BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF AVELUMAB, AXITINIB AND ITS APPLICATION TO PHARMACOKINETIC STUDIES IN RABBIT PLASMA BY USING LCMS/MS

Objective: An easy, quick, precise, active and reproducible LC-MS/MS technique was developed for the bioanalytical method of Avelumab and Axitinib using Cytarabine as an internal standard. Methods: This article summarizes the recent progress on bioanalytical LC-MS/MS methods using waters x-bridge phenyl column (150x4.6 mm, 3.5µ) column and organic mobile phase of 0.1% Tri fluoro acetic acid and Acetonitrile in 50:50 ratio. Results: The calibration curve was linear in the range of 2-40 ng/ml for avelumab and 0.5-10 ng/ml axitnib. Accuracy, precision, recovery, matrix effect and stability results were found to be within the suitable limits. Simple and efficient method was developed and utilized in pharmacokinetic studies to see the investigated analyte in body fluids. Conclusion: The application denotes all the parameters of system suitability, specificity, linearity and accuracy are in good agreement with USFDA guidelines and applied effectively for the investigation of pharmacokinetic studies in rabbit.

Effect of Sustained Administration of Thymol on Its Bioaccessibility and Bioavailability in Rabbits

The objective of this study was the detection of thymol in rabbit plasma, tissues, large intestinal content, and faeces. Forty-eight rabbits were divided into control and experimental groups (thymol 250 mg/kg feed). Thymol was administered for 21 days and then withdrawn for 7 days. Concentration of thymol in the intestinal wall (IW) was significantly higher than in plasma (p < 0.05) and liver (p < 0.05); in the kidneys it was significantly higher than in plasma (p < 0.05) and liver (p < 0.05) during thymol addition. Thymol in IW was significantly higher than in plasma also after withdrawal (p < 0.01). Significant correlation (rs = −1.000, p < 0.01) between IW and plasma points to the intensive absorption of thymol from the intestine, while the correlation between plasma and liver (rs = 0.786, p < 0.05) indicates intensive biotransformation and excretion processes in liver. Significant correlation between liver and kidney (rs = 0.738, p < 0.05) confirms the intensive metabolism of thymol in the kidney. During the withdrawal period, thymol was detected above trace amounts only in faeces, and was significantly higher than in the colon during both periods (p < 0.01). Results show intensive biotransformation of thymol in the rabbit organism.

Lipemia in the Plasma Sample Affects Fentanyl Measurements by Means of HPLC-MS2 after Liquid-Liquid Extraction

Examination of fentanyl levels is frequently performed in certain scientific evaluations and forensic toxicology. It often involves the collection of very variable blood samples, including lipemic plasma or serum. To date, many works have reported the methods for fentanyl detection, but none of them have provided information about the impact on the assay performance caused by an excessive amount of lipids. This aspect may be, however, very important for highly lipophilic drugs like fentanyl. To address this issue, we developed the liquid chromatography method with mass spectrometry detection and utilized it to investigate the impact of lipids presence in rabbit plasma on the analytical method performance and validation. The validation procedure, conducted for normal plasma and lipemic plasma separately, resulted in good selectivity, sensitivity and linearity. The limits of detection and quantification were comparable between the two matrices, being slightly lower in normal plasma (0.005 and 0.015 µg/L) than in lipemic plasma (0.008 and 0.020 µg/L). Liquid–liquid extraction provided a low matrix effect regardless of the lipid levels in the samples (<10%), but pronounced differences were found in the recovery and accuracy. In the normal plasma, this parameter was stable and high (around 100%), but in the lipemic matrix, much more variable and less efficient results were obtained. Nevertheless, this difference had no impact on repeatability and reproducibility. In the present work, we provided reliable, convenient and sensitive method for fentanyl detection in the normal and lipemic rabbit plasma. However, construction of two separate validation curves was necessary to provide adequate results since the liquid-liquid extraction was utilized. Therefore, special attention should be paid during fentanyl quantification that involves lipemic plasma samples purified by this technique.

Pharmacokinetic Properties of the Novel Synthetic Cannabinoid 5F-APINAC and Its Influence on Metabolites Associated with Neurotransmission in Rabbit Plasma

The strong psychoactive effects of synthetic cannabinoids raise the need for the deeper studying of their neurometabolic effects. The pharmacokinetic properties of 5F-APINAC and its influence on metabolomics profiles associated with neurotransmission were investigated in rabbit plasma. Twelve rabbits divided into three groups received 1-mL 5F-APINAC at 0.1, 1 and 2 mg/kg. The intervention groups were compared with the controls. Sampling was performed at nine time points (0–24 h). Ultra-high-performance liquid chromatography–tandem mass spectrometry was used. The pharmacokinetics were dose-dependent (higher curve at a higher dose) with a rapid biotransformation, followed by gradual elimination within 24 h. The tryptophan concentrations abruptly decreased (p < 0.05) in all tested groups, returning to the basal levels after 6 h. 5-hydroxylindole acetic acid increased (p < 0.05) in the controls, but this trend was absent in the treated groups. The aspartic acid concentrations were elevated (p < 0.001) in the treated groups. L-kynurenine was elevated (p < 0.01) in the intervention groups receiving 1 mg/kg to 2 mg/kg. Dose-dependent elevations (p < 0.01) were found for kynurenic acid, xanthurenic acid and quinolinic acid (p < 0.01), whereas the anthranilic acid trends were decreased (p < 0.01). The indole-3-propionic acid and indole-3-carboxaldehyde trends were elevated (p < 0.05), whereas the indole-3-lactic acid trajectories were decreased (p < 0.01) in the intervention groups. 5F-APINAC administration had a rapid biotransformation and gradual elimination. The metabolites related to the kynurenine and serotonergic system/serotonin pathways, aspartic acid innervation system and microbial tryptophan catabolism were altered.

Simultaneous quantification of dexamethasone and 6β-hydroxydexamethasone in rabbit plasma, aqueous and vitreous humor, and retina by UHPLC–MS/MS

Aim: To develop and validate a fit for purpose method for the simultaneous determination of dexamethasone and its major metabolite, 6β-hydroxydexamethasone, in rabbit plasma and ocular matrices to measure the in vivo release and distribution profile of dexamethasone from intravitreal implants. Materials & methods: An UHPLC–MS/MS system was employed to perform the bioanalysis. The method was validated according to the US FDA Bioanalytical Method Validation Guidance for Industry. Results & conclusion: The method was found to be fit-for-purpose for the described biological matrices and had a LLOQ of 0.1 ng/ml.

Determination of esomeprazole in rabbit plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS)

A rapid extraction method was developed and validated for esomeprazole determination in rabbit plasma by liquid chromatography tandem mass spectrometry (LC&ndash;MS/MS). Esomeprazole in rabbit plasma was extracted and then cleaned up using QuEChERS technique. The analyte was separated using C18 column and quantified by MS/MS detector. Pantoprazole was used as the internal standard. The positive ESI source was used in this study together with the multi&shy;-reactive ion monitoring mode. The validity of the method has been confirmed in accordance with ICH Harmonized guidelines on bioanalytical method validation. The method showed good specificity, good stability, with the linearity varying from 0.1 ng/mL to 20 ng/mL, the lower limit of quantification was 0.1 ng/mL, the accuracy and precision were within 85% and 115% which achieved the ICH Harmonized guideline requirements, FDA guideline and AOAC International requirements. The method has been applied to quantify the concentration of esomeprazole in rabbit plasma, and then to compare the bioavailability of two preparations containing esomeprazole.