High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

High‐performance liquid chromatography assay using ultraviolet detection for urinary quantification of milrinone concentrations in cardiac surgery patients undergoing cardiopulmonary bypass

An analytical assay using liquid–liquid extraction and high‐performance liquid chromatography with ultraviolet detection was developed for the quantification of total (conjugated and unconjugated) urinary concentrations of milrinone after the inhalation of a 5 mg dose in 15 cardiac patients undergoing cardiopulmonary bypass. Urine samples (700 μL) were extracted with ethyl‐acetate and subsequently underwent acid back‐extraction before and after deconjugation by mild acid hydrolysis. Milrinone was separated on a strong cation exchange analytical column. The mobile phase consisted of a constant mixture of acetonitrile:tetrahydrofurane–NaH₂PO₄ buffer (40:60 v/v, pH 3.0). Thirteen calibration curves were linear in the concentration range of 31.25–4000 ng/mL, using olprinone as the internal standard (r² range 0.9911–0.9999, n = 13). Mean milrinone recovery and accuracy were respectively 85.2 ± 3.1% and ≥93%. Intra‐ and inter‐day precisions (coefficients of variation) were ≤5% and ≤8%, respectively. Over a 24 h collection period, the cumulative urinary milrinone recovered from 15 patients was 26.1 ± 7.7% of the nominal 5 mg dose administered. The relative amount of milrinone glucuronic acid conjugate was negligible in the urine of patients undergoing cardiopulmonary bypass This method proved to be reliable, specific and accurate to determine the cumulative amount of total milrinone recovered in urine after inhalation. Copyright © 2014 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography method for determination of carnosic acid in rat plasma and its application to pharmacokinetic study

A sensitive and reproducible high‐performance liquid chromatography method with ultraviolet detection (UV) was developed for the determination of carnosic acid (CA) in rat plasma. After simple acidification and liquid–liquid extraction of plasma samples using gemfibrozil as an internal standard, the supernatant was evaporated to dryness under a gentle stream of nitrogen. The residue was reconstituted in 200 µL before being injected into the chromatographic system. The analysis was performed on a C₁₈ column protected by an ODS guard column using acetonitrile–0.1% phosphoric acid (55:45, v/v) as mobile phase, and the wavelength of the UV detector was set at 210 nm. The calibration curve was linear over the range of 0.265–265.0 µg/mL with a correlation coefficient of 0.9997. The recovery for plasma samples of 0.530, 13.25, 132.5 and 265.0 µg/mL was 72.2, 87.9, 90.4 and 94.7%, respectively. The intra‐day and inter‐day relative standard deviations for the measurements of quality control samples were less than 3.1%. The stability of the plasma samples was also validated. This method was successfully used to study the pharmacokinetics and bioavailability of CA in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved highly sensitive method to determine low oxyresveratrol concentrations in rat plasma and its pharmacokinetic application

Existing methods to determine oxyresveratrol, a trans‐polyphenolic stilbene, lack selectivity, require large plasma sample volumes or have time‐consuming sample preparation and chromatographic isolation. Here an improved highly sensitive liquid chromatography–tandem mass spectrometry method was developed to determine low oxyresveratrol concentrations in rat plasma. The plasma samples were prepared by liquid–liquid extraction with acetoacetate. The analyte s were separated on Venusil hydrophilic interaction chromatography (HILIC) column (2.1 × 50 mm, 5.0 µm) guarded by a HILIC column (4 × 3.0 mm, 5.0 µm). The mobile phase consisted of acetonitrile–water (containing 1 mmol/L ammonium formate) at gradient elution mode with a flow rate of 0.3 mL/min. Resveratrol was used as the internal standard. An electrospray ionization source was applied and operated in the negative multiple reaction monitoring (MRM) mode. Oxyresveratrol and resveratrol were detected on MRM by the transitions from the precursor to the product ion (m/z 243.1 → 175.1 and 227.1 → 143.0). The total running time was 5 min and the retention times of oxyresveratrol and resveratrol were 1.97 and 1.82 min. Chromatograms showed no endogenous interfering peaks with blank samples. The linear calibration curve was obtained over the concentration range of 1–500 ng/mL. The injection volume was 10 μL and the limit of quantification was 1 ng/mL. The extraction recovery varied from 78.2 to 84.3% for low, medium and high quality control samples. At the same time, the intra‐ and inter‐day relative standard deviations were <6.78 and <10.02%, respectively, while the corresponding intra‐ and inter‐day accuracy relative error values fell in the range of 3.75–6.67%. The HPLC‐MS/MS method was successfully applied to a pharmacokinetics study, in which the experimental rats received a single dose of oxyresveratrol (10 mg/kg, intragastric administration). The pharmacokinetic results are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

High‐performance liquid chromatographic method for the quantification of Mitragyna inermis alkaloids in order to perform pharmacokinetic studies

In Africa, Mitragyna inermis (Willd.) O. Kuntze (Rubiaceae) is commonly used in traditional medicine to treat malaria. Antimalarial activity is mostly due to the hydromethanolic extract of M. inermis leaves and especially to the main alkaloids, uncarine D and isorhynchophilline. In the present study, we describe for the first time an HPLC method for the simultaneous quantification of uncarine D and isorhynchophylline in biological matrices. SPE was used to extract the components and the internal standard naphthalene from human and pig plasma samples. Chromatographic separation was performed on a C‐18 reversed column at a flow rate of 1 mL/min, using methanol–phosphate buffer (10:90, pH 7), as a mobile phase. Good linearity was observed over the concentration ranges of 0.0662–3.31 μg/mL for uncarine D and 0.0476–2.38 μg/mL for isorynchophylline. The precision was less than 12% and the accuracy was from 86 to 107% without any discrepancy between the two species. Uncarine D and isorhynchophylline recoveries were over 80%. These results allowed the quantification of both uncarine D and isorhynchophylline in pig plasma after intravenous administration of M. inermis extract.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of carboplatin in canine plasma by high‐performance liquid chromatography

Carboplatin is an antineoplastic drug administered to treat different tumoral conditions in canine oncology. The objective of this study was to validate a high‐performance chromatographic (HPLC) method which could be applied in canine pharmacokinetic studies. Following ultrafiltration using a Centrifree device, standards, quality controls and plasma samples were separated by isocratic reversed‐phase HPLC on an Inertsil ODS‐2 (250 × 4.6 mm i.d.) analytical column and quantified using UV detection at 220 nm. The mobile phase was potassium phosphate (pH 4.5), with a flow‐rate of 1.0 mL/min. The procedure produced a linear curve (r² > 0.999) over the concentration range 1–200 μg/mL. The lower limit of quantification was 1 μg/mL. The intra‐assay and inter‐assay precision was ～90%. The overall recovery was ～90%. The method was illustrated with a preliminary pharmacokinetic analysis on nine dogs treated with carboplatin at our hospital. Carboplatin disposition followed a monocompartmental structure in dogs and was characterized by a short half‐life (50 min). Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of seven ginsenosides in rat plasma by high‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry: application to pharmacokinetics of Shenfu injection

A high‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (HPLC‐TOF MS) method was successfully developed and validated for the identification and determination of seven ginsenosides, R_e, R_f, R_b1, R_c, R_b2, R_o and R_d, in a Chinese herbal preparation, Shenfu injection, and rat plasma. Based on the method, the pharmacokinetic profiles of the seven ginsenosides were investigated following intravenous administration of single dose of Shenfu injection to six rats. The established method had high linearity, selectivity, sensitivity, accuracy and precision. The pharmacokinetic results showed that R_b1, R_c and R_b2 had similar pharmacokinetic profiles and relatively long half‐life values (19.29 ± 6.36, 29.54 ± 22.91 and 35.60 ± 30.66 h). The half‐lives of R_f and R_d were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they could be metabolized more rapidly than R_b1, R_c and R_b2. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of ibudilast in human serum by high‐performance liquid chromatography for pharmacokinetic study

A simple, accurate, precise and cost effective reversed‐phase HPLC method was developed to determine the concentration of ibudilast in human serum. Ibudilast and an internal standard, butyl 4‐hydroxybenzoate, were extracted by liquid–liquid extraction with methyl tert‐butyl ether. HPLC analysis was carried out under the following conditions: a Luna C₁₈(2) 5 μm column, a mobile phase of acetonitrile–0.02% phosphoric acid (50 : 50, v/v, adjusted to pH 6.0 with triethylamine) and a UV detector at 319 nm. The chromatograms showed good resolution and sensitivity as well as no interference from the human serum. The calibration curves were linear over the concentration range, 1–100 ng/mL, for serum with correlation coefficients >0.999. The intra‐ and inter‐day assay precision as well as the accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 101.7 ± 6.1%. The lower limit of quantitation in human serum was 1 ng/mL, which is sensitive enough for pharmacokinetic studies. Stability studies revealed that ibudilast in human serum was stable during storage as well as during the assay procedure. This method was applied successfully to an examination of the pharmacokinetics of ibudilast in human subjects following a single oral dose of an ibudilast (10 mg) capsule. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Identification of seven metabolites of oxyresveratrol in rat urine and bile using liquid chromatography/tandem mass spectrometry

Oxyresveratrol (trans‐2,4,3′,5′‐tetrahydroxystilbene) is a major compound isolated from Smilax china, a Chinese herbal medicine. The rat urine and bile samples were pretreated by solid‐phase extraction method after oral administration at a dose of 100 mg/kg of oxyresveratrol. Seven metabolites were identified by LC‐MS/MS method with electrospray ionization in negative ion mode. The results indicated that main metabolites of oxyresveratrol were monoglucuronided and monosulfated oxyresveratrol. Based on the results, the metabolic pathway of oxyresveratrol in rat urine and bile was proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

High‐performance liquid chromatographic enantioseparation of amino alcohol analogues possessing 1,2,3,4‐tetrahydroisoquinoline skeleton on polysaccharide‐based chiral stationary phases

The stereoisomers of 1,2,3,4‐tetrahydroisoquinoline amino alcohol analogues and derivatives thereof were separated in normal‐phase mode on chiral stationary phases based on preprepared silica coated with cellulose tris‐(3,5‐dimethylphenyl carbamate), cellulose tris‐(3‐chloro‐4‐methylphenyl carbamate), cellulose tris‐(4‐methylbenzoate) or cellulose tris‐(4‐chloro‐3‐methylphenyl carbamate). On all the investigated chiral columns, the retention and the enantioseparation were influenced by the nature and the concentrations of the mobile phase components and additives, and also the temperature. Experiments were performed in the temperature range 10–50°C. Thermodynamic parameters were calculated from plots of lnα vs 1/T. On these polysaccharide‐based chiral columns, both enthalpy‐driven separations and entropy‐controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. The sequence of elution of the stereoisomers was determined in all cases. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of oxyresveratrol analog trans-2,4,3',5'-tetramethoxystilbene in rat plasma by a rapid HPLC method: application in a pre-clinical pharmacokinetic study

A rapid HPLC method was developed and validated for the quantification of oxyresveratrol analog trans‐2,4,3′,5′‐tetramethoxystilbene (oxyresveratrol tetramethyl ether, OTE) in rat plasma. Chromatographic separation was achieved on an RP‐HPLC column, which was protected by a guard column through a 12 min gradient delivery of a mixture of acetonitrile–water at 50°C. The UV absorbance at 325 nm was recorded. The retention time of OTE and trans‐stilbene (internal standard) was about 7.7 and 8.4 min, respectively. The calibration curves were linear (R² ≥ 0.9986) with a lower limit of quantification of 15 ng/mL. The intra‐ and inter‐day variations, in terms of RSD, were all lower than 9.8% while the intra‐day and inter‐day bias ranged from ?8.3 to +9.2%. The pharmacokinetics of OTE was assessed in rats using 2‐hydroxypropyl‐β‐cyclodextrin as a dosing vehicle. After intravenous administration, OTE possessed a long terminal elimination half‐life (t_{1/2 λz} = 481 ± 137 min) and slow clearance (Cl = 29.1 ± 3.7 mL/min/kg). Upon oral administration, OTE was rapidly absorbed. However, it only displayed minimal plasma exposure and its absolute oral bioavailability (F) was as low as 4.5 ± 3.2%. Fortunately, the levels of OTE after single oral administration were sufficient to inhibit human cytochrome P450 1B1. Copyright © 2010 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic analysis of lacosamide in canine serum using ultraviolet detection: application to pre-clinical pharmacokinetics in dogs

A method for analysis of lacosamide [(R)‐2‐acetamido‐N‐benzyl‐3‐methoxypropionamide] is needed for both human and veterinary pharmacokinetic investigations. While lacosamide is currently used to manage partial‐onset seizures in humans suffering from epilepsy, it is also presently being investigated for use in the treatment of canine epilepsy in veterinary medicine. Currently, no dosing regimen for the drug exists in dogs. A novel and simple high‐performance liquid chromatography method was developed for determination of lacosamide in dog serum. Serum proteins (0.1 mL) were precipitated with ?20.0°C acetonitrile after addition of the internal standard, daidzein. Separation was achieved with a Phenomenex® Luna® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 210 nm. The calibration curves were linear ranging from 0.5 to 25 µg/mL. Precision of the assay was <13% (RSD) and was within 12% for all points in the calibration curve. The limit of quantitation for this method was 0.5 µg/mL. The assay was applied successfully to a pre‐clinical study of lacosamide pharmacokinetics in dogs. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of 3‐methoxypterostilbene in biological fluids by high‐performance liquid chromatography: application to pre‐clinical pharmacokinetics

A method of analysis for 3‐methoxypterostilbene [trans‐3,3′5‐trimethoxy‐4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high‐performance liquid chromatographic method was developed for the determination of 3‐methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at ?20°C). Separation was achieved with a Phenomenex® C₁₈ (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3‐methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.     

High‐performance liquid chromatography with solid‐phase extraction for the quantitative determination of nilotinib in human plasma

A simple, rapid and sensitive high‐performance liquid chromatography (HPLC)‐based method with ultraviolet detection was developed for the quantitation of nilotinib, a tyrosine kinase inhibitor, in human plasma. Nilotinib and the internal standard dasatinib were separated using a mobile phase of 0.5% KH₂PO₄ (pH2.5)–acetonitrile–methanol (55:25:20, v/v/v) on a Capcell Pak MG II column (250 × 4.6 mm) at a flow rate of 0.5 mL/min and optical measurement at 250 nm. Analysis required only 100 μL of plasma and involved a rapid and simple solid‐phase extraction with an Oasis HLB cartridge, which gave recoveries from 72 to 78% for nilotinib and from 74 to 76% for dasatinib. The lower limit of quantification for nilotinib was 10 ng/mL. The linear range of this assay was between 10 and 5000 ng/mL (r² > 0.9992 for the regression line). Intra‐ and inter‐day coefficients of variation were less than 10.0% and accuracies were within 10.4% over the linear range. Our results indicate that this method is applicable to the monitoring of plasma levels of nilotinib in a clinical setting. Copyright © 2009 John Wiley & Sons, Ltd.     

Pharmacokinetics of dronedarone in rat using a newly developed high‐performance liquid chromatographic assay method

A sensitive and selective high‐performance liquid chromatographic method for the determination of dronedarone in rat plasma was developed. Dronedarone was extracted using one‐step liquid–liquid extraction. The separation of dronedarone was accomplished using a C₁₈ analytical column. The mobile phase was composed of a combination of monobasic potassium phosphate and acetonitrile. The UV detection was at 254 nm for ethopropazine, the internal standard, and after its elution, changed to 290 nm for dronedarone detection. The total analytical run time was 20 min. Mean recovery was >80%; the assay had excellent linear relationships (>0.999) between peak height ratios and plasma concentrations; the lower limit of quantification 25 was ng/mL, based on 100 μL of rat plasma. Accuracy and precision were <18% over the concentration range of 25–500 ng/mL. The assay was applied successfully to the measurement of dronedarone plasma concentrations in rats given the drug orally. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of iguratimod in rat plasma by high performance liquid chromatography: method and application

A high-performance liquid chromatographic method with UV detection has been developed for the determination of iguratimod (T-614) in rat plasma. Plasma was precipitated with acetonitrile after the addition of the internal standard (IS), N-[4-(2-formylaminoacetyl)-5-methoxy-2-phenoxyphenyl]-methanesulfonamide. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase acetonitrile-acetic acid aqueous solution, pH 4.5 (40:60, v/v), at a flow rate of 1 mL/min, and the UV detection wavelength was set at 257 nm. The calibration curve was linear over the range 0.10-50.0 microg/mL, and the lower limit of quantification was 0.10 microg/mL. The intra- and inter-day relative standard deviations were all less than 11.5%. The method has been successfully applied to study the pharmacokinetics of iguratimod in rats. A single 10 mg/kg dose of iguratimod was given to the rats by intragastric administration. The mean maximum plasma concentration of iguratimod for the six rats was 14.5 microg/mL, and the mean elimination half-life of iguratimod was 4.0 h.     

High‐performance liquid chromatographic column packings with different particle sizes: Chromatographic behavior for the quality analysis of HuanglianShangqing pill

The chromatographic separation of traditional Chinese medicines is still a highly challenging task in analytical science with respect to its hundreds and thousands of chemical compounds, while increase of separation efficiency can greatly improve the separation power of chromatographic column for traditional Chinese medicine. In this study, 13 bioactive components in HuanglianShangqing pill were selected as an index to optimize the separation conditions and evaluate the system suitability of three commercially available columns packed with 1.8, 3.5, and 5.0 μm particles. The chromatographic separations were obtained by the most appropriate Eclipse Plus C₁₈ column (100 × 2.1 mm, 3.5 μm) within 45 min using gradient elution with aqueous‐ammonium acetate (10 mmol/L, pH 5.0) and acetonitrile, at a flow rate of 0.3 mL/min and an operating temperature of 30°C. The quality of HuanglianShangqing pill was assessed through combining simultaneous quantification of 13 compounds with fingerprint analysis. For the qualitative analysis, mass spectrometry was used to confirm the 13 compounds. All the validation data conformed to the acceptable requirements. For the fingerprint analysis, 32 peaks were selected as the common peaks at 254 nm to evaluate the similarities among HuanglianShangqing pills obtained from ten manufacturers.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.