Determination of Metoprolol in human blood plasma by LC-MS/MS

Abstract

A high performance LC-MS/MS method for determination of Metoprolol in human plasma was
developed and successfully validated. Metoprolol is a β1-selective aryloxy propanolamine used in
the treatment of cardiovascular disorders such as hypertension, arrhythmia and heart failure.
Metoprolol (analyte) and the Metoprolol d7 (Internal Standard) were extracted from K3EDTA based
Human Plasma samples by Liquid-liquid extraction method. Chromatographic separation was
achieved on Gemini 5m C18 110Å (150x4.6mm) column using Methanol: 10mM Ammonium
Formate containing 0.1% Formic Acid Solution (95:5 v/v) as a mobile Phase at a flow rate of 0.800
mL/min with 80% flow splitting. The retention time of Metoprolol and Metoprolol d7 was found to
be 1.49 minute and 1.49 minute respectively. The standard curve was linear (R2 > 0.99) over the
concentration range of 0.68 ng/mL to 399.22 ng/mL. All the bioanalytical validation parameters were
determined as per US FDA guidelines. The developed bioanalytical method was sensitive with a
limit of detection below 1 ng/mL, with good linearity (r2 >0.99). The peaks obtained for the
Metoprolol and Metoprolol d7 were symmetrical in nature and well resolved from each other without
any interferences from human plasma.All the validation data, such as accuracy, precision, and
percent recovery, lipemic effect, haemolysis effect, matrix factor, etc. were within the required limits
as stated by guidelines. The method can be used for pharmacokinetic as well as Bioavailability, and
bioequivalence studies.