LC-MS-MS Determination of (S,R)-Penehyclidine in Rat Plasma

A sensitive and selective method for determination of (S,R)-penehyclidine in rat plasma by liquid chromatography-tandem mass spectrometry is described. The procedure employed the use of an internal standard (I.S.) and a simple protein precipitation step. The method developed was linear from 0.1 to 100 ng mL⁻¹, with a sensitivity of 0.1 ng mL⁻¹ as the lower limit of quantification. The intra- and inter-day assay accuracy (relative error) was within 8.27% and precision (RSD) was below 6.7%. It was successfully applied to pharmacokinetic studies of (S,R)-penehyclidine in rat plasma.

     

Simple, Sensitive, High-Throughput Method for the Quantification of Mitragynine in Rat Plasma Using UPLC-MS and Its Application to an Intravenous Pharmacokinetic Study

A simple, sensitive and rapid ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed and validated for the quantification of mitragynine in rat plasma using amitriptyline hydrochloride as an internal standard. Sample preparation involved a one-step liquid?Cliquid extraction using methyl t-butyl ether. Mitragynine was separated on an Acquity UPLC^? BEH HILIC column using isocratic elution with a mobile phase of 10 mM ammonium formate buffer containing 0.1% formic acid:acetonitrile (15:85, v/v). At a flow rate of 0.2 mL min^?1, the retention time of mitragynine was found to be 1.3 min. Ionization was performed in the positive ion electrospray mode. The selected mass-to-charge (m/z) ratio transition of mitragynine ion [M + H]⁺ used in the selected ion recording (SIR) was 399.1. The calibration curve was found to be linear over a concentration range of 1?C5,000 ng mL^?1 (r = 0.999) with a lower limit of quantification (LLOQ) of 1 ng mL^?1. Intra- and inter-day assay variations were found to be less than 15%. The extraction recoveries ranged from 85?C93% at the three concentrations (2, 400 and 4,000 ng mL^?1) in rat plasma. This method was successfully used to quantify mitragynine in rat plasma following intravenous administration of the compound.     

Determination of Tangeretin in Rat Plasma by LC-Electrospray-Ion Trap MS

A selective, sensitive, and accurate method has been developed and validated for the quantification of tangeretin in rat plasma. The application of LC-electrospray-ion trap mass spectrometry in full scan and multiple reactions monitoring modes were investigated. Following solid phase extraction using a hydrophilic–lipophilic balance cartridge, the analytes were separated on a C₁₈ column using an isocratic mobile phase composed of acetonitrile/water (50:50, v/v) containing 0.3% formic acid. In full scan mode, the LOQ was 2 ng mL^?1. The standard calibration curve was linear (R ² = 0.9999) over the concentration range 2–200 ng mL^?1. The precision over the concentration range was within 15% (RSD) and the accuracy was ranged from 86 to 115%. In multiple reaction monitoring mode, the LOQ was 1 ng mL^?1 and the standard calibration curve was linear (R ² = 0.9976) over the concentration range 1–100 ng mL^?1 with a precision of 12% and accuracy rangeing from 91 to 113%.     

Enantiomeric Separation of Moxifloxacin and Its (R,R)-Isomer by Ligand-Exchange Chiral Chromatography

A new stereospecific LC method for the separation and quantification of moxifloxacin and its (R,R)-enantiomer in bulk drug was developed and validated by ligand-exchange liquid chromatography on a reversed phase column using aqueous mobile phase containing the chiral reagent l-isoleucine-Cu(II). The UV detector was operated at 293 nm. The flow rate of mobile phase was set at 0.9 mL min^?1. The achiral ODS column offers good separation of the two enantiomers in less than 20 min. The test concentration was 1,000 μg mL^?1 in the mobile phase. This method was capable of detecting the (R,R)-enantiomer of moxifloxacin up to 0.1 μg mL^?1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with the (R,R)-enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the (R,R)-enantiomer in bulk drug samples ranged from 98.1 to 104.4%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

N-(1-Naphthyl)ethylenediamine, a New UV Labeling Reagent Used for LC Determination of Valproic Acid in Human Plasma

A high performance liquid chromatography method is presented for the determination of valproic acid levels in human plasma. The method was based on pre-column derivatization using N-(1-naphthyl)ethylenediamine as a new labeling agent. The calibration curve was linear in the investigated concentration range between 0.1 and 100 μg mL^?1 and showed good accuracy and reproducibility. The assay provided a limit of quantification of 0.1 μg mL^?1 for valproic acid and a limit of detection of 10 ng mL^?1, respectively. The presented method was successfully applied to the determination of valproic acid levels in plasma after oral administration of 600 or 800 mg of sodium valproate.     

An Effective High-Performance Liquid Chromatographic–Mass Spectrometric Assay for Catecholamines, as the N(O,S)-Ethoxycarbonyl Ethyl Esters, in Human Urine

The purpose of this study was to develop a simple and accurate analytical method for determination of norepinephrine, epinephrine, and dopamine in urine. The method involves liquid–liquid extraction then liquid chromatography–mass spectrometry (LC–MS). Alkyl chloroformate derivatives were prepared, as the N(O,S)-alkoxycarbonyl alkyl esters of the analytes, in the aqueous samples. The optimum derivatizing reagent for preparation of the N(O,S)-alkoxycarbonyl alkyl esters was chosen by comparing the efficiency of LC of the derivatized analytes after liquid–liquid extraction. The optimum conditions for liquid–liquid extraction from the aqueous matrix were pH 3.0, no salt, and diethyl ether as extraction solvent. Limits of detection (LOD) were 0.5 ng mL^?1 for dopamine and epinephrine and 0.1 ng mL^?1 for norepinephrine. Limits of quantification (LOQ) for urine samples were 1.0 ng mL^?1 for all three compounds. The precision of intra- and inter-day assays was 1.65–581 and 7.17–9.73% (relative standard deviation, RSD), respectively. The range of inaccuracy for intra- and inter-day assays was ?6.47 to 11.9% and ?7.5 to 7.76% (bias) at concentrations of 5 and 50 ng mL^?1, respectively.     

Extraction of Azole Antifungal Drugs from Milk and Biological Fluids Using a New Hollow Fiber Liquid-Phase Microextraction and Analysis by GC-FID

A simple, rapid and sensitive high-performance liquid chromatography LC (HPLC) method of determining the concentration of the novel betulinic acid derivative DRF-4012 (5??-chloro-2,3-didehydroindolo[2??,3??:2,3]betulinic acid) in rat plasma for pharmacokinetic and toxicokinetic purposes has been developed and validated. A simple and fast protein precipitation was performed, and then an extraction using an ethyl acetate:methanol (75:25 v/v) mixture was used to extract DRF-4012 and an internal standard (IS, DRF-4015) from rat plasma. Chromatographic separation was achieved using a Zorbax Eclipse XDB-C8 reversed-phase column with UV detection at 235 nm. The isocratic mobile phase, phosphate buffer (water adjusted to pH 3 with 20% o-phosphoric acid) and acetonitrile (15:85, v/v), was run at a flow rate of 1.2 mL min^?1. The assay was linear (r ² > 0.99) over the concentration range 0.040?C75.0 ??g mL^?1, and presented limits of detection and quantification of 0.020 and 0.040 ??g mL^?1, respectively, in rat plasma. The absolute recovery of both the analyte and the IS was >85% from rat plasma. The intraday accuracy ranged from 99.25 to 102.67% with a precision of 2.62?C4.48%, and the interday accuracy ranged from 98.48 to 104.56% with a precision of 3.87?C5.68%. This developed and validated method was successfully used to determine the DRF-4012 concentration in rat plasma for a pharmacokinetic and toxicokinetic study after the intravenous administration of a 1 mg mL^?1 DRF-4012 nanoparticle formulation at doses of 2?C10 mg kg^?1 in Wistar rats.     

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–MS Determination of p-(1-Dimethylamino ethylimino)aniline: a Metabolite of Tribendimidine in Human Plasma

A sensitive and selective liquid chromatography–mass spectrometric method was developed and validated for the determination of p-(1-dimethylamino ethylimino)aniline (dADT), a metabolite of tribendimidine, in human plasma. The analyte was separated on a Hypersil C₁₈ column (250 × 4.6 mm ID, 5 μm) by isocratic elution with 10 mM ammonium acetate (containing 0.1% triethylamine)-acetonitrile (20:80, v/v) at a flow rate of 1.0 mL min^?1 and measured by electrospray ionization source in positive selective ion monitoring mode at m/z 178. The weighted (1/x ²) calibration curve was linear within a concentration range of 0.5–1,000 ng mL^?1 and displayed a correlation coefficient (r) of 0.9996. The lower limit of quantification was determined to be at 0.5 ng mL^?1. The inter and intra-day precisions (%RSD) were less than 8% and the extraction recoveries ranged from 84.21 to 85.20%. The developed method was successfully applied to the determination of dADT in human plasma as part of a clinical pharmacokinetic study.     

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.     

LC Method for Quantification of Lutein in Rat Plasma: Validation, and Application to a Pharmacokinetic Study

A reversed-phase LC method has been developed for quantitative analysis of lutein in rat plasma and applied to a study of the pharmacokinetics of lutein in rats. From a variety of compounds and solvents tested, astaxanthin was selected as the internal standard. n-Hexane was found to be the best solvent for extracting lutein from plasma. LC analysis of the extracts was performed on a C₁₈ column equipped with a guard pre-column. Linearity was good (r > 0.99) over the range 10–100 ng mL^?1. Recovery from plasma was 82.7–92.9% the intra-day and inter-day precision were always better than 3%. The limits of detection (LOD) and quantification (LOQ) were 2.5 and 8.3 ng mL^?1, respectively. The LC method was used to quantify lutein and zeaxanthin in rat plasma in a 36-h pharmacokinetic study in which experimental rats received a single oral dose of lutein (20 mg kg^?1). The results are presented.     

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.     

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.     

Quantitative Determination of Anti-Inflammatory Columbianetin in Rat Plasma by LC-ESI-MS/MS for Pharmacokinetic Studies after Oral Administration of Duhuo Extract

Specific LC-ESI-MS/MS method or procedure was developed and validated for columbianetin quantification in rat plasma using epicatechin as an internal standard (IS). Chromatographic separation was performed on an Eclipse plus C18 (150 × 4.6 mm, 1.8 ??m) at a flow rate of 0.300 mL min^?1, and water-acetonitrile was used as mobile phase. The calibration curve of the method was linear in the concentration range of 5?C1,000 ng mL^?1. The lower limit of quantification (LLOQ) was 5 ng mL^?1. The intra- and inter-day precision of the quality control samples was within 15.0%, and the accuracy was within 90.0?C110%. The recoveries were more than 90.0% for columbianetin at concentrations of 10, 200 and 1,000 ng mL^?1, respectively. This method was successfully applied for evaluation of the pharmacokinetics of columbianetin after oral doses of 0.60 g kg^?1 Angelica pubescence extract in rats.     

LC Quantification of RT-B: The Active Metabolite of a New Resveratrol Derivative RT-A in Rat Plasma

RT-A, a new prodrug based on resveratrol, is currently under investigation. Preclinical studies in rats indicate that RT-A is readily absorbed and rapidly split into an active metabolite RT-B by lysase of the ester bond. An LC method was developed for the determination of RT-B in rat plasma. The assay was performed on a 5 μm Elite C₁₈ column (200 mm × 4.6 mm) with a mobile phase consisting of acetonitrile–0.1% phosphoric acid (28:72, v/v, pH 1.8) at a flow rate of 1.0 mL min^?1. Detection was at 318 nm, and baicalin was used as an internal standard. Calibration was linear over the range of 0.04–10 μg mL^?1 with a correlation coefficient of 0.9994. The mean extraction recoveries of RT-B determined over the concentrations of 0.1, 1.0, and 5.0 μg mL^?1 were (86.5 ± 6.8) %, (82.6 ± 2.0) %, and (92.7 ± 7.9) %. The RSD of intra- and inter-day precisions were all less than 10%. This method was successfully applied to evaluate the pharmacokinetics of RT-B after intravenous administration of RT-A.     

LC–MS Method for the Quantification of Clonazepam in Rat Plasma

A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method has been developed and validated for the determination of clonazepam in rat plasma. Clonazepam and internal standard diazepam were extracted from plasma samples by a single-step protein precipitation. The chromatographic separation was performed on a Dikma ODS-C18 reversed-phase column at 40 °C. The mobile phase composed of a premix of solvent A (0.1% formic acid–4 mM ammonium acetate–water)–solvent B (acetonitrile) (13:87, v/v) at a flow-rate of 0.7 mL min^?1. Positive electrospray ionization was utilized as the ionization source. Clonazepam and the internal standard were determined using multiple reaction monitoring of precursor → product ion transitions at m/z 316.0 → 270.0 and m/z 285.1 → 193.2, respectively. The lower limit of quantification was 0.25 ng mL^?1 using 50 μL plasma samples and the linear calibration range was from 0.25 to 128 ng mL^?1. The within- and between-batch RSDs were lower than 15% and the relative recoveries of clonazepam ranged from 97.4 to 104.7%. The mean extraction recoveries of clonazepam and IS were 79.7 and 77.6%, respectively. The method has been successfully applied to the pharmacokinetic studies in rat after oral administration of clonazepam.     

LC–MS–MS Simultaneous Determination of Paracetamol, Pseudoephedrine and Chlorpheniramine in Human Plasma: Application to a Pharmacokinetic Study

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma. Diphenhydramine was used as the internal standard. Analytes were extracted from alkalized human plasma by liquid–liquid extraction (LLE) using ethyl acetate. After electrospray ionization positive ion fragments were detected in the selected reaction monitoring (SRM) mode with a triple quadrupole tandem mass spectrometer. The method was linear in the concentration range of 20.0–10000.0 ng mL^?1 for paracetamol, 1.0–500.0 ng mL^?1 for pseudoephedrine and 0.1–50.0 ng mL^?1 for chlorpheniramine. The intra- and inter-day precisions were below 14.5% and the bias was between ?7.3 and +2.8% for all analytes. The validated LC–MS–MS method was applied to a pharmacokinetic study in which each healthy Chinese volunteer received a tablet containing 300 mg benorylate, 30 mg pseudoephedrine hydrochloride and 2 mg chlorpheniramine maleate. This is the first assay method described for the simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma samples.     

Development of a Sensitive LC Assay with Fluorescence Detection for the Determination of Zearalenone in Rat Serum

A simple and sensitive liquid chromatographic assay with fluorescence detection assay was developed for the determination of zearalenone levels in rat serum. The assay utilized a single liquid–liquid extraction with t-butyl methyl ether and isocratic elution using a mobile phase consisting of acetonitrile and 0.1% triethylamine in distilled water (pH = 6) (50:50, v/v). Linearity was observed over a concentration range from 10 to 1,000 ng mL^?1 (r = 0.9995), with the limit of quantification at 10 ng mL^?1 with 100 μL of rat serum. The validated assay was applied to a pharmacokinetic study in rats.     

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.