Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection

Profiling of the main metabolites from several licorice (Glycyrrhiza glabra) samples collected at different locations is carried out in this work by using comprehensive two-dimensional liquid chromatography (LC × LC) coupled to diode array (DAD) and mass spectrometry (MS) detectors. The optimized method was based on the application of a HILIC-based separation in the first dimension combined with fast RP-based second dimension separation. This set-up was shown to possess powerful separation capabilities allowing separating as much as 89 different metabolites in a single sample. Identification and grouping of metabolites according to their chemical class were achieved using the DAD, MS and MS/MS data. Triterpene saponins were the most abundant metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as expected, was confirmed as the main component in all the studied samples. LC × LC-DAD-MS/MS was able to resolve these complex licorice samples providing with specific metabolite profiles to the different licorice samples depending on their geographical origin. Namely, from 19 to 50 specific compounds were exclusively determined in the 2D-chromatograms from the different licorice samples depending on their geographical origin, which can be used as a typical pattern that could potentially be related to their geographical location and authentication.     

Simultaneous determination of the novel tyrosine kinase inhibitor meditinib and its active metabolite demethylation meditinib in monkey plasma by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic studies

Meditinib (ME) is a novel tyrosine kinase inhibitor used as an antichronic myeloid leukemia drug. A simple, sensitive and specific LC/MS/MS method was developed and validated for the analysis of ME and its metabolite demethylation meditinib (PI) in monkey plasma using naltrexone as the internal standard. Sample preparation involved protein precipitation with methanol. The analysis was carried out on an Agilent C₈ column (3.5 µm, 2.1 × 50 mm). Elution was achieved with a mobile phase gradient varying the proportion of a water solution containing 0.1% formic acid (solvent A) and a 0.1% formic acid in methanol solution (solvent B) at a flow rate of 300 μL/min. The method had a linear calibration curve over the concentration range of 2–1000 ng/mL for ME and 2–1000 ng/mL for PI. The lower limits of quantification of ME and PI were 2 and 2 ng/mL, respectively. The intra‐ and inter‐day precision values were <15% and accuracy values were within ±10.0%. The mean recoveries of ME and PI from plasma were >85%. The assay has been successfully used for pharmacokinetic evaluation of ME and PI using the monkey as an animal model, and those data are reported for the first time. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly efficient and sensitive LC‐MS/MS method for the determination of afatinib in human plasma: application to a metabolic stability study

Afatinib (AFT) is a new tyrosine kinase inhibitor approved for the treatment of nonsmall cell lung cancer. In the present study, a simple, specific, rapid and sensitive liquid chromatography tandem mass‐spectrometric method for the quantification of AFT in human plasma, was developed and validated. Chromatographic separation of the analytes was accomplished on a reversed‐phase Luna^®‐PFP 100 Å column (50 × 2.0 mm; 3.0 μm) maintained at ambient temperature. Isocratic elution was carried out using acetonitrile–water (40:60, v/v) containing 10 mm ammonium formate buffer (pH 4.5) adjusted with formic acid at a flow rate of 0.4 mL min^?1. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring mode. The method yields a linear calibration plot (r² = 0.9997) from a quantification range of 0.5–500 ng mL^?1 with the lower limit of quantification and lower limit of detection of 1.29 and 0.42 ng mL^?1, respectively. The intra‐ and inter‐day precision and accuracy were estimated and found to be in the ranges of 1.53–4.11% for precision and ?2.80–0.38% for accuracy. Finally, quantification of afatinib in a metabolic stability study in rat liver microsomes was achieved through the proposed method. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantitative determination of euphol in rat plasma by LC‐MS/MS and its application to a pharmacokinetic study

Euphol is a potential pharmacologically active ingredient isolated from Euphorbia kansui. A simple, rapid, and sensitive method to determine euphol in rat plasma was developed based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for the first time. The analyte and internal standard (IS), oleanic acid, were extracted from plasma with methanol and chromatographied on a C₁₈ short column eluted with a mobile phase of methanol–water–formic acid (95:5:0.1, v/v/v). Detection was performed by positive ion atmospheric pressure chemical ionization in selective reaction monitoring mode. This method monitored the transitions m/z 409.0 → 109.2 and m/z 439.4 → 203.2 for euphol and IS, respectively. The assay was linear over the concentration range 27–9000 ng/mL, with a limit of quantitation of 27 ng/mL. The accuracy was between –7.04 and 4.11%, and the precision was <10.83%. This LC‐MS/MS method was successfully applied to investigate the pharmacokinetic study of euphol in rats after intravenous (6 mg/kg) and oral (48 mg/kg) administration. Results showed that the absolute bioavailability of euphol was approximately 46.01%. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioanalysis of tolvaptan,a novel AVP‐V2 receptor antagonist in human plasma by a novel LC‐ESI‐MS/MS method: a pharmacokinetic application in healthy South Indian male subjects

A simple, rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) assay method is proposed for the determination of tolvaptan in human plasma samples using tolvaptan d₇ as internal standard (IS). Analyte and the IS were extracted from 100 μL of human plasma via simple liquid–liquid extraction. The chromatographic separation was achieved on a C₁₈ column using a mixture of methanol and 0.1% formic acid buffer (80:20, v/v) as the mobile phase at a flow rate of 1.0 mL/min. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.05–501 ng/mL. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The intra‐day and inter‐day precision (coefficient of variation) and accuracy results in three validation batches across five concentration levels were well within the acceptance limits. A run time of 2.0 min for each sample made it possible to analyze more samples in a short time, thus increasing the productivity. The proposed method was successfully applied to a pharmacokinetic study of 15 mg and 60 mg tolvaptan tablet formulation in healthy South Indian male subjects under fasting condition. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated LC‐MS/MS assay for the quantitative determination of curdione in rabbit plasma and its application to a pharmacokinetic study after administration of zedoary turmeric oil and bioavailability of the oil

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the first time for the identification and quantification of curdione in rabbit plasma after vaginal drug administration and intravenous administration of zedoary turmeric oil (ZTO) solution (10 mg/kg). The analysis was performed on a triple‐quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive ionization mode. After mixing with internal standard diazepam, plasma samples were extracted with ethyl ether–acetic ether (1:1, v/v). Chromatographic separation was carried out on a C₁₈ column with gradient elution using a mixture of water and acetonitrile (both containing 0.1% formic acid) as mobile phases. Linearity ranged over 1.06–106 and 10.6–530 ng/mL (r ≥ 0.995) with the lower limit of quantfication 1.06 ng/mL. The intra‐ and inter‐day precision relative standard deviation values were <12% and the accuracy relative error was from ?10.6 to ?6.1% at all quality control sample levels. The method was applied to a study of the pharmacokinetics of curdione after vaginal drug administration and intravenous administration of ZTO. Copyright © 2014 John Wiley & Sons, Ltd.     

Analysis and pharmacokinetic study of polyphyllin H in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC‐MS/MS

A highly sensitive, rapid assay method has been developed and validated for the analysis of polyphyllin H in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of polyphyllin H and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB‐C₁₈ (100 × 2.1 mm, 1.8μm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.5 min. The MS/MS ion transitions monitored were 870.46 → 869.6 for polyphyllin H and 947.12 → 969.60 for IS. Linear responses were obtained for polyphyllin H ranging from 1 to 50 ng/mL. The intra‐and inter‐day precisions (RSDs) were <1.77 and 3.39% and the extraction recovery ranged from 91.89 to 93.33% with RSD <2.68%. Stability studies showed that polyphyllin H was stable in the preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration–time profiles of polyphyllin H. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of an LC‐MS/MS method for the determination of a novel thienoquinolin urea transporter inhibitor PU‐48 in rat plasma and its application to a pharmacokinetic study

A specific, sensitive and stable high‐performance liquid chromatographic–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantitative determination of methyl 3‐amino‐6‐methoxythieno [2,3‐b]quinoline‐2‐carboxylate (PU‐48), a novel diuretic thienoquinolin urea transporter inhibitor in rat plasma. In this method, the chromatographic separation of PU‐48 was achieved with a reversed‐phase C₁₈ column (100 × 2.1 mm, 3 μm) at 35°C. The mobile phase consisted of acetonitrile and water with 0.05% formic acid added with a gradient elution at flow rate of 0.3 mL/min. Samples were detected with the triple‐quadrupole tandem mass spectrometer with multiple reaction monitoring mode via electrospray ionization source in positive mode. The retention time were 6.2 min for PU‐48 and 7.2 min for megestrol acetate (internal standard, IS). The monitored ion transitions were mass‐to‐charge ratio (m/z) 289.1 → 229.2 for PU‐48 and m/z 385.3 → 267.1 for the internal standard. The calibration curve for PU‐48 was linear over the concentration range of 0.1–1000 ng/mL (r² > 0.99), and the lower limit of quantitation was 0.1 ng/mL. The precision, accuracy and stability of the method were validated adequately. The developed and validated method was successfully applied to the pharmacokinetic study of PU‐48 in rats.     

Chromatographic methods in HIV medicine: Application to therapeutic drug monitoring

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC‐UV and LC‐MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid–liquid extraction or solid‐phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C₁₈). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co‐infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.     

Validation of a sensitive LC/MS/MS method for the determination of telaprevir and its R‐isomer in human plasma

The purpose of this study was to validate a reversed‐phase high‐performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R‐diastereomer (VRT‐127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir‐R was performed on a Waters XBridge^TM BEH Shield C₁₈, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir‐d11). An internal standard for the telaprevir‐R (telaprevir‐R‐d11) was also prepared by incubating telaprevir‐d11 in basic solution, which facilitated isomer inter‐conversion. The detection and quantitation of telaprevir, telaprevir‐R, telaprevir‐IS and telaprevir‐R‐IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.