Anti-fibrosis attributes: UHPLC-MS/MS-based pharmacokinetics profiling of a novel autotaxin inhibitor with excellent in vivo efficacy in rat

3,4-Difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a small molecule with outstanding therapeutic effectiveness to anti-pulmonary fibrosis, was developed as an autotaxin inhibitor candidate compound. To evaluate the pharmacokinetics and plasma protein binding of NAI59, a UPLC–MS/MS method was developed to quantify NAI59 in plasma and phosphate-buffered saline. The calibration curve linearity ranged from 9.95 to 1990.00 ng/mL in plasma. The accuracy was −6.8 to 5.9%, and the intra- and inter-day precision was within 15%. The matrix effect and recovery, as well as dilution integrity, were within the criteria. The chromatographic and mass spectrometric conditions were also feasible to determine phosphate-buffered saline samples, and it has been proved that this method exhibits good precision and accuracy in the range of 9.95–497.50 ng/mL in phosphate-buffered saline. This study is the first to determine the pharmacokinetics, absolute bioavailability, and plasma protein binding of NAI59 in rats using this established method. Therefore, the pharmacokinetic profiles of NAI59 showed a dose-dependent relationship after oral administration, and the absolute bioavailability in rats was 6.3%. In addition, the results of protein binding showed that the combining capacity of NAI59 with plasma protein attained 90% and increased with the increase in drug concentration.     

Development and validation of LC–MS/MS method for amlexanox in rat plasma and its application in preclinical pharmacokinetics

Amlexanox, an anti-inflammatory and anti-allergic agent, has been widely used clinically for the treatment of canker sores, asthma, and allergic rhinitis. Recently, amlexanox has received considerable attention in curing nonalcoholic fatty liver diseases and hepatitis virus infection. Herein, we first established a sensitive high-performance liquid chromatography-tandem mass spectrum (LC–MS/MS) method for the determination of amlexanox in rat plasma. Propranolol was used as the internal standard (IS). Using a simple protein precipitation method, the amlexanox and IS were separated with Capcell Pak C18 column (2.0 × 50 mm, 5 μm) and eluted with water and acetonitrile each containing 0.1% formic acid using gradient elution condition at a flow rate of 0.4 mL·min⁻¹. Amlexanox and IS were detected by a triple quadrupole mass in multiple reactive monitoring (MRM) under the transitions of m/z 299.2 → 281.2 and m/z 259.9 → 116.1 with positive electrospray ionization, respectively. The calibration curves of amlexanox were established with the range of 50 to 2000 ng·mL⁻¹ (r² > 0.99). The validation method consisted of selectivity, accuracy, precision, carryover effect, matrix effect, recovery, dilution effect, and stability. The fully validated method was successfully applied to the pharmacokinetic study of amlexanox in Wistar rats.     

Comparative bioavailability of two zolpidem hemitartrate formulations in healthy human Brazilian volunteers using high-performance liquid chromatography coupled to tandem mass spectrometry

To assess the bioequivalence of two zolpidem hemitartrate formulations in 30 healthy volunteers. Plasma samples were obtained over a 24 h period. Plasma concentrations of zolpidem were analyzed by liquid chromatography coupled to tandem mass spectrometry with positive ion electrospray ionization using multiple reaction monitoring. Values of peak concentration (C_max), area under curve (AUC), half-life, elimination constant, volume of distribution and clearance showed statistically significant differences when comparing women (604.34 ng h/ml, 127.36 ng/ml, 4.4 h, 0.18 1/h, 50.56 L and 8.55 L/h, respectively) and men (276.1 ng h/ml, 70.9 ng/ml, 3.3 h, 0.26 1/h, 91.42 L and 24.34 L/h, respectively), receiving the same dose (5 mg), respectively. The geometric means with corresponding 90% confidence interval for Test/Reference percentage ratios were 99.73% (CI 93.69–106.16) for C_max, 97.44% (90% CI = 91.85–103.37%) for area under curve of plasma concentration until the last concentration observed (AUC_last) and 98.30% (90% CI = 92.48–104.49) for the area under curve between the first sample (pre-dosage) and infinity (AUC_0–inf). Since the 90% CI for AUC_last, AUC_0–inf and C_max ratios were within the 80–125% interval proposed by the US Food and Drug Administration, it was concluded that zolpidem hemitartrate formulation (5 mg orodispersible tablet) is bioequivalent to the zolpidem hemitartrate formulation (Patz SL 5 mg sublingual tablet) with regard to both the rate and the extent of absorption. A new formulation of zolpidem 2.5 mg may be useful in women for the same clinical benefits as the 5 mg formulation in men.     

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.     

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.     

Development of an ultra-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry method for determination of xanthopurpurin in rat plasma and its application to pharmacokinetic study

A rapid and sensitive method was developed and validated for the quantitative determination of xanthopurpurin (XPP) in rat plasma using ultra-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. XPP inhibits IgE production and prevents peanut-induced anaphylaxis. The XPP and emodin (internal standard) were determined in negative ion mode with m/z 239.0350 → 211.0400 and 269.0455 → 241.0507, respectively. The separation process was achieved using an ACQUITY UPLC HSS T₃ column with acetonitrile and 0.1% formic acid in water (85:15). The linear range was 0.5–100 ng/mL, and the correlation coefficient (r²) was > 0.993. The inter-day and intra-day precision was within an acceptable range of 15%. The extraction recovery and matrix effect were 78.9–87.2% and 94.3–98.5%, respectively. Under different conditions, the XPP was stable in the range of 5.6–10.6%. This method was successfully applied to study the pharmacokinetics of XPP with an oral dose of 10.0 mg/kg and intravenous dose of 2.0 mg/kg in rats. The absolute oral bioavailability of XPP was 4.6%.     

An improved LC–MS/MS method for determination of docetaxel and its application to population pharmacokinetic study in Chinese cancer patients

Because of its unpredictable side effects and efficacy, the anticancer drug docetaxel (DTX) requires improved characterisation of its pharmacokinetic profiles through population pharmacokinetic studies. A sensitive and rugged LC–MS/MS method for the detection of DTX in human plasma was developed and optimised using paclitaxel as an internal standard (IS). The plasma samples underwent rapid extraction using hybrid solid-phase extraction-protein precipitation. The analyte and IS were separated with an isocratic system on a Zorbax Eclipse Plus C₁₈ column using water containing 0.05% acetic acid along with 20 μM of sodium acetate and methanol (30/70, v/v) as the mobile phase. Quantification was performed using a triple quadrupole mass spectrometer through multiple reaction monitoring in positive mode, using the m/z 830.3 → 548.8 and m/z 876.3 → 307.7 transitions for DTX and paclitaxel, respectively. The range of the calibration curve was 1–500 ng/mL for DTX, and the linear correlation coefficient was >0.99. The accuracies ranged from −4.6 to 4.2%, and the precision was no higher than 7.0% for the analytes. No significant matrix effect was observed. Both DTX and the IS showed considerable recovery. This method was finally applied to the establishment of a population pharmacokinetic model to optimise the clinical use of DTX.     

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%).     

Pharmacokinetic study of tanshinol and ligustrazine in rat plasma after intravenous administration of tanshinol and Danshen Chuanxiongqin Injection

To investigate the effect of ligustrazine on the pharmacokinetic profile of tanshinol after intravenous administration in rats, a sensitive liquid chromatography tandem mass spectrometry method was developed and validated for quantitative determination of tanshinol and ligustrazine in rat plasma. After prepared by protein precipitation, the analytes were separated on a Waters Acquity HSS T3 column (100 × 2.1 mm, 1.8μm) and eluted by 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 ml/min. The precursor–product ion transitions were m/z 197.0 → 135.0 for tanshinol, m/z 417.1 → 255.1 for liquiritin (internal standard) in negative ion mode and m/z 137.1 → 55.0 for ligustrazine in positive ion mode. To avoid the interference of tanshinol metabolite transformation, the stability of analytes in samples collected after administration was assessed. The validated method was successfully applied to a pharmacokinetic study after intravenous administration of single tanshinol and Danshen Chuanxiongqin Injection. After Danshen Chuanxiongqin injection administration, the values of elimination half-time, area under the concentration–time curve and C_o were 0.36 ± 0.13 h, 1.29 ± 0.37 μg/ml h and 10.51 ± 2.58 μg/ml for male rats, respectively. In the single tanshinol group, the corresponding values were 0.56 ± 0.24 h, 1.85 ± 0.44 μg/ml h and 14.11 ± 2.26 μg/ml for male rats—30–40% higher than those for the Danshen Chuanxiongqin Injection group. There was a significant different between male and female rats. This study provided information on the influence of ligustrazine on the pharmacokinetic characteristics of tanshinol after intravenous administration of Danshen Chuanxiongqin Injection in rats, which will be helpful for its clinical application.     

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.