Determination of rosamultin in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

A specific and reliable LC–MS/MS method for the determination of rosamultin in rat plasma was validated. Plasma samples were prepared with protein precipitation method, and chromatographic separation was performed on a Thermo C₁₈ analytical column (4.6 mm × 50 mm, 3.0 μm). The mass spectrometry (MS) analysis was conducted in positive SRM mode for the transitions of m/z 673.2 → 511.1 for rosamultin and m/z 601.1 → 330.9 for IS. The method validation was conducted over the calibration range of 1.0–500 ng/mL with the precision ≤11.03% and accuracy within ±14.64%. The assay was applied to the pharmacokinetic study after oral administration of rosamultin at a dose of 20 mg/kg in rats.     

Quantitative determination of bilobetin in rat plasma by HPLC–MS/MS and its application to a pharmacokinetic study

Although bilobetin, a biflavone isolated from the leaves of Ginkgo biloba, represents a variety of pharmacological activities, to date there have been no validated determination methods for bilobetin in biological samples. Thus, we developed a liquid chromatographic method using a tandem mass spectrometry for the determination of bilobetin in rat plasma. After protein precipitation with acetonitrile including diclofenac (internal standard), the analytes were chromatographed on a reversed-phased column with a mobile phase of purified water and acetonitrile (3:7, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally deprotonated ions [M − H]⁻ at m/z 551.2 → 519.2 for bilobetin and 296.1 → 251.7 for the IS. The accuracy and precision of the assay were in accordance with US Food and Drug Administration regulations for the validation of bioanalytical methods. This analytical method was successfully applied to monitor plasma concentrations of bilobetin over time following intravenous administration in rats.     

Determination of ginsenoside Rh3 in rat plasma by LC–MS/MS and its application to a pharmacokinetic study

Ginsenoside Rh3 (GRh3) is a bacterial metabolite of ginsenoside Rg5, which is the main component of hot-processed ginseng. A simple, efficient and sensitive method was developed and validated for the determination of GRh3 in rat plasma by LC–tandem mass spectrometry. After protein precipitation with methanol/acetonitrile (1:1, vol/vol) using propranolol as the internal standard, the target analytes were separated on an XDB C₁₈ column, with methanol containing 0.1% formic acid and water containing 0.1% formic acid used as mobile phases for gradient elution. Mass spectrometry was performed in electrospray ion source–positive ion mode and multiple reaction monitoring mode, monitoring the transitions m/z 622.5 → 425.5 and m/z 260.1 → 116.1 for GRh3 and internal standard, respectively. The concentration range of GRh3 was 20–20,000 ng/mL and the correlation coefficient (r²) was greater than 0.99. The accuracy error and relative standard deviation were below 15%. The extraction recovery and matrix effect were 74.2% to 78.7% and 96.9% to 108.4%, respectively. Under different conditions, GRh3 was stable in the range of 1.8%–8.7%. This method has been successfully applied to study the pharmacokinetics of GRh3 with an oral dose of 10.0 mg/kg and an intravenous dose of 2.0 mg/kg in rats, respectively. The absolute bioavailability of GRh3 was 37.6%.     

LC–MS/MS method for determination of kansuinine a in rat plasma and its application to a rat pharmacokinetic study

Kansuinine A is a macrocyclic jatrophane diterpene isolated from the plant Euphorbia kansui Liou. It exhibits many pharmacological activities including cytoxic, antitumor, antiallergic and proinflammatory effects. In the present study, a simple and sensitive LC–MS/MS method was established and validated for the determination of kansuinine A in rat plasma. After methanol-mediated protein precipitation, chromatographic separation was achieved on an Acquity BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) using acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. Kansuinine A and IS were quantified in negative multiple reaction monitoring mode with ion transitions at m/z 731.1–693.2 for kansuinine A and m/z 723.2–623.1 for IS. The method showed excellent linearity over the range 1–500 ng/ml. The intra- and inter-day precisions (relative standard deviation) were 2.13–4.28 and 3.83–7.67%, respectively, whereas accuracy (relative error) ranged from −4.17 to 3.73%. The extraction recovery, stability and matrix effect met the requirement of the regulations issued by the US Food and Drug Administration. The validated method was successfully applied to the pre-clinical pharmacokinetic study of kansuinine A in rats after oral administration (20 mg/kg) and intravenous administration (2 mg/kg). This study provides valuable reference for the further study of E. kansui liou, especially for the drug development and clinical application of kansuinine A.     

LC–MS/MS assay for the quantification of foretinib in rat plasma and its application to preclinical pharmacokinetic study

A simple and sensitive ultra-high-performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of foretinib in rat plasma. The analyte and internal standard were extracted from the bio-samples with acetonitrile and then separated on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% formic acid aqueous and acetonitrile as mobile phase, at a flow rate of 0.4 ml/min. The mass detection was performed in positive selected reaction monitoring mode with precursor-to-product transitions at m/z 317.1 > 128.1 for foretinib and m/z 502.2 > 323.1 for internal standard. The assay was demonstrated to be linear in the concentration range of 0.5–1000 ng/ml, with correlation coefficient >0.999. The mean extraction recovery of foretinib from rat plasma was within the range of 84.55–88.09%, while the matrix effect was in the range of 88.56–99.21%. The intra- and inter-day precisions were <12.95% and the accuracy ranged from −7.55 to 8.57%. Foretinib was stable in rat plasma under the tested storage conditions. The validated assay was successfully applied to the pharmacokinetic study of foretinib in the rats. The results revealed that foretinib showed moderate elimination half-life, low clearance and dose-independent pharmacokinetic profiles inrats.     

Determination of meloxicam in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to detect meloxicam in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source was used in positive ion mode. Protein precipitation with acetonitrile was used for sample preparation. Meloxicam and ¹³C₆-meloxicam internal standard were analyzed on an Acquity CSH C₁₈ column with a mobile phase of acetonitrile and water in 0.1% formic acid using a gradient program for separation. The retention time of meloxicam was 1.1 min and the total run time was only 2.0 min. Detection was performed in multiple reaction monitoring mode using an electrospray ionization source with optimized mass spectrometry parameters. The calibration curves were linear in the range 10.0–3.00 × 10³ ng/ml (r ≥ 0.99). The within-run and between-run RSDs were ≤14.8%. The within-run and between-run REs ranged from −4.6 to 10.7%. There was no significant matrix effect, and the recovery rate was high. This method was fully validated, including reinjection reproducibility in human plasma. The method was applied to the pharmacokinetic study. All of the incurred sample reanalysis methods met the criteria.     

Determination of mitragynine in plasma with solid-phase extraction and rapid HPLC–UV analysis, and its application to a pharmacokinetic study in rat

A new solid phase extraction method for rapid high performance liquid chromatography–UV determination of mitragynine in plasma has been developed. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile–ammonium acetate buffer, 50 mM at pH 5.0 (50:50, v/v). The method had limits of detection and quantification of 0.025 and 0.050 μg/mL, respectively. The method was accurate and precise for the quantitative analysis of mitragynine in human and rat plasma with within-day and between-day accuracies between 84.0 and 109.6%, and their precision values were between 1.7 and 16.8%. Additional advantages over known methods are related to the solid phase extraction technique for sample preparation which yields a clean chromatogram, a short total analysis time, requires a smaller amount of plasma samples and has good assay sensitivity for bioanalytical application. The method was successfully applied in pharmacokinetic and stability studies of mitragynine. In the present study, mitragynine was found to be fairly stable during storage and sample preparation. The present study showed for the first time the detailed pharmacokinetic profiles of mitragynine. Following intravenous administration, mitragynine demonstrated a biphasic elimination from plasma. Oral absorption of the drug was slow, prolonged and was incomplete, with a calculated absolute oral bioavailability value of 3.03%. The variations observed in previous pharmacokinetic studies after oral administration of mitragynine could be attributed to its poor bioavailability rather than to the differences in assay method, metabolic saturation or mitragynine dose.     

LC–MS/MS analysis of puerarin and 18β-glycyrrhetinic acid in human plasma after oral administration of Samso-eum and its application to pharmacokinetic study

The aim of this study was to confirm pharmacokinetic screening of multiple components in healthy Korean subjects after oral administration of Samso-eum and perform quantitation of active components in the human plasma. Thirteen potential bioactive components [puerarin (PRR), daidzin, nodakenin, ginsenoside Rb1, 18β-glycyrrhetinic acid (18β-GTA), 6-shogaol, naringin, glycyrrhizin, hesperidin, platycodin D, naringenin, hesperetin, and 6-gingerol] were screened based on literature. The results showed that three analytes (daidzin, naringenin, and hesperetin) were detected in trace amounts. In addition, PRR and 18β-GTA were detected in human plasma after the oral administration of Samso-eum. In this study, a liquid chromatography–electrospray ionization-tandem mass spectrometry method was validated for the simultaneous determination of PRR and 18β-GTA in human plasma. This was the first study to evaluate pharmacokinetics of PRR and 18β-GTA after the usual oral dose of Samso-eum (30 g containing 102.48 mg PRR, 48.18 mg glycyrrhizin) in human subjects.     

A rapid UHPLC–MS/MS method for the quantification of ARQ531 in rat plasma: Validation and its application to a pharmacokinetic study

A simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) method was developed and validated for the determination of ARQ531, a Bruton’s tyrosine kinase inhibitor in rat plasma. After protein precipitation with acetonitrile, the samples were separated on a UPLC BEH C₁₈ column with 0.1% formic acid in water and acetonitrile as mobile phase at a flow rate of 0.4 ml/min. The mass detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring with precursor-to-product ion transitions of m/z 479.1 > 365.1 and m/z 441.2 > 138.1 for ARQ531 and internal standard, respectively. Good linearity (correlation coefficient > 0.9988) was achieved over the concentration range of 0.5–1,000 ng/ml and the lower limit of quantitation was 0.5 ng/ml. The accuracy ranged from −13.50 to 11.35% and the precision was <8.87%. The extraction recovery was >85.56%. ARQ531 was demonstrated to be stable under the tested conditions. The validated method was further applied to a pharmacokinetic study of ARQ531 in rats after intravenous (1 mg/kg) and oral (1, 3 and 10 mg/kg) administration. The results demonstrated that ARQ531 displayed linear pharmacokinetic profiles over the oral dose range of 1–10 mg/kg and good oral bioavailability (>50%).     

Development and validation of a rapid and sensitive LC–MS/MS method for the determination of polyphyllin II in rat plasma and its application in a pharmacokinetic study

Polyphyllin II, a major steroidal saponin isolated from Paris polyphylla, exhibits significant pharmacological activities. In this study, a rapid and sensitive liquid chromatography–tandem mass spectrometry method was established and validated for the determination of polyphyllin II in plasma. Polyphyllin II and polyphyllin VII (internal standard) were separated on a Waters Acquity™ HSS T3 column and the mass analysis was performed in a triple quadrupole mass spectrometer equipped with an electrospray ionization ion source. Results showed that the method was sensitive (lower limit of quantitation 0.5 ng/ml), precise (<15%) and linear in the range of 0.5–500 ng/ml (r > 0.99). Interestingly, the sensitivity in current study was ~10 times higher than that in the previous study. The results of the pharmacokinetic study of polyphyllin II in rats suggested that polyphyllin II was poorly absorbed into blood and reached its highest concentration at ~3.67–5.00 h with a slow elimination half-life of 8.34–13.37 h. The bioavailability was 6.1–8.2%. The results indicated that the absorption of polyphyllin II may primarily occur via passive diffusion in rats. This study provides valuable information that can be used as a reference for the pharmacokinetic investigation of other steroidal saponins.     

A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC–MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC₁₈ column using 10 mm ammonium formate–acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent–daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06–5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.     

Establishment and validation of an SIL-IS LC–MS/MS method for the determination of ibuprofen in human plasma and its pharmacokinetic study

In this work, we developed and validated a highly sensitive, rapid and stable LC–MS/MS method for the determination of ibuprofen in human plasma with ibuprofen-d3 as a stable isotopically labeled internal standard (SIL-IS). Human plasma samples were prepared by one-step protein precipitation. The chromatographic separation was achieved on a Poroshell 120 EC-C₁₈ (2.1 × 50 mm, 2.7 μm). Aqueous solution (containing 0.05% acetic acid and 5 mm NH₄Ac) and methanol were selected as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in negative ion mode. Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 205.0 → 161.1 for ibuprofen and m/z 208.0 → 164.0 for SIL-IS, respectively. This method exhibited a linear range of 0.05–36 μg/ml for ibuprofen with correlation coefficient >0.99. Mean recoveries of ibuprofen in human plasma ranged from 78.4 to 80.9%. The RSD of intra- and inter-day precision were both < 5%. The accuracy was between 88.2 and 103.67%. The matrix effect was negligible in human plasma, including lipidemia and hemolytic plasma. A simple, efficient and accurate LC–MS/MS method was successfully established and applied to a pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ibuprofen granules.     

Determination of oleandrin and adynerin in rat plasma by UPLC–MS/MS and their pharmacokinetic study

Oleandrin and adynerin are the main toxic components of oleander, an evergreen shrub or a small tree of the oleander family, which belongs to the class of cardiac glycosides exhibiting delayed action. The pharmacokinetic differences of oleandrin and adynerin in rats were studied by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) under two different administration modes: oral (5 mg/kg) and sublingual intravenous injection (1 mg/kg). The chromatographic column was UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm), and the column temperature was set at 40 °C. The mobile phase was acetonitrile–water (containing 0.1 % formic acid), with gradient elution, the flow rate was 0.4 mL/min, and the elution time was 4 min. Electrospray (ESI) positive ion mode detection with multiple reaction monitoring mode (MRM) was used for quantitative analysis: oleandrin m/z 577 → 145, adynerin m/z 534 → 113, and internal standard m/z 237 → 135. The established UPLC–MS/MS method was successfully applied to the pharmacokinetics in rats after administering oleandrin and adynerin. The bioavailability of oleandrin and adynerin was found to be low, 7.0 % and 93.1 %; respectively.     

Determination of phenazopyridine in human plasma via LC–MS and subsequent development of a pharmacokinetic model

This paper describes a new LC–MS method for the determination of phenazopyridine and the subsequent development of a pharmacokinetic model for phenazopyridine in vivo. Phenazopyridine hydrochloride is a strong analgesic used in the treatment of urinary tract infections. Although it has been used as a clinical treatment for a very long time, pharmacokinetic data and suitable methods for its determination in plasma are currently lacking. The study described in this paper used high performance liquid chromatography–mass spectrometry, HPLC–MS, to determine the plasma concentrations of phenazopyridine in human subjects after oral administration. After liquid–liquid extraction, the phenazopyridine in the plasma was analyzed on a C₁₈ column under SIM mode. A double-peak phenomenon was observed in most of the concentration–time profiles of the subjects. Although some drugs are known to cause this phenomenon, phenazopyridine has not been reported to do so. Several possible causes were analyzed in order to obtain an explanation. We proposed a two-site absorption compartment model to fit the concentration data in vivo, which has one more absorption site than the classical one-compartment model. The model describes the concentration profiles in different dose groups well and could provide an explanation for the double-peak phenomenon. The three dose groups exhibited similar model parameters and a linear pharmacokinetic process over the dose range used.     

Validated HPLC–MS–MS method for simultaneous determination of atorvastatin and 2-hydroxyatorvastatin in human plasma—pharmacokinetic study

Cholesterol-reducing statin drugs are the most frequently prescribed agents for reducing morbidity and mortality related to coronary heart disease. In this publication a validated, highly sensitive, and selective isocratic HPLC method is reported for quantitative determination of the major statin drug atorvastatin (ATV) and its metabolite 2-hydroxyatorvastatin (HATV). Detection was performed with an electrospray ionization triple-quadrupole mass spectrometer equipped with an ESI interface operating in positive-ionization mode. Multiple reaction monitoring (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 0.10–40.00 ng mL⁻¹ for both ATV and HATV. Inter-day and intra-day precision and accuracy of the proposed method were characterized by measurement of relative standard deviation (RSD) and percentage deviation, respectively; both were less than 8% for both analytes. The limit of quantitation was 0.02 ng mL⁻¹ for ATV and 0.07 ng mL⁻¹ for HATV. The method was used for pharmacokinetic study of ATV and HATV. Pharmacokinetic data for all analytes are also reported.     

Determination of acamprosate in human plasma by UPLC–MS/MS: Application to therapeutic drug monitoring

Acamprosate is a medication used to treat alcohol dependence. Therapeutic drug monitoring is important in drugs for the treatment of substance-related disorders. Therefore, in this study, a new selective, very simple and rapid ultra-performance liquid chromatography–tandem mass spectrometer method was developed for the therapeutic drug monitoring of acamprosate. The developed method allows the determination of acamprosate in human plasma. The method was validated in terms of selectivity and linearity, which was in the range of 100–1,200 ng/ml for acamprosate. Intra-assay and inter-assay accuracy and precision were within the acceptable limits of the Eueopean Medicines Agency guideline. The lower limit of quantitation was 100 ng/ml for acamprosate. The developed method was successfully applied for therapeutic drug monitoring in patient plasma samples.     

Simultaneous determination of eight bioactive components in Marsdenia tenacissima extract in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

As a traditional Chinese medicine, Marsdenia tenacissima (Roxb.) Wight et Arn. plays an indispensable role in clinical practice owing to its specific efficacy in treating malignant tumors, leukocythemia, cystitis and asthma. This study aimed to establish a novel and scientific LC–MS/MS approach to simultaneously determine tenacissoside B, H, G and I, caffeic acid, cryptochlorogenic acid, chlorogenic acid and neochlorogenic acid from M. tenacissima extract within the rat plasma samples. Digoxin was used as the internal reference. All determinations were carried out using the Eclipse Plus C₁₈ column, and water (containing 0.1% formic acid) was used as the mobile phase A, while acetonitrile was the mobile phase B for gradient elution. The UPLC methods were validated, including calibration curves, accuracy, precision, stability and recovery of the total eight analytes, in accordance with the requirements for biopharmaceutical analysis. Moreover, the proposed approach was also used in comprehensive pharmacokinetic research on those eight analytes in rats following M. tenacissima extract gavage. According to the pharmacokinetic parameters, tenacissoside B, I, H and G are the long-acting and primary bioactive constituents in M. tenacissima extract, with long mean residence times and high concentrations. Our findings shed light on the absorption mechanism and provide significant information for the clinical application of M. tenacissima.     

A novel LC–MS/MS method for the determination of ziritaxestat in rat plasma and its pharmacokinetic study

Ziritaxestat is a first-in-class autotoxin inhibitor. The purpose of this study was to develop a liquid chromatography/electrospray ionization tandem mass spectrometric (LC–MS/MS) method for the determination of ziritaxestat in rat plasma. The plasma sample was deproteinated using acetonitrile and then separated on an Acquity BEH C₁₈ column with water containing 0.1% formic acid and acetonitrile as mobile phase, which was delivered at 0.4 ml/min. Ziritaxestat and the internal standard (crizotinib) were quantitatively monitored with precursor-to-product transitions of m/z 589.3 > 262.2 and m/z 450.1 > 260.2, respectively. The total running time was 2.5 min. The method showed excellent linearity over the concentration range 0.5–2000 ng/ml, with correlation coefficient >0.9987. The extraction recovery was >82.09% and the matrix effect was not significant. Inter- and intra-day precisions (RSD) were <11.20% and accuracies were in the range of −8.50–7.45%. Ziritaxestat was demonstrated to be stable in rat plasma under the tested conditions. The validated LC–MS/MS method was successfully applied to study the pharmacokinetic profiles of ziritaxestat in rat plasma after intravenous and oral administration. Pharmacokinetic results demonstrated that ziritaxestat displayed a short half-life (~3 h) and low bioavailability (20.52%).     

Enantioselective LC–ESI–MS/MS method for quantitation of (−)-lumefantrine and (+)-lumefantrine in mice plasma and application to a pharmacokinetic study

We developed and validated a simple, sensitive, selective, and reliable LC–MS/MS–ESI method for the direct quantitation of lumefantrine (LFN) enantiomers [(−)-LFN and (+)-LFN] in mice plasma as per regulatory guideline. LFN enantiomers and carbamazepine (internal standard) were extracted from mice plasma using Strata X SPE (solid-phase extraction) cartridges. Good resolution between enantiomers was achieved on a Chiralpak IA-3 column using an isocratic mobile phase (0.1% of diethyl amine in methanol), which was delivered at a flow rate of 0.8 mL/min. Detection and quantitation were performed using multiple reaction monitoring mode following the transitions m/z 530.27 → 512.30 and 237.00 → 194.00 for LFN enantiomers and the internal standard, respectively, in the positive-ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 2.39–895 ng/mL for each enantiomer. The intra- and inter-day precisions were in the range of 1.03–6.14 and 6.36–8.70 and 2.03–4.88 and 5.82–11.5 for (−)-LFN and (+)-LFN, respectively. Both (−)-LFN and (+)-LFN were found to be stable under different stability conditions. The method was successfully used to delineate stereoselective pharmacokinetics of LFN enantiomers in mice after an oral administration of rac-LFN (20 mg/kg). The pharmacokinetic results indicated that the disposition of LFN enantiomers was stereoselective in mice.     

Validated LC–MS/MS method for quantitation of a selective JAK1 inhibitor,filgotinib in rat plasma,and its application to a pharmacokinetic study in 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.