Determination of Geniposide in Rats Plasma. Application to Pharmacokinetic Studies of Sendeng-4 Decoction

A sensitive and simple high-performance liquid chromatographic (HPLC) method has been developed for determination of geniposide in rat plasma. The plasma was extracted with acetonitrile. Separation of the main effective constituent, geniposide, was accomplished on a reversed-phase ODS C₁₈ column (250 mm × 4.6 mm i.d., 5 µm particles) with acetonitrile-water, 12:88 (v/v), as mobile phase and UV detection at 238 nm. Paeoniflorin was used as the internal standard (IS). The calibration plot was linear over the range 0.0848–7.42 µg mL^?1. The lower limit of quantification was 0.0848 µg mL^?1. Intra-day precision was better than 11.4% and inter-day precision was better than 9.3%. Mean extraction recovery was 87.1%. The validated method was successfully used in pharmacokinetic studies of geniposide in rat plasma after oral administration of Sendeng-4 decoction. The pharmacokinetic study indicated that absorption of geniposide from Sendeng-4 decoction was rapid, as also was its subsequent elimination.     

Simultaneous Estimation of Atorvastatin Calcium, Ramipril and Aspirin in Capsule Dosage Form by RP-LC

A simple, sensitive, precise and accurate reversed phase liquid chromatographic method has been developed for the simultaneous estimation of atorvastatin (AT) calcium, ramipril (RA) and aspirin (AS) from capsule dosage form. The method was developed using a Phenomenex Luna C₁₈ (250 mm, 4.6 mm i.d., 5 µm) column with a mobile phase consisting of 0.1%, orthophosphoric acid buffer:acetonitrile:methanol (45:50:5 v/v/v), pH 3.3, at a flow rate of 1 mL min^?1. Detection was carried out with ultra-violet detection at 210 nm. The retention times were about 12.19, 2.35, and 3.95 min for AT calcium, RA and AS, respectively. The developed method was validated for linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linearity ranges were 1–6 µg mL^?1 for AT calcium, 0.5–3 µg mL^?1 for RA and 7.5–45 µg mL^?1 for AS with mean recoveries of 100.59 ± 0.68, 100.62 ± 0.83 and 100.49 ± 0.73% for AT calcium, RA and AS, respectively. Limit of detection obtained were 29.85 ng mL^?1 for AT calcium, 4.71 ng mL^?1 for RA and 85.13 ng mL^?1 for AS. Impurity of salicylic acid was found in capsule dosage form at the retention time of about 4.84 min. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

LC Determination of a Novel Synthetic Thiazolidinedione (BP-1107) in Rat Plasma and Its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatographic method for the quantification of BP-1107 in rat plasma has been established. Plasma samples were prepared by extraction with tert-butyl methyl ether, and troglitazone was used as an internal standard. The analytical separation was performed on a C₁₈ column using acetonitrile–0.3% phosphoric acid in water (pH 4.00 adjusted with triethylamine) (75:25, v/v) as a mobile phase. A detailed validation of the method was performed as per USFDA guidelines. For BP-1107 at the concentrations of 2.42, 16.11 and 32.22 μg mL^?1 in rat plasma, the extraction recoveries were 114.14 ± 9.75, 95.37 ± 12.06 and 90.00 ± 6.46%, respectively. The mean recovery for internal standard was 91.96 ± 2.51%. The lower limit of quantitation of BP-1107 was 16 ng. The linear quantification range of the method was 0.81–53.70 μg mL^?1 in rat plasma with a correlation coefficient greater than 0.999. The intra-day and inter-day accuracy for BP-1107 at 2.42, 16.11 and 32.22 μg mL^?1 levels in rat plasma fell between 97.10–110.02 and 97.52–108.04%. The intra-day and inter-day precision were in the ranges of 1.91–5.63 and 4.43–6.28%, respectively. The method was successfully applied to a pharmacokinetic study of BP-1107 in rats after an intravenous administration.     

LC–ESI–MS Determination of Imperatorin in Rat Plasma After Oral Administration and Total Furocoumarins of Radix Angelica dahuricae and its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatography–electrospray ionization mass spectrometry method for the quantification of imperatorin in rat plasma after oral administration and total furocoumarins of Radix Angelica dahuricae has been established. The plasma samples were deproteinized by adding internal standard (IS) osthole solution, which was prepared by acetonitrile. The analysis was performed on a Shim-pack C₁₈ column (150 × 2.0 mm i.d., 5 μm) using acetonitrile and 0.5% formic acid solution (70:30, v/v) as a mobile phase. The detection was performed on a quadrupole mass spectrometer detector with an ESI interface operated in the selected ion monitoring mode. The linear quantification range of the method was 2–4000 ng mL^?1 in rat plasma with a correlation coefficient greater than 0.99, the limit of detection (LOD) was 0.5 ng mL^?1 and the lower limit of quantification (LLOQ) 2 ng mL^?1. The intra- and inter-day relative standard deviations (RSD) were less than 2.5 and 3.5%, respectively. The recoveries were above 90%. The validated method was successfully applied to a pharmacokinetic study of imperatorin in rats after oral administration and total furocoumarins of Radix Angelica dahuricae.     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL^?1 for ferulic acid and 1.740–348.0 ng·mL^?1 for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL^?1 for ferulic acid and 1.740 ng·mL^?1 for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.     

HPLC Analysis and Pharmacokinetic Study of Paeoniflorin after Intravenous Administration of a New Frozen Dry Powder Formulation in Rats

A simple and specific high performance liquid chromatographic (HPLC) method with UV detection using picroside II as the internal standard was developed and validated to determine the concentration of paeoniflorin in rat plasma and study its pharmacokinetics after an single intravenous administration of 40 mg kg^?1 paeoniflorin to Wistar rats. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 0.05 mol L^?1 NaH₂PO₄ solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB C₁₈ column (250 × 4.6 mm I.D., 5 μm) with a Shim-pack GVP-ODS C₁₈ guard column (10 × 4.6 mm I.D., 5 μm) 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^?1. The UV detection was performed at a wavelength of 230 nm. The linear calibration curves were obtained in the concentration range of 0.05–200.0 μg mL^?1 in rat plasma with the lower limit of quantification (LLOQ) of 0.05 μg mL^?1. The intra- and inter-day precisions in terms of % relative standard deviation (RSD) were lower than 5.7 and 8.2% in rat plasma, respectively. The accuracy in terms of % relative error (RE) ranged from ?1.9 to 2.6% in rat plasma. The extraction recoveries of paeoniflorin and picroside II were calculated to be 69.7 and 56.9%, respectively. This validated method was successfully applied to the pharmacokinetic study of a new paeoniflorin frozen dry power formulation. After single intravenous administration, the main pharmacokinetic parameters t _1/2, AUC_0-∞, CL_TOT, V _Z, MRT_0-∞ and V _ss were 0.739 ± 0.232 h, 43.75 ± 6.90 μg h mL^?1, 15.50 ± 2.46 L kg^?1 h^?1, 1.003 ± 0.401 L kg^?1, 0.480 ± 0.055 h and 0.444 ± 0.060 L kg^?1, respectively.     

Rapid and Sensitive RP-LC Method for the Quantification and Pharmacokinetic Study of p-Hydroxyphenethyl Anisate in Rat Plasma

A rapid, sensitive and specific reversed-phase liquid chromatographic method was developed and validated for the quantification of p-hydroxyphenethyl anisate (HPA), which is one of the main constituents of Notopterygium Radix (underground parts of Notopterygium incisum and N. forbesii), in rat plasma, and study its pharmacokinetics after the intravenous administration of 40 mg kg^?1 HPA to rats. The method involves a plasma clear-up step using liquid–liquid extraction by ethyl acetate, followed by RP-LC separation and detection. Separation of HPA was performed on an analytical Diamonsil ODS C₁₈ column equipped with a Dikma ODS C₁₈ EasyGuard column using a mobile phase consisting of MeOH–H₂O (75:25, v/v) at a flow-rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 256 nm. The linear calibration curves were obtained in the concentration range of 0.05–5.0 μg mL^?1 (r = 0.9992, n = 5) in rat plasma with the lower limit of detection of 0.01 μg mL^?1 and the lower limit of quantification of 0.04 μg mL^?1, and the extraction recovery of HPA was calculated to be the range of 82.01–86.66%. The intra- and inter-day precisions in terms of % relative standard deviation were lower than 2.33 and 3.99% in rat plasma, respectively, with accuracies ranging from 91.22 to 110.5%. The developed method was suitable for the determination and pharmacokinetic study of HPA in rat plasma.     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma, and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min^?1. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL^?1 for wogonin and 10.8–271 ng mL^?1 for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL^?1 for wogonin and 10.8 ng mL^?1 for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min^?1. 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 in the concentration range of 1.00–400 ng mL^?1 in rat plasma, with a 1.00 ng mL^?1 lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from ?2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.     

Sensitive and Selective LC–ESI–MS Analysis of Aesculin in Rat Plasma

A rapid and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the determination of aesculin in rat plasma. The analyses were chromatographed on a Zorbax Extend-C18 analytical column (150 × 2.1 mm I.D., 5 µm) with 30:70 (v/v) methanol–0.1% formic acid as mobile phase. Detection was performed by triple-quadrupole tandem mass spectrometry in multi-reaction-monitoring mode with an electrospray ionization source. The method was validated for accuracy and precision, and linearity in the two matrices was good. The assay was linear in the range 12.5–1,800 ng mL^?1. The lower limit of quantification of aesculin (LLOQ) was 12.5 ng mL^?1. The recovery of aesculin and tinidazole (IS) were well above 85%. The within- and between-batch accuracy was 100–104% and 97–109%, respectively. There were no stability-related problems in the procedure for the analysis of aesculin. The method was successfully used in a preclinical study of the pharmacokinetics of aesculin in rats.     

Determination of Ranolazine in Rat Plasma by Liquid Chromatography–Electrospray Ionization Mass Spectrometry

A rapid and sensitive liquid chromatographic–mass spectrometric (LC–MS) method, with phenoprolamine hydrochloride (DDPH) as internal standard, has been developed and validated for determination of ranolazine in rat plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with methanol–10 mM ammonium acetate, 76:24 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI–MS). The protonated analyte was quantified by selected-ion monitoring (SIM) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 0.046–12 μg mL⁻¹. Inter and intra-day precision (CV%) and accuracy (RE%) for quality-control samples (0.187, 1.5, and 12 μg mL⁻¹) ranged between 2.96 and 13.38% and between −11.23 and 12.67%, respectively. Extraction recovery of RAN from plasma was in the range 82.77–86.54%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of ranolazine in rat plasma.     

LC Method for Studies on the Stability of Lopinavir and Ritonavir in Soft Gelatin Capsules

This study describes the development and full validation of a stability-indicating HPLC method to quantify ritonavir (RTV) and lopinavir (LPV) in soft gelatin capsules. The method uses a LiChrospher^® 100 RP-18 (250 mm × 4.6 mm, 5 µm, Merck) column and isocratic elution. The mobile phase consisted of a mixture of acetonitrile-water-methanol (53:37:10, v/v/v), pumped at a flow-rate of 1.0 mL min^?1 and UV detection at 210 nm using a photodiode array detector. LPV and RTV were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The response was linear over a range of 40-360 µg mL^?1 for LPV and 10–90 µg mL^?1 for RTV (r > 0,999 for both drugs). The mean recoveries were 99.46 and 100.81% for LPV and RTV, respectively. The RSD values for intra- and inter-day precision studies were < 0.70% for both drugs. Degradation studies showed that lopinavir is stable in thermal, alkaline and oxidative conditions, while ritonavir degraded under these conditions. The method was found to be stability-indicating and can be used for the routine analysis of the association LPV/RTV in soft gelatin capsules.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

A sensitive RP-HPLC method for estimation of artemether from polymeric nanoparticles after pre-column acid treatment using UV-visible detector

Abstract

Artemether; a sesquiterpene lactone is widely used for the treatment of malaria as artemisinin-based combination therapy (ACT). The present work involves the development and validation of sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method for quantification of artemether (ART) in polymeric nanoparticles. ART was transformed to α, β-unsaturated decalones by pre-column acid treatment to enhance the sensitivity of chromophoric group lacking ART for quantification by HPLC-UV. Waters Spherisorb^® 5?µm ODS(C18) column (4.6*250?mm) with gradient elution by mobile phase comprising of ACN and PBS (10?mM; pH 6.0) was used to separate acid-treated ART. The analysis was carried at λ_max of 253?nm with 20?min and 20?µL run time and injection volume, respectively. The method was found to be linear in the concentration range of 0.5–10?µg mL^?1 with 0.09?µg mL^?1 and 0.27?µg mL^?1 as LOD and LOQ respectively. Further, the method was also found to be specific for ART in presence of blank polymeric nanoparticles, accurate (% average recovery rate 101.7?±?1.68%), precise (RSD <2%), and robust. The method was successfully used to determine % entrapment efficiency and in vitro release of ART-loaded polymeric nanoparticles with HPLC using a UV-visible detector.     

LC Determination of Ketorolac in Human Plasma and Urine for Application to Pharmacokinetic Studies

A simple, specific and sensitive liquid chromatographic method has been developed for the assay of ketorolac in human plasma and urine. The clean-up of plasma and urine samples were carried out by protein precipitation procedure and liquid–liquid extraction, respectively. Separation was performed by a Waters sunfire C₁₈ reversed-phase column maintained at 35 °C. The mobile phase was a mixture of 0.02 M phosphate buffer (pH adjusted to 4.5 for plasma samples and to 3.5 for urine samples) and acetonitrile (70:30, v/v) at a flow rate of 1.0 mL min^?1. The UV detector was set at 315 nm. Nevirapine was used as an internal standard in the assay of urine sample. The method was validated over the concentration range of 0.05–8 and 0.1–10 μg mL^?1 for ketorolac in human plasma and urine, respectively. The limits of detection were 0.02 and 0.04 μg mL^?1 for plasma and urine estimation at a signal-to-noise ratio of 3. The limits of quantification were 0.05 and 0.1 μg mL^?1 for plasma and urine, respectively. The extraction recoveries were found to be 99.3 ± 4.2 and 80.3 ± 3.7% for plasma and urine, respectively. The intra-day and inter-day standard deviations were less than 0.5. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay demonstrated to be applicable for clinical pharmacokinetic studies.     

LC Determination of Icariside II in Rat Plasma and Tissues: Application to a Tissue Distribution Study

A sensitive and reliable reversed-phase liquid chromatography (RP-LC) with ultraviolet (UV) detection has been developed and validated for the quantification of Icariside II in rat plasma and tissues using Fermononetin as the internal standard. Protein precipitation and liquid?Cliquid extraction were utilized for plasma and tissue sample preparation, respectively. The analysis was successfully carried out on an Agilent SB-C₁₈ column (5 ??m, 4.6 × 250 mm) with the implementation of the following conditions: a mobile phase of phosphoric acid solution (0.1%, v/w)?CAcetonitrile (55:45, v/v), a flow rate of 1 mL min^?1, a column temperature of 25 °C and a detection wavelength of 270 nm. Good linear relationships of calibration curves were obtained (r ² > 0.9906) over the investigated concentration range with plasma and tissue samples. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 0.1 and 0.02 ??g g^?1, respectively (for plasma sample, they were 0.05 and 0.1 ??g mL^?1, respectively). The developed method which was embodied with good precision, accuracy, recovery and stability was corroborated to satisfy the requirements for biomedical sample analysis. This method has been successfully applied to tissue distribution study of Icariside II in rats after a single intravenous dose at 12.5 mg kg^?1. Results suggested that Icariside II was distributed to rat tissues rapidly with greater initial concentrations in kidney, lung and liver. Moderate initial distributions were obtained in rat muscle, heart, bone, spleen and plasma. Low amount of Icariside II was detected in testes, and no Icariside II could be detected in the brain.     

A Rapid Tandem Mass Spectrometric Assay for Determination of Ursolic Acid – Application to Analysis of Ursolic Acid in Four Species of Staphylea L. and Leaves of Staphylea Pinnata L. Gathered During Ontogenesis

Plant extracts of Staphylea L. exhibit a number of biological activities which are presumably attributed to ursolic acid. A rapid and specific tandem mass spectrometric (MS-MS) assay for the quantification of ursolic acid in the leaves of four species of Staphylea L. (Bladdernut) and in the leaves of S. pinnata L. during ontogenesis, was developed and validated. The samples were analyzed by flow injection analysis without chromatographic separation using a transport liquid of methanol/water/formic acid (80:20:0.1 v/v/v) at a flow-rate of 0.2 mL min⁻¹. The run cycle time was ~2-3 min injection-to-injection. Quantification was achieved using multiple reaction monitoring at MRM transition m/z 439 > 203. Calibration curves were linear over the concentration range of 2–20 μg mL⁻¹ with a lower limit of quantification of 2 μg mL⁻¹ (1.8 ± 0.297, RSD: 0.165). Validation data showed that the RSD% values were in the range of 1.8 to 6.8%, whereas the % DEVs ranged from −18 to −2% indicating reasonable and acceptable precision and accuracy, respectively. A recovery percent of 106.8 ± 10.3 of ursolic acid from spiked extracts samples, indicated the specificity and reliability of tandem mass procedure for determination of ursolic acid in the plant extracts. The derived data of sample analysis showed different contents of ursolic acid among various Staphylea species. The highest content of ursolic acid was found in the leaves extract of S. pinnata L. Additionally, the highest amount of ursolic acid accumulated in the leaves of S. pinnata L. was in the August /September period of the year. Smaller amounts of ursolic acid were found in samples collected before and after that time. The results obtained serve as a justification of determining the most appropriate time for collecting plant material as a source of ursolic acid.     

LC Analysis and Pharmacokinetic Study of Pachymic Acid After Intravenous Administration to Rats

A simple and sensitive high-performance liquid chromatographic method with UV detection was developed and validated to investigate the concentration of pachymic acid (PA) in rat plasma. The sample preparation was a liquid-liquid extraction and chromatographic separation was achieved with a Dikma Diamonsil^TM C₁₈ column (250 × 4.6 mm I.D.) with a C₁₈ guard column (8 × 4 mm I.D.) using a mobile phase consisting of MeOH-MeCN-aq. 0.45% H₃PO₄ (45:40:22) at a flow rate of 1.0 mL min^?1. The UV detection was at 210 nm. Standard curves were linear (r = 0.9998) in plasma over the concentration range of 0.5–50 μg mL^?1 and had acceptable accuracy and precision. Intra- and inter-day precisions expressed as the relative standard deviation (RSD) were 0.26–1.60% and 1.24–2.31%. The lower limit of quantification and lower limit of detection were 0.45 and 0.17 μg mL^?1. The method has been used successfully to study the pharmacokinetics of PA. After a dose of 30 mg kg^?1 by intravenous administration, the main pharmacokinetic parameters t _1/2, AUC_0-∞, CL, V_ss and MRT_0-∞ were 8.79 ± 6.80 h, 18.90 ± 9.39 μg h mL^?1, 0.53 ± 0.28 L h^?1, 5.60 ± 4.60 L and 12.58 ± 9.95 h, respectively.