HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver |
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| Authors: | Vincenzo Pucci Claudio Giuliano Rena Zhang Kenneth Alan Koeplinger Joseph F Leone Edith Monteagudo Fabio Bonelli |
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| Institution: | 1. Department of Drug Metabolism and Pharmaceuticals, Merck & Co., Inc., Boston, MA, USA. Fax: +1‐617‐9822485;2. Department of Drug Metabolism and Pharmacokinetics, Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Merck Research Laboratories, Pomezia, Roma, Italy;3. Department of Drug Metabolism and Pharmacokinetics, Merck & Co., Inc., West Point, PA, USA;4. Department of Medicinal Chemistry, Merck & Co., Inc., Rahway, NJ, USA |
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| Abstract: | A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples. |
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| Keywords: | Hydrophilic interaction chromatography LC‐MS/MS Liver Nucleotide |
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