Advanced UPLC-MS/MS Method for the Quantification of SIPI6398 in Rat Plasma and Its Pharmacokinetic Characterization.

SIPI6398 is a novel anti-schizophrenia agent with a new mechanism of action and demonstrates better target selectivity and safety compared to its competitors. However, few in vivo studies on the pharmacokinetics and bioavailability of SIPI6398 have been performed. A rapid and simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was developed for accurate quantification of SIPI6398 in rat plasma. A simple protein precipitation of acetonitrile-methanol (9 : 1, v/v) was used to treat plasma. Chromatography was performed on a UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm) at a flow rate of 0.4 ml/min. The mobile phase consisted of acetonitrile-water (with 0.1% formic acid) and gradient elution was used, and the elution time was 4 minutes. Quantitative analysis was performed using electrospray ionization (ESI) in positive ion detection mode with multiple reaction monitoring (MRM) mode. To evaluate the pharmacokinetics and bioavailability, SIPI6398 was administered to rats in two different ways: oral (4 mg/kg) and intravenous (2 mg/kg) administration. The calibration curve for the UPLC-MS/MS approach shows excellent linearity in the range of 1-2000 ng/mL with an r value above 0.99. The precision, accuracy, recovery, matrix effect, and stability results all meet the criteria established for biological analytical methods. The UPLC-MS/MS method was successfully applied it to pharmacokinetics study of SIPI6398. The bioavailability of SIPI6398 was calculated to be 13.2%. These studies have the potential to contribute towards a more comprehensive comprehension of the pharmacokinetics and bioavailability of SIPI6398.

Pharmacokinetics of Ziyuglycoside I and Ziyuglycoside II in Rat Plasma by UPLC-MS/MS.

Ziyuglycoside I and ziyuglycoside II are important active components of Sanguisorba officinalis L., which have excellent pharmacological effects, such as antioxidant and anticancer effects. However, the bioavailability of ziyuglycoside I and ziyuglycoside II has not been reported. This work aims to establish a UPLC-MS/MS method to study the pharmacokinetics of ziyuglycoside I and ziyuglycoside II in rats under different administration routes (intragastric and intravenous administration) and to calculate the bioavailability. The concentration of ziyuglycoside I and ziyuglycoside II in rat plasma in the range of 2-2000 ng/mL showed a good linear relationship (r > 0.99). The intra-day accuracies of ziyuglycoside I and ziyuglycoside II ranged from 87% to 110%, and the inter-day accuracies ranged from 97% to 109%. The intra-day precision was less than 15% and the inter-day precision was less than 14%. The matrix effects ranged from 88% to 113%. The recoveries were all above 84%. The developed UPLC-MS/MS method for the determination of ziyuglycoside I and ziyuglycoside II in rat plasma was applied to pharmacokinetics. The bioavailability of ziyuglycoside I and ziyuglycoside II was measured at 2.6% and 4.6%, respectively.

A newly developed UPLC-MS/MS method for simultaneous quantitative analysis of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-acetylharpagide derived from Ajuga plants in mice blood and the in vivo pharmacokinetics.

OBJECTIVE: To develop a sensitive and fast detection method via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to assess the concentration of ajuforrestin A, ajuforrestin B, ajugamacrin and 8-O-Acetylharpagide primarily derived from Ajuga plants in mice blood and their pharmacokinetics. METHODS: Single protein precipitation with high-proportioned acetonitrile is chosen for sample clean-up. The UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column with a mobile phase in gradient elution mode at the flow rate of 0.4 mL/min was used for sample separation. Acetonitrile was selected as the organic phase solution and water containing 0.1% formic acid was chosen as the aqueous solution. A tandem mass spectrometer containing an electrospray ionization (ESI) source in the positive ionization mode was used to detect four compounds via multiple reaction monitoring (MRM). RESULTS: The calibration curves (5-1000 ng/mL) of four compounds were linear with correlation coefficients > 0.997. The matrix effects, accuracy, precision, and recovery were all within permissible scope. CONCLUSIONS: In this approach, the corresponding pharmacokinetic parameters were successfully clarified in mouse for the first time, which provided a theoretical basis for the improvement of the standard of Ajuga plants and the safety of clinical medication. Furthermore, this method may provide the UPLC-MS/MS evidence for the differentiation of the main close relative varieties of genus Ajuga according to these plants contain different mixtures of the four marker compounds.

Herb-drug interactions of silybinin and cilofexor in beagle dogs based on pharmacokinetics by UPLC-MS/MS.

Objective: A remarkably sensitive, accurate, and efficient ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach was developed as a facile and expeditious method for measuring cilofexor concentration in beagle dogs, the herb-drug interactions between silybinin and cilofexor was explored based on pharmacokinetics. Methods: The plasma sample protein of the beagles were rapidly sedimented with acetonitrile, and cilofexor and tropifexor (internal standard, ISTD) were separated by gradient elution using a 0.1% formic acid aqueous solution and acetonitrile as the mobile phase. The concentrations were detected using positive ion multiple reaction monitoring (MRM) mode. Mass transfer pairs were m/z 587.91→267.91 for cilofexor and m/z 604.08→228.03 for ISTD, respectively. A two-period self-controlled experimental design was adopted for the HDIs experiment. In the first period (Group A), six beagle dogs were orally administered cilofexor at a dose of 1 mg/kg. In the second period (Group B), silybinin (3 mg/kg) was orally administered to the six beagle dogs twice a day for seven consecutive days, after which cilofexor was orally administered. The cilofexor concentration in beagle dogs was determined, and HDIs were evaluated based on their pharmacokinetics. Results: The accuracy and precision of cilofexor were both less than 15%, and the recoveries, matrix effects, and stability met the relevant requirements. The Cmax of cilofexor in group B was 49.62% higher than that in group A, whereas the AUC(0-t) and AUC(0-∞) of cilofexor in group B were 47.85% and 48.52% higher, respectively, than those in group A. Meanwhile, the t1/2 extended from 7.84 h to 9.45 h, CL and Vz decreased in Group B. Conclusion: A novel UPLC-MS/MS approach was successfully applied for the measurement of cilofexor in beagle dog plasma. Silybinin can alter the pharmacokinetics of cilofexor in beagle dogs, thereby increasing plasma exposure to cilofexor.

Development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma and application in their pharmacokinetics.

In this paper, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying the levels of crassicauline A, fuziline, karacoline, and songorine in rat plasma. After processing the rat plasma, the proteins in the plasma were separated by extracting the analytes with acetonitrile-methanol (9:1, v/v). The chromatographic column used was the UPLC HSS T3 column, and the mobile phase (methanol-water with 0.1% formic acid) under a gradient elution profile was used to separate the four compounds, with elution times for each analyte being less than 5 min. Electrospray ionization in positive-ion mode and operating in multiple reaction monitoring mode was used for quantitative analysis. Crassicauline A, fuziline, karacoline, and songorine were administered to 48 rats (n = 6 per group) orally (5 mg/kg) and intravenously (0.5 mg/kg). The standard curves demonstrated excellent linearity in the range of 1-2500 ng/mL, wherein all r values were greater than 0.99. The UPLC-MS/MS method for the determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma was successfully applied in determining their pharmacokinetics parameters, from which their oral bioavailabilities were calculated to be 18.7%, 4.3%, 6.0%, and 8.4%, respectively.

Determination of Tenacissoside G, Tenacissoside H, and Tenacissoside I in Rat Plasma by UPLC-MS/MS and Their Pharmacokinetics.

An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of tenacissoside G, tenacissoside H, and tenacissoside I in rat plasma. The rat plasma was treated with liquid-liquid extraction using ethyl acetate. The determination was performed on the UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm) with a mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) and gradient elution at a flow rate of 0.4 mL/min. Electrospray (ESI) positive ion mode detection and multireaction monitoring (MRM) quantitative analysis were performed. A total of 36 rats were given tenacissoside G, tenacissoside H, and tenacissoside I, respectively, orally (5 mg/kg) and intravenously (1 mg/kg), with 6 rats in each group, to evaluate the pharmacokinetic difference of tenacissoside G, tenacissoside H, and tenacissoside I in rats. The calibration curves showed good linearity in the range of 5-2000 ng/mL, where r was greater than 0.99. The results of precision, accuracy, recovery, matrix effect, and stability met the requirements of biological sample detection methods. The established UPLC-MS/MS method was successfully applied to pharmacokinetic studies of tenacissoside G, tenacissoside H, and tenacissoside I, and the bioavailability was 22.9%, 89.8%, and 9.4%, respectively.

A Methodology for Quantitation of Dictamnine and Fraxinellone and its Application to Study Pharmacokinetics and Bioavailability in Rats Via Oral and Intravenous Administration.

The pharmacological activities of dictamnine and fraxinellone have been well reported; however, only a few studies have focused on the pharmacokinetics and bioavailability of concomitant delivery of these drugs in vivo. To shed light on this neglected area, we developed a rapid and sensitive UPLC-MS/MS method that quantified the levels of dictamnine and fraxinellone simultaneously in rat plasma. This method was initiated by a one-step protein precipitation strategy to purify plasma samples collected from rats treated with either oral or intravenous administration of dictamnine and fraxinellone. The mobile phase contained acetonitrile and 0.1% formic acid at a steady flow rate of 0.6 mL/min. As a result, an excellent analyte peak resolution was achieved, and the entire process took only 3 min per sample. The results were indicative of the desired linearity (r2 ≥ 0.999), precision (RSD% was within 15%), accuracy (RE% was within 15%), recoveries (≥80.66 and 68.15% for dictamnine and fraxinellone, respectively) and matrix effects (≥94.66 and 91.37% for dictamnine and fraxinellone, respectively). Additionally, the detectable limits of these two compounds were both low even when they reached 5 ng/mL. Taken together, these findings contribute to a better understanding of the pharmacokinetics and bioavailability properties of concomitant delivery of dictamnine and fraxinellone.

Determination of Senegenin and Tenuifolin in Mouse Blood by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry and Their Pharmacokinetics.

An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of senegenin and tenuifolin in mouse blood was developed. The pharmacokinetics of senegenin and tenuifolin in mice after intravenous (5 mg/kg) and oral (60 mg/kg) administration were studied, and the absolute bioavailability was calculated. A CORTECS T3 column was used, with a column temperature set at 40°C. The mobile phase was acetonitrile and 0.1% formic acid. Gradient elution was adopted, using a flow rate of 0.4 mL/min and an elution time of 4 min. Quantitative analysis was performed using electrospray ionization (ESI) with multiple reaction monitoring (MRM) in negative ion mode. Institute of Cancer Research (ICR) mice were bled from the tail vein after intravenous or oral administration of senegenin and tenuifolin. A UPLC-MS/MS method was established to determine the blood concentrations of each drug in mice, and the noncompartmental model was used to fit the pharmacokinetic parameters. Senegenin and tenuifolin showed a good linear relationship (r > 0.995) within a concentration range of 5-400 ng/mL in mouse blood. The intraday precision was <12%, the interday precision was <14%, and the accuracy was 87-109%. The recovery was >88%, and the matrix effect was 87-94%. The oral bioavailability of senegenin and tenuifolin in mice was 8.7% and 4.0%, respectively. The established UPLC-MS/MS method is suitable for pharmacokinetic studies of senegenin and tenuifolin in mice.

Toxicokinetics of 11 Gelsemium Alkaloids in Rats by UPLC-MS/MS.

Gelsemium elegans (Gardn. & Champ.) Benth. is a plant belonging to the genus Gelsemium (family Gelsemiaceae), and its main components are alkaloids. It is a Chinese traditional medicinal plant and notoriously known as a highly toxic medicine. However, a method has not yet been found for the simultaneous detection of 11 Gelsemium alkaloids in rat plasma, and the toxicokinetics of 11 Gelsemium alkaloids after intravenous administration has not been reported. In this work, we have developed a sensitive and rapid method of ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) for the detection of 11 Gelsemium alkaloids in rat plasma. The toxicokinetic behavior was also investigated, so as to provide a reference of the scientific properties of Gelsemium elegans and improve the efficacy and safety of drugs. Sixty-six Sprague-Dawley rats were randomly divided into 11 groups, six rats in each group. Each group was intravenously given one alkaloid (0.1 mg/kg), respectively. A Waters UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 µm) was used for chromatographic separation. Methanol and water (containing 0.1% formic acid) were used for the mobile phase with gradient elution. Multiple reactions were monitored, and positive electrospray ionization was used for quantitative analysis. The precision was less than 16%, and the accuracy was between 86.9% and 113.2%. The extraction efficiency was better than 75.8%, and the matrix effects ranged from 88.5% to 107.8%. The calibration curves were in the range of 0.1-200 ng/mL, with a correlation coefficient (R 2) greater than 0.995. The UPLC-MS/MS method was successfully applied to the toxicokinetics of 11 Gelsemium alkaloids in rats after intravenous administration (0.1 mg/kg for each alkaloid). The results of the toxicokinetics provide a basis for the pharmacology and toxicology of Gelsemium alkaloids and scientific evidence for the clinical use of Gelsemium alkaloids.

The Effect of Grapefruit Juice on the Pharmacokinetics of Tadalafil in Rats.

We developed and validated a novel, sensitive, selective, and inexpensive high-performance liquid chromatography (HPLC) method for the determination of tadalafil in rats plasma and to investigate the effect of grapefruit juice on the pharmacokinetics of tadalafil in rats. The ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm) chromatography column can be used to separate tadalafil and carbamazepine (internal standard, IS). A mixture of acetonitrile-0.2% trifluoroacetic acid-water (48 : 10 : 42, V/V/V) was used as the mobile phase with a flow rate of 1.0 mL/min. The column temperature was set at 35.0°C. The detection wavelength was set at 286 nm. The tadalafil was extracted by ethyl acetate from plasma at the alkaline condition. 12 healthy male Sprague-Dawley (SD) rats were randomly divided into two groups, Group A (experimental group, received grapefruit juice 5 mL/kg for 7 days) and Group B (control group, received normal saline for 7 days). All the rats were given a single dose of tadalafil (5 mg/kg) after the last administration. The main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the conditions of this experiment, the plasma concentrations of tadalafil in the range of 10-2000 ng/ml had a good linear relationship. The intra- and interday precision for tadalafil in plasma were less than 15%, and the relative recovery rate was good at low, medium, and high QC levels. The C max of tadalafil in the control group and the experimental group was (725.89 ± 161.59) ng/mL and (1271.60 ± 179.31) ng/mL, t 1/2 was (9.28 ± 2.07) h and (11.70 ± 1.47) h, AUC (0-t) was (7399.61 ± 696.85) ng·h/mL and (9586.52 ± 2048.81) ng·h/mL, and AUC(0-∞) was (7995.50 ± 707.23) ng·h/mL and (10639.43 ± 2235.94) ng·h/mL, respectively. Results show that the C max of tadalafil in group A was 75.17% higher than that in group B, the Vz/F was also reduced, and the t 1/2 was increased by 2.42 h. The developed HPLC-DAD method for the determination of tadalafil in rats plasma was accurate, reproducible, specific, and it was found to be suitable for the pharmacokinetics of tadalafil and food-drug interactions. Grapefruit juice can inhibit the metabolism of tadalafil and increase the exposure of tadalafil in rats.

Quantification of Lappaconitine in Mouse Blood by UPLC-MS/MS and Its Application to a Pharmacokinetic Study.

Lappaconitine is extracted from Aconitum sinomontanum Nakai, which belongs to the Ranunculaceae. Lappaconitine is as a diterpenoid alkaloid used as a nonaddictive analgesic. To assure the rational use of the drug, ultrahigh-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was conducted to determine lappaconitine in mouse blood and its application to pharmacokinetics. In this study, khasianine was used as internet standard (IS). A UPLC BEH C18 column was used for chromatographic separation and the mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid). The flow rate of was 0.4 mL/min. Quantitative detection was performed in a multiple reaction monitoring (MRM) mode using an electrospray ionization source in positive mode. Twenty-four mice were randomly divided into four groups, three of which received 2, 4, and 8 mg/kg lappaconitine by intragastric administration, while the other group received 1 mg/kg lappaconitine by intravenous administration. After 0.0833, 0.5, 1, 1.5, 2, 3, 4, and 8 h, blood samples were collected and acetonitrile was used for protein precipitation. A linear calibration relationship (R2 = 0.9979) in the range of 0.1-500 ng/mL in mouse blood indicated good results. The lower limit of quantitation was 0.1 ng/mL and the limit of detection was 0.04 ng/mL. The intra-day and inter-day precision were below 13% and 14%, respectively. The accuracy was 90.1-107.2%, and the recovery exceeded 81.1%. The matrix effect ranged between 102.1 and 108.8%. The absolute bioavailability of lappaconitine was 2.0%. UPLC-MS/MS achieved high sensitivity, speed, and selectivity. Methodological verification indicated this method as suitable for determination of lappaconitine in mouse blood.

Effects of Fungicides on Rat's Neurosteroid Synthetic Enzymes.

Exposure to environmental endocrine disruptors may interfere with nervous system's activity. Fungicides such as tebuconazole, triadimefon, and vinclozolin have antifungal activities and are used to prevent fungal infections in agricultural plants. In the present study, we studied effects of tebuconazole, triadimefon, and vinclozolin on rat's neurosteroidogenic 5α-reductase 1 (5α-Red1), 3α-hydroxysteroid dehydrogenase (3α-HSD), and retinol dehydrogenase 2 (RDH2). Rat's 5α-Red1, 3α-HSD, and RDH2 were cloned and expressed in COS-1 cells, and effects of these fungicides on them were measured. Tebuconazole and triadimefon competitively inhibited 5α-Red1, with IC50 values of 8.670 ± 0.771 × 10-6 M and 17.390 ± 0.079 × 10-6 M, respectively, while vinclozolin did not inhibit the enzyme at 100 × 10-6 M. Triadimefon competitively inhibited 3α-HSD, with IC50 value of 26.493 ± 0.076 × 10-6 M. Tebuconazole and vinclozolin weakly inhibited 3α-HSD, with IC50 values about 100 × 10-6 M, while vinclozolin did not inhibit the enzyme even at 100 × 10-6 M. Tebuconazole and triadimefon weakly inhibited RDH2 with IC50 values over 100 × 10-6 M and vinclozolin did not inhibit this enzyme at 100 × 10-6 M. Docking study showed that tebuconazole, triadimefon, and vinclozolin bound to the steroid-binding pocket of 3α-HSD. In conclusion, triadimefon potently inhibited rat's neurosteroidogenic enzymes, 5α-Red1 and 3α-HSD.

[The study of effects of pirfenidone on the pulmonary fibrosis induced by paraquat in mice].

OBJECTIVE: To study the curative effects of pirfenidone (PF) on pulmonary fibrosis induced by paraquat (PQ) in mice and to provide the theoretical basis for clinical treatment. METHODS: Ninety adult healthy male ICR mice were randomly divided into six groups: control group, PQ group, 2 mg/kg Dexamethasone group, 25 mg/kg PF group, 50 mg/kg PF group and 100 mg/kg PF group, there were 15 mice in each group. The corresponding volume of normal saline was given to the each mouse in control group according to the weight, after 2 h 0.1% CMC was given to the each mouse of control group one time by intragastric administration, then the CMC was administrated at regular time until sacrifice. All mice for other 5 groups were exposed to 100 mg/kg PQ by intragastric administration. At 2 h after exposure to PQ, 0.02 ml/10 g dexamethasone and 25, 50, 100 mg/kg PF were given to mice for dexamethasone group and for 3 PF groups by intragastric administration each day for 49 days, respectively. The lung coefficient was calculated and pathological changes of lung tissue were observed by HE staining for each mouse. The hydroxyproline (HYP) level in lung tissue was measured for each mouse. The mRNA level of and the protein level of TGF-ß(1) in lung tissue for each mouse were determined, and the protein level of TGF-ß(1) in the bronchus-alveolus lavage fluid (BALF) of each mouse was detected. RESULTS: The survival rates on the 3rd day in PQ group, 3 PF groups and dexamethasone group were 53.33%, 46.67%, 73.33%, 86.67% and 80%, respectively. The survival rates on the 3rd day in dexamethasone group, 50 mg/kg and 100 mg/kg PF groups were significantly higher than those of PQ group and 25 mg/kg PF group (P < 0.05). The lung coefficients of 3 PF groups were significantly lower than that of the PQ group (P < 0.05). The lung tissue HYP levels of dexamethasone group and 3 PF groups were 50.95 ± 11.65, 44.52 ± 9.48, 43.27 ± 6.01 and 40.82 ± 5.90 mg/g respectively, which were significantly lower than that (74.27 ± 3.68) of PQ group (P < 0.01). The TGF-ß(1) protein levels of BALF in dexamethasone group, 50 and 100 mg/kg PF groups were 22.03 ± 7.27, 27.75 ± 5.84 and 21.31 ± 6.82 ng/ml respectively, which were significantly lower than that (52.52 ± 15.51) ng/ml of PQ group (P < 0.01) The expression level of TGF-ß(1) mRNA in 100 mg/kg PF group decreased significantly, as compared with PQ group (P < 0.01). CONCLUSION: PF could reduce the collagen deposition and pulmonary fibrosis induced by PQ in mice lungs.