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%).     

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.     

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.     

Protein precipitation method for determination of clobazam and N-desmethylclobazam in human plasma by LC–MS/MS

A protein precipitation method for the determination of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS) was established. CLB and N-CLB were extracted from human plasma samples by protein precipitation with methanol. Analyte separation was done using a Phenomenex Kinetex™ Biphenyl (50 × 2.1 mm, 1.7 μm) column using isocratic elution with a mobile phase of 5 mm ammonium formate with 0.01% ammonium hydroxide (40%) and methanol (60%) at a flow rate of 0.4 mL/min and an injection volume of 10 μL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 301.1 → 259.0, 306.0 → 263.9 for CLB and CLB-D5 and 287.0 → 245.0, 292.0 → 250.0 for N-CLB and N-CLB-D5 in positive electrospray ionization mode, respectively. The method was validated over a concentration range of 2.0–750 ng/mL for CLB and 0.7–200 ng/mL for N-CLB on SCIEX Triple Quad 4500 MS System. Total run time was 5 min. This method has been designed for bioequivalence study for formulations containing 20 mg of CLB.     

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%).     

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.     

A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

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.     

Rapid and simple method for determination of hexabromocyclododecanes and other LC–MS–MS-amenable brominated flame retardants in fish

In this study, a novel analytical approach for simultaneous determination of hexabromocyclododecane isomers (HBCDs), tetrabromobisphenol A (TBBPA), three brominated phenols, and four hydroxylated derivatives of polybrominated diphenyl ethers (OH-PBDEs) was developed and validated for muscle tissue of both lean and fatty fish. The rapid, simple, and high-throughput sample-preparation procedure was based on acetonitrile extraction then purification by dispersive solid-phase extraction (d-SPE) with a combination of C₁₈ and primary–secondary amine (PSA) sorbents. Ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS–MS) was used for identification and quantification of the analytes. Method recovery for both matrices ranged from 80 to 115 % with relative standard deviations (RSDs) <13 % for all analytes. Limits of quantification (LOQs) were in the range 0.1–1 μg kg^?1 wet weight. The validated method was used for analysis of brominated compounds in 32 fish and five bivalve samples collected from different European markets within the monitoring survey organized in the framework of the CONffIDENCE project. Of the 12 targeted analytes, only α-HBCD, 2,4-dibromophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP) were quantified in the samples. α-HBCD was found in six fish samples (herring and mackerel) in the range of 0.8–2.5 μg kg^?1 wet weight. 2,4-DBP and 2,4,6-TBP were found in three blue mussel samples in the range of 19.6–43.5 and 2.3–7.5 μg kg^?1 wet weight, respectively.     

A validated LC–MS/MS method for simultaneous quantification of simvastatin and simvastatin acid in beagle plasma: Application to an absolute bioavailability study

A highly sensitive LC–MS/MS method for simultaneous detection of both simvastatin (SV) and simvastatin acid (SVA) in beagle plasma was developed and successfully applied to an absolute bioavailability study. Lovastatin (LV) was used as internal standard (IS). The analysis was performed using electrospray ionization and selective reaction monitoring in positive mode at m/z 441.0 → 325.0 for SV, 459.0 → 343.0 for SVA and 427.0 → 325.0 for the IS, respectively. The assay procedure involved a simple liquid–liquid extraction of SV, SVA and LV from beagle plasma into methyl tert-butyl ether. Separation of SV, SVA and the IS was achieved on a Shim-pack VP-ODS column (150 × 2.0 mm, 5 μm) with a binary gradient solvent system of 0.1% formic acid in water and methanol (15:85, v/v) as the mobile phase. The method was validated over the range of 0.25–500 ng/ml for SV (r² ≥ 0.9923) and 0.24–481.23 ng/ml for SVA (r² ≥ 0.9987). The results of method validation for accuracy, precision, extraction recovery, matrix effect and stability were within the acceptance criteria. The values of absolute bioavailability of SV and SVA in beagles were 2.97 and 25.40%, respectively. It is the first study developed for the measurement of absolute bioavailability of SV and SVA acid in beagles.     

New UPLC–MS/MS method for simultaneous determination of irbesartan and hydrochlorthiazide in human plasma

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) is a preeminent analytical tool for rapid biomedical analysis with the objective of reducing analysis time and maintaining good efficiency. In this study a simple, rapid, sensitive and specific ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for quantification of the angiotensin II receptor antagonist, irbesartan and hydrochlorthiazide in human plasma. After a simple protein precipitation using methanol and acetonitrile, irbesartan, hydrochlorthiazide and internal standard (IS) telmisartan were separated on Acquity UPLC BEH? C18 column (50 × 2.1 mm, i.d. 1.7 μm, Waters, USA) using a mobile phase consisting of acetonitrile:10 mM ammonium acetate:formic acid (85:15:0.1 % v/v/v) pumped at a flow rate of 0.3 mL/min and detected by tandem mass spectrometry with negative ion mode. The ion transitions recorded in multiple reaction monitoring mode were m/z 427.2 → 193.08 for irbesartan, m/z 295.93 → 268.90 for hydrochlorthiazide and m/z 513.2 → 287.14 for IS. The assay exhibited a linear dynamic range of 30–500 ng/mL for irbesartan and 1–500 ng/mL in human plasma with good correlation coefficient of (0.996) and (0.997) and with a limit of quantitation of 30  and 1 ng/mL for irbesartan and hydrochlorthiazide, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 10.13 % for irbesartan and 11.14 % for hydrochlorthiazide. The proposed UPLC–MS/MS method is simple, rapid and highly sensitive, and hence it could be reliable for pharmacokinetic and toxicokinetic study in both animals and humans.     

Validated HPLC–MS–MS method for determination of azithromycin in human plasma

A validated, highly sensitive, and selective HPLC method with MS–MS detection has been developed for quantitative determination of azithromycin (AZI) in human Na₂EDTA plasma. Roxithromycin (ROX) was used as internal standard. Human plasma containing AZI and internal standard was ultrafiltered through Centrifree Micropartition devices and the concentration of AZI was determined by isocratic HPLC–MS–MS. Multiple reaction monitoring mode (MRM) was used for MS–MS detection. The calibration plot was linear in the concentration range 2.55–551.43 ng mL⁻¹. Inter-day and Intra-day precision and accuracy of the proposed method were characterized by R.S.D and percentage deviation, respectively; both were less than 8%. Limit of quantification was 2.55 ng mL⁻¹. The proposed method was used to determine the pharmacokinetic profile of AZI (250-mg tablets).     

Simultaneous and sensitive LC–MS/MS determination of tetrahydrocannabinol and metabolites in human plasma

Cannabis is not only a widely used illicit drug but also a substance which can be used in pharmacological therapy because of its analgesic, antiemetic, and antispasmodic properties. A very rapid and sensitive method for determination of ?⁹-tetrahydrocannabinol (THC), the principal active component of cannabis, and two of its phase I metabolites in plasma has been developed and validated. After solid-phase extraction of plasma (0.2 mL), the clean extracts were analyzed by tandem mass spectrometry after a 5-min liquid chromatographic separation. The linear calibration ranges were from 0.05 to 30 ng?mL^?1 for THC and 11-nor-?⁹-carboxy-tetrahydrocannabinol (THC-COOH) and from 0.2 to 30 ng?mL^?1 for ?⁹-(11-OH)-tetrahydrocannabinol (11-OH-THC). Imprecision and inaccuracy were always below 7 and 12 % (expressed as relative standard deviation and relative error), respectively. The method has been successfully applied to determination of the three analytes in plasma obtained from healthy volunteers after oral administration of 20 mg dronabinol.     

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.     

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.     

A validated method for the determination of traces of UV filters in fish using LC–MS/MS

An analytical method for the determination of UV filter substances in fish tissue has been developed and validated using benzophenone-3, 3-(4-methylbenzylidene)-camphor, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propenoate and 2-ethylhexyl 3-(methoxyphenyl)-2-propenoate as target analytes. The fish fillets were homogenised and extracted by Soxhlet extraction. The extracts were run through a clean-up process including gel permeation chromatography followed by solid-phase extraction. Quantification of the compounds was performed using liquid chromatography with tandem mass spectrometric detection. Blank fish as well as spiked blank fish were analysed to validate the analytical method. The analytical method developed has the multiple advantages of enabling separation, simultaneous identification and quantification of each of the four selected compounds in a single run. Contamination of blank samples and abnormally high concentrations in spiked samples were avoided by taking extensive precautions during the fish preparation procedure. The method was validated in accordance with internationally accepted criteria, such as specificity, accuracy and repeatability. The combination of LC with tandem mass spectrometry ensures a high level of specificity. The accuracy of the method was reported as the mean recovery rate for the analytes in the sample matrix. Mean recoveries were in the range 86–108%. The precision is expressed as the relative standard deviation, and in all but one of the cases was 20% or below. The accuracy of the method allows residue analyses to be performed on biological matrices at ng/g levels. The determined limit of quantification for each analyte was 8 ng/g fish. For all spiking levels ≥8 ng/g, relative standard deviations were ≤ 20%.     

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.