Fast, Sensitive Bioanalytical Method Development and Validation for the Determination of Escitalopram in Human Plasma by Liquid Chromatography- Electrospray, Tandem Mass Spectrometry

A simple, reproducible and fast (1.5 min chromatogram) bioanalytical method of liquid chromatography and tandem with mass detector (LC/MS-MS) was developed and validated to determine escitalopram (ESP) in human plasma using liquid-liquid extraction technique. Imipramine (IMP) was used as internal standard (IS) and K2 EDTA was used as anti-coagulant. Analytes were extracted by methyl-tertbutyl ether (MTBE) and subsequent separation on a Luna 3 mu m C18, 100 x 4.60 mm column using acetonitrile:10 mM ammonium formate pH 4.5 (90:10 v/v) as mobile phase at a flow rate of 1 mL/min and 30 +/- 3o C column oven temperature. Analytes were monitored with electrospray ionization in positive multiple reaction mode (MRM) for both ESP and IMP using quadrupole MS/MS spectrometer. ESP and IMP were detected with proton adducts at m/z 324.20 -> 110.13 for ESP and 280.8/86.0 for IMP. ESP and IMP were eluted at 0.655 and 0.745 min, respectively. The method was validated over a linear (r(2) = 0.9979) concentration range of 306.022 to 199205.354 pg/mL. The inter-day and intra-day precisions were found to be less than 15% and the accuracy was all within +/- 15% (at LLOQ +/- 20%). The developed LC-MS/MS method was fully validated for all the other parameters as per FDA guidelines like selectivity, matrix effect, recovery and stability as well. Due to the high degree of sensitivity, very less time consuming, easy extraction procedure and low requirement of sample volume, the method will be applicable for therapeutic drug monitoring.