Quantitative determination of Phakellistatin 13, a new cyclic heptapeptide,in rat plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study |
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Authors: | Hua Wei Jun Wen Rui Xie Houwen Lin Guorong Fan Yutian Wu |
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Institution: | (1) Shanghai Key Laboratory for Pharmaceutical Metabolite Research, Shanghai Research Center for Drug (Chinese Materia Medica) Metabolism, School of Pharmacy, Second Military Medical University, No.325 Guohe Road, Shanghai, 200433, China;(2) Department of Pharmaceutical Analysis, School of Pharmacy, Second Military Medical University, No.325 Guohe Road, Shanghai, 200433, China;(3) Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China; |
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Abstract: | A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, followed by a 96-well protein precipitation,
has been developed and fully validated for the determination of Phakellistatin 13 (PK13), a new cyclic heptapeptide isolated
from the sponge Phakellia fusca Thiele, in rat plasma. After protein precipitation of the plasma samples (50 μL) in a 96-well plate by methanol (200 μL)
containing the internal standard Pseudostellarin B (20 ng/mL), the plate was vortex mixed for 3 min. Following filtration
for 5 min, the filtrate was directly injected into the LC-MS/MS system. The analytes were separated on an XB-C18 analytical
column (5 μm, 50 mm × 4.6 mm i.d.) using an eluent of methanol–water (85:15, v/v) and detected by electrospray ionization mass spectrometry in the negative multiple reaction monitoring mode with a chromatographic
run time of 5.0 min. The method was sensitive with a lower limit of quantification (LLOQ) of 0.1 ng/mL, with good linearity
(r > 0.999) over the quantitation range of 0.1–5 ng/mL. The validation results demonstrated that this method was significantly
specific, accurate, precise, and was successfully applied in measuring levels of PK13 in rat plasma following intravenous
administration of 20, 50, and 100 μg/kg of peptide in rats, respectively, which was suitable for the preclinical pharmacokinetic
studies on PK13. |
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