increasing sensitivity on lc-ms/ms for lower detection limit

[wigette]

Hello,

I am new to lc-ms/ms but I am working on a method development project in hopes that I can increase the detection limit of a certain compound that is part of a group of compounds.

The method is currently done with gc-ms but I am trying to switch it to lc-ms/ms while hoping to lower the detection limit.

I have been working on optimising the run conditions for the 6 compounds that I am working with for now: CV and CE voltages, a good additive for the mobile phase, a good gradient program (for good separation of peaks as they elute fairly close together), dwell time, etc. Now, I am trying to make a good calibration curve (with internal standards) with 8 different concentrations points. I am not seeing peaks at the lower concentrations (which is the range that I need to see it in to meet the goals of this project). With this, I am also trying to decide what is the best concentration of the internal standards? I am not sure if I should try to keep it within the range of my 8-point concentration series or if I should go higher?

My main concerns right now are what I could be trying or changing to try and increase the sensitivity of detection at the lower concentrations?

I would really appreciate ANY input. Thank you very much for your time!!

[Alp]

It would help to know where the analytes are coming from (matrix, urine, blood, water, soil, food....) and the method of analyte isolation or extraction. Liquid/liquid, SPE, dilute and shoot....
There are many parameters to consider in lowering the LOQ of a method.

What you want to do is increase instrument sensitivity, increase ionization effeciency of your analyte or increase the amount of analyte reaching the instrument (or all three).

If you reconstitute the sample, it might be possible to lower the volume used to reconstitute, thereby increasing the concentration of the injected (assuming HPLC or such) sample. You could modify your method to allow for a larger starting volume (of blood, urine, water, soil....).

If you are running HPLC or such you can try a narrower bore column.

Is the lack of signal at lower concentration due to ionaization suppression? if so a cleaner extract might help. Maybe it is due to non-optimal sample prep and analyte is "sticking" somewhere or maybe getting washed away in a step.
Are you using SPE, liq/liq, dilute and shoot or some other form of extraction/sample prep?

Do you have the correct/optimal instrument settings, precursor/product ion etc?

Many things to consider.

Alp

[Camisotro]

Also, when you selected an additive for the mobile phase - did you optimize it for MS sensitivity (or signal-to-noise) first and then try and make it work in LC-MS? Or did you select it based on the LC separation you could get? When you're aiming for the best LOD possible you may want to start with the former and then see where that leads you separation-wise.

As long as your compounds' MS/MS signals don't exhibit crosstalk (e.g. compound A elutes and you see an echo in the MRM channel for compound B), then the separation between compounds is not as important as a good separation of all compounds from the matrix effects.

[wigette]

Thanks so much for your reply Alp.

Currently, I am only directly injecting diluted standards onto the machine, to try and optimize the conditions of the instrument. The matrix will be surface/ambient water. The current method uses liquid-liquid for the unfiltered samples and SPE for the filtered samples. When I do start the extraction optimisation, I will start with liquid-liquid on spiked water samples.

Lowering the reconstitution volume is a good idea, although currently the volume is low (200 ul) and I am not sure how much lower I will/can go.

Being new to LC-MS/MS and instruments in general, I am unsure of all the different parameters that I can play with while trying to optimise instrument sensitivity. I believe that the CE and CV voltages are pretty good as is the dwell time for the transitions. I have chosen two transitions for each of the compounds and they seem to be working. This is an MRM method. I am working with ESI-negative ionisation. I have chosen a basic (pH8) additive, which is 4-methylmorpholine for the mobile phase. I am using a extend-C18, 2.1 x 100mm (3.5 um pore size) column.

Is there something else here that I could be playing with to try and improve the instrument sensitivity to these compounds?

Also, is there something that could be affecting the reproducibility of the injections? I am trying to get a good calibration curve and it is not good enough and I am not sure what I could be improving here.

And thanks so much to you ctroster.

When I chose the additive, I chose it based on MS sensitivity, and after this, I have been working on LC separation. I am reasonably happy with LC separation at this point.