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Pharm Methods. 2011 Jul;2(3):157-60. doi: 10.4103/2229-4708.90354.

A validated RP-HPLC method for quantitation of trigonelline from herbal formulations containing Trigonella foenum-graecum (L.) seeds.

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Herbal Research Lab, Ramnarain Ruia College, Matunga (E), Mumbai, India.



Trigonella foenum-graecum (L.) (Fabaceae, Fenugreek) is an important ingredient of Ayurvedic and other marketed herbal formulations. Fenugreek seeds are employed in many traditional systems as an antibacterial and antidiabetic agent, gastric stimulant and galactogogue. Trigonelline, a major phytoconstituent found in fenugreek seeds, shows estrogenic, anti-diabetic and anti-invasive activity. Therefore, it is a suitable bioactive marker to establish the quality of crude drug and its formulations.


To develop an efficient and effective RP-HPLC method for estimation of trigonelline from Trigonella foenum-graecum seeds and its marketed herbal formulations.


Separation and detection of trigonelline was carried out on a Cosmosil CN-MS column eluted with methanol:distilled water [95:5, v/v; pH 3.5 using hydrochloric acid]. Detection was carried out at 267 nm using a Photo Diode Array detector. Fenugreek seeds and two marketed herbal formulations were subjected for HPLC analysis of Trigonelline.


The RP-HPLC method was validated as per ICH guidelines and the content of trigonelline in marketed polyherbal formulations such as Dibet powder and Amyron syrup was determined. The LOD and LOQ were found to be 5.00 ng/mL and 50.00 ng/mL, respectively. Detector response was linear from 100.00 to 8000.00 ng/mL. The method was found to be simple, sensitive, accurate, reproducible and rugged.


This work can be recommended for quality assurance and marker-based standardization of formulations containing fenugreek seeds.


Amyron syrup; RP-HPLC; Trigonella foenum-graecum (L.); dibet powder; trigonelline

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