A simple, accurate and reproducible LCMS/MS method has been developed for the simultaneous quantification of Harmine, Kaempferol, Oleic acid and Diosgenin from the methanolic extract of fruits of plant Tribulus terrestris L. and its marketed formulation. The Agilent 1100 series HPLC system and 3200 Q Trap LCMS/MS system of Applied Biosystem MDS/SCIEX was used for the analysis. The optimized conditions [Mobile phase A: 0.01 M Ammonium Acetate buffer; pH 7.5 adjusted with ammonia And Mobile phase B: Acetonitrile: Methanol (80:20)] gradient elution system on a C8 reversed-phase column with a flow rate of 1 mL/min. The separated compounds were detected as peak response of precursor ion (Q1)/ product ion (Q3) for Harmine 213.1/198.1 and Diosgenin 415.2/271.2 by positive mode(+MRM) and for Kaempferol 284.9/284.9 and Oleic acid 281.2/281.2 by negative mode (-MRM) in single injection. The quantity of Harmine was found to be 0.016 % and 0.008%, Kaempferol was 0.013% and 0.008%, Oleic acid was 0.254 % and 0.112 % and Diosgenin was 0.086 % and 0.019% in plant and formulation respectively. The developed method was then validated in accordance with the ICH guidelines in terms of specificity, linearity, LOD, LOQ, precision and recovery.
