Comparative pharmacokinetic study of six lignans in normal and diabetic rats after oral administration of Saururus chinensis extract by LC–MS/MS

Saururus chinensis (SC) possesses significant anti-diabetic activity and lignans were its major bioactive compounds. In this study, a rapid and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was established for simultaneous quantification of six lignans, namely (-)-(7R,8R)-machilin D ( 1 ), verrucesin ( 2 ), rel-(7S,8S,7′R,8′R)-3,3′,4,4′,5,5′-hexamethoxy-7.O.7′,8.8′-lignan ( 3 ), manassantin A ( 4 ), manassantin B ( 5 ), and saucerneol F ( 6 ) in rat’s plasma. It was validated with acceptable linearity (r ≥ 0.9922), accuracy (80.42–95.17%), precision (RSD ≤ 12.08%), and extraction recovery (80.36–93.45%). The method was successfully applied to the comparative pharmacokinetic study of the six lignans in normal and diabetic rats after oral administration of SC extract. Results showed that the areas under the plasma concentration-time curve (AUC_{0 → t} and AUC_{0 → ∞}) of (-)-(7R,8R)-machilin D, rel-(7S,8S,7′R,8′R)-3,3′,4,4′,5,5′-hexamethoxy-7.O.7′,8.8′-lignan, manassantin B, and saucerneol F in diabetic rats were significantly increased, and the plasma clearance (CL) of (-)-(7R,8R)-machilin D in diabetic rats was significantly decreased. However, the AUC_{0 → t} and AUC_{0 → ∞} of verrucesin were significantly decreased, and its CL was significantly increased in diabetic rats compared with those in normal rats. These results indicated that there were remarkable differences in the pharmacokinetic parameters between the normal and diabetic rats. The pharmacokinetic studies might be beneficial for the clinical use of SC as hypoglycemic agent.     

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.     

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.     

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC–MS/MS

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075–20.0 ng/mL with the R² value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the C_max increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5–1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.     

Comparative pharmacokinetics of the main active components in normal and ulcerative colitis rats after oral administration of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair and its single herb extracts by LC–MS/MS

Zingiberis Rhizoma and Ginseng Radix et Rhizoma are usually used together for the treatment of ulcerative colitis in clinical practices. However, their compatibility mechanism remains unclear. In this study, a rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed for simultaneous quantification of ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, and 6-gingerol in rat plasma after oral administration of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair and its single herb extracts. The calibration curves exhibited good linearity, with correlation coefficients of more than 0.993. The precision deviations of intra- and interday analysis were within 10.66%, and accuracy error ranged from −12.74 to 11.56%. The average recoveries of analytes were higher than 76.60% and the matrix effects were minimal. Thus, the validated method was successfully applied to a pharmacokinetic study of four ingredients in normal and ulcerative colitis rat plasma. The results indicated that the pharmacokinetic parameters of four analytes in normal and model groups showed significant differences. The larger exposure (the mean AUC_0-t of ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, and 6-gingerol were increased by 50.93, 141.90, 3.68, and 37.25%, respectively) and slower elimination (the CLz/F of ginsenoside Re, ginsenoside Rg1, and 6-gingerol were decreased by 52.94, 83.64, and 32.18%, respectively) were observed in ulcerative colitis rats. Furthermore, compared with single herbs, the analytes in rat plasma after oral administration of combined extracts presented relatively high systemic exposure levels with AUC_0-t > 2000 h·ng/mL and C_max > 200 ng/mL. Collectively, the differences of pharmacokinetic characteristics revealed the synergistic effect of Zingiberis Rhizoma–Ginseng Radix et Rhizoma herb pair, which provided a valuable and reliable basis for its clinical application in the treatment of ulcerative colitis.     

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.     

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.     

Simultaneous quantification of vortioxetine,fluoxetine and their metabolites in rat plasma by UPLC–MS/MS

In this study, a specific and quick ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was fully developed and validated for simultaneous measurement of the rat plasma levels of vortioxetine (VOR), Lu AA34443 (the major metabolite of VOR), fluoxetine and its metabolite norfluoxetine with diazepam as the internal standard (IS). After a simple protein precipitation with acetonitrile for sample preparation, the separation of the analytes were performed on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) column, with acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. The detection was achieved on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via an electrospray ionization source. Good linearity was observed in the calibration curve for each analyte. The data of precision, accuracy, matrix effect, recovery and stability all conformed to the bioanalytical method validation of acceptance criteria of US Food and Drug Administration recommendations. The newly developed UPLC–MS/MS method allowed simultaneous quantification of VOR, fluoxetine and their metabolites for the first time and was successfully applied to a pharmacokinetic study in rats.     

Effects of voriconazole and fluconazole on the pharmacokinetics of almonertinib in rats by UPLC–MS/MS

Almonertinib was included in the first-line treatment of non-small cell lung cancer with EGFR T790M mutations by the Chinese Society of Clinical Oncology in 2021. Considering that immunocompromised lung cancer patients are prone to opportunistic fungal infections, and most triazole antifungal drugs are moderate or strong inhibitors of CYP3A4, this study was conducted to develop and validate an accurate and rapid ultra-performance liquid chromatography tandem mass spectrometry method for quantifying almonertinib in plasma and for investigating the pharmacokinetic changes of almonertinib caused by voriconazole and fluconazole in rats. After liquid–liquid extraction with tert-butyl methyl ether, an XSelect HSS T3 column (2.1 × 100 mm, 2.5 μm, Waters) was used for the chromatographic separation of almonertinib and sorafenib-D₃ (internal standard). The analytes were detected using an AB Sciex Triple Quad 5,500 mass spectrometer in the positive ionization mode. The method exhibited great linearity (0.5–200 ng/ml, r > 0.997) and stability under the established experimental conditions. All validation experiments were in accordance with the guidelines, and the results were all within the acceptable limits. This method was successfully applied to the researches of pharmacokinetics and drug interactions for almonertinib in rats. Voriconazole and fluconazole significantly altered the pharmacokinetic profiles of almonertinib and increased the systemic exposure of almonertinib in rats to different degrees, but further human trials should be conducted to validate the results.     

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.     

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%).     

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.     

Determination of atorvastatin and metabolites in human plasma with solid-phase extraction followed by LC–tandem MS

The aim of the present study was to develop a chromatographic method for the analysis of atorvastatin, o- and p-hydroxyatorvastatin (acid and lactone forms) in human plasma after administration of atorvastatin at the lowest registered dose (10 mg) in clinical studies. Sample preparation was performed by solid-phase extraction and was followed by separation of the analytes on an HPLC system with a linear gradient and a mobile phase consisting of acetonitrile, water and formic acid. Detection was achieved by tandem mass spectrometry operated in the electrospray positive ion mode. Validation of the method for the compounds for which reference compounds were available (acid forms of atorvastatin, o- and p-hydroxyatorvastatin) showed linearity within the concentration range (0.2–30 ng/ml for atorvastatin acid and p-hydroxyatorvastatin acid, and 0.5–30 ng/ml for o-hydroxyatorvastatin acid) (r²0.99, n=5 for all analytes). Accuracy and precision (evaluated at 0.5, 3 and 30 ng/ml for atorvastatin, p-hydroxyatorvastatin and 1, 3 and 30 ng/ml for o-hydroxyatorvastatin) were both satisfactory. The detection limit was 0.06 ng/ml for atorvastatin and p-hydroxyatorvastatin, and 0.15 ng/ml for o-hydroxyatorvastatin. The method has been successfully applied in a clinical study where atorvastatin, o- and p-hydroxyatorvastatin (both acid and lactone forms) could be detected in a 24-h sampling interval after administration of the lowest registered dose of atorvastatin (10 mg) for one week.     

Pharmacokinetic studies of a novel tubulin inhibitor SK1326 in rat plasma by UPLC–MS/MS

A sensitive method for quantitation of SK1326 in rat plasma has been established using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI/MS/MS). SK1326 and the internal standard (tramadol) in plasma sample were extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with a mobile phase of 0.5% formic acid–acetonitrile (35:65, v/v). The reconstituted samples were injected into a C₁₈ reversed-phase column. Using MS/MS in the multiple reaction monitoring mode, SK1326 and tramadol were detected without severe interference from the rat plasma matrix. SK1326 produced a protonated precursor ion ([M + H]⁺) at m/z 432.3 and a corresponding product ion at m/z 114.4. The internal standard produced a protonated precursor ion ([M + H]⁺) at m/z 264.4 and a corresponding product ion at m/z 58.1. Detection of SK1326 in rat plasma by the UPLC–ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK1326 in rat plasma. The pharmacokinetic parameters of SK1326 were evaluated after intravenous (at a dose of 10 mg/kg) and oral (at a dose of 20 mg/kg) administration of SK1326 in rats. After oral administration (20 mg/kg) of SK1326, the F (fraction absorbed) value was ~77.1%.     

Packed in-tube SPME–LC–MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC–MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2⁴⁻¹ and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME–LC–MS/MS method took another 12 min, with a sample throughput of 3 samples h⁻¹. The developed method presented adequate precision, accuracy and linearity; R² values ranged from 0.990 to 0.997, in the range of 10–1000 ng ml⁻¹. The lower limits of quantification varied from 10 to 25 ng ml⁻¹. Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.     

An analytical strategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC–MS/MS analysis of triptorelin and endogenous testosterone in rat plasma

Triptorelin, a gonadotropin-releasing hormone agonist, has been used in the treatment of hormone-responsive prostate cancer by inducing testosterone suppression. Research on the relationship between the time courses of triptorelin and testosterone is very important, but accurate quantification of triptorelin and testosterone simultaneously in biological specimens is a challenging analytical problem. In the present study, a rapid, sensitive, and selective method for simultaneous determination of triptorelin and testosterone in rat plasma by solid-phase extraction and liquid chromatography–tandem mass spectrometry was developed using a ZORBAX RRHD Eclipse Plus C8 column (2.1?×?50 mm, 1.8 μm) with a 0.05 % propionic acid/methanol gradient. In view of the polarity difference between the two analytes, two internal standards, i.e., leuprolide and testosterone-¹³C₃, were used for individual quantitation of triptorelin and testosterone. Endogenous testosterone was determined by reference to a calibration curve prepared using testosterone-D₃ as a surrogate analyte. The method exhibits excellent linearity over three orders of magnitude for each analyte. The lower limit of quantification was 0.01 ng/mL for triptorelin and 0.05 ng/mL for testosterone, with consumption of 100 μL of plasma. The method was successfully applied to characterize the pharmacokinetics and pharmacodynamics of slow-release 28-day form triptorelin acetate biodegradable microspheres in rats after intramuscular injections of three consecutive doses of 0.6 mg/kg per 28 days. The results revealed that the pharmacokinetic profile of triptorelin produced an initial flare-up in testosterone levels, rapid castration within 5 days after injection, and long-term castration until the next dose.

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.