Chromatography Forum

Hi I am just getting up to speed with the technique of LC-MS-MS which I am using to separate and detect malachite green and its leuco metabolite.
Could anyone give me some pointers on tuning. I have established a hplc separation and I am using a C18 3.9 X150 mm i.d column. Flow rate is 0.6 ml/min using gradient mobile phase of ammonium acetate:acetonitrile.
The retention time of malachite green is 3.5 and of leuco malachite green in 7.2 mins (also retention time of deuterated leuco malachite green which I am using as internal standard( maybe to check sample preparation procedure aswell).
Where to from here, just trying to get a logical plan of action!!
G
Bit embarrassed to ask such a basic question but have to start somewhere

Hi Gerrie,

Since you are doing MS/MS you have to try to check which precursor ion and the product ions to use. Normally what one does is to infuse the compound mix into the MS (dissolved preferentially in the mobile phase to be used). Run a single MS scan to determine which precursor ion is the best. Not always the M+1 or M-1 are the best ions. After that run and MS in single ion monitoring using the best mass found previously followed by a second MS in scan mode to determine the best product (daughter) ion. After having your "mother/daughter" pair you can start optimising the instrumental parameters for better sensitivity.
This is one way of doing it.

I hope you understood what I was trying to say.
Regards
JNF

JNF, I will try that thank you for your help. I will let you know how I get on

Both leuco and malachite green (MG) "light up" in positive ESI mode on LC-MS. To custom tune (almost not necessary...) insert a Tee between your column and MS. Run your mobile phase into MS while infusing analyte (usually 10ug/mL) at around 10uL/min. Leuco shows up in MS1 at m/z 331 (M+H+); MG at 329 (M+--it's already ionized). The specifics of custom tuning will depend on your instrument. Generally, you can start with a tune file that works for your calibration solution and modify it. Your manual will have directions for doing this. Your LC flowrate is high, so you should probably adjust your capillary temp and gases first.

The trick with leuco and MG is getting them to fragment--all that conjugation makes them very stable. Leuco will produce an m/z 216 (and almost nothing else) in an ion trap, but MG hardly fragments at all.

I'm pretty sure there's published LC-MS-MS methods for these drugs.

Who is the supplier of your deuterated MG?