A Rapid and Sensitive UPLC-MS/MS Method for Quantification of Bruceantinol in Rat Plasma and Its Application to a Pharmacokinetic Study.

Bruceantinol (BOL), a quassinoid compound isolated from the fruits of Brucea javanica, has been reported to have cytotoxic and antibacterial effects. In this study, a rapid, sensitive, and specific ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of BOL in rat plasma. The samples were treated by simple liquid-liquid extraction with ethyl acetate and separated on an UPLC BEH C18 column (2.1 mm × 50 mm) using a 3-min gradient elution scheme, which consists of water (0.1% v/v, formic acid) and methanol (0.1%, v/v, formic acid) to achieve the separation of BOL and sinomenine (IS) with high selectivity. The electrospray ionization source was used in positive ion mode; the multiple reaction monitoring quantified the target fragment ions m/z 629.6 → 569.5 for BOL and m/z 330.5 → 207.3 for IS. This work was evaluated with regards to the specificity, extraction recovery, matrix effect, linearity, accuracy, precision, stability, and dilution integrity. This approach was used to examine the pharmacokinetics of BOL in rats after oral (0.3 mg/kg) and intravenous (0.15 mg/kg) administration. BOL presented fast excretion and very low oral bioavailability.

HPLC-MS/MS method for bioavailability study of bruceines D & E in rat plasma.

Bruceines D and E are quassinoids from seeds of Brucea javanica (L.) Merr. exhibiting hypoglycemia effect. The crude drug is used as a traditional medicine by diabetes patients. The aim of this study is to understand the bioavailability and pharmacokinetics of both the bruceines D & E. A rapid and sensitive HPLC-MS/MS method was developed and validated for the quantification of both quassinoids, bruceines D & E in rat plasma. Both the bruceines D & E were separated with the Zorbax SBC-18 column with gradient elution and mobile phase system of acetonitrile and deionized water with 0.1% formic acid at a flow rate of 0.5mL/min. Analytes were detected in multiple reaction monitoring (MRM) mode with electrospray positive ionization. The quassinoids, namely bruceines D & E were detected with transitions of m/z 411.2→393.2 and m/z 395.2→377.2, respectively. Another quassinoid, eurycomanone was used as the internal standard with transition of m/z 409.2→391.2. The method was validated and conformed to the regulatory requirements. The validated method was applied to pharmacokinetic and bioavailability studies in rats. The pharmacokinetic study indicated both bruceine D and E were rapidly absorbed into the circulation system and reached its peak concentration at 0.54±0.34h and 0.66±0.30h, respectively. Bruceine E was eliminated slower than Bruceine D with t1/2 value almost increased two-fold compared to Bruceine D. In conclusion, a rapid, selective and sensitive HPLC-MS/MS method was developed for the simultaneous determination of both the bruceines D and E in rat plasma. Both bruceines D and E displayed poor oral bioavailability.

Determination of brusatol in plasma and tissues by LC-MS method and its application to a pharmacokinetic and distribution study in mice.

OBJECTIVES: The quassinoid brusatol, which can be isolated from Brucea javanica (L.) Merr., becomes popularly studied because of its anti-tumor activity. In order to further investigate brusatol and extend its applications, a sensitive analytical method for determination of brusatol in biological samples is essential. However, few methods had been reported until now. In this study, a highly sensitive and reproducible LC-MS method for simultaneous quantification of brusatol in mouse plasma and tissues was developed and validated. METHOD: Plasma samples and tissue homogenate were extracted with diethyl ether after addition of the internal standard solution(IS). The supernatant was blown to dryness with nitrogen and residual was reconstituted with 100µl of methanol. The separation was performed on an Intersil ODS-3 column and gradient elution was conducted with the mobile phase of water and methanol (0-5min 47:53, 5-5.5min 47:53-10:90, 5.5-9min 10:90, posttime 4min 47:53) at a flow rate of 0.8mL/min. Quantification was performed in the selected ion monitoring (SIM) mode at m/z 543.2 for brusatol and 220.0 for IS (ornidazole). The method was validated by analyzing quality control plasma and tissue homogenate samples, and was applied to analyze samples obtained from mice after injections of brusatol via the tail vein. RESULTS: With ornidazole as the internal standard, calibration curve of the method ranged from 10 to 320ng/ml for plasma and 10-240ng/ml for tissues. Recovery rate of brusatol from plasma and tissues were between 71.09%-94.91%. Relative standard deviation (RSD) for inter- and intra-day precision was less than 15%, and the accuracy was between 96.1%-111.8%. The pharmacokinetics and distribution study of brusatol in mice after three single doses via the tail vein were carried out based on this method. The concentration of brusatol in plasma decreased rapidly and a more than 10 fold concentration of brusatol was found as compared to that in other tissues. CONCLUSIONS: This is the first reported LC-MS method for detecting brusatol in tissues and can accurately determine the concentrations of these compounds in plasma and different tissues. Further research on the metabolism of brusatol in vivo is still needed.

Determination of eurycomanone in rat plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry for pharmacokinetic study.

In this study, a rapid, sensitive, and reliable hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the determination of eurycomanone in rat plasma was developed and validated. Plasma samples were pretreated with a protein precipitation method and quercitrin was used as an internal standard (IS). A HILIC silica column (2.1 × 100 mm, 3 µm) was used for hydrophilic-based chromatographic separation, using the mobile phase of 0.1% formic acid with acetonitrile in gradient elution at a flow rate of 0.25 mL/min. Precursor-product ion pairs for multiple-reaction monitoring were m/z 409.1 → 391.0 for eurycomanone and m/z 449.1 → 303.0 for IS. The linear range was 2-120 ng/mL. The intra- and inter-day accuracies were between 95.5 and 103.4% with a precision of <4.2%. The developed method was successfully applied to the pharmacokinetic analysis of eurycomanone in rat plasma after oral dosing with pure compound and E. longifolia extract. The Cmax and AUC0-t , respectively, were 40.43 ± 16.08 ng/mL and 161.09 ± 37.63 ng h/mL for 10 mg/kg eurycomanone, and 9.90 ± 3.97 ng/mL and 37.15 ± 6.80 ng h/mL for E. longifolia extract (2 mg/kg as eurycomanone). The pharmacokinetic results were comparable with each other, based on the dose as eurycomanone.

Development and validation of a sensitive LC-MS/MS assay for quantification of bruceine D in rat plasma.

A sensitive LC-MS/MS method for the determination of bruceine D in rat plasma was developed. The analyte and IS were separated on a Luna C18 column (2.1 × 50 mm, 1.7 µm) using a mobile phase of acetonitrile and 0.1% formic acid in water (40:60, v/v) at a flow rate of 0.25 mL/min. The selected reaction monitoring mode was chosen to monitor the precursor-to-product ion transitions of m/z 409.2 → 373.2 for bruceine D and m/z 469.2 → 229.3 for IS using a negative ESI mode. The method was validated over a concentration range of 0.5-2000 ng/mL for bruceine D. Total chromatography time for each run was 3.5 min. The method was successfully applied to a pharmacokinetic study of bruceine D in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of a potential antitumor quassinoid in rat plasma by UPLC-MS/MS and its application in a pharmacokinetic study.

A sensitive UPLC-MS/MS method was developed and validated for the determination of brusatol in rat plasma. Chromatographic separation was carried out on a C18 column using methanol and 10mM ammonium acetate containing 0.1% (v/v) formic acid (55:45, v/v). The lower limit of quantification (LLOQ) was 1.0 ng/mL for brusatol in plasma. The intra- and inter-day precision for the analyte ranged from 3.2% to 9.2% and 1.3% to 7.8%, and the accuracy was between 97.3% and 108.5%. The method was successfully applied in a pharmacokinetic study of brusatol following intravenous injection (0.5, 1.0, and 2.0mg/kg) of brusatol.

Development of a validated LC-MS/MS method for the determination of ailanthone in rat plasma with application to pharmacokinetic study.

Ailanthone, a natural compound isolated from Chinese herb Ailanthus altissima, has drawn a lot of attention for its antitumor activity. In this study, a simple and sensitive method for determination of ailanthone in rat plasma was developed for the first time, using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Brusatol was used as an internal standard. Separation was achieved on an Agilent Zorbax Eclipse Plus C18 column with gradient elution using water-methanol as mobile phase at a flow rate of 0.2 mL/min. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring (MRM) was used to detect ailanthone and IS transitions of 375.2 → 301.1 and 519.1 → 437.4, respectively. The lower limit of quantification was 5 ng/mL with a linear range of 5-2000 ng/mL. The intra- and inter-day accuracy (RE) ranged from -3.6 to 1.5% and -0.7 to 4.7% and the intra- and inter-day precision (RSD) was between 2.8-6.7% and 3.1-8.0%. The validated method has been successfully applied to a pharmacokinetic study of ailanthone in rats. The elimination half-lives (t1/2) were 105.5 ± 13.6, 113.3 ± 39.6, and 95.8 ± 23.9 min after single intravenous administration of 0.5, 1.0, and 1.5mg/kg ailanthone, respectively. The area under the plasma concentration versus time curve (AUC0-6h) and initial plasma concentration (C0) were linearly related to dose.

Development and validation of liquid chromatography combined with tandem mass spectrometry methods for the quantitation of simalikalactone E in extracts of Quassia amara L. and in mouse blood.

INTRODUCTION: Simalikalactone E (SkE) from Quassia amara, has been proved to be a valuable anti-malarial and anti-cancer compound. As SkE is very scarce, methods of quantitation are needed in order to optimise its isolation process and to determine pharmacokinetic data. OBJECTIVE: To validate methods using liquid chromatography coupled to mass spectrometry for the quantitation of SkE in plant extracts and in biological fluids. METHODS: High- and ultrahigh-performance liquid chromatography (UHPLC) coupled to ion trap mass spectrometry (MS) with single ion monitoring detection and to triple quadrupole-linear ion trap tandem mass spectrometry with multiple reaction monitoring detection methods were developed. Validation procedure was realised according to the International Conference on Harmonisation guideline. Methanol extracts of dried Quassia amara leaves, and mouse-blood samples obtained after various routes of administration, were analysed for SkE. RESULTS: Methods were validated and gave similar results regarding the content of SkE expressed per kilogram of dry leaves in the traditional decoction (160 ± 12 mg/kg) and in the methanol extract (93 ± 2 mg/kg). The recovery of the analyte from mouse blood ranged from 80.7 to 119.8%. Simalikalactone E was only detected using UHPLC-MS/MS (0.2 ± 0.03 mg/L) in mouse blood after intravenous injection: none was detected following intraperitoneal or oral gavage administration of SkE. CONCLUSION: The LC-MS methods were used for the quantitation of SkE in plant extracts and in mouse blood. These methods open the way for further protocol optimisation of SkE extraction and the determination of its pharmacokinetic data.