Development of an SPE-LC-MS/MS method for simultaneous quantification of bosentan and its active metabolite hydroxybosentan in human plasma to support a bioequivalence study.
A highly sensitive, selective and rapid bioanalytical method has been developed for the simultaneous determination of bosentan and hydroxybosentan in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytes and their deuterated analogs were quantitatively extracted from 100 μL human plasma by solid phase extraction. The chromatographic separation of analytes was achieved on a Thermo Hypurity C18 (100 mm × 4.6 mm, 5 μ) analytical column with a resolution factor of 2.4 under isocratic conditions. The method was validated over a dynamic concentration range of 0.4-1600 ng/mL for bosentan and 0.2-250 ng/mL for hydroxybosentan. Ion-suppression effects were investigated by post-column infusion of analytes. The precision (%CV) values for the calculated slopes of calibration curves, which would reflect the relative matrix effect, were less than 1.2% for both the analytes. The intra-batch and inter-batch precision (%CV) across quality control levels was ≤4.0% and the mean relative recovery was >94% for both the analytes. The method was successfully applied to a bioequivalence study of 125 mg tablet formulation (test and reference) in 12 healthy Indian male subjects under fasting condition. The ratios of mean log-transformed values of C(max), AUC(0-t) and AUC(0-inf) and their 90% CIs varied from 91.3 to 104.7%. The percentage change for incurred sample reanalysis (ISR) was within ±13.0%.
Chemistry Department, Kadi Sarva Vishwavidyalaya, Sarva Vidyalaya Campus, Sector 15/23, Gandhinagar 382015, Gujarat, India.
Jignesh M
JM
Parekh
Dhaval K
DK
Shah
Mallika
M
Sanyal
Manish
M
Yadav
Pranav S
PS
Shrivastav
D000284:Administration, Oral; D000328:Adult; D000959:Antihypertensive Agents / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics*; D019540:Area Under Curve; D001711:Biotransformation; D000077300:Bosentan; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D056148:Chromatography, Reverse-Phase / Q000592:standards; D018592:Cross-Over Studies; D005215:Fasting / Q000097:blood; D006207:Half-Life; D006801:Humans; D006900:Hydroxylation; D007194:India; D008297:Male; D008657:Metabolic Clearance Rate; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000592:standards; D013449:Sulfonamides / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics*; D013607:Tablets; D053719:Tandem Mass Spectrometry / Q000592:standards; D013810:Therapeutic Equivalency; D055815:Young Adult
D000959:Antihypertensive Agents; D013449:Sulfonamides; D013607:Tablets; D000077300:Bosentan