV detector was set at 338 nm. The calibration curve was linear over the range of 0.105-13.125 microg/mL with a correlation coefficient of 0.9998. The retention times of aesculin and scopolin were 10.4 and 12.8 min, respectively. The recoveries for plasma samples of 0.105, 4.725 and 13.125 microg/mL were 91.08, 95.30 and 96.10%, respectively. The RSD of intra- and inter-day assay variations was less than 7.35%. The lower limit of detection was 0.03 microg/mL .This HPLC assay is a simple, sensitive and accurate and was successfully applied to the pharmacokinetic study of scopolin in rats.     

A validated high-performance liquid chromatographic method for the determination of moclobemide and its two metabolites in human plasma and application to pharmacokinetic studies

A rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with ultraviolet detection has been developed for the determination of moclobemide and its metabolites, p-chloro-N-(-2-morpholinoethyl)benzamide N'-oxide (Ro 12-5637) and p-chloro-N-[2-(3-oxomorpholino)ethyl]-benzamide (Ro 12-8095), in human plasma. The assay was performed after single liquid-liquid extraction with dichloromethane at alkaline pH using phenacetin as the internal standard. Chromatographic separation was performed on a C(18) column using a mixture of acetonitrile and water (25:75, v/v), adjusted to pH 2.7 with ortho-phosphoric acid, as mobile phase. Spectrophotometric detection was performed at 239 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The quantification limit for moclobemide and Ro 12-8095 was 10 ng/mL, and for Ro 12-5637 was 30 ng/mL. Linearity of the method was confirmed for the range 20-2500 ng/mL for moclobemide (r = 0.9998), 20-1750 ng/mL for Ro 12-8095 (r = 0.9996) and 30-350 ng/mL for Ro 12-5637 (r = 0.9991). Moreover, within-day and between-day precisions and accuracies of the method were established. The described method was successfully applied in pharmacokinetic studies of parent drug and its two metabolites after a single oral administration of 150 mg of moclobemide to 20 healthy volunteers.     

Determination of fenbendazole and its metabolites in trout by a high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for the simultaneous determination of fenbendazole (FBZ), fenbendazole sulfoxide (FBZ-SO) and fenbendazole sulfone (FBZ-SO2) in trout muscle and skin tissues. The compounds were extracted with acetonitrile and the extract was concentrated and cleaned up by solid phase extraction on C18 and CN cartridges. The extract was analysed by reversed-phase gradient HPLC on a C18 column followed by ultraviolet detection at 290 nm. The method's detection limits were 4.0 micrograms kg-1 for FBZ, 4.5 micrograms kg-1 for FBZ-SO and 3.8 micrograms kg-1 for FBZ-SO2. The mean recovery in muscle was 88% for FBZ, 94% for FBZ-SO and 92% for FBZ-SO2 at a level of 5-150 micrograms kg-1. The corresponding mean recoveries in skin tissue were 88, 81 and 86% at a level of 10-100 micrograms kg-1. The mean relative repeatability standard deviation was 9.2% at a level of 5 micrograms kg-1, 5.9% at a level of 10-100 micrograms kg-1 and 2.3% at a level of 150 micrograms kg-1. The method was applied to trout given feed containing FBZ in an aquaculture pilot plant. The three compounds FBZ, FBZ-SO and FBZ-SO2 were all detected in muscle and skin tissues shortly after administration. The concentrations were generally highest in skin tissue.     

Simultaneous determination of mexiletine and four hydroxylated metabolites in human serum by high-performance liquid chromatography and its application to pharmacokinetic studies.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of mexiletine and its four hydroxylated metabolites in human serum. The method involves a single-step extraction of mexiletine, hydroxymethylmexiletine, p-hydroxymexiletine and their corresponding alcohols with diisopropyl ether-dichloromethane-propan-2-ol (2.5:1.5:0.5, v/v). Separation of the compounds on a deactivated Supelcosil LC8-DB column is accomplished by high-performance liquid chromatography with ultraviolet detection at 203 nm. Overall the recovery of each compound is reproducible and greater than 75%. The lower limit of detection is 2 ng/ml for mexiletine and its metabolites. The application of the method is shown by measuring the concentrations in serum of mexiletine and its metabolites over 24 h in a healthy volunteer after a single intravenous injection of the drug and by monitoring serum concentrations in patients receiving long-term treatment by mouth of the drug.     

Development and validation of a reliable high-performance liquid chromatographic method for determination of nodakenin in rat plasma and its application to pharmacokinetic study

A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed for the determination of nodakenin in rat plasma. The concentration of nodakenin was determined in plasma samples after deproteinization with methanol using hesperidin as internal standard. HPLC analysis was performed on a Diamonsil C(18) analytical column using acetonitrile-water (25:75, v/v) as the mobile phase and a UV detection at 330 nm. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day variation). The extraction recoveries were 91.3 ± 10, 87.8 ± 4.8 and 92.6 ± 5.1 at concentrations of 0.500, 5.00 and 40.0 μg/mL, respectively. The standard curve for nodakenin was linear (r(2) ≥ 0.99) over the concentration range 0.250-50.0 μg/mL with a lower limit of quantification of 0.250 μg/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were not higher than 12% and the accuracy (relative error, RE) was within ± 5.8% at three quality control levels. The validated method was successfully applied for the evaluation of the pharmacokinetics of nodakenin in rats after oral administration of Rhizoma et Radix Notopterygii decoction and nodakenin solution.     

Determination of uracil in 5-fluorouracil substance by high-performance liquid chromatography

Separation of 5-fluorouracil and uracil in chromatographic systems consisting of a silica column as a stationary phase and ethyl acetate or ethyl acetate partially saturated with water as a mobile phase has been studied. The results indicated that when saturation of ethyl acetate with water was chosen to reach more than 60%, such mobile phases [e.g., 2.4% (v/v) water in ethyl acetate] are useful in determining uracil in 5-fluorouracil substances.     

Determination of betaxolol enantiomers by high-performance liquid chromatography. Application to pharmacokinetic studies

A high-performance liquid chromatographic (HPLC) method is described for the determination of (R)- and (S)-enantiomers of betaxolol in blood and other biological fluids. Separation of the enantiomers is performed after preparation of diastereomeric derivatives with the chiral reagent R(-)-naphthylethylisocyanate by reversed-phase HPLC. Fluorimetric detection allows the quantification of betaxolol enantiomers down to 0.5 ng/ml. This method was used to evaluate the pharmacokinetic profile of the betaxolol enantiomers in three subjects following one single oral dose (20 mg) of racemic betaxolol. No significant difference was observed in blood levels of the isomers.     

Determination of sulpiride and sultopride by high-performance liquid chromatography for pharmacokinetic studies

A high-performance liquid chromatographic method with UV detection (226 nm) for the analysis of sulpiride and sultopride in body fluids has been developed. Plasma, red blood cell (RBC) and urine samples were extracted by chloroform at pH 10. Internal standards were a new substituted benzamide (N-[(ethyl-1-pyrrolidinyl-2)methyl] methoxy-2-ethylsulphonyl-5-benzamide, DAN) for the sulpiride assay and sulpiride for the sultopride assay. The detection limit in plasma and RBC was 10 ng/ml for sulpiride and 15 ng/ml for sultopride. The proposed techniques were selective, reliable and sensitive enough to be used for pharmacokinetic studies and drug monitoring. Some plasma and RBC data from pharmacokinetic studies in healthy volunteers (sulpiride) or patients (sultopride) are presented. Half-lives determined from either plasma or RBC concentrations were similar (7 h for sulpiride and 5 h for sultopride).     

Determination of paeonol in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies following oral administration of Moutan cortex decoction

Quantification of paeonol, the principal bioactive component of Moutan cortex, in rat plasma following oral administration of Moutan cortex decoction was achieved by using a simple and sensitive high-performance liquid chromatographic method. The calibration curves for paeonol were linear in both the low (25-200 ng/mL) and the high concentration range (200-4000 ng/mL) with r(2) values of 0.9928 and 0.9993, respectively. The coefficients of variation of intra- and inter-day assays were 14.36, 6.52, 1.76, 1.25, 5.36, 3.30 and 1.42% and 12.70, 1.19, 2.98, 1.91, 1.75, 1.78 and 0.96% at concentrations of 25, 50, 100, 200, 500, 1000 and 2000 ng/mL, respectively. The recoveries of paeonol from rat plasma were found to be 101.9, 104.5, 105.4 and 101.2% for concentrations of 50, 500, 1000 and 2000 ng/mL, respectively. The paeonol plasma concentrations were fitted to two-compartment model with fi rst order absorption. The mean terminal half-lives (t(1/2)) of paeonol was 80.9 min.     

Effective high-performance liquid chromatographic determination of glafenine in plasma: pharmacokinetic application

A high-performance liquid chromatographic method for an effective determination of glafenine and its main metabolite, glafenic acid, is described. The assay involves separate extraction procedures for glafenine and for its metabolite, but the same internal standard (floctafenine) and the same chromatographic conditions (including a 5-micron C8 column, a quaternary solvent mixture of water-acetonitrile-diethylamine-acetic acid and an ultraviolet detector set at 360 nm). For 1 ml of plasma, the detection limit is 0.05 mg/l for glafenine and 0.25 mg/l for glafenic acid. Compared with previously described techniques, this assay uses a very low glafenine linearity range, which allows the true pharmacokinetics of this drug to be described for the first time.     

Simultaneous determination of cocaine and its metabolites with caffeine in rat serum microsamples by high-performance liquid chromatography

A single, isocratic high-performance liquid chromatographic method is described for the determination of cocaine and three of its metabolites along with caffeine in serum microsamples (50 microliters). The small sample size permits the tracking of pharmacokinetic data over time in individual, small animals. The method also was used to demonstrate that cocaine, benzoylecgonine and norcocaine in rat serum samples were stable for at least a month without the presence of sodium fluoride.