A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for simultaneous quantification of methotrexate (MTX) and tofacitinib (TFB) in rat plasma (50 μL) using phenacetin as an internal standard (IS), as per the US Food and Drug Administration guidelines. After a solid-phase extraction procedure, the separation of the analytes and IS was performed on a Chromolith RP₁₈e column using an isocratic mobile phase of 5 m m ammonium acetate (pH 5.0) and acetonitrile at a ratio of 25:75 (v/v) using flow-gradient with a total run time of 3.5 min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 455.2 → 308.3, m/z 313.2 → 149.2 and m/z 180.3 → 110.2 for MTX, TFB and IS, respectively. The calibration curves were linear over the range of 0.49-91.0 and 0.40-74.4 ng/mL for MTX and TFB, respectively. The intra- and interday accuracy and precision values for MTX and TFB were <15% at low quality control (QC), medium QC and high QC and <20% at lower limit of quantification. The validated assay was applied to derive the pharmacokinetic parameters for MTX and TFB post-dosing of MTX and TFB orally and intravenously to rats.

Filgotinib is a selective JAK1 (Janus kinase) inhibitor, filed in Japan for the treatment of rheumatoid arthritis. In this paper, we report a validated liquid chromatography coupled with tandem mass spectrometry for the quantification of filgotinib in rat plasma using tofacitinib as an internal standard (IS) as per the Food and Drug Administration regulatory guidelines. Filgotinib and the IS were extracted from rat plasma using ethyl acetate as an extraction solvent and chromatographed using an isocratic mobile phase (0.2% formic acid:acetonitrile; 20:80, v/v) at a flow rate of 0.9 mL/min on a Gemini C18 column. Filgotinib and the IS were eluted at ~1.31 and 0.89 min, respectively. The MS/MS ion transitions monitored were m/z 426.3 → 291.3 and m/z 313.2 → 149.2 for filgotinib and the IS, respectively. The calibration range was 0.78–1924 ng/mL. No matrix effect and carryover were observed. Intra‐ and inter‐day accuracies and precisions were within the acceptance range. Filgotinib was stable for three freeze–thaw cycles: on bench‐top up to 6 h, in an autosampler up to 21 h, and at −80°C for 1 month. This novel method has been applied to a pharmacokinetic study in rats.

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guideline for the estimation of tofacitinib on mice dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electro spray ionization in the positive-ion mode. The method employs liquid extraction of tofacitinib from DBS disk of mice whole blood followed by chromatographic separation using 5 mM ammonium acetate (pH 6.5):acetonitrile (20:80, v/v) at a flow rate of 0.60 mL/min on an X-Terra Phenyl column with a total run time 2.5 min. The MS/MS ion transitions monitored were 313→149 for tofacitinib and 316→149 for the internal standard (CN-tofacitinib). The assay was linear in the range of 0.99-1980 ng/mL. The intra- and inter-day precision was in the range of 1.17-10.3 and 3.37-10.9%, respectively. Stability studies showed that tofacitinib was stable on DBS cards for one month. This novel method has been applied to analyze the DBS samples of tofacitinib obtained from a pharmacokinetic study in mice.

Different options on performing incurred sample reanalysis (ISR) on dried blood spot (DBS) cards were investigated using drugs belonging to various therapeutic areas: (a) darolutamide (to treat prostate cancer) and (b) filgotinib (to treat rheumatoid arthritis). The proposed novel methodology included the generation of half-DBS and quarter-DBS discs after initial blood collection using the full-DBS discs. Accordingly, blood collection via DBS was performed in male BALB/c mice following intravenous and oral dosing of darolutamide; in male Sprague Dawley rats following intravenous and oral dosing of filgotinib. The ISR data generated from the full-DBS disc, half-DBS disc and quarter-DBS disc were compared for the assessment of the proposed methodology. Quantification of darolutamide and filgotinib was accomplished using liquid chromatography-electrospray ionization/tandem mass spectrometry methods. Darolutamide and filgotinib ISR samples, which were collected and prepared using full-, half-and quarter-DBS discs, met the acceptance criteria for ISR analysis. In conclusion, this is the first report showing a viable tool for the performance of ISR on DBS cards. The use of quarter-or half-DBS discs would aid in not only ISR but also in long-term storage experiments of analytes because it would avoid the need for additional blood sampling in patients.

In order to solve the problem of insufficient adsorption rate of droplets on the target back via aerial electrostatic spray, this study proposed a high-voltage electrostatic generator to charge the liquids in two isolated water tanks with positive and negative charges respectively. A charge transfer loop was developed in space between the aerial electrostatic spray system and the ground. This method greatly enhanced the adsorption performance under outdoor conditions that 16.7% droplets density increased on the target front, a nearly fourfold destiny increased on the target back compared with the conventional UAV spray system. The target back-to-front ratio of droplet density was improved from 6.1% to 25.7%, which validated the satisfactory performance of the developed system.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of rocilinostat in small volume mouse plasma (20 μL) using vorinostat as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation procedure with acetonitrile. Chromatography was achieved on Prodigy ODS-2 column using a binary gradient using mobile phase A (0.2% formic acid in water) and B (acetonitrile) at a flow rate of 0.38 mL/min. The total chromatographic run time was 4.1 min and the elution of rocilinostat and IS occurred at ~3.2 and 2.9 min, respectively. A linear response function was established in the concentration range of 0.28-1193 ng/mL in mouse plasma. The intra- and inter-day accuracy and precisions were in the ranges of 3.12-8.93 and 6.41-11.6%, respectively. This novel method has been applied to a pharmacokinetic study in mice. Copyright © 2016 John Wiley & Sons, Ltd.

A highly sensitive and rapid assay method has been developed and validated for the estimation of S-(-)-raclopride (S-RCP) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive ion mode. The assay procedure involves a simple liquid-liquid extraction technique for extraction of S-RCP and phenacetin (internal standard, IS) from rat plasma. Chromatographic separation was achieved with 0.2% formic acid : acetonitrile (80:20, v/v) at a flow rate of 0.30 mL/min on a Phenomenex Prodigy C(18) column with a total run time of 4.5 min. The MS/MS ion transitions monitored were 347.2 → 112.1 for S-RCP and 180.1 → 110.1 for IS. Method validation and pre-clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range was extended from 0.05 to 152 ng/mL in rat plasma. The intra-day and inter-day precisions were 0.23-10.5 and 3.74-7.29%, respectively. This novel method was applied to a pharmacokinetic study of S-RCP in rats.

<p class="ADMETabstracttext">Filgotinib is a selective JAK1 (Janus kinase) inhibitor, showed efficacy in patients suffering from moderate-to-severe rheumatoid arthritis. In this paper, we present the data on the development and validation of a sensitive, selective and high-throughput LC-MS/MS (liquid chromatography with tandem mass spectrometry) method for the quantitation of filgotinib from rat dried blood spot (DBS) cards. To the DBS disc cards, 0.2 % formic acid enriched with internal standard (IS) was added and sonicated. Thereafter the extraction of filgotinib and the IS (tofacitinib) was accomplished using ethyl acetate as an extraction solvent. The resolution of filgotinib and the IS was achieved on a Gemini C₁₈ column with an isocratic<strong> </strong>mobile phase, which is a mixture of 0.2 % formic acid:acetonitrile (20:80, v/v) at a flow-rate of 0.9 mL/min. The total run time was 2.90 min and the retention time of filgotinib and the IS was ~1.31 and 0.89 min, respectively. Filgotinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transition of 426.3®291.3 and m/z 313.2®149.2, respectively, for quantitation. The calibration range was 1.37-1937 ng/mL. No matrix effect and carry over were observed. All the validation parameters met the acceptance criteria. The validated method has been applied to a pharmacokinetic study in rats. A good correlation between DBS and plasma concentrations for filgotinib was observed.</p>