LC-MSMS, MRM method for reserpine [Solved, thx you all>^<]

[catlover]

Hi everyone,

I am having training on LC-MSMS and need to get familiar with developing MRM method.
I followed some research papers related to MRM method for reserpine, but somehow there is no peak shown (or say the noise is quite high?)

Here is my setting of MRM, (using Waters TQ detector)
Ionization mode: ESI+
Cone: 35 V
Capillary: 3.5 kV
Collision energy: 35 eV
Parent ion: 610
Daughter ion: 195
Desolvation Temp: 350
Desolvation gas: 650
Cone gas: 50

Since my adviser only give me few mL of samples of 500 ppt, so I guess it is impossible to have autotuning.
In these days, I tried to start with MS scan, followed by daughter scan and parent scan.
But in the MS scan, there are somehow so many other peaks around 700....

I will try my best doing the MRM method, but still would like to discuss and learn more here.
Please kindly reply. Thanks~

[Loekie]

Hi Catlover,

The molecular formular of reserpine is C33H40N2O9 giving a monoisotopical macular weight of 608 Da. so for the protonated species you might want to use the 609 ion for your precursor ion and not the 610 ion. 500 ppt might not be enough for a full scan method like the Q1ms, product and precursor ion scans.

[lmh]

Reserpine is very cheap and easily available, and it's also the standard chemical used by mass spec manufacturers to assess sensitivity, which means you could probably get a set of optimised conditions for it by phoning the manufacturer of your instrument! Don't let your adviser get away with giving you too little; to optimise it yourself you will need to be able to infuse either from a syringe or from the instrument's fluidics if present (I notice you're using Waters equipment?), which needs more material than if you're merely injecting a typical sample. Also Loekie is quite right that for full scan mode you'll need a lot more than you'd need to detect a peak by a SRM method.

[catlover]

Reserpine is very cheap and easily available, and it's also the standard chemical used by mass spec manufacturers to assess sensitivity, which means you could probably get a set of optimised conditions for it by phoning the manufacturer of your instrument! Don't let your adviser get away with giving you too little; to optimise it yourself you will need to be able to infuse either from a syringe or from the instrument's fluidics if present (I notice you're using Waters equipment?), which needs more material than if you're merely injecting a typical sample. Also Loekie is quite right that for full scan mode you'll need a lot more than you'd need to detect a peak by a SRM method.

Yes, there is a fluidics for optimization, I learn how to use it.
The syringe volume is 250 microlitre, so is it possible to success if a low volume (say 3mL) but higher concentration (ppm) standard is used?
I will have a try today (finally get a 5 ppm reserpine ), and I would like to reply with my result tonight~

[catlover]

I finished the tuning by myself, I am sure that there is a 609.38 prent peak and it gives both 195 174daughter peak. I also try autotuning in the Intellistart, but it stated that no parent ion is found.
I am going to try the Intellistart again, but if it keeps fail, I will start the analysis to see if my tuning is ok~

By the way, there is somehow always a peak in the blank around 0.4 mins. I tried to wash the column and needle, also tried to load the blank several times. But the peak is still there...

[lmh]

My personal opinion is still that you need a more concentrated sample.

With the fluidics, you can either infuse directly, or combine with the LC flow. From the point of view of optimising the tune file, it is best to combine with the LC flow because this mimics the situation during a real chromatography run. You can choose an LC flow representative of where the analyte should elute (if you are using a gradient, and don't know the likely retention time, it probably makes sense to pick a mixture about half-way up the gradient). But combining with LC flow dilutes your sample drastically; say 5uL per min from fluidics into 500uL per min from LC is a 100-fold dilution, so if your adviser gave you 500ppt you now have 5ppt. It's hardly surprising Intellistart couldn't find a parent ion!

If you infuse directly, you will get a bigger signal, but it's not the same as chromatography and needs lower gas flows and temperatures, so the tune file you get will need modifying to suit chromatography.

Now, does it matter about the parent ion? If Intellistart couldn't find it, either you gave Intellistart the wrong mass (remember, Intellistart wants you to type the neutral mass, and expects to add adduct masses, so if, like me, you tend to remember the mass of the ion you expect to see, it's easy to type the wrong thing in!). Or Intellistart didn't find it because it's too dilute. If the latter, then how sure are you of your 609 parent peak? If it doesn't disappear when you turn off the fluidics, it may be a background ion and not reserpine, in which case you might be setting up an MRM to detect a background ion... this is one reason why Intellistart wants to see a good parent ion. You can, of course, find out: set up the chromatography with your manual MRMs, and run blanks and standards and check that the standards have a good peak that is absent from the blanks. If you are setting up a method by direct infusion without combining with LC flow, you cannot tell if the ion is background or sample, because if you stop the fluidics, nothing is going into the spray chamber at all, and the background stops too.

But I still think that the fact Intellistart won't help is a sign that you're trying to work below the concentrations the manufacturers expected to be available for a good set-up of the instrument, and this is bound to influence the quality of the method you develop.

[Camisotro]

For analytes of good sensitivity, 500 ppt can definitely be observed in MRM mode, but is typically lost in the noise in full-scan. I usually start with 250-1000 ppb for full-scan, and then once I know what I'm seeing and where to see it, I can go lower.

Also sometimes you can put the horse before the cart... if you know a mass transition that will work, you can jump right in to MRM with a few guesses for the required energies, optimize the energies, and then backtrack to product ion mode, and backtrack to full scan.

[catlover]

I really need to thank you all of your responds, it does help me optimizing MS method.
It is a really nice experience to learn here~ Thank you so much!!! >^<

Well, back to the topic. I tried as what you all said, I got a 5 ppm reserpine and tried the tuning (infusion mode because I did not get the rely yesterday morning, sorry).
Then, I changed the solvent composition from 75%H2O in ACN to 1:1 H2O : 0.1% acetic acid in ACN.
Finally there is a very strong peak observed at 0.51 mins (because I use 2 x 50 mm column, so the retention time is mostly quite short~)
I guess the problem is the solvent composition, I will try calibration and may be also try the MS scan, parent scan and daughter scan to double check my result if I have time~

Next week, I have to really get into my project!! Hope everything will be ok!!

[Camisotro]

I really need to thank you all of your responds, it does help me optimizing MS method.
It is a really nice experience to learn here~ Thank you so much!!! >^<

Well, back to the topic. I tried as what you all said, I got a 5 ppm reserpine and tried the tuning (infusion mode because I did not get the rely yesterday morning, sorry).
Then, I changed the solvent composition from 75%H2O in ACN to 1:1 H2O : 0.1% acetic acid in ACN.
Finally there is a very strong peak observed at 0.51 mins (because I use 2 x 50 mm column, so the retention time is mostly quite short~)
I guess the problem is the solvent composition, I will try calibration and may be also try the MS scan, parent scan and daughter scan to double check my result if I have time~

Next week, I have to really get into my project!! Hope everything will be ok!!

For a 2.1 x 50 mm column, the theoretical void volume is 173 uL. What is your flow rate? If it's, say, 400 uL/min, then reserpine is unretained. Which is fine for just the sake of MRM development, but if you are going to do any actual chromatography you'll want retention. That means trying higher aqueous composition until the retention time increases, and/or using a longer/more retentive column.