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KMID : 0369820170470060531
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Jorunal of Korean Pharmaceutical Sciences
2017 Volume.47 No. 6 p.531 ~ p.540
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Development of new clean-up method for UPLC?MS/MS analysis of leuprolide
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Lee Dong-Seok
Heo Seo-Hee
Kim Sook-Jin
Lee Yong-Bok
Cho Hea-Young
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Abstract
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In this study, a sensitive, accurate and cost-effective ultra-performance liquid chromatography-tandem mass spectrometric method was developed using a new clean-up process for the quantification of leuprolide in rat plasma. Chromatography was carried on a HALO peptide ES-C18 column using water and acetonitrile (containing 1% acetic acid) as the mobile phase with a gradient elution. Electrospray ionization was applied and operated in the multiple reaction monitoring positive mode. Leuprolide and an internal standard (leuprolide-d5) were extracted by the liquid?liquid extraction with methylene chloride after protein precipitation by the new clean-up process. In the clean-up process, acetic acid was used for higher reproducibility, and then 10 μL of cleaned aqueous phase was finally injected into the UPLC?MS/MS system. The chromatograms showed good resolution, and no interfering peaks to plasma constituents were observed. The lower limit of quantification in rat plasma was 20 pg/mL for leuprolide. The calibration curves for leuprolide in rat plasma were linear over all the concentration range 20?10,000 pg/mL with a correlation coefficient of 0.991. The intra- and inter-day assay for precision and accuracy fulfilled the international guideline requirements. This validated method was successfully applied to characterize the pharmacokinetics of leuprolide in rats after an intravenous administration of 0.1 mg/kg. Our method allows laboratory researchers to simply determine leuprolide in plasma.
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KEYWORD
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Leuprolide, Clean-up, Ultra-performance liquid chromatography-tandem mass spectrometry, Pharmacokinetics, Rat
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