Exploring the Effect of Compound Glycyrrhizin and Silybinin on the Metabolism of Pexidartinib in Rats Based on CYP3A4 and CYP2C9.

Pexidartinib offered a new therapeutic option for adult patients with symptomatic tenosynovial giant cell tumor (TGCT) who were refractory to surgical treatment and had severe morbidity or functional limitations. Meanwhile, the metabolism of pexidartinib was mainly mediated through the oxidation of cytochrome P450 (CYP) 3A and glucuronidation by uridine glucuronosyltransferase (UGT) 1A4 and attention shall be paid to CYP450-based drug-drug interactions during therapeutic dosing. This study aimed to examine the changes in the pharmacokinetics of pexidartinib by silymarin and compound glycyrrhizin on pexidartinib in vivo in rats by high-performance liquid chromatography (HPLC)-UV approach and to detect its expression in CYP3A4 and CYP2C9 using the western blot. The findings of chromatography experiments revealed that silybinin as well as compound glycyrrhizin increased the exposure of pexidartinib in rats and had a significant inhibitory effect on the metabolism of pexidartinib. The results of immunoblotting assays suggested that silybinin as well as compound glycyrrhizin inhibited the protein expression of CYP3A4 and CYP2C9 in rats. Therefore, the combination of pexidartinib with silybinin and compound glycyrrhizin should be monitored to avoid clinical adverse effects.

Pharmacokinetics of Herb-Drug Interactions of Plumbagin and Tazemetostat in Rats by UPLC-MS/MS.

Objective: A sensitive and rapid UPLC-MS/MS method for determination of tazemetostat in rat plasma was developed, and the pharmacokinetics of herb-drug interactions (HDIs) of plumbagin (PLB) and tazemetostat was investigated. Methods: After the rat plasma samples were precipitated by acetonitrile, tazemetostat and verubecestat (ISTD) were detected. Gradient elution was performed with 0.1% formic acid and acetonitrile as mobile phases. The multi-reaction monitoring was used with ESI+ source, and the ion pairs for tazemetostat and ISTD were m/z 573.12→135.99 and m/z 410.10→124.00, respectively. 12 SD rats were randomly divided into the control group and the experimental group, 6 rats in each group. The rats in the experimental group were given PLB 100 mg/kg by gavage once a day for 7 consecutive days. The rats in the control group were given the same amount of 0.1% sodium carboxymethyl cellulose solution by gavage once a day for 7 consecutive days. At the seventh day, tazemetostat (80 mg/kg) was given and the blood was collected at different time points. The main parameters of pharmacokinetics were calculated and the herb-drug interactions (HDIs) were evaluated. Results: In the calibrated range of 1-1000 ng/mL, tazemetostat had a good linearity. The extraction recovery was more than 84%, and the RSD of intra-batch and inter-batch precision were both less than 15%. The Cmax of tazemetostat in the experimental group was 32.48% higher than that in the control group, and the AUC(0-t) and AUC(0-∞) of tazemetostat in the experimental group were 46.24% and 46.67% higher than that in the control group, respectively, and the t1/2 was prolonged from 10.56 h to 11.73 h. Conclusion: A simple, rapid and sensitive UPLC-MS/MS method for the determination of tazemetostat in rat plasma was established. PLB can inhibit the metabolism of tazemetostat and increase the plasma exposure of tazemetostat in rats.

Determination of Tropifexor in Beagle Dog Plasma by UPLC-MS/MS and Its Application in Pharmacokinetics.

The primary objective of this study was to develop and validate an efficient and accurate ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach as a means to detect tropifexor plasma concentrations in beagle dogs and to study its pharmacokinetic profile in beagle dogs. The chromatographic separation of tropifexor and oprozomib (internal standard, ISTD) on the column, with the addition of acetonitrile for rapid precipitation and protein extraction, was achieved with 0.1% formic acid aqueous solution-acetonitrile for the mobile phase. A Xevo TQ-S triple quadrupole tandem mass spectrometer, under the selective reaction monitoring (SRM) mode, for the determination of the concentrations in the positive ion mode. The mass transfer pairs of tropifexor and oprozomib (ISTD) were m/z 604.08 ⟶ 228.03 and m/z 533.18 ⟶ 199.01, respectively. The profile displayed well linearity with calibration curves for tropifexor and oprozomib (ISTD) ranging from 1.0 to 200 ng/mL. In parallel, the lower limit of quantification (LLOQ) value for tropifexor could be measured with the aid of this novel technique at 1.0 ng/mL. In addition, the scope of intraday and interday for analyte accuracy was between -4.86% and 1.16%, with a precision of <7.31%. The recoveries of the analytes were >88.13% and were free of significant matrix effects. The stability met the requirements for the quantification of plasma samples under various conditions. Finally, the pharmacokinetic profile of tropifexor in beagle dog plasma following oral administration of 0.33 mg/kg tropifexor was determined by using the method facilitated in this work.

Effect of Chaihu Shugan Pills on the Pharmacokinetics of Duloxetine and its Metabolite 4-Hydroxyduloxetine in Beagle Dogs: A Herb-Drug Interaction Study.

The effect of Chaihu Shugan pills (CHSG) on the pharmacokinetics of duloxetine and its metabolite 4-hydroxyduloxetine in beagle dogs was investigated by establishing an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to simultaneously measure the concentrations of duloxetine and 4-hydroxyduloxetine in beagle dog plasma. Duloxetine and 4-hydroxyduloxetine were separated on the UPLC-C18 column after acetonitrile precipitation and detected by mass spectrometry with multireaction detection mode (MRM). Six adult healthy beagle dogs (weighing 7-9 kg, male and female) were randomly selected and examined for a single-dose administration of duloxetine hydrochloride (2 mg/kg, control group) and oral administration of CHSG (0.3 g/kg) twice daily for 15 consecutive days followed by a single-dose administration of duloxetine hydrochloride (2 mg/kg, experimental group) using the self-control method. All plasma samples were treated in the same way, and then the concentrations of duloxetine and 4-hydroxyduloxetine were determined using the established UPLC-MS/MS method. The obtained data were subjected to DAS 2.0 software to calculate the pharmacokinetic parameters, and SPSS 20.0 software was used to compare the differences between the two groups. Duloxetine and 4-hydroxyduloxetine had a good linear relationship in the ranges of 1-1000 ng/ml and 0.1-100 ng/ml, and the lower limits of quantification (LLOQ) were 1 ng/mL and 0.1 ng/ml, respectively. The precision, accuracy, extraction recovery, matrix effect, and stability meet the requirements of the guiding principles. After combination with CHSG, C max and AUC0⟶t of duloxetine decreased by 49.33% and 13.08%, respectively, and t 1/2 was shortened to 10.17 h; C max and AUC0⟶t of 4-hydroxyduloxetine decreased by 71.47% and 48.78%, respectively, and t 1/2 was shortened to 7.97 h. The UPLC-MS/MS method was fully developed to simultaneously measure the plasma concentration of duloxetine and its metabolite 4-hydroxyduloxetine in beagle dogs. CHSG could slow down the absorption of duloxetine, induce the metabolism of duloxetine and 4-hydroxyduloxetine in beagle dogs, and reduce plasma exposure.

The Effect of Posaconazole and Isavuconazole on the Pharmacokinetics of Erdafitinib in Beagle Dogs by UPLC-MS/MS.

Objective: A robust, quick, and reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of erdafitinib in beagle dog plasma was developed and validated to evaluate the changes of posaconazole and isavuconazole on the pharmacokinetics of erdafitinib in beagle dogs, respectively. Methods: This experiment adopted a three-period self-control experimental design. In the first period (group A), erdafitinib was orally administered to six beagle dogs at a dose of 4 mg/kg. In the second period (group B), the same six beagle dogs were orally given posaconazole at a dose of 7 mg/kg, and after 30 min, erdafitinib was orally given. In the third period (group C), isavuconazole at a dose of 7 mg/kg was given orally, and then, erdafitinib was orally given. At the different time points after erdafitinib was given in the three periods, the blood samples were collected. The concentration of erdafitinib was detected by the developed UPLC-MS/MS method. DAS 2.0 was used to calculate the pharmacokinetic parameters of erdafitinib. Results: Erdafitinib had a good linear relationship in the range of 1-500 ng/ml, and the lower limit of quantification was 1 ng/ml. The precision, accuracy, extraction recovery, matrix effect, and stability meet the requirements of the guiding principles. After erdafitinib was combined with posaconazole, the Cmax and AUC0→t of erdafitinib increased by 27.19% and 47.62%, respectively, and the t1/2 was prolonged to 6.33 h. After erdafitinib was combined with isavuconazole, the Cmax and AUC0→t of erdafitinib increased by 23.13% and 54.46%, respectively, and the t1/2 was prolonged to 6.31 h. Conclusion: A robust and reliable UPLC-MS/MS method was fully optimized and developed to detect the plasma concentration of erdafitinib in beagle dogs. Posaconazole and isaconazole could inhibit the metabolism of erdafitinib in beagle dogs and increase the plasma exposure of erdafitinib.

Effect of Sijunzi Pills on Pharmacokinetics of Omeprazole in Beagle Dogs by HPLC-UV: A Herb-Drug Interaction Study.

A sensitive high-performance liquid chromatography (HPLC-UV) method for determination of omeprazole in beagle dog plasma was developed and to investigate the effect of Sijunzi pills (SJZPs) on the pharmacokinetics of omeprazole in beagle dogs. The beagle dog plasma was extracted with ethyl acetate and n-hexane under alkaline conditions. Omeprazole and internal standard (IS, fluconazole) were separated on an XDB-C18 column, and acetonitrile and 0.1% trifluoroacetic acid were used as the mobile phase. Omeprazole and IS were detected by using a diode array detector. This experiment adopts the experimental design of double-cycle self-control. In the first cycle (group A), six beagle dogs were given omeprazole 0.67 mg/kg orally in a single dose. In the second period (group B), the same six beagle dogs were orally given SJZPs 0.2 g/kg twice a day for 7 consecutive days, and then, omeprazole was orally given. At the different time points after omeprazole was given in the two periods, the blood samples were collected. The concentration of omeprazole was detected by the developed HPLC method. DAS 2.0 was used to calculate the pharmacokinetic parameters of omeprazole. Under the current experimental conditions, this UPLC method showed good linearity in the detection of omeprazole. Interday and intraday precision did not exceed 10%, and the range of accuracy values were from -1.43% to 2.76%. The results of extraction recovery and stability met the requirements of FDA approval guidelines of bioanalytical method validation. The C max of omeprazole in group B was 61.55% higher than that in group A, and the AUC(0-t) and AUC(0-∞) of omeprazole in group B were 63.96% and 63.65% higher those that in group A, respectively. At the same time, the clearance (CL) and apparent volume of distribution (Vd) decreased in group B. In this study, an HPLC method for the determination of plasma omeprazole concentration was established. SJZPs could inhibit the metabolism of omeprazole and increase the concentration of omeprazole in beagle dogs. It is suggested that when SJZPs are combined with omeprazole, attention should be paid to the herb-drug interactions and possible adverse reactions.

The Pharmacokinetic Effect of Itraconazole and Voriconazole on Ripretinib in Beagle Dogs by UPLC-MS/MS Technique.

BACKGROUND: A new UPLC-MS/MS technique for the determination of ripretinib in beagle dog plasma was developed, and the pharmacokinetic effects of voriconazole and itraconazole on ripretinib in beagle dogs were studied. METHODS: After extraction with ethyl acetate under alkaline conditions, ripretinib was detected using avapritinib as the internal standard (IS). The mobile phases were 0.1% formic acid-acetonitrile. The scanning method was multi-reaction monitoring using ESI+ source, and the ion pairs for ripretinib and IS were m/z 509.93→416.85 and 499.1→482.09, respectively. This animal experiment adopted a three period self-control experimental design. In the first period, ripretinib was orally administered to six beagle dogs at a dose of 5 mg/kg. In the second period, the same six beagle dogs were orally given itraconazole at a dose of 7 mg/kg, after 30 min, ripretinib was orally given. In the third period, voriconazole at a dose of 7 mg/kg was given orally, and then ripretinib was orally given. At different time points, the blood samples were collected. The concentration of ripretinib was detected, and the pharmacokinetic parameters of ripretinib were calculated. RESULTS: Ripretinib had a good linear relationship in the range of 1-1000 ng/mL. The precision, accuracy, recovery, matrix effect and stability met the requirements of the guiding principles. After erdafitinib combined with itraconazole, the Cmax and AUC0→t of ripretinib increased by 38.35% and 36.36%, respectively, and the t1/2 was prolonged to 7.53 h. After ripretinib combined with voriconazole, the Cmax and AUC0→t of ripretinib increased by 37.44% and 25.52%, respectively, and the t1/2 was prolonged to 7.33 h. CONCLUSION: A new and reliable UPLC-MS/MS technique was fully optimized and developed to detect the concentration of ripretinib in beagle dog plasma. Itraconazole and voriconazole could inhibit the metabolism of ripretinib in beagle dogs and increase the plasma exposure of ripretinib.

Development of an UPLC-MS/MS Method for the Quantitative Analysis of Upadacitinib in Beagle Dog Plasma and Pharmacokinetics Study.

BACKGROUND: Upadacitinib, a novel selective Janus kinase 1 (JAK1) inhibitor, has been recently approved by the US FDA for the treatment of adult patients with moderately to severely active rheumatoid arthritis (RA). An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the quantitative analysis of upadacitinib in beagle dog plasma was developed and validated. METHODS: Upadacitinib and fedratinib (internal standard, IS) were extracted with ethyl acetate under alkaline condition and then separated and detected. The chromatographic column was Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm), the mobile phase was acetonitrile and 0.1% formic acid in water with gradient elution procedure, and the flow rate was 0.40 mL/min. Under the positive ion mode, upadacitinib and IS were monitored by multiple reaction monitoring (MRM) as the following mass transition pairs: m/z 447.00 → 361.94 for upadacitinib and m/z 529.82 → 141.01 for IS. RESULTS: In the concentration range of 1-500 ng/mL, upadacitinib had good linearity, and the lower limit of quantification (LLOQ) was 1 ng/mL. The RSD of the intra- and inter-day precision was less than 10.03%, and the RE of accuracy was -3.79% to 2.58%. The extraction recovery of upadacitinib was more than 80%, the matrix effect was around 100%, and upadacitinib was found to be stable. CONCLUSION: The novel optimized UPLC-MS/MS assay was an effective tool for the determination of upadacitinib and had been successfully applied to the pharmacokinetic study of upadacitinib in beagle dogs, and this method would also be used to study DDIs.

Simultaneous Determination of Celecoxib, Dezocine and Dexmedetomidine in Beagle Plasma Using UPLC-MS/MS Method and the Application in Pharmacokinetics.

BACKGROUND: An efficient, fast and sensitive ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for simultaneous determination of celecoxib (CEL), dezocine (DEZ) and dexmedetomidine (DEX) in beagle plasma were established. METHODS: The beagle dogs plasmawas precipitated by acetonitrile. The column was Acquity UPLC BEH C18 column and the mobile phase was acetonitrile-formic acid with gradient mode, and the flow rate was set at 0.4 mL/min. Under the positive ion mode, CEL, DEZ, DEX and Midazolam (internal standard, IS) were monitored by multiple reaction monitoring (MRM) as the following mass transition pairs: m/z 381.10→282.10 for CEL, m/z 246.20→147.00 for DEZ, m/z 201.10→94.90 for DEX, and m/z 326.10→291.10 for IS. RESULTS: This UPLC-MS/MS method had good linearity for CEL, DEZ and DEX. The RSDs of inter-day and intra-day precision were the values of 0.31-7.66% and 0.11-9.63%, respectively; the RE values were from -6.05% to 10.98%. The extraction recovery was more than 79%, and the matrix effect was around 100%. The RSDs of stability were less than 8.96%. All of them met the acceptance standard of biological analysis method recommended by FDA. CONCLUSION: This UPLC-MS/MS method is an effective tool for the simultaneous determination of CEL, DEX and DEX, and has been successfully applied to the study of pharmacokinetics in beagle dogs.

Establishment and Verification of UPLC-MS/MS Technique for Pharmacokinetic Drug-Drug Interactions of Selinexor with Posaconazole in Rats.

BACKGROUND: A method for the determination of selinexor by UPLC-MS/MS was established to study the effect of posaconazole on the pharmacokinetics of selinexor in rats. METHODS: The experiment rats were divided into group A (0.5% CMC-Na) and group B (posaconazole, 20 mg/kg), 6 rats in each group. 30 minutes after administration of 0.5% CMC-Na or posaconazole, all the rats were given selinexor (8 mg/kg), and plasma samples were collected. The plasma samples underwent acetonitrile protein precipitation, and were separated by UPLC on an Acquity UPLC BEH C18 column with gradient elution. Acetonitrile and 0.1% formic acid were used as the mobile phases. The analyte detection was used a Xevo TQ-S triple quadrupole tandem mass spectrometer and multiple reaction monitoring (MRM) for analyte monitoring. We use acetonitrile for protein precipitation. RESULTS: Selinexor had good linearity (1.0-1000 ng/mL, r2 =0.996 2), and the accuracy and precision, recovery rate and matrix effects(ME) were also met the FDA approval guidelines. Compared with group A, the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group B increased by 60.33%, 48.28% and 48.27%, and Tmax increased by 53.92%, CLz/F reduced by 32.08%. CONCLUSION: This bioanalysis method had been applied to the study of drug interactions in rats. It was found that posaconazole significantly increased the concentration of selinexor in rats. Therefore, when selinexor and posaconazole are combined, we should pay attention to the possible drug-drug interactions to reduce adverse reactions.

UPLC-MS/MS for the Herb-Drug Interactions of Xiao-Ai-Ping Injection on Enasidenib in Rats Based on Pharmacokinetics.

OBJECTIVE: To develop and validate a sensitive and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of enasidenib in rat plasma and to investigate the effect of Xiao-ai-ping injection (XAPI) on the pharmacokinetics of enasidenib in rats. METHODS: The rat plasma was precipitated with acetonitrile, enasidenib and internal standard (IS) were separated on an Acquity UPLC BEH C18 column, and acetonitrile and 0.1% formic acid were used as the mobile phase in gradient mode. Enasidenib and IS were monitored and detected by multiple reaction monitoring (MRM) using tandem mass spectrometry in positive ion mode. 12 Sprague-Dawley (SD) rats were randomly divided into control group (group A) and experimental group (group B), 6 rats in each group. Group B was intramuscularly injected with XAPI (0.3 mL/kg) every morning, 7 days in a row. Group A was intramuscularly injected with normal saline, 7 days in a row. On the seventh day, enasidenib (10 mg/kg) was given to both groups 30 min after injection of normal saline (group A) or XAPI (group B), and the blood was collected at different time points such as 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, and 48 h. The concentration of enasidenib was detected by UPLC-MS/MS, and the main parameters of pharmacokinetic of enasidenib were calculated using the DAS 2.0 software. RESULTS: Under the current experimental conditions, this UPLC method showed good linearity in the detection of enasidenib. Interday and intraday precision did not exceed 10%, the range of accuracy values were from -1.43% to 2.76%. The results of matrix effect, extraction recovery, and stability met the requirements of FDA approval guidelines of bioanalytical method validation. The C max of enasidenib in the group A and the group B was (458.87 ± 136.02) ng/mL and (661.47 ± 107.32) ng/mL, t 1/2 was (7.74 ± 0.91) h and (8.64 ± 0.42) h, AUC(0 - t) was (4067.24 ± 1214.36) ng·h/mL and (5645.40 ± 1046.30) ng·h/mL, AUC(0 - ∞) was (4125.79 ± 1235.91) ng·h/mL and (5759.61 ± 1078.59) ng·h/mL, respectively. The C max of enasidenib in group B was 44.15% higher than that in group A, and the AUC(0 - t) and AUC(0 - ∞) of enasidenib in group B were 38.80% and 39.60% higher than that in group A, respectively, and the t 1/2 was prolonged from 7.74 h to 8.64 h. CONCLUSION: An UPLC-MS/MS method for the determination of enasidenib in rat plasma was established. XAPI can inhibit the metabolism of enasidenib and increase the concentration of enasidenib in rats. It is suggested that when XAPI was combined with enasidenib, the herb-drug interaction and adverse reactions should be paid attention to, and the dosage should be adjusted if necessary.

UPLC-MS/MS Measurement of the Effect of Isavuconazole, Itraconazole and Fluconazole on the Pharmacokinetics of Selinexor in Rats.

OBJECTIVE: An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of selinexor was established to investigate the effects of isavuconazole, itraconazole and fluconazole on the pharmacokinetics of selinexor in rats, respectively. METHODS: Twenty-four healthy male rats were randomly divided into four groups: group A, normal saline; group B, isavuconazole (20 mg/kg); group C, itraconazole (20 mg/kg); and group D, fluconazole (20 mg/kg). After 30 min of oral administration of normal saline, isavuconazole, itraconazole, and fluconazole, all the rats were given selinexor (8 mg/kg). The plasma concentration of selinexor was estimated by UPLC-MS/MS, and the pharmacokinetic parameters of selinexor were calculated by Drug and Statistics (DAS) 2.0 software. RESULTS: Under these experimental conditions, the method showed good linearity and stability. Intraday and interday accuracy and sample recovery were acceptable. Compared with group A, the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group B increased by 59.05%, 31.69%, and 31.45%; the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group C increased by 56.14%, 25.34%, and 25.08%; the Cmax, AUC(0-t) and AUC(0-∞) of selinexor in group D increased by 43.44%, 29.16%, and 31.96%, respectively. The Tmax of the experimental groups were extended, and CLz/F was also significantly reduced. CONCLUSION: These results indicated that isavuconazole, itraconazole, and fluconazole have significant inhibitory effects on selinexor pharmacokinetics and increased selinexor plasma exposure in rats. Therefore, when these drugs were used in combination, clinicians should pay attention to the changes in treatment effects and the occurrence of adverse reactions caused by the drug-drug interactions.

Simultaneous Determination of Five Components of Chaihu-Shugan-San in Beagle Plasma by HPLC-MS/MS and Its Application to a Pharmacokinetic Study after a Single Dose of Chaihu-Shugan-San.

Chaihu-shugan-san (CHSGS) has been widely used in China to treat depression and gastrointestinal diseases for thousands of years, but little is known about its pharmacokinetic properties. The purpose of our study is to develop a reliable and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to detect five components in beagle plasma and study their pharmacokinetic after oral administration of CHSGS in beagles. An Agilent C18 column (2.1 × 150 mm, 3.5 µm) was used to separate the analytes, and the column temperature was maintained at 40°C. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1% formic acid, aqueous) as mobile phases. The elution procedure was 60% B-10% B (0-3 min) and 10% B-60% B (3.1-4 min). The flow rate was 0.3 mL/min, and the total measurement time was 4 min. Within the determined range, the standard calibration curves of the five analytes had a satisfactory linear relationship (r 2 ≥ 0.9923). The recovery rate (n = 6) of the five analytes was between 85.42% and 90.85%, and the matrix effects (n = 6) were between 94.52% and 103.91%. These results show that the validated method could be successfully applied to study the pharmacokinetic in beagles after a single dose of CHSGS.

Effects of Dexmedetomidine on the Pharmacokinetics of Dezocine, Midazolam and Its Metabolite 1-Hydroxymidazolam in Beagles by UPLC-MS/MS.

OBJECTIVE: We developed and validated a sensitive and reliable UPLC-MS/MS method for simultaneous determination of dezocine (DEZ), midazolam (MDZ) and its metabolite 1-hydroxymidazolam (1-OH-MDZ) in beagle plasma and investigated the effect of dexmedetomidine (DEX) on the pharmacokinetics of DEZ, MDZ and 1-OH-MDZ in beagles. MATERIALS AND METHODS: Diazepam was used as the internal standard (IS); the three analytes and IS were extracted by acetonitrile precipitation and separated on an Acquity UPLC BEH C18 column using acetonitrile-0.1% formic acid as mobile phase in gradient mode. In positive ion mode, the three analytes and IS were monitored by multiple reaction monitoring (MRM). Six beagles were designed as a double cycle self-control experiment with 0.15 mg/kg in the first cycle (Group A). After a 1-week washout period, the same six beagles were slowly injected intravenously with 2 µg/kg DEX in the second cycle (Group B), with continuous injection for 7 days. On the seventh day, 0.5 hr after intravenous injection of 2 µg/kg DEX, the six beagles were intramuscularly given with DEZ 0.33 mg/kg and MDZ 0.15 mg/kg. RESULTS: Under the conditions of this experiment, this method exhibited a good linearity for each analyte. The accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery, matrix effect and stability have also met the requirements. CONCLUSION: The developed UPLC-MS/MS method for simultaneous determination of DEZ, MDZ and 1-OH-MDZ in beagles plasma was accurate, reproducible, specific, and suitable. DEX could inhibit the metabolism of DEZ and MDZ and increase the exposure of DEZ and MDZ in beagles. Therefore, the change of therapeutic effect and the occurrence of adverse reactions caused by drug-drug interaction should be paid attention to when the drugs were used in combination.

Effects of Dexmedetomidine on the Pharmacokinetics of Parecoxib and Its Metabolite Valdecoxib in Beagles by UPLC-MS/MS.

A sensitive and reliable ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagles. The effects of dexmedetomidine on the pharmacokinetics of parecoxib and valdecoxib in beagles were studied. The plasma was precipitated by acetonitrile, and the two analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm); the mobile phase was acetonitrile and 0.1% formic acid with gradient mode, and the flow rate was 0.4 mL/min. In the negative ion mode, the two analytes and internal standard (IS) were monitored by multiple reaction monitoring (MRM), and the mass transition pairs were as follows: m/z 369.1 → 119.1 for parecoxib, m/z 313.0 → 118.0 for valdecoxib, and m/z 380.0 → 316.0 for celecoxib (IS). Six beagles were designed as a double cycle self-control experiment. In the first cycle, after intramuscular injection of parecoxib 1.33 mg/kg, 1.0 mL blood samples were collected at different times (group A). In the second cycle, the same six beagles were intravenously injected with 2 µg/kg dexmedetomidine for 7 days after one week of washing period. On day 7, after intravenous injection of 2 µg/kg dexmedetomidine for 0.5 hours, 6 beagle dogs were intramuscularly injected with 1.33 mg/kg parecoxib, and blood samples were collected at different time points (group A). The concentration of parecoxib and valdecoxib was detected by UPLC-MS/MS, and the main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the experimental conditions, the method has a good linear relationship for both analytes. The interday and intraday precision was less than 8.07%; the accuracy values were from -1.20% to 2.76%. C max of parecoxib in group A and group B was 2148.59 ± 406.13 ng/mL and 2100.49 ± 356.94 ng/mL, t 1/2 was 0.85 ± 0.36 h and 0.85 ± 0.36 h, and AUC(0-t) was 2429.96 ± 323.22 ng·h/mL and 2506.38 ± 544.83 ng·h/mL, respectively. C max of valdecoxib in group A and group B was 2059.15 ± 281.86 ng/mL and 2837.39 ± 276.78 ng/mL, t 1/2 was 2.44 ± 1.55 h and 2.91 ± 1.27 h, and AUC(0-t) was 4971.61 ± 696.56 ng·h/mL and 6770.65 ± 453.25 ng·h/mL, respectively. There was no significant change in the pharmacokinetics of parecoxib in groups A and B. C max and AUC(0 - ∞) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t 1/2 was increased from 2.44 h to 2.91 h. V z /F and CL z /F were correspondingly reduced, respectively. The developed UPLC-MS/MS method for simultaneous determination of parecoxib and valdecoxib in beagle plasma was specific, accurate, rapid, and suitable for the pharmacokinetics and drug-drug interactions of parecoxib and valdecoxib. Dexmedetomidine can inhibit the metabolism of valdecoxib in beagles and increase the exposure of valdecoxib, but it does not affect the pharmacokinetics of parecoxib.

Metabolomics analysis of the antidepressant prescription Danzhi Xiaoyao Powder in a rat model of Chronic Unpredictable Mild Stress (CUMS).

ETHNOPHARMACOLOGICAL RELEVANCE: Danzhi Xiaoyao Powder (DZXY) is a classical prescription, that has been extensively used in traditional Chinese medicine (TMC) to treat depression for many years. However, the mechanism of DZXY is still unclear. AIM OF THE STUDY: The aim was to investigate the mechanism of the antidepressant effect of DZXY on a rat model of chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: Forty male SD (Sprague-Dawley) rats with similar open field test (OFT) results were randomLy divided into a control group (n = 10) and an experimental group (n = 30). A depression model was established in the experimental group using the CUMS method. After the CUMS model was established successfully, the rats were randomLy divided into a depression model group and a DZXY group. The DZXY group was fed DZXY, while the depression model group and control group were given an equal amount of 0.5% sodium carboxymethyl cellulose suspension. Intragastric administration was performed once daily for 14 consecutive days. Animal weight, the sugar preference test, the open field test and the forced swimming test were used to evaluate the modeling effect and the antidepressant effect of DZXY. After the experiment, the plasma of rats was collected and the changes in plasma metabolites were analyzed by UPLC/Q-TOF-MS. The UPLC/Q-TOF-MS spectra data were evaluated by pattern recognition analysis to determine the changes in endogenous metabolites in the rat plasma samples. RESULTS: The results of the behavioral investigation showed that the rat model of depression was successfully replicated and that DZXY had an antidepressant effect. Using the UPLC-MS/MS metabolomics platform, partial least squares (PLS) and orthogonal partial least squares (OPLS), metabolic profile models (R2 and Q2 ≥ 0.5) of rat plasma were successfully constructed. The model could distinguish among the control group, the depression model group and the DZXY group. Finally, 38 differential metabolites were identified in the plasma. According to KEGG (http://www.kegg.jp) pathway analysis, amino acid metabolism, lipid metabolism, purine metabolism, the prolactin signaling pathway and bile secretion were enriched and represented the main metabolic pathways influenced in the plasma. CONCLUSIONS: This study successfully established a CUMS depression model. A total of 38 differential metabolites associated with depression were identified in the plasma of rats, 24 of which were modulated by DZXY. These results suggest that DZXY can improve excitability and play an antidepressant role by regulating phenylalanine metabolism, arachidonic acid metabolism, porphyrin metabolism, D-arginine and D-ornithine metabolism, steroid hormone biosynthesis, unsaturated fatty acid biosynthesis and steroid biosynthesis.

Development and Validation of UPLC-MS/MS Method for Determination of Enasidenib in Rat Plasma and Its Pharmacokinetic Application.

In our research, a straightforward UPLC-MS/MS method, with diazepam as the internal standard (IS), was proposed and acknowledged to determine the concentrations of enasidenib in rat plasma. When preparing the sample, we used acetonitrile for protein precipitation. The gradient elution method was used, and the mobile phase was acetonitrile and 0.1% formic acid. Diazepam was used as the IS. We used the Acquity UPLC BEH C18 column to separate enasidenib and IS. Under the positive ion electrospray ionization (ESI) source conditions, the mass transfer pairs of enasidenib were monitored by multiple reaction monitoring (MRM) to be m/z 474.2 ⟶ 456.1 and m/z 474.2 ⟶ 267.0, and the IS mass transfer pairs were m/z 285.0 ⟶ 154.0. Enasidenib had good linearity (r 2 = 0.9985) in the concentration range of 1.0-1000 ng/mL. Besides, the values of intraday and interday precision were 2.25-8.40% and 3.94-5.46%, respectively, and the range of the accuracy values varied from -1.44 to 2.34%. Matrix effect, extraction recovery, and stability were compliant with FDA approval guidelines in terms of bioanalytical method validation. We had established a new method that had been applied to the pharmacokinetic study of enasidenib in rats.

Simultaneous determination of six components of Danzhi Xiaoyao Pill in beagle plasma by HPLC-MS/MS and a study of pharmacokinetic of paeoniflorin and geniposide after single-dose administration.

This study was to develop a reliable and simple high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to detect paeoniflorin, geniposide, saikosaponin b2, liquiritin, paeonol and atractylenolide Ⅲ in beagle plasma and to study pharmacokinetic of paeoniflorin and geniposide after single-dose administration of Danzhi Xiaoyao Pill (DZXY). Chromatographic separation was performed using an Agilent C18 column, and multiple reaction monitoring (MRM) mode was used. A gradient elution procedure was used with solvent A (acetonitrile) and solvent B (0.1 % formic acid-water) as mobile phases. The elution procedure was as follows: 85 % B-30 % B (0-7 min) and 30 % B-30 % B (7.1-8 min). The flow rate was 0.3 mL/min. The column temperature was 40 ℃, and the injection volume was 10 µL. The main analytical parameters of paeoniflorin, geniposide, saikosaponin b2, liquiritin, paeonol and atractylenolide Ⅲ were m/z 525→449, m/z 433→224, m/z 780→617, m/z 417→254, m/z 167→43 and m/z 249→231, respectively. Ethyl acetate was used to extract the analytes in the plasma. Standard calibration curves of six analytes showed satisfactory linearity (r2≥0.99 2) within the determined ranges. The lower limits of quantification were 0.5 ng/mL for paeoniflorin and liquiritin, 2.5 ng/mL for geniposide and saikosaponin b2 and 1.0 ng/mL for atractylenolide Ⅲ and paeonol, respectively. The intra-day and inter-day precision (RSD %) were all below 6.94 %, and the intra-day and inter-day accuracy (RE %) were within ± 6.10 %. The recovery and ME of six analytes were 85.99 %-98.10 % and 95.78%-108.06%, respectively. Additionally, the method we established in this experiment can be successfully used to study the pharmacokinetics of paeoniflorin and geniposide in beagle plasma.

Simultaneous Determination of Parecoxib and Its Metabolite Valdecoxib Concentrations in Beagle Plasma by UPLC-MS/MS and Application for Pharmacokinetics Study.

A method for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagle plasma by UPLC-MS/MS was developed and validated. After the plasma was extracted by acetonitrile precipitation, the analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) using acetonitrile-formic acid as the mobile phase in gradient mode. The analytes were monitored by multiple reaction monitoring (MRM) in electrospray negative ion mode. The mass transfer pairs were m/z 368.97→119.01 for parecoxib, m/z 312.89→118.02 for valdecoxib, and m/z 379.98→316.02 for celecoxib (internal standard, IS). The correlation coefficients of parecoxib and valdecoxib ranged from 5 to 4000 ng/mL were greater than 0.9998. The recovery of parecoxib and valdecoxib was greater than 82.54%. The inter- and intra-day precision RSD values were 1.36~3.65% and 2.28~5.91%, respectively. The accuracy of RE values were -1.38%~1.96%. Finally, the matrix effect (ME) and stability were also within acceptable criteria. This method had been successfully applied to the pharmacokinetics of parecoxib and valdecoxib in beagle plasma after injection of parecoxib (1.33 mg/kg, intramuscular injection).

Effect Of Chinese Herb Danzhi Xiaoyao Pills On Pharmacokinetics Of Venlafaxine In Beagles.

OBJECTIVE: To investigate the effects of Chinese herb Danzhi Xiaoyao pills on the pharmacokinetics of venlafaxine and its metabolites O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) in beagles by using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: Six beagles (half male, half female) were chosen to test, being fasted before the experiment but having free access to drinking water 1 day before being fed drugs. After oral administration of venlafaxine hydrochloride tablets (10.28 mg/kg), the blood samples were collected in succession at different points in time. After 1-week washout period, Danzhi Xiaoyao pills (0.6g/kg) were given through oral administration to the six beagles every morning until the 7th day, venlafaxine hydrochloride tablets (10.28 mg/kg) were given after feeding Danzhi Xiaoyao pills (0.6g/kg) half an hour and blood samples were collected continuously at different points. All samples were analyzed by UPLC-MS/MS, and the main pharmacokinetic parameters of venlafaxine, ODV and NDV were computed by DAS 2.0. RESULTS: The Cmax of the venlafaxine group (control group) and the combination group (experimental group) were (2267.26±252.89) ng/mL and (1542.64±190.73) ng/mL, respectively. The AUC(0-∞) of the two groups were (13,934.79±3609.23) ng·h/mL and (8001.91±2167.58) ng·h/mL, respectively. The ODV Cmax of the two groups were (2253.80±215.81) ng/mL and (2721.37±118.20) ng/mL, and AUC(0-∞) were (13,974.99±2784.04) ng·h/mL and (17,539.44±1894.29) ng·h/mL, respectively. The NDV Cmax of the two groups were (50.98±5.76) ng/mL and (58.74±12.33) ng/mL, and AUC(0-∞) were (179.26±34.94) ng·h/mL and (220.68±51.41) ng·h/mL, respectively. After administration of Danzhi Xiaoyao pills, the Cmax and AUC(0-∞) of venlafaxine decreased significantly, indicating that the plasma exposure of venlafaxine decreased. The increase of Cmax and AUC(0-∞) of ODV and NDV indicated a rise in plasma exposure. CONCLUSION: Danzhi Xiaoyao pills can accelerate the metabolism of venlafaxine in beagles. In clinical, when venlafaxine was co-administrated with Danzhi Xiaoyao pills, dose adjustment of venlafaxine should be taken into account.