Development and validation of novel LC–MS/MS method for determination of Lusutrombopag in rat plasma and its application to pharmacokinetic studies

A novel, simple and sensitive method for the determination of Lusutrombopag in rat plasma using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated. The determination was performed on an API4000 triple quadrupole mass spectrometry in the multiple reaction monitoring mode using the respective [M+H]⁺ ions m/z 593.1 → 272.3 for Lusutrombopag. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 2.0 ng/mL in rat plasma. Good linearity was obtained over the range of 2.0–150.0 ng/mL and the correlation coefficient was found to be 0.9998. The intra and inter-day precisions were found to be 3.8–6.9% and 6.8–10.5%, respectively. The intra and inter-day accuracy derived from QC samples was found to be 2.5–4.9% and 5.5–7.2%, respectively. The analyte was stable under various conditions (at room temperature, during freeze-thaw, in the autosampler and under deep-freeze conditions). The F-test and t-test at 95% confidence level were subjected on data for statistical analysis. The developed method was successfully applied to the pharmacokinetic study in rats.     

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.     

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.     

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.     

Development,validation and application of a UHPLC–MS/MS method for quantification of the adiponectin-derived active peptide ADP355 in rat plasma

A UHPLC–MS/MS method for the quantification of ADP355, an adiponectin-derived active peptide, was developed and validated. The extraction method employed simple protein precipitation using methanol and chromatographic separation was achieved on anAccucore™ RP-MS C₁₈ column (100 × 2.1 mm, 2.6 μm, 80 Å), using 0.1% formic acid in both water and acetonitrile with gradient elution at the flow rate of 400 μl/min within 4.0 min. Detections were performed under positive ion mode with multiple reaction monitoring ion transitions m/z 1109.2 → 309.8 and 871.4 → 310.1 for ADP355 and Jt003 respectively at unit resolution. The linearity range of the calibration curve was 2–1,000 ng/ml with a lower limit detection of 0.5 ng/ml. The selectivity, linearity, precision, accuracy, recovery, matrix effect and stability were validated, and all items met the requirement of US Food and Drug Administration guidance. This method was successfully applied to an intravenous pharmacokinetic study of ADP355 in rats and the in-vitro stability in rat serum, plasma and whole blood was also assessed.     

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%).     

A rapid and sensitive UPLC–MS/MS method for the determination of zanubrutinib in beagle plasma and its application in pharmacokinetics

A reliable and sensitive ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed for the determination of zanubrutinib in the plasma of beagle dogs. The column used was an Acquity BEH C₁₈ column (2.1 mm × 50 mm, 1.7 μm), maintained at 40°C with an injection volume of 2 μl. The gradient elution program was as follows: 0–1 min, 10–10% A; 1–1.1 min, 10–90% A; 1.1–2.1 min, 90–90% A; 2.1–2.2 min, 90–10% A; 2.2–3.0 min, 10–10% A. Mobile phase A was 0.1% formic acid, B was acetonitrile, and the total analysis time was 3 min. The mass spectrometry was performed in positive ion mode, and the scanning mode was multi-reaction monitoring mode with electrospray ionization as the ion source; m/z 472.2 → 455.01 for zanubrutinib and m/z 441.03 → 137.99 for ibrutinib (internal standard). The plasma samples were processed by protein precipitation. The standard curve showed good linearity (r² = 0.999 8) in the range of 1.0–1,000 ng/ml (zanubrutinib) with a low limit of quantification of 1 ng/ml. Also, the intra-day and inter-day precision (RSD) was <5.88% and the accuracy (RE) ranged from −1.56 to 1.08%; the recoveries of zanubrutinib in beagle plasma ranged from 90.12 to 93.53% (RSD 1.67–6.42%) and the ME values of zanubrutinib were 98.70–101.06% (RSD 5.37–8.49%, n = 6). All values meet US Food and Drug Administration requirements. A rapid, highly selective and sensitive method for the determination of zanubrutinib concentration in plasma by UPLC–MS/MS was successfully developed. This method is suitable for pharmacokinetic studies in beagle dogs by following oral administration of zanubrutinib.     

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.     

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.     

Development and validation of an LC–MS/MS method for the quantification of artificial sweeteners in human matrices

Artificial sweeteners are widely used as substitutes for sugar. The sweeteners are generally considered safe, however their whereabouts during pregnancy and lactation and the effect on child development are poorly explored. There is a need for new tools to measure these substances during pregnancy and lactation. Here, we describe the development and validation of a sensitive liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of acesulfame, cyclamate, saccharin and sucralose in human plasma, umbilical cord blood, amniotic fluid and breast milk. The samples were prepared by protein precipitation and separated on a Luna Omega Polar C₁₈ column (2.1 × 50 mm, 1.6 μm). Electrospray ionization in negative mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1 to 500 ng/ml (10–500 ng/ml for sucralose). Interassay precisions were all ≤15% and the accuracies were within ±15%. Stability, linearity, dilution integrity, carryover and recovery were also examined and satisfied the validation criteria. Finally, this analytical method was successfully applied on spiked samples of plasma, umbilical cord blood, amniotic fluid and breast milk, proving its suitability for use in clinical studies on artificial sweeteners, including during pregnancy and lactation.     

Validation and application of a novel UHPLC–MS/MS method for the measurement of furanodienone in rat plasma

A sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established to analyze furanodienone in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for furanodienone and patchouli alcohol (internal standard, IS) were m/z 231.1 → 83.2 and m/z 205.1 → 95.1, respectively. Great linearity of furanodienone in plasma samples was found in the corresponding concentration range (r > 0.995). Intra- and inter-day precisions (RSD, %) were <11.3% in plasma, and the accuracy (RE, %) was within ±10.7%. This method was used to the furanodienone study on rat pharmacokinetics after a single oral dose of 10 mg/kg of furanodiene. The results indicated that the maximum observed plasma concentration was 52.4 ± 19.1 ng/ml at 1.2 ± 0.7 h with an elimination half-life of 2.2 ± 0.7 h. The obtained data indicated that furanodienone could be moderately distributed and eliminated.     

A validated LC–MS/MS method for the quantification of capivasertib in dog plasma: Application to its pharmacokinetics study

In this study, a simple and reliable liquid chromatography tandem mass spectrometric method was first developed for the determination of capivasertib in dog plasma with ipatasertib as internal standard. The plasma samples were deproteinated using acetonitrile. An Acquity BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) maintained at 40°C was used for chromatographical separation, with water containing 0.1% formic acid and acetonitrile as mobile phase. Multiple reaction monitoring transitions were m/z 429.2 > 135.1 for capivasertib and m/z 458.2 > 387.2 for ipatasertib, respectively. Excellent linearity was achieved in the concentration range of 1–1,000 ng/ml with a correlation coefficient of >0.9981. The lower limit of quantification was 1 ng/ml. The extraction recovery of capivasertib from dog plasma was >85.81% and no significant matrix effect was found. The intra- and inter-day precision was <9.58% and the accuracy ranged from −10.60% to 12.50%. The validated method was further applied to the pharmacokinetic study of capivasertib in dog plasma after single oral (5 mg/kg) and intravenous (1 mg/kg) administrations. The results revealed that capivasertib was rapidly absorbed into plasma with good bioavailability (47.04%) and low clearance.     

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–QToF–MS method for quantification of ethambutol,isoniazid, pyrazinamide and rifampicin in human plasma and its application

In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC–QToF–MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C₁₈ column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2–5 μg ml⁻¹ for ethambutol, 0.2–7.5 μg ml⁻¹ for isoniazid, 1–40 μg ml⁻¹ for pyrazinamide and 0.25–2 μg ml⁻¹ for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC–QToF–MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.     

Development and validation of an LC–MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside,and its application in a pharmacokinetic study of normal and diabetic nephropathy mice

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5′-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC–MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an Acquity HSS T₃ column. The run time was 3.2 min. The linearity ranges of the analytes were 0.5–1,000 ng/ml for RDV and 5–10,000 ng/ml for both RM-442 and RN. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442 and RSD < 7.2% for RN) and accuracy (91.0–106.3% for RDV, 92.5–98.6% for RM-442 and 87.5–98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in the blood of normal and diabetic nephropathy DBA/2 J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration–time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).     

Development of an LC–MS/MS method for quantifying two main metabolites of abivertinib in human plasma

Abivertinib represents a highly selective irreversible epidermal growth factor receptor tyrosine kinase inhibitor. Two major metabolites of abivertinib, M7 and MII-6, were detected in human plasma, which are recommended to be monitored for safety reasons in clinical trial. A high-throughput quantification method utilizing liquid chromatography–tandem mass spectrometry was designed and verified to quantify abivertinib's primary metabolites in human plasma. Solid-phase extraction was used to process the plasma, and then the analytes underwent a gradient elution separation in an Aquity UPLC BEH C₁₈ column (1.7 μm, 2.1 × 50 mm) with mobile phase A (10 mm ammonium acetate containing 0.1% formic acid) and mobile phase B (methanol–acetonitrile, 2:8, v/v, with 0.1% formic acid). Ion transitions of M7 (m/z 490.2 → 405.1) and MII-6 (m/z 476.2 → 391.1) were monitored under multiple reaction monitoring mode and electrospray ionization in positive ion mode. This simultaneous determination method was found to have acceptable precision, accuracy and linearity in the 0.5–500 ng/mL range for M7 and the 0.5–500 ng/mL range for MII-6, accompanied by a mild matrix effect but high recovery. Further stability assessments indicated that both analytes remained stable throughout the entire experimental process from harvesting whole blood to plasma extraction and analysis.     

Development and validation of a LC–MS/MS method for the simultaneous determination of aflatoxins, dyes and pesticides in spices

Based on several alerts from European countries over the last years concerning spices, we have been encouraged to establish an accurate method for the determination of dyes, aflatoxins and pesticides in various types of spices using reversed-phase (RP) liquid chromatography–tandem mass spectrometry interfaced with electrospray (LC–ESI–MS/MS). A simple sample treatment procedure entailing the use of an extraction step with acetonitrile without further cleanup has been developed. A C18 column with an aqueous ammonium formate/methanol mixture as the mobile phase was used, and gradient elution was performed. Mass spectral acquisition was done in positive ion mode by applying multiple reaction monitoring of at least two fragmentation transitions per compound to provide a high degree of selectivity. The method was in-house validated in terms of linearity, sensitivity, repeatability, recovery and selectivity on six kinds of spices. Satisfactory results in the majority of the cases were obtained for all analytes and matrices, with practical limits of quantitation acceptable for routine monitoring purposes. Extraction recoveries for most of the compounds ranged from 60% to 140% at spiking levels of 0.05 and 0.5 mg kg⁻¹. The applicability of the method for the simultaneous determination of dyes, aflatoxins and pesticides in several types of spices was demonstrated, and the method successfully applied to a limited number of products from the local market.