Search

Background: The number of validated quantification methods for rifampicin, a prototypical Oatp inhibitor, in biological rat samples was limited. Objective: This study was conducted to validate a modified reversed-phase liquid chromatographic method for the determination of rifampicin in rat liver tissue according to the current ICH M10 Bioanalytical Method Validation Draft Guideline (2019) for application to samples of in situ rat liver perfusion studies. Methods: Liver tissue samples were obtained from recirculatory in situ rat liver perfusion studies. The analysis was performed on a C18 column with a mobile phase composed of 0.05 M phosphate buffer (pH 4.58): acetonitrile (55:45, v/v). The assay was validated for selectivity, calibration curve and range, matrix effect, carry-over, accuracy and precision, reinjection reproducibility, and stability. Results: he method was considered selective and stable, without having carry-over and matrix effects. The calibration curve was linear (R2: 0.9983) within the calibration range (0.5-60 ppm). Accuracy and precision values fulfilled the required limits. Liver concentrations of rifampicin in liver tissue, obtained after 60 min perfusion with 10 μM and 50 μM of rifampicin, were 45.1 ± 11.2 and 313.4 ± 84.4 μM, respectively. Conclusion: The bioanalytical method validation was completed and the method was successfully applied for the determination of rifampicin in rat liver tissue.

Background: Metabolomics is one of the main areas to understand cellular process at molecular level by analyzing metabolites. In recent years metabolomics has emerged as a key tool to understand molecular basis of diseases, to find diagnostic and prognostic biomarkers and develop new treatment opportunities and drug molecules. Objective: In this study, untargeted metabolite and lipid analysis were performed to identify potential biomarkers on premature ovarian insufficiency plasma samples. 43 POI subject plasma samples were compared with 32 healthy subject plasma samples. Methods: Plasma samples were pooled and extracted using chloroform:methanol:water (3:3:1 v/v/v) mixture. Agilent 6530 LC/MS Q-TOF instrument equipped with ESI source was used for analysis. A C18 column (Agilent Zorbax 1.8 μM, 50 x 2.1 mm) was used for separation of the metabolites and lipids. XCMS, an “R software” based freeware program, was used for peak picking, grouping and comparing the findings. Isotopologue Parameter Optimization (IPO) software was used to optimize XCMS parameters. The analytical methodology and data mining process were validated according to the literature. Results: 83 metabolite peaks and 213 lipid peaks were found to be in semi-quantitatively and statistically different (fold change >1.5, p Conclusion: According to the results, two groups were successfully separated through principal component analysis. Among the peaks, phenyl alanine, decanoyl-L-carnitine, 1-palmitoyl lysophosphatidylcholine and PC(O-16:0/2:0) were identified through auto MS/MS and matched with human metabolome database and proposed as plasma biomarker for POI and monitoring the patients in treatment period.

Background: Phenylalanine is a significant biomarker for various diseases like phenylketonuria, gastric cancers, and ischemic stroke according to recent studies. Methods: In the present study; a simple, sensitive, selective and novel analytical method was validated by using an ultrafiltration-based extraction and LC-MS/MS quantification of phenylalanine in human plasma using 13C phenylalanine heavy isotope. Amicon® Ultra Centrifugal Filter was used for ultrafiltration. Parameters affecting LC separation and MS/MS detection were investigated and optimized. Chromatographic separation was achieved on a Merck SeQuant ZIC-HILIC (100x4.6 mm, 5 μm) at a column temperature of 40°C using a mobile phase of mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (50:50 v/v) at a flow rate of 0.35 mL/min. The transitions m/z 167→121 for 13C phenylalanine, m/z 166→120 for phenylalanine itself were monitored using the MRM mode. Result: The assay was linear concentration range of 0.0025 μg/mL to 1.20 μg/mL (R2=0.999). The developed method was validated according to FDA guidelines. The method was found linear, sensitive, precise, accurate, and selective.