Liquid–liquid extraction

A rapid and sensitive LC-MS/MS method for the quantification of nebivolol using d4- nebivolol as internal standard has been developed and validated. The nebivolol and d4- nebivolol were extracted by liquid- liquid extraction using tert-butyl ethyl ether and separated on Kromasil 100-5C 4.6x100mm column using a mixture of 0.1% formic acid in 5 mM ammonium acetate, methanol and acetonitrile at composition of (20:20:60 v/v) at a flow rate of 0.5 mL/min. Detection involved an API-4000 LC-MS/MS with electrospray ionization in the positive mode.  The method was validated as per the FDA guidelines and shown to provide an intra and inter day precision and accuracy within the acceptable limit with in a run time of 3.0 min. The proposed method can adopt for the regular bioequivalence study analysis and also can easily adoptable for clinical drug monitoring due to its simplicity and ruggedness.