Small Molecule Quan using light:heavy ratio without deuterated (heavy) version of each analyte (light) kbrady  2019-10-08


The assay I am working with quantifies 5 metabolites of methotrexate (MTXPG1, MTXPG2, etc), but rather than using the deuterated version of each compound as internal standard for each metabolite, the method cheats a bit and quantifies MTXPG1 and MTXPG2 as the ratio of MTXPG1:MTXPG1-d3 and MTXPG2:MTXPG1-d3, respectively. MTXPG3-5 are quantified as the ratio of MTXPG3:MTXPG3-d3, MTXPG4:MTXPG3-d3, and MTXPG5:MTXPG3-d3, respectively. (Yes I realize this is less than ideal, but I did not make the method :) )

Following your advice here (, I formatted my transition list as shown in the attached translist.csv ('True Identity' and 'True Identity Purpose' columns are just for clairity, I'm not pasting those columns). Can skyline handle an experiement set up like this?

Also attached is the document grid generated by Skyline through Edit -> Insert -> Transition List. You can see Skyline has not kept the m/z as provided in the transition list, and has instead constrained 'heavy' MTXPG2, MTXPG4, and MTXPG5 to be similar in mass to the 'light' precursors. I am guessing you have some internal check along the lines 'A heavy transition must be heavier than the light transition it is associated with'? Is there a way around this? I would like to use light:heavy ratio for my calibration curve, but this currently only works for MTXPG1 and MTXPG3 (which are actually matched with a deuterated compound, unlike MTXPG2, MTXPG4, and MTXPG5).

kbrady responded:  2019-10-10

I have solved this and will post instructions soon.