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.
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, 50mmx2.1mm, 1.9microm) using a mobile phase composed of acetonitrile and 20mM ammonium acetate, which was pumped in a gradient mode at a flow rate of 450microl/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 2.5-500ng/ml. Total runtime for the method was 5min, which was suitable to produce high-throughput results for pharmacokinetic evaluation.
Mayatra
Sujal J
SJ
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab, India.
Prasad
Bhagwat
B
Jain
Meenakshi
M
Jain
Rahul
R
Singh
Saranjit
S
D000818:Animals; D000962:Antimalarials / Q000097:blood* / Q000737:chemistry / Q000493:pharmacokinetics; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D015507:Drugs, Investigational; D005615:Freezing; D008297:Male; D015394:Molecular Structure; D011319:Primaquine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D011786:Quality Control; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D013997:Time Factors
C482216:2-tert-butylprimaquine; D000962:Antimalarials; D015507:Drugs, Investigational; D011319:Primaquine