Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC-MS/MS.
A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid-liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20  mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow-rate of 1 mL/min on a Kromasil KR-100. The method was in linear range from 50 to 5000  ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC-PDA and LC-MS/MS and it was found that one metabolite is novel.
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, India.
Karthik
K
Arumugam
Mallikarjuna Rao
MR
Chamallamudi
Ravindranath Reddy
RR
Gilibili
Ramesh
R
Mullangi
Subramanian
S
Ganesan
Sidhartha S
SS
Kar
Ranjithkumar
R
Averineni
Gopal
G
Shavi
Nayanabhirama
N
Udupa
D000818:Animals; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D016018:Least-Squares Analysis; D048448:Phenylcarbamates / Q000737:chemistry / Q000652:urine*; D051381:Rats; D015203:Reproducibility of Results; D000068836:Rivastigmine; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002800:Cholinesterase Inhibitors; D048448:Phenylcarbamates; D000068836:Rivastigmine