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.     

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).     

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.     

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.     

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.     

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.     

Development of an LC–MS/MS method for quantification of two pairs of isomeric flavonoid glycosides and other ones in rat plasma: Application to pharmacokinetic studies

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of six flavonoid glycosides – isoorientin ( 1 ), orientin ( 2 ), 2″‐O‐β ‐d ‐xylopyranosyl isoorientin ( 3 ), 2″‐O‐β ‐d ‐xylopyranosyl isovitexin ( 4 ), 6‐C‐l ‐α ‐arabipyranosyl vitexin ( 5 ) and vitexin ( 6 ) – in rat plasma using isoquercitrin as the internal standard (IS). Plasma samples were prepared by a one‐step protein precipitation with acetonitrile. Chromatographic analysis was carried out on a 25 cm C₁₈ column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. Six analytes and IS were detected through electrospray ionization in negative‐ion selection reaction monitoring mode. The mass transitions were as follows: m/z 447.2 → 327.0 for 1 , m/z 447.2 → 327.0 for 2 , m/z 579.3 → 458.9 for 3 , m/z 563.0 → 293.1 for 4 , m/z 563.0 → 353.0 for 5 , m/z 431.1 → 311.1 for 6 , and m/z 463.1 → 300.2 for IS. Calibration curves exhibited good linearity (r ² > 0.9908) over a wide concentration range for all compounds. Intra‐ and inter‐day precision (RSD, %) at four different levels were both <14.2% and the accuracy (RE, %) ranged from −11.9 to 12.0%. The extraction recoveries of the six components ranged from 88.2 to 103.6%. The validated assay was successfully applied to the pharmacokinetic studies of the six components in male rat plasma after intravenous administration of total flavonoids of Scorzonera austriaca Wild.     

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.     

An LC–MS/MS method for simultaneous determination of hosenkoside A and hosenkoside K from Semen Impatientis in rat plasma and its application to a pharmacokinetic study

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of two baccharane glycosides (hosenkoside A and hosenkoside K) of total saponins of Semen Impatientis in rat plasma using mogroside V as the internal standard (IS). The analytes were separated using a C₁₈ RP Agilent XDB column (1.8 μm, 50 × 2.1 mm i.d.) and detection of the compounds was done using a TSQ Quantum triple quadrupole mass spectrometer coupled with a negative electrospray ionization source under selection reaction monitoring mode. According to the US Food and Drug Administration guidelines, the established method was fully validated and the results were proved within acceptable limits. The lower limits of quantification of both analytes were 5 ng/mL. The validated method was successfully applied to a pharmacokinetic study of orally administered the total saponins of Semen Impatientis in rats.     

UPLC–MS/MS determination of flavokawain B,a novel anti‐tumor chemotherapeutic agent in rat plasma and its application to a pharmacokinetic study in rats

A sensitive, selective and rapid ultra‐performance liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of flavokawain B in rat plasma using myrislignan as an internal standard. Sample preparation was accomplished through a protein precipitation extraction process. Chromatographic resolution of flavokawain B and the IS was achieved on an Agilent XDB‐C₁₈ column (2.1 × 100 mm, 1.8 μm) using a gradient mobile phase comprising 0.1% formic acid in water and acetonitrile delivered at a flow rate of 0.5 mL/min. Flavokawain B and the IS eluted at 3.27 and 1.96 min, respectively. The total chromatographic run time was 6.0 min. A linear response function was constructed in the concentration range 0.524–1048 ng/mL. Method validation was performed as per the US Food and Drug Administration guidelines and the results met the acceptance criteria. Intra‐ and inter‐day accuracy and precision were in the ranges of ?14.3–13.2 and 3.4–11.8%, respectively. Flavokawain B was demonstrated to be stable under various stability conditions. This method has been applied to a pharmacokinetic study in rats.     

An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study

A rapid, simple, selective and sensitive LC‐MS/MS method was developed for the determination of curculigoside in rat plasma. The analytical procedure involves extraction of curculigoside and syringin (internal standard, IS) from rat plasma with a one‐step extraction method by protein precipitation. The chromatographic resolution was performed on an Agilent XDB‐C₁₈ column (4.6 × 50 mm, 5 µm) using an isocratic mobile phase of methanol with 0.1% formic acid and H₂O with 0.1% formic acid (45:55, v/v) at a flow rate of 0.35 mL/min with a total run time of 2.0 min. The assay was achieved under the multiple‐reaction monitoring mode using positive electrospray ionization. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over 4.00–4000 ng/mL (R = 0.9984) for curculigoside with a lower limit of quantification of 4.00 ng/mL in rat plasma. The intra‐ and inter‐day precisions and accuracies were 3.5–4.6 and 0.7–9.1%, in rat plasma, respectively. The validated LC‐MS/MS method was successfully applied to a pharmacokinetic study of curculigoside in rats after a single intravenous and oral administration of 3.2 and 32 mg/kg. The absolute bioavailability of curculigoside after oral administration was 1.27%. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Simultaneous determination of baicalin,baicalein, wogonoside,wogonin, scutellarin,berberine, coptisine,ginsenoside Rb1 and ginsenoside Re of Banxia xiexin decoction in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A rapid and high‐sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous determination of nine active constituents (baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re) in rat plasma after oral administration of Banxia xiexin decoction (BXD). Biological samples were processed wtih acetone–ethyl acetate (4:1, v/v). The mobile phase consisted of methanol and water (containing 0.1% formic acid) with gradient elution at a flow rate of 0.3 mL/min. Detection was performed on a triple quadrupole mass spectrometer using positive ion and negative ESI in the multiple reaction monitoring mode. The calibration curves for all analytes had good linearity (r > 0.9933). The mean recovery of all the nine active ingredients was >75.2%, and the intra‐ and inter‐day precisions were within 12.0%; the accuracy was between 87.4 and 110.4%. This method was successfully applied to a pharmacokinetic study after administration of BXD. The results of the pharmacokinetic study might be helpful for BXD reasonable clinical application and further studies on mechanism.     

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.     

Simultaneous determination of lamivudine,stavudine and nevirapine in human plasma by LC–MS/MS and its application to pharmacokinetic study in clinic

A new high‐throughput LC–MS/MS method for the simultaneous determination of lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma is presented, with zidovudine as an internal standard. The analytes were extracted from plasma by protein precipitation and only 150 μL plasma was needed. Chromatographic separation was achieved on a Shiseido C₈ column (150 × 2.0 mm, 5 μm) with a total run time of 6 min. A tandem mass spectrometric detection was conducted using multiple reaction monitoring under positive ionization mode with an electrospray ionization interface. The method was developed and validated over the concentration range of 25–5000 ng/mL for 3TC and NVP and 20–4000 ng/mL for d4T. The method was validated in terms of intra‐ and inter‐day precision (≤8.6%), accuracy (within ± 8.4%), linearity and specificity. The method has been successfully applied to the pharmacokinetic study of a combination treatment of 300 mg lamivudine, 30 mg stavudine and 200 mg nevirapine in 22 healthy male volunteers under fasting conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

A sensitive LC–MS/MS method for simultaneous quantification of geniposide and its active metabolite genipin in rat plasma and its application to a pharmacokinetic study

Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH₃COO]⁻ m/z 447.3 → 225.3 for GE and [M − H]⁻ m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE.     

An LC–MS/MS method for quantification of HR011303, a novel highly selective urate transporter 1 inhibitor in beagle dogs and the application to a pharmacokinetic study

HR011303 is a novel and highly selective urate transporter 1 (URAT1) inhibitor. In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of HR011303 in beagle dog plasma. Plasma samples were pretreated with protein‐precipitation extraction by acetonitrile and added with a trifluoromethyl substituted analog of HR011303 as internal standard. The chromatographic separation was performed on a Shiseido C₁₈ column (100 × 4.6 mm, i.d., 5 μm) by mobile phases consisting of 5 mm ammonium–formic acid (100:0.1) and acetonitrile–formic acid (100:0.1) solutions in gradient elution. The MS detection was conducted in electrospray positive ionization with multiple reactions monitoring at m/z 338 → 240 for HR011303 and m/z 328 → 230 for the internal standard using 25 eV argon gas collision induced dissociation. The established LC–MS/MS method showed good selectivity, sensitivity, precision and accuracy. The plasma pharmacokinetics of HR011303 in beagle dogs following both oral and intravenous administration were then successfully evaluated using this LC–MS/MS method.     

Liquid chromatography–tandem MS/MS method for simultaneous quantification of paracetamol,chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study

A facile, fast and specific method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid–liquid extraction. The analysis was performed on a reversed‐phase C₁₈ HPLC column (5 μm, 4.6 × 50 mm) using acetonitrile–10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 μL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple‐reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03–30.0, 0.015–15.00 and 0.15–15.00 μg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers.