lf you use a mass spectrometer and a calibration curve, you should be able to quantify them since they differ by 2 Daltons in m/z.

My experience is that steroids are visible using APCI rather than ESI, and they tend to lose water in positive ion. Stigmasterol [M+1] is 413, but may show as 395 after loss of water. β-Sitosterol [M+1] is 415, but may show as 397 with loss of water.

Have you tried silver nitrate impregnated silica gel? the double bond difference between them may be enough to resolve them.

Hi Nurkhalida 

Although not exactly the same but the link attached may be helpful to you as it was a discussion about the two molecules in question and problems pertaining their separation on same TLC plate. Hope it helps. 

Regards

https://www.researchgate.net/post/How_to_separate_beta-sitosterol_and_stigmasterol_on_same_TLC_plate

I think the best way to separate sterols ( cholesterol, stigmasterol, campesterol,  sitosterol etc.) by using GC (GCMS). I think you can many methods for analyzing sterols by GC (MS) in Google. good luck.