کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1199526 | 1493581 | 2014 | 11 صفحه PDF | دانلود رایگان |
• PPE and LLE methods for extraction of bortezomib from in vitro systems.
• Fast and ultra-sensitive UPLC/MS/MS with extremely low LLOQ (0.5 pg/100 μL).
• Bortezomib rapidly degrades in cell culture media and in myeloma cells.
• The decay strongly depends on the pH value.
• Calculation method for correct quantification of bortezomib in in vitro systems.
Bortezomib represents the first clinically approved proteasome inhibitor for multiple myeloma. Research conducted on its intracellular kinetics in target cells and on possibly related mechanisms of resistance is sparse so far. We therefore developed and validated a highly sensitive ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) method for bortezomib quantification within cultured myeloma cells and media. Fast gradient UPLC based on a BEH C18 column (1.7 μm particle size) with aqueous formic acid and acetonitrile as mobile phase. Selective extraction procedures using protein precipitation extraction (PPE) and liquid–liquid extraction (LLE) were established and compared. Extracted bortezomib was quantified by positive electrospray tandem mass spectrometry using deuterated D8-bortezomib as internal standard. The calibrated ranges were 0.5–2500 pg per sample. For LLE, overall accuracies varied between 99.2% and 112% (medium) and 89.9% and 111% (cells), while overall precision ranged from 1.13% to 13.0% (medium) and 2.80% to 12.7% (cells), respectively. Recovery rates (cells/medium) were >77%/>65% for LLE and >89%/63% for PPE. Matrix effects were generally lower for LLE compared to PPE. Regardless of the extraction method, retrievable amounts of bortezomib were considerably reduced after 24 h of incubation (0.2, 1, 5, and 25 nM). Revealing greater dependence on the extent of acidification, retrieval of bortezomib can be increased distinctly in acidified solution or acidified culture medium. Thus, particular attention needs to be paid to the occurring bortezomib degradation in neutral culture medium since correct quantification of intracellular bortezomib can only be achieved in relation to the corresponding extracellular concentration.
Journal: Journal of Chromatography A - Volume 1345, 6 June 2014, Pages 128–138