ABSTRACT
A simple, selective and reliable LC-MS/MS method was developed and validated for the simultaneous quantitation of darolutamide diastereomers (diastereomer-1 and diastereomer-2) and its active metabolite i. e. ORM-15341 in mice plasma using warfarin as an internal standard (IS) as per the regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mM ammonium acetate:absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397â202, 395â202 and 307â250 for darolutamide diastereomers, ORM-15341 and the IS, respectively in the negative ionization mode. The calibration curves were linear (r>0.992) in the range of 100-2400 ng/mL for all the analytes. The intra- and inter-day precisions were in the range of 1.25-10.2 and 1.58-12.3; 2.85-5.68 and 1.85-9.58; 2.34-12.1 and 2.58-7.38 for diastereomer-1, diastereomer-2 and ORM-15341, respectively. Both diastereomers and ORM-15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.
Subject(s)
Androgen Antagonists/blood , Liquid-Liquid Extraction/methods , Pyrazoles/blood , Androgen Antagonists/chemistry , Androgen Antagonists/pharmacokinetics , Androgen Antagonists/therapeutic use , Animals , Calibration , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Drug Stability , Liquid-Liquid Extraction/instrumentation , Male , Mice , Mice, Inbred BALB C , Prostatic Neoplasms, Castration-Resistant/drug therapy , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/therapeutic use , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization/instrumentation , Spectrometry, Mass, Electrospray Ionization/methods , Stereoisomerism , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/methods , Warfarin/blood , Warfarin/pharmacokineticsABSTRACT
A simple, selective and reliable LC-MS/MS method was validated for simultaneous quantitation of darolutamide diastereomers in 50 µL mouse plasma using warfarin as an internal standard (IS) as per regulatory guidelines. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Chiralpak IA column with an isocratic mobile phase 5 mm ammonium acetate-absolute alcohol (20:80, v/v) at a flow rate of 1.0 mL/min. Detection and quantitation was done in multiple reaction monitoring mode following the transitions m/z 397 â 202 and 307 â 250 for darolutamide diastereomers and the IS, respectively, in the negative ionization mode. The linearity range was 100-2400 ng/mL for each diastereomer. The intra- and inter-day precisions were in the ranges of 1.78-4.20 and 4.34-14.6, and 3.63-4.74 and 4.78-5.15 for diastereomer-1 and diastereomer-2, respectively. Both diastereomers were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice. Following oral administration of darolutamide at 10 mg/kg, maximum concentration in plasma was 4189 and 726 ng/mL for diastereomer-1 and diastereomer-2, respectively. The terminal half-life was found to be ~0.50 h for both the diastereomers. The AUC(0-t) was found to be 18,961 ng*h/mL for diastereomer-1 and 1340 ng*h/mL diastereomer-2.
Subject(s)
Chromatography, Liquid/methods , Pyrazoles , Tandem Mass Spectrometry/methods , Animals , Linear Models , Male , Mice , Mice, Inbred BALB C , Pyrazoles/blood , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , StereoisomerismABSTRACT
Crossbred cattle in some sectors of the world have a significant role in enhancing milk production thereby enhancing the per capita milk availability as a human food source. However, there are certain constraints associated with crossbred animals, such as disease susceptibility, increased reproductive problems, repeat breeding and poor seminal quality. The semen of crossbred bulls has a poor freezing capacity, increased cryo-damage, poor mass cell motility, greater percentages of dead/abnormal sperm and poor initial and post-freeze cell motility. The rejection rate of crossbred bulls for cryostorage of semen has been reported to be as great as 50% as a result of unacceptable semen quality. The identification of superior bulls using molecular technologies is needed which necessitates identification of the genes having a role in sperm function. The present study was, therefore, conducted to gain information on identification and expression of genes having a role in sperm motility in crossbred bulls. The gene transcripts in bulls with sperm of superior and inferior quality were profiled in Vrindavani crossbred cattle by microarray analyses and the results were verified by real time-quantitative PCR. Microarray analyses revealed 19,454 genes which were differentially expressed. At a two-fold cut off, 305 genes were differentially (P<0.01) expressed with 160 genes upregulated and 145 genes down regulated. Some of the upregulated candidate genes were further validated by RT-qPCR. These genes had a four to 16 fold upregulation in sperm with inferior motility as compared to sperm of crossbred bulls with superior motility.
