Development of an HPLC method for the determination of salidroside in beagle dog plasma after administration of salidroside injection: application to a pharmacokinetics study

A simple RP-HPLC method was established for the determination of salidroside in dog plasma. Salidroside is one of the most active ingredients of Rhodiola L. The method had within-run precision values in the range of +/- 2.3 to +/- 9.1% (n = 5) and between-run precision in the range of +/- 3.2 to +/- 9.8%. A simple protein precipitation for salidroside extraction was processed using ACN at precipitant-to-plasma volume ratio (P-P ratio) of 3:2. The extraction recoveries of salidroside at seven concentrations were higher than 63.2%. There was a linear relationship between chromatographic area and concentration over the range of 0.83-520 microg/mL for salidroside in plasma (R = 0.9926). The LOQ (S/N = 10) of the method was 0.83 microg/mL. The method was applied in a study of the pharmacokinetics of salidroside injection in six beagle dogs. The major pharmacokinetic parameters of C(max), AUC(0-24), AUC(0-infinity), and t(1/2) of salidroside in beagle dogs after i.v. administration of a single 75 mg/kg (5 mL/kg) dose were 96.16 +/- 8.59 microg/mL, 180.3 +/- 30.6 microg h/mL, 189.3 +/- 32.1 microg h/mL, and 2.006 +/- 0.615 h, respectively.     

Determination of glycyrrhizin in dog plasma by liquid chromatography-mass spectrometry and its application in pharmacokinetic studies

A sensitive liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method was established and validated for the determination of glycyrrhizin in dog plasma. After treatment with methanol to precipitate proteins, plasma samples were analyzed on a reversed-phase C18 (ODS) column with a mobile phase of methanol:1% formic acid solution (75:25, v/v). MS determination was performed using negative electrospray ionization (negative ESI) in the selected ion monitoring mode. Glycyrrhizin was monitored at the m/z 821 channel and internal standard (gliquidone) at the m/z 526 channel. The calibration curve was linear over the range from 0.05 μg mL(-1) to 10 μg mL(-1) with a correlation coefficient above 0.99. This method was successfully applied to the pharmacokinetic studies in beagle dogs. The absolute bioavailability of glycyrrhizin in beagle dogs was 3.24%.     

Development and validation of a simple and rapid HPLC method for the quantitative determination of voriconazole in rat and beagle dog plasma

A simple and rapid high-performance liquid chromatographic method with UV detection is developed and validated to determine the concentration of voriconazole in rat and beagle dog plasma. After protein precipitation using acetonitrile, the supernatant solution is chromatographed on a Diamonsil C(18) column (250 x 4.6-mm i.d., 5 microm). The mobile phase used is a combination of acetonitrile-water-acetic acid (55:45:0.25, v/v/v) with a pH of 4.0. Detection is achieved by a UV detector monitored at a wavelength of 256 nm. The matrix calibration curves are obtained both in the concentration range of 0.10-50.0 microg/mL in rat and beagle dog plasma, with the lower limit of quantitation of 0.10 microg/mL. The intra- and inter-assay precisions in terms of % relative standard deviation are lower than 8.6% and 6.0% in rat and beagle dog plasma, respectively. The accuracy in terms of % relative error ranged from -0.5% to 8.0% and -0.5% to 6.0% in rat and beagle dog plasma, respectively. This validated method is successfully applied to determine the concentration of voriconazole in plasma after intravenous administration of 36 mg/kg voriconazole to rats and 10 mg/kg voriconazole to beagle dogs, respectively.     

Determination of nifedipine in dog plasma by high‐performance liquid chromatography with tandem mass spectrometric detection

Nifedipine is a dihydropyridine calcium channel blocker used widely in the management of hypertension and other cardiovascular disorders. In this work, a simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated to determine nifedipine in dog plasma using nimodipine as the internal standard. Chromatographic separation was carried out on a C₈ column. The mobile phase consisted of a mixture of acetonitrile, water and formic acid (60:40:0.2, v/v/v) at a flow rate of 0.5 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode via an atmospheric pressure chemical ionization source. The method has a lower limit of quantification of 0.20 ng/mL with consumption of plasma as low as 0.05 mL. The linear calibration curves were obtained in the concentration range of 0.20–50.0 ng/mL (r = 0.9948). The recoveries of the liquid extraction method were 74.5–84.1%. Intra‐day and inter‐day precisions were 4.1–8.8 and 6.7–7.4%, respectively. The quantification was not interfered with by other plasma components and the method was applied to determine nifedipine in plasma after a single oral administration of two controlled‐release nifedipine tablets to beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Application of a sensitive and specific LC‐ESI‐MS/MS method for the simultaneous quantification of twelve bioactive components in dog plasma for an intravenous pharmacokinetic study of Yiqifumai Injection in beagle dogs

Yiqifumai Injection is a lyophilized powder preparation widely used to treat coronary heart disease. However, its in vivo bioactive components and pharmacokinetic behavior remain unknown. Therefore a sensitive and specific LC–MS/MS was developed and validated for the simultaneous quantification of eight saponins and four lignans in beagle dog plasma. The plasma samples were pretreated by protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation of all the 12 analytes and estazolam (internal standard, IS) was successfully accomplished on an Ultimate® XB‐C₈ column (100 × 2.1 mm, 3 μm) with a gradient elution system. The total running time was 8 min with a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed via positive electrospray ionization in multiple reaction monitoring mode. The assay was fully validated in terms of selectivity, linear range, lower limit of quantitation, precision, accuracy, matrix effect, recovery and stability. This validated method was successfully applied to the pharmacokinetics of 12 bioactive components after intravenous administration of Yiqifumai Injection to beagle dogs at a dose of 0.541 g/kg.     

Development and validation of a rapid and high‐sensitivity liquid chromatography–tandem mass spectrometry assay for the determination of neostigmine in small‐volume beagle dog plasma and its application to a pharmacokinetic study

A simple, rapid and high sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the determination of neostigmine in small‐volume beagle dog plasma was developed to assess the plasma pharmacokinetics of neostigmine. After protein precipitation in a Sirocco 96‐well filtration plate, the filtrate was directly injected into the LC‐MS/MS system. The analytes were separated on a Hanbon Hedera CN column (100 × 4.6 mm, 5 µm) with a mobile phase composed of methanol–water (60:40, v/v) and the water containing 0.01% formic acid at a flow rate of 0.6mL/min, with a split ratio of 1:1 flowing 300 μL into the mass spectrometer. The run time was 3 min. Detection was accomplished by electrospray ionization source in multiple reactions monitoring mode with the precursor‐to‐product ion transitions m/z 223.0 → 72.0 and 306.0 → 140.0 for neostigmine and anisodamine (internal standard), respectively. The method was sensitive with a lower limit of quantitation of 0.1 ng/mL, and good linearity in the range 0.1–100ng/mL for neostigmine (r ≥ 0.998). All the validation data, such as accuracy, intra‐run and inter‐run precision, were within the required limits. The method was successfully applied to pharmacokinetic study of neostigmine methylsulfate injection in beagle dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

Determination of asperosaponin VI in dog plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pilot pharmacokinetic study

A sensitive and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the determination of asperosaponin VI in beagle dog plasma using glycyrrhizic acid as the internal standard (IS). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on a Hedera ODS‐2 column using mobile phase of methanol–10 mm ammonium acetate buffer solution containing 0.05% acetic acid (71:29, v/v) at a flow rate of 0.38 mL/min. Asperosaponin VI and the IS were eluted at 2.8 and 1.9 min, respectively, ionized in negative ion mode, and then detected by multiple reaction monitoring. The detection used the transitions of the deprotonated molecules at m/z 927.5 → 603.4 for asperosaponin VI and m/z 821.4 → 645.4 for glycyrrhizic acid (IS). The assay was linear over the concentration range of 0.15–700 ng/mL and was successfully applied to a pilot pharmacokinetic study in beagle dogs. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of picroside II in dog plasma by HPLC and its application in a pharmacokinetics study

A sensitive and simple high-performance liquid chromatography method with UV detection was developed and validated for determining picroside II in dog plasma. Paeoniflorin was employed as internal standard and the sample pre-treatment procedure consists of deproteinization by addition of acetonitrile. Chromatographic separations were performed on a Shimadzu VP-ODS column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-0.1% acetic acid aqueous (v/v), 23:77, v/v, at a rate of 1 mL/min. Detection was carried out at a wavelength of 266 nm. Calibration standards ranged from 0.25 to 500 microg/mL in dog plasma and the mean correlation coefficient of 0.9981 was found for the linear calibration curves (n = 6). The limit of quantification (LOQ) was 0.25 microg/mL. Intra- and inter-assay RSD ranged from 0.70 to 7.5%. Accuracy (%bias) ranged from -6.3 to 6.0%. This method was applied to the pharmacokinetic study of picroside II in dogs. The study demonstrated the plasma picroside II concentration-time curves were fitted to the two-compartment open model and showed linear pharmacokinetics.     

HPLC determination and pharmacokinetic study of tenatoprazole in dog plasma after oral administration of enteric-coated capsule

A simple, sensitive and selective high-performance liquid chromatographic (HPLC) method with UV detection (306 nm) was developed and validated for determination of tenatoprazole, a novel proton-pump inhibitor, in dog plasma. Tenatoprazole and internal standard (pantoprazole) were extracted into diethyl ether and separated using an isocratic mobile phase of 10 mm phosphate buffer (pH4.7)-acetonitrile (70:30, v/v) on a Diamonsil C(18) column (150 x 4.6 mm, 5 microm). The retention times for tenatoprazole and internal standard were 7.1 and 12.3 min, respectively. No endogenous interferences were observed. This HPLC method was fully validated. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 20%. A linear range of 0.02-5.0 microg/mL was established. The interday and intraday precisions were within RSD 13.4-10.1 and 4.6-1.4%, respectively. This method developed can be easily applied to the pharmacokinetic study of tenatoprazole in dog plasma after oral administration of an enteric-coated capsule. The plasma concentration of tenatoprazole from six dogs showed a mean C(max) of 2.63 microg/mL at T(max) of 1.89 h. The bioavailability of tenatoprazole was improved by administration of enteric-coated capsule.     

Quantitation of oxcarbazepine and its metabolites in human plasma by micellar electrokinetic chromatography

A reliable micellar electrokinetic chromatographic method for the determination of oxcarbazepine and its two main metabolites, 10-hydroxycarbamazepine and 10,11-trans-dihydroxy-10,11-dihydroxycarbamazepine, in human plasma was developed. The separation and determination of the analytes was achieved using a system consisting of 60 mM SDS in phosphate buffer (30 mM, pH 8.0), to which 20% (v/v) methanol was added. Separation was carried out in an uncoated fused-silica capillary with a separation voltage of 25 kV and currents typically less than 40 microA. Spectrophotometric detection was at 205 nm. Isolation of oxcarbazepine and its metabolites from plasma was accomplished by a solid-phase extraction procedure. The mean extraction yield of the analytes from plasma was higher than 94%. The linear correlation coefficients were better than 0.994 for all analytes. The limit of detection was 0.05 microg/mL, the limit of quantitation 0.15 microg/mL. The repeatability for the spiked blank plasma samples was lower than 1.9% and the intermediate precision lower than 2.1%, both expressed as RSD%. The results obtained analysing real plasma samples from epileptic patients under therapy with Tolep were satisfactory in terms of precision, accuracy and detectability.     

Determination of gambogic acid in dog plasma by high-performance liquid chromatography for a pharmacokinetic study

A rapid, simple and sensitive high-performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed-phase C(18) analytical column. The mobile phase consisted of a mixture of methanol-0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35 degrees C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156-20 microg/mL. The intra-assay and inter-assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs.     

Pharmacokinetics of methyl salicylate‐2‐O‐β‐D‐lactoside,a novel salicylic acid analog isolated from Gaultheria yunnanensis,in dogs

Methyl salicylate‐2‐O‐β‐D‐lactoside (MSL), a natural salicylate derivative of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis), has been shown to provide a beneficial anti‐inflammatory effect in animal models. Studies on the pharmacokinetics and bioavailability of MSL can provide both a substantial foundation for understanding its mechanism and empirical evidence to support its use in clinical practice. A simple and sensitive high‐performance liquid chromatography (HPLC) method, coupled with ultraviolet analyte detection, was developed for determining the concentration of MSL and its metabolite in beagle plasma. Chromatographic separation was achieved on a Agilent Zorbax SB‐C₁₈ column (5 μm ,4.6 × 250 mm). The mobile phase consisted of aqueous solution containing 0.1% phosphoric acid and acetonitrile (82:90, v/v), at a flow rate of 1 mL/min. Validation of the assay demonstrated that the developed HPLC method was sensitive, accurate and selective for the determination of MSL and its metabolite in dog plasma. After orally administering three doses of MSL, it could no longer be detected in dog plasma and its metabolite, salicylic acid, was detected. Salicylic acid showed a single peak in the plasma concentration–time curves and linear pharmacokinetics following the three oral doses (r² > 0.99). In contrast, only MSL was detected in plasma following intravenous administration. These results will aid in understanding the pharmacological significance of MSL. The developed method was successfully used for evaluation of the oral and intravenous pharmacokinetic profile of MSL in dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Development and validation of a simple,sensitive and accurate LC‐MS/MS method for the determination of guanfacine,a selective α2A‐adrenergicreceptor agonist,in plasma and its application to a pharmacokinetic study

A simple, practical, accurate and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and fully validated for the quantitation of guanfacine in beagle dog plasma. After protein precipitation by acetonitrile, the analytes were separated on a C₁₈ chromatographic column by methanol and water containing 0.1% (v/v) formic acid with a gradient elution. The subsequent detection utilized a mass spectrometry under positive ion mode with multiple reaction monitoring of guanfacine and enalaprilat (internal standard) at m/z 246.2 → 159.0 and m/z 349.2 → 205.9, respectively. Good linearity was obtained over the concentration range of 0.1–20 ng/mL for guanfacine in dog plasma and the lower limit of quantification of this method was 0.1 ng/mL. The intra‐ and inter‐day precisions were <10.8% relative standard deviation with an accuracy of 92.9–108.4%. The matrix effects ranged from 89.4 to 100.7% and extraction recoveries were >90%. Stability studies showed that both analytes were stable during sample preparation and analysis. The established method was successfully applied to an in vivo pharmacokinetic study in beagle dogs after a single oral dose of 4 mg guanfacine extended‐release tablets. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of tandospirone and its active metabolite, 1‐[2‐pyrimidyl]‐piperazine in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1‐[2‐pyrimidyl]‐piperazine (1‐PP) in Sprague–Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C₁₈ column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000–500.0 ng/mL for TDS and 10.00–500.0 ng/mL for 1‐PP. Total time for each chromatograph was 3.0 min. The intra‐day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter‐day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1‐PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1‐PP in plasma necessary for the pharmacokinetic investigation.     

Precolumn fluorescence labeling method for simultaneous determination of hydroxyzine and cetirizine in human serum

A highly selective and sensitive method was developed for simultaneous determination of the antihistaminic drug hydroxyzine (HZ) and its pharmacologically active metabolite cetirizine (CZ) in human serum using haloperidol as internal standard. The method was based on fluorescence labeling of both drugs with a fluorescent arylboronic acid 4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl boronic acid followed by separation on silica column using a mobile phase consisting of acetonitrile and water (90:10, v/v%) containing triethylamine and acetic acid. The labeling reaction conditions were optimized and the liquid-liquid extraction method was successfully applied to extract the both drugs from serum. The linearity range was 0.025-2.00 microg/mL for HZ and CZ. The limit of detection (S/N = 3) was 10 and 5 ng/mL for HZ and CZ, respectively.     

An HPLC method for the quantitation of 3‐pentylbenzo[c]thiophen‐1(3H)‐one in dog plasma and its application to a pharmacokinetic study

A simple, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method was developed for the estimation of 3‐pentylbenzo[c]thiophen‐1(3H)‐one (S₅), a potential anti‐ischemic stroke agent, in dog plasma. The analytical procedure involves protein precipitation of S₅ and nobiletin (internal standard) from dog plasma with acetonitrile. Chromatographic separation was achieved on Sapphire C18 analytical column with methanol–water (80:20, v/v) as mobile phase. The eluate was monitored using a UV detector set at 260 nm. The calibration curves were linear over the range of 0.2–20 µg/mL. Absolute recoveries of S₅ were 79.2–86.1% from dog plasma. The intra‐ and inter‐day relative standard deviation precisions were <7 and 5%, respectively. The method was successfully applied to the pharmacokinetic study of S₅ in beagle dogs. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Liquid chromatography/tandem mass spectrometry for pharmacokinetic studies of 20(R)-ginsenoside Rg3 in dog

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-ginsenoside Rg3 in dog. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. Dioscin was used as the internal standard. The method had a lower limit of quantitation of 0.5 ng/mL for Rg3 in 200 microL of plasma or 2 ng/mL in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of Rg3 in plasma. The intra- and inter-day precisions were measured to be below 8% and accuracy between -1.5 and 1.4% for all quality control samples. This quantitation method was successfully applied to pharmacokinetic studies of Rg3 after both an oral and an intravenous administration to beagle dogs. No Rh2 and protopanaxadiol were detected in plasma.