The problem with falcarinol is that the peak showing up does not make any sense to the molecular weight. For falcarinol, I have 157, 254, 276, 322, 354 m/z in positive mode (water/MeOH), and the molecular weight is 244. Even in the literature, the MS data varies a lot. It seems every paper has its own set of values and none of their meet mine.

I have analyst. Should I use Q3 for the optimization or use the MRM mode?

This not a good ESI compound. Its proton affinity is low. Try to make ammonium adducts with amm formate or acetate. APCI or HePI should work better.

I had issues with similar compounds on an API 4000. I know it might not be the answer you were looking for, but I would use GC/MS. The hard ionization will produce fragmentation that is more consistent, and probably higher magnitude.

Yes, I agree with Jonathan that it will be hard to quantify this type of compound on your API2000. Certainly you have an APCI probe, which could be better for ionisation of the compound. Don't hesitate to infuse a higher concentration, and to try different solvent for this infusion (MeOH, Meoh with formic acid, with acetic acid, 0.1%, 1%). I agree this Pawel that's you will possibly just observed the M-H20. Hope this help. Fabrice

Thanks everyone for helping me with my problem. I guess I will have to try everything I can and work with the instrument I have. I will update my finds asap

Do you use any material cleaned with detergent? This could contaminate your solutiong making adducts appear easily. Try to clean all the stuff with MeOH:H2O.

As it was said before, adding formic acid should be a solution.

You definitely would be better of with APCI than with ESI. Forget negative ion. Use 1% formic acid in the mobile phase. Manual optimization is essential. Optimize until you get a good spectrum for both standards on Q1. Don't fuss with the chromatography, just get your compounds to the MS. Once you set MRM up properly, chromatographic separation will not be necessary. This is doable, but definitely not a "piece of cake". It took me a month to get a similar MRM analysis going with a Shimadzu HPLC interfaced through APCI to an API 3000 triquad running Analyst. Good luck.

Hi John,

Do you mean 1% FA or 0.1% FA? I will have to run LC/MS for the crude plant extracts that contain the two compounds, so I need to at least separate the two compounds. Also, the C17 diynes are so unstable, and formic acid can change the compounds before they even reach MS. That's always my concern with using buffers.

I agree with John, that you need to isolate these compounds or order standards and optimize the MS by itself. The API-2000 MS/MS I used to run had a direct infusion pump on the side that I could access through the tuning menu of Analyst 1.5.2 (assuming you first had chosen the instrument configuration with the infusion pump rather than my HPLC). I'm assuming you've optimized the Q1 scan conditions for the flow you are running into the source (not more than 0.5 mL/min, though 0.2-0.3 mL/min seems to be a good place), meaning the curtain gas, nebulizer gas, temperature, etc, but you may be having problems with the stability of the compound. Directly infuse the compound in solvent and/or solvent:buffer mix appropriate to the HPLC eluting conditions and look into starting the temperature low and ramping up to see how the signal/sensitivity changes, also try the same technique with the ionization power.

Once you have the source conditions optimized through a direct infusion (finished with the solvent:buffer mix) move onto performing an MS/MS experiment with Q1 selecting the parent ion and Q3 scanning to see the daughter ions. You can use the compound optimization tool (also found in the tuning menu) and let the computer run through an optimization, but be warned that Analyst (1.5 at least) performs this test using the default source conditions. The other option is to optimize yourself, doing an MS/MS experiment and ramping the collision conditions to change the fragmentation patterns and ion levels. If you are able to make this all work and select some possible MRM transitions, run multiple when you are quantifying so you can bring down your signal to noise ratio.

Finally, as the API-2000 has the flow pointed at the curtain plate and not perpendicular, as the later API series instruments have, make sure your source stays clean as you go through the concentrated buffers being suggested above. There might be some good ideas there, but that source can be a pain to keep clean. The other thing mentioned above that I would try is running a APCI if you have that source since the ionization conditions are gentler. Otherwise, look into performing some derivitization to enhance the ionizability of falcarinol, I like this paper Santa, T.; Al-Dirbashi, O. Y.; Fukushima, T. Drug discoveries & therapeutics 2007, 1, 108–18. as a place to start.