Method development and validation for naratriptan determination in human plasma by HPLC with tandem mass spectrometry detection, and its application to bioequivalence study

Abstract

The authors developed a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of naratriptan (NP) in human plasma using naratriptan-d3 (NPD3) as an internal standard (IS). Chromatographic separation was performed on a Zorbax SB-C18, 75 x 4.6 mm, 3.5 µm column with an isocratic mobile phase composed of 0.1% formic acid : acetonitrile (50:50 v/v), at a flow-rate of 0.6 mL/min. NP and NPD3 were detected with proton adducts at m/z 336.5→98.0 and 339.4→101.0 in selected reaction monitoring (SRM) positive mode, respectively. The liquid-liquid extraction method was used to extract the NP and NPD3. This method was validated over a linear concentration range of 0.1-25.0 ng/mL with a correlation coefficient of (r2) >; 0.9998. The Intra-day and Interday precision was found to be 1.8 to 3.6%, and 2.3 to 2.6%, and accuracy to be 101.7- 104.2% and 101.8 to 102.9%, respectively. NP was found to be stable throughout freeze-thaw (three cycles), bench top and auto sampler stability studies. This method was successfully applied for the analysis of plasma samples following oral administration of NP (2.5 mg) in 31 healthy Indian male human volunteers under fasting conditions.