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Development and validation of a sensitive and selective UHPLC–MS/MS method for quantitation of an investigational anti-malarial compound, 2- tert-butylprimaquine (NP-96) in rat plasma, and its application in a preclinical pharmacokinetic study
Ist Teil von
Journal of pharmaceutical and biomedical analysis, 2010-07, Vol.52 (3), p.410-415
Ort / Verlag
Amsterdam: Elsevier B.V
Erscheinungsjahr
2010
Quelle
MEDLINE
Beschreibungen/Notizen
An ultra-high performance liquid chromatographic tandem mass spectroscopy (UHPLC–MS/MS) method was developed and validated for the quantification of an investigational anti-malarial entity, 2-
tert-butylprimaquine (NP-96), in rat plasma. Simple protein precipitation by acetonitrile was used for the sample preparation. Effective separation of NP-96, internal standard (IS) and matrix components were achieved on an UHPLC column (Hypersil Gold C18, 50
mm
×
2.1
mm, 1.9
μm) using a mobile phase composed of acetonitrile and 20
mM ammonium acetate, which was pumped in a gradient mode at a flow rate of 450
μl/min. Selective reaction monitoring (SRM) was utilized for quantitation of the molecules. To increase sensitivity of the method, two ions of
m/
z 299 and
m/
z 231 were selected for NP-96, while IS was monitored for an ion of
m/
z 489. The method was validated according to FDA guideline on bioanalytical method validation and showed good compliance. The intra-day and inter-day precision expressed as R.S.D. was lower than 15% at all the tested quality control levels, including upper and lower limits of quantification. The calibration range was 2.5–500
ng/ml. Total runtime for the method was 5
min, which was suitable to produce high-throughput results for pharmacokinetic evaluation.