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.     

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1–500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C_max; 616 ± 73 ng/mL) was achieved at 0.5 h (T_max) and area under curve (AUC_0–24) was 1841 ± 308 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials. Copyright © 2011 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

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.     

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 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.     

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 rapid and sensitive method for quantification of ibrutinib in rat plasma by UPLC–ESI–MS/MS: validation and application to pharmacokinetic studies of a novel ibrutinib nanocrystalline

Ibrutinib has an excellent effect in the treatment of mantle cell lymphoma so it has attracted much attention. A novel ibrutinib nanocrystalline was exploited in our study to improve the bioavailability. A fast and reliable UPLC–MS/MS method was established for the accurate quantification of ibrutinib in rat plasma. The chromatographic separation was achieved by an Agilent zorbax SB-C₁₈ rapid solution HD column (2.1 × 50 mm, 1.8 μm). The mobile phase consisted of deionized water (containing 10 mm ammonium acetate and 0.1% formic acid) and pure acetonitrile. Isocratic elution (water–acetonitrile 10:90, v/v) was adopted and the flow rate was 0.4 mL/min. Column temperature was set to 40°C. Vilazodone was used as the internal standard in this analytical method. Multiple reaction monitoring mode with positive electrospray ionization was selected to detect ibrutinib and vilazodone. Acetonitrile was used to precipitate protein to extract plasma samples. There was no endogenous interference for both ibrutinib and vilazodone and the linear range of this method was 1–2000 ng/mL. The recoveries were 98.4, 97.4 and 102.7% at low, medium and high concentrations. Accordingly, the matrix effect was 96.6, 111.1 and 99.6%. The pharmacokinetic difference between ibrutinib crude and a novel ibrutinib nanocrystalline in rats was investigated by this validated method successfully. The peak concentration and area under the concentration–time curve showed significant differences in gender and the bioavailability was improved after oral administration of ibrutinib nanocrystalline.     

Method development and validation of LC–MS/MS-based assay for the simultaneous quantitation of trastuzumab and pertuzumab in cynomolgus monkey serum and its application in pharmacokinetic study

We present a simple and robust LC–MS/MS assay for the simultaneous quantitation of an antibody cocktail of trastuzumab and pertuzumab in monkey serum. The LC–MS/MS method saved costs, decreased the analysis time, and reduced quantitative times relative to the traditional ligand-binding assays. The serum samples were digested with trypsin at 50°C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated using a C18 column (2.1 × 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC–MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 μg/mL. The intra- and inter-assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single-dose intravenous drip administration to cynomolgus monkeys.     

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.     

Method development and validation for rat serum fingerprinting with CE–MS: application to ventilator-induced-lung-injury study

In the search for a noninvasive and reliable rapid screening method to detect biomarkers, a metabolomics fingerprinting approach was developed and applied to rat serum samples using capillary electrophoresis coupled to an electrospray ionization-time of flight-mass spectrometer (CE–TOF-MS). An ultrafiltration method was used for sample pretreatment. To evaluate performance the method was validated with carnitine, choline, ornithine, alanine, acetylcarnitine, betaine, and citrulline, covering the entire electropherogram of pool of rat serum. The linearity for all metabolites was >0.99, with good recovery and precision. Approximately 34 compounds were also confirmed in the pool of rat serum. The method was successfully applied to real serum samples from rats with ventilator-induced lung injury, an experimental rat model for acute lung injury (ALI), giving a total of 1163 molecular features. By use of univariate and multivariate statistics 18 significant compounds were found, of which five were confirmed. The involvement of arginase and nitric oxide synthase has been proved for other lung diseases, meaning the increase of asymmetric dimethyl arginine (ADMA) and ornithine and the decrease of arginine found were in accordance with published literature. Ultimately this fingerprinting approach offers the possibility of identifying biomarkers that could be regularly screened for as part of routine disease control. In this way it might be possible to prevent the development of ALI in patients in critical care units.

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%).     

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.     

LC–MS/MS method for the determination of erianin in rat plasma: Application to a pharmacokinetic study

Erianin is one of the bibenzyl ingredients isolated from Dctidrobium chrysotoxum Lindl. In recent years, erianin has attracted attention owing to its antitumor activity. In this study, an LC–MS/MS method was established to measure erianin in rat plasma. Gigantol was used as the internal standard. A Waters Acquity UPLC BEH C₁₈ column was employed for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 ml/min. Selective reaction monitoring mode was used for quantitative analysis of erianin in positive electrospray ionization. In the concentration range of 0.1–1200 ng/ml, erianin in rat plasma was linear with correlation coefficient >0.999. The lowest limit of quantification was 0.1 ng/ml. The intra- and inter-day RSDs were <9.69%, while the RE was in the range of −8.59–11.24%. The mean recovery was >85.37%. Erianin was stable in rat plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive and reliable, and has been successfully applied to pharmacokinetic study of erianin in rat plasma. Erianin was rapidly eliminated from rat plasma with a short half-life (〜1.5 h) and low oral bioavailability (8.7%).     

Simultaneous determination of FL118 and W34 in rat Blood by LC–MS/MS: Application to pharmacokinetic studies

W34 is a prodrug of FL118, and it can be converted to FL118 via a hydrolysis reaction. In this report, a highly sensitive LC–MS/MS method using a C₁₈ column was validated and used for the simultaneous determination of W34 and FL118 in rat blood. A stepwise gradient elution with 0.1% formic acid in water and acetonitrile was employed. The assays were linear over a concentration range of 0.50–50.0 ng/ml for both W34 and FL118. The accuracy of the validation method ranged from 89.74 to 98.94% for W34 and from 88.61 to 94.60% for FL118. The precision was within 7.15% for W34 and 9.63% for FL118. Extraction recoveries of W34 were 94.56–100.49 and 87.67–106.32% for FL118. No significant matrix effects for both W34 and FL118 were observed in blood. The assay has been successfully applied to biological samples obtained from a stability and pharmacokinetic study of W34 and FL118.     

UPLC–MS/MS simultaneous determination of seven active ingredients of Yaobitong capsule in rat plasma and its integrated pharmacokinetic application

A reliable and sensitive UPLC–MS/MS method was first established and validated for the simultaneous determination of seven active ingredients of Yaobitong capsule in rat plasma: ginsenoside Rg1, ginsenoside Rb1, osthole, tetrahydropalmatine, paeoniflorin, albiflorin, and ferulic acid. And this method was further applied for the integrated pharmacokinetic study of Yaobitong capsule in rats after oral administration. Plasma samples (100 μL) were precipitated with 300 μL of methanol using carbamazepine as internal standard. Chromatographic separation was achieved using an Aquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 μm), with the mobile phase consisting of 0.1% formic acid and acetonitrile. The method was validated using a good linear relationship (r ≥ 0.991), and the lower limit of quantification of the analytes ranged from 0.5 to 40 ng/mL. In the integrated pharmacokinetic study, the weight coefficient was calculated by the ratio of AUC_0–∞ of each component to the total AUC_0–∞ of the seven active ingredients. The integrated pharmacokinetic parameters C_max, T_max, and t_1/2 were 81.54 ± 9.62 ng/mL, 1.00 ± 0.21 h, and 3.26 ± 1.14 h, respectively. The integration of pharmacokinetic parameters showed a shorter t_1/2 because of fully considering the contribution of the characteristics of each active ingredient to the overall pharmacokinetics.     

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.