Development of an LC–MS/MS method for quantification of two isomeric phenylpropenes and the application to pharmacokinetic studies in rats

Isomers β‐asarone and α‐asarone have recently been demonstrated to have differential pharmacological activities . Here, we report an LC–MS/MS method developed using acetonitrile to extract two isomeric phenylpropenes from rat plasma. Separation was achieved using a XDB‐C₁₈ column (100 × 2.1 mm; i.d., 1.8 μm) with a mobile phase of methanol–0.1% formic acid (55:45, v/v) at a flow rate of 0.3 mL/min. Calibration curves ranging from 5.20 to 2080 ng/mL for β‐asarone and from 3.68 to 1470 ng/mL for α‐asarone were linear (r² ≥ 0.9938) with the lower limits of quantification being 5.20 and 3.68 ng/mL for both isomers. Intravenous administration of β‐asarone (2.22 mg/kg) and α‐asarone (2.36 mg/kg) in rats yielded half‐lives of 13.40 ± 4.11 and 28.88 ± 7.82 min with clearance values of 0.196 ± 0.062 mL/min/kg and 0.112 ± 0.012 mL/min/kg for β‐asarone and α‐asarone, respectively.     

An LC–MS/MS method for quantification of demethylzeylasteral,a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study

In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC–MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.     

Development of a targeted method for quantification of gypenoside XLIX in rat plasma,using SPE and LC–MS/MS

A sensitive, selective and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation . Plasma samples were prepared by a simple solid‐phase extraction. Separation was performed on a Waters XBridgeTM BEH C₁₈ chromatography column (4.6 × 50 mm, 2.5 μm) using a mobile phase of acetonitrile and water (62.5:37.5, v /v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R ² > 0.990) over a concentration range of 10–7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra‐ and inter‐day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC–MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).     

Validated LC–MS/MS method for the quantification of sciadopitysin in rat plasma and its application to pharmacokinetic and bioavailability studies in vivo

A sensitive and rapid LC–MS/MS method was developed and validated for quantitation of sciadopitysin in rat plasma using amentoflavone as an internal standard. Sample processing was accomplished after deproteinization with 150 μL aliquot of acetonitrile. Chromatographic separation was achieved using an Agela C₁₈ column with an isocratic mobile phase comprising 2 mm ammonium acetate–acetonitrile (35:65, v/v) at a flow rate of 0.4 mL/min. Detection was performed by selection reaction monitoring on a triple‐quadrupole mass spectrometer following the transitions m/z 579 → 547 and 537 → 375 for sciadopitysin and internal standard, respectively, in the negative ionization mode. The calibration curve was linear from 2.90 to 1160 ng/mL for sciadopitysin. Intra‐ and inter‐day precisions were in the ranges 4.1–11.4 and 5.7–9.1% for sciadopitysin. Sciadopitysin was stable under different stability conditions. The validated assay was applied to pharmacokinetic and bioavailability studies in rats.     

Development and validation of an LC–MS/MS method for the determination of hinokiflavone in rat plasma and its application to a pharmacokinetic study

Hinokiflavone has drawn a lot of attention for its multiple biological activities. In this study, a sensitive and selective method for determination of hinokiflavone in rat plasma was developed for the first time, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Amentoflavone was used as an internal standard. Separation was achieved on a Hypersil Gold C₁₈ column with isocratic elution using methanol–water (65:35, v /v) as mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray mode with selected reaction monitoring was used to detect the transitions of m/z 537 → 284 for hinokiflavone and m/z 537 → 375 for IS. The LOQ was 0.9 ng/mL with a linear range of 0.9–1000 ng/mL. The intra‐ and inter‐day accuracy (RE%) ranged from −3.75 to 6.91% and from −9.20 to 2.51% and the intra‐ and inter‐day precision (RSD) was between 0.32–14.11 and 2.85–10.04%. The validated assay was successfully applied to a pharmacokinetic study of hinokiflavone in rats. The half‐life of drug elimination at the terminal phase was 6.10 ± 1.86 h, and the area under the plasma concentration‐time curve from time zero to the time of last measurable concentration and to infinity values obtained were 2394.42 ± 466.86 and 2541.93 ± 529.85 h ng/mL, respectively.     

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC–MS/MS after oral administration of Rubus chingii Hu extract

A simple and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol‐3‐O‐rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C₁₈ column (2.1 × 50 mm, i.d., 3.0 μm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r² > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol‐3‐O‐rutinoside and tiliroside, respectively. Intra‐ and inter‐day precisions were <8.2% and accuracy ranged from −11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats.     

Validated LC–ESI–MS/MS method for the determination of tunicamycin in rat plasma: Application to a pharmacokinetic study

A liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the quantification of tunicamycin in rat plasma as per regulatory guideline. Chromatography of tunicamycin and the IS in the processed plasma samples was achieved on an X‐Terra phenyl column using a binary gradient (mobile phase A, acetonitrile and mobile phase B, 5 mm ammonium formate) elution at a flow rate of 0.6 ml/min. LC–MS/MS was operated under the multiple reaction monitoring mode using the electrospray ionization technique in positive ion mode and the transitions of m/z 817.18 → 596.10, 831.43 → 610.10, 845.29 → 624.10, 859.23 → 638.10 and 309.24 → 163.20 were used to quantitate homologs A–D and the IS, respectively. The total chromatographic run time was 4.5 min. The correlation coefficient (r²) was >0.99 for all homologs with accuracy 90.7–107.4% and precision 0.74–15.1%. The recovery of homologs was 78.6–90.2%. No carryover was observed and the matrix effect was minimal. Tunicamycin four homologs were found to be stable on the bench‐top for 6 h, for up to three freeze–thaw cycles, in the injector for 24 h and for 1 month at ?80 ° C. The applicability of the validated method has been demonstrated in a rat pharmacokinetic study.     

A simple LC‐MS method for determination of cyasterone in rat plasma: application to a pilot pharmacokinetic study

A simple, specific, and sensitive liquid chromatography–mass spectrometry (LC‐MS) method for determination of cyasterone in rat plasma was developed in our laboratory. Cucurbitacin B was used as an internal standard (IS). After protein precipitation with twofold volume of acetonitrile, the analyte and IS were separated on a Luna C₁₈ column (100 × 4.6 mm, i.d., 3.0 µm; Phenomenex) by isocratic elution with acetonitrile–water (80:20, v/v) as the mobile phase at a flow rate of 0.4 mL/min. An electrospray ionization source was applied and operated in the positive ion mode; selected ion monitoring scan mode was used for quantification, and the target ions m/z 543.3 for cyasterone and m/z 581.3 for IS were chosen. Good linearity was observed in the concentration range of 0.40–400 ng/mL for cyasterone in rat plasma. Intra‐day and inter‐day precision were both <7.4%. This method was proved to be suitable for pharmacokinetic studies after oral (5.0 mg/kg) or intravenous (0.5 mg/kg) administration of cyasterone in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification of morusin using LC–MS in rat plasma: Application to a pharmacokinetic study

A sensitive LC–MS method was developed for the quantification of morusin in rat plasma using praeruptorin C as internal standard. After extraction with diethyl ether, post‐treatment samples were chromatographed on a Hypersil C₁₈ column. An isocratic mobile phase consisting of methanol–water (70:30, v /v) was applied at a flow rate of 0.4 mL/min. Detection was performed via electrospray ionization source with positive ion mode using selected ion monitoring mode at m/z 443.1 for morusin and m/z 451.0 for IS. Acceptable linearity (r ² ≥ 0.99) was observed over the concentration range of 1.5–800 ng/mL. This method was successfully applied in the pharmacokinetics study of morusin in rats.     

Determination of piracetam in rat plasma by LC–MS/MS and its application to pharmacokinetics

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of piracetam in rat plasma was developed and validated over the concentration range of 0.1–20 µg/mL. After addition of oxiracetam as internal standard, a simplified protein precipitation with trichloroacetic acid (5%) was employed for the sample preparation. Chromatographic separation was performed by a Zorbax SB‐Aq column (150 × 2.1 mm, 3.5 µm). The mobile phase was acetonitrile–1% formic acid in water (10:90 v/v) delivered at a flow rate of 0.3 mL/min. The MS data acquisition was accomplished in multiple reaction monitoring mode with a positive electrospray ionization interface. The lower limit of quantification was 0.1 µg/mL. For inter‐day and intra‐day tests, the precision (RSD) for the entire validation was less than 9%, and the accuracy was within the 94.6–103.2% range. The developed method was successfully applied to pharmacokinetic studies of piracetam in rats following single oral administration dose of 50 mg/kg. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of β‐eudesmol in rat plasma using LC–MS/MS and its application to a pharmacokinetic study

A sensitive and specific LC–MS/MS assay for determination of β ‐eudesmol in rat plasma was developed and validated. After liquid–liquid extraction with ethyl ether , the analyte and IS were separated on a Capcell Pak C₁₈ column (50 × 2.0 mm, 5 μm) by isocratic elution with acetonitrile—water–formic acid (77.5:22.5:0.1, v /v/v) as the mobile phase at a flow rate of 0.4 mL/min. An ESI source was applied and operated in positive ion mode; a selected reaction monitoring scan was used for quantification by monitoring the precursor–product ion transitions of m/z 245.1 → 163.1 for β ‐eudesmol and m/z 273.4 → 81.2 for IS. Good linearity was observed in the concentration range of 3–900 ng/mL for β ‐eudesmol in rat plasma. Intra‐ and inter‐day precision and accuracy were both within ±14.3%. This method was applied for pharmacokinetic studies after intravenous bolus of 2.0 mg/kg or intragastric administration of 50 mg/kg β ‐eudesmol in rats.     

Validation of an LC–MS/MS method for simultaneous detection of diverse components of Qinxing Qingre Zhike Granule in rat plasma and its application to pharmacokinetic study after oral administration to rats

A sensitive and validated method of liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established to test the plasma concentrations of active ingredients in Qinxing Qingre Zhike Granule, namely geniposide, liquiritin, isoliquiritin, baicalin, wogonoside, baicalein, liquiritigenin, isoliquiritigenin and glycyrrhetinic acid. The analysis was performed on an Ultimate XB‐C₁₈ column at the flow rate of 0.4 mL min^?1 in a single run of 18 min. The mobile phase was composed of 0.05% formic acid in water and acetonitrile with gradient elution. Positive and negative scanning and selected multiple reaction monitoring modes were applied for quantization. The proposed method showed good linearity in the given ranges from 0.6800–340.0 to 3.920–1960 ng mL^?1 with r² > 0.9917 for all the analytes. The precision (RSD) was no more than 12%, and the accuracy (RE) was less than ±11% for intra‐ and inter‐day. The extract recovery and matrix effect were acceptable for the requirements of biological sample analysis. Moreover, the developed method was effectively applied to the pharmacokinetic investigation of Qinxing Qingre Zhike Granule after oral administration in rats.     

A robust LC–MS/MS method for the simultaneous determination of docetaxel and voriconazole in rat plasma and its application to pharmacokinetic studies

Opportunistic fungal infections are common in immunocompromised cancer patients, especially patients undergoing chemotherapy. Because antitumor agents are possible to combine with antifungal agents in clinical, it is necessary to study drug–drug interaction between antitumor agents and antifungal agents. The aim of the study was to explore a method for the simultaneous determination of voriconazole and docetaxel in plasma and investigate pharmacokinetic interaction of voriconazole and docetaxel in rats. A precise and reliable method using liquid chromatography tandem mass spectrometry (LC–MS/MS) was established for the simultaneous measure of docetaxel and voriconazole in rat plasma after liquid–liquid extraction with ethyl acetate. The method was fully validated and successfully applied to a pharmacokinetic interaction study of docetaxel and voriconazole in rats after single or combined administration. We found that the AUC of each drug after coadministration increased compared with that after the single administration, which might be caused by interaction at the absorption stage or the competitive inhibition on the metabolic enzymes. This established method can be utilized to study the detailed mechanism of the drug–drug interaction and guide rational drug use in the clinic.     

LC–MS/MS method for the simultaneous determination of ethyl gallate and its major metabolite in rat plasma

A simple, rapid and sensitive liquid chromatography–tandem mass spectroscopy (LC–MS/MS) method was developed and validated for the determination of ethyl gallate, a pharmacologically active constituent isolated from Lagerstroemia speciosa (Linn.) Pers. This method was used to examine the pharmacokinetics of ethyl gallate and its major metabolite gallic acid in rat plasma using propyl gallate as an internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a Zorbax SB‐C₁₈ column (3.5 μm, 2.1 × 50 mm) with an isocratic mobile phase consisted of methanol–acetonitrile–10 mM ammonium acetate (10 : 25 : 65, v/v/v) containing 0.1% formic acid at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple‐reaction monitoring mode using the electrospray ionization technique in negative mode. The lower limits of quantification of gallic acid and ethyl gallate of the method were 0.5 and 1.0 ng/mL. The intra‐day and inter‐day accuracy and precision of the assay were less than 8.0%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of ethyl gallate to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

A validated LC–MS/MS method for the simultaneous determination of thalidomide and its two metabolites in human plasma: Application to a pharmacokinetic assay

An accurate and sensitive LC–MS/MS method for determining thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in human plasma was developed and validated using umbelliferone as an internal standard. The analytes were extracted from plasma (100 μL) by liquid–liquid extraction with ethyl acetate and then separated on a BETASIL C₁₈ column (4.6 × 150 mm, 5 μm) with mobile phase composed of methanol–water containing 0.1% formic acid (70:30, v/v) in isocratic mode at a flow rate of 0.5 mL/min. The detection was performed using an API triple quadrupole mass spectrometer in atmospheric pressure chemical ionization mode. The precursor‐to‐product ion transitions m/z 259.1 → 186.1 for thalidomide, m/z 273.2 → 161.3 for 5‐hydroxy thalidomide, m/z 273.2 → 146.1 for 5′‐hydroxy thalidomide and m/z 163.1 → 107.1 for umbelliferone (internal standard, IS) were used for quantification. The calibration curves were obtained in the concentrations of 10.0–2000.0 ng/mL for thalidomide, 0.2–50.0 ng/mL for 5‐hydroxy thalidomide and 1.0–200.0 ng/mL for 5′‐hydroxy thalidomide. The method was validated with respect to linear, within‐ and between‐batch precision and accuracy, extraction recovery, matrix effect and stability. Then it was successfully applied to estimate the concentration of thalidomide, 5‐hydroxy thalidomide and 5′‐hydroxy thalidomide in plasma samples collected from Crohn's disease patients after a single oral administration of thalidomide 100 mg.     

Quantitative bioanalysis of bavachalcone in rat plasma by LC–MS/MS and its application in a pharmacokinetics study

This study aims to develop and validate a simple and sensitive liquid chromatography with tandem mass spectrometry (LC–MS/MS) method for investigating the pharmacokinetic characteristics of bavachalcone. Liquid–liquid extraction was used to prepare plasma sample. Chromatographic separation of bavachalcone and IS was achieved using a Venusil ASB C₁₈ (2.1 × 50 mm, 5 μm) column with a mobile phase of methanol (A)–water (B) (70:30, v /v). The detection and quantification of analytes was performed in selected‐reaction monitoring mode using precursor → product ion combinations of m/z 323.1 → 203.2 for bavachalcone, and m/z 373.0 → 179.0 for IS. Linear calibration plots were achieved in the range of 1–1000 ng/mL for bavachalcone (r ² > 0.99) in rat plasma. The recovery of bavachalcone ranged from 84.1 to 87.0%. The method was precise, accurate and reliable. It was fully validated and successfully applied to pharmacokinetic study of bavachalcone.     

Determination of pethidine in human plasma by LC–MS/MS

A sensitive and selective liquid chromatography–tandem mass spectrometry method for the determination of pethidine in human plasma was developed and validated over the concentration range of 4–2000 ng/mL. After addition of ketamine as internal standard, liquid–liquid extraction was used to produce a protein‐free extract. Chromatographic separation was achieved on a 100 × 2.1 mm, 5 µm particle, Allure^TM PFP propyl column, with 45:40:15 (v/v/v) acetonitrile–methanol–water containing 0.2% formic acid as mobile phase. The MS data acquisition was accomplished by multiple reactions monitoring mode with positive electrospray ionization interface. The lower limit of quantification was 4 ng/mL; for inter‐day and intra‐day tests, the precision (RSD) for the entire validation was less than 7%, and the accuracy was within 95.9–106.5%. The method is sensitive and simple, and was successfully applied to analysis of samples of clinical intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of epalrestat and puerarin in rat plasma by UHPLC–MS/MS: Application to their pharmacokinetic interaction study

In the present study, a simple, rapid and reliable ultrahigh‐performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated to determine simultaneously epalrestat (EPA) and puerarin (PUE) in rat plasma for evaluation of the pharmacokinetic interaction of these two drugs. Both the analytes and glipizide (internal standard, IS) were extracted using a protein precipitation method. The separation was performed on a C18 reversed phase column using acetonitrile and 5 mmol/L ammonium acetate in water as the mobile phase with a gradient elution program. The analytes, including IS, were quantified with multiple reaction monitoring under negative ionization mode. The optimized mass transition ion pairs (m /z ) were 318.1 → 274.0 for EPA, 415.1 → 266.9 for PUE and 444.2 → 166.9 for IS. The linear calibration curves for EPA and PUE were obtained in the concentration ranges of 10–4167 and 20–8333 ng/mL, respectively (r > 0.99). The current method was successfully applied for the pharmacokinetic interaction study in rats following administration of EPA and PUE alone or co‐administration (EPA 15 mg/kg, oral; PUE 30 mg/kg, intravenous). The results showed that the combination of EPA and PUE could increase t _1/2 of EPA and reduce T _max of EPA. These changes indicated that EPA and PUE might cause drug–drug interactions when co‐administrated.     

A reliable LC–MS/MS method for the quantification of five bioactive saponins of crude and processed Bupleurum scorzonerifolium in rat plasma and its application to a pharmacokinetic study

A simple and reliable liquid chromatography–mass spectrometry (LC–MS) method was developed for simultaneous determination of saikosaponin A, saikosaponin B₁, saikosaponin C, saikosaponin D and saikosaponin F in rat plasma using glycyrrhetinic acid as an internal standard (IS). The separation was operated on a Waters BEH C₁₈ column. The mobile phases of gradient elution consisted of acetonitrile (A) and 0.1% aqueous acetic acid (B). The mass spectrometric detection was accomplished in multiple reaction monitoring mode. The five saponins displayed good linearity (r² > 0.9996). The lower limits of quantitation of saikosaponin A, saikosaponin B₁, saikosaponin C, saikosaponin D and saikosaponin F were determined to be 2.9, 2.3, 3.5, 2.9 and 3.1 ng/mL, respectively. Moreover, the intra‐ and inter‐day precisions of the five saponins showed an RSD within 2.96%, whereas the accuracy (RE) ranged from ?2.28 to 2.78%. Finally, the developed method was fully validated and applied to a comparative pharmacokinetic study of the five bioactive saponins in rats following oral administration of crude and vinegar‐processed Bupleurum scorzonerifolium.