Maximum concentration for LC/MSMS !??????

[catlover]

Hi everyone, it's me again >^<
I am currently doing pesticide analysis via LC/MSMS, it's petty easy to do the MS tuning.
The main problem is that the calibration is a kind of not quite stable...
I already finish 7 pesticide standard, and 3 of them is not quite good...geez...
Those 3 pesticide are Fenitrothion, Quinalphos and Diazinion.

For Fenitrothion and Quinalphos, both the MS tuning is quite ok.
(one is around 10^4 respond, another is 10^5 respond)
The calibration is okay from 10 ppb to 50 ppb, however,
peak area of 100 ppb sample drops in those 2 standard,
peak area drop more obviously in higher concentration (150 ppb and 200 ppb).

I think it may due to decomposition at the first place but it seems not quite reasonable.

And for Diazinon, ah...I really get lost of what I should do,
the respond is very nice and give signal up to 10^7,
I predicted that the calibration can be easily done,
but sadly, the signal doesn't vary much with concentration.
Is it related to selective problem?
Should I just accept that calibration because the signal does increase with concentration?????

By the way, the system I used are Waters UPLC with Waters TQ detector.
Mobile phase is mainly around 85% ACN : water with buffer and acetic acid.
I will further optimize the flow rate after the individual calibration....

I need to thank you all first~! Because I know I must learn something here!!
I love this forum and am glad to be one here.

[James_Ball]

I have used and ABI3200 for those compound a few years ago. Did you try optimizing using infusion coupled with a flow of the mobile phase at the normal flow rate of the analysis or just the flow from the infusion syringe? Sometimes when coupled with mobile phase flow the sensitivity changes.

ESI is affected by analyte concentration, so maybe you are exceeding the ionization potential for those analytes as the concentration increases.

With the Diazinion being flat for several levels before increasing, could it be you have an interference at low levels? Not so likely with MRM but not impossible.

[Camisotro]

Hi everyone, it's me again >^<
I am currently doing pesticide analysis via LC/MSMS, it's petty easy to do the MS tuning.
The main problem is that the calibration is a kind of not quite stable...
I already finish 7 pesticide standard, and 3 of them is not quite good...geez...
Those 3 pesticide are Fenitrothion, Quinalphos and Diazinion.

For Fenitrothion and Quinalphos, both the MS tuning is quite ok.
(one is around 10^4 respond, another is 10^5 respond)
The calibration is okay from 10 ppb to 50 ppb, however,
peak area of 100 ppb sample drops in those 2 standard,
peak area drop more obviously in higher concentration (150 ppb and 200 ppb).

I think it may due to decomposition at the first place but it seems not quite reasonable.

And for Diazinon, ah...I really get lost of what I should do,
the respond is very nice and give signal up to 10^7,
I predicted that the calibration can be easily done,
but sadly, the signal doesn't vary much with concentration.
Is it related to selective problem?
Should I just accept that calibration because the signal does increase with concentration?????

By the way, the system I used are Waters UPLC with Waters TQ detector.
Mobile phase is mainly around 85% ACN : water with buffer and acetic acid.
I will further optimize the flow rate after the individual calibration....

I need to thank you all first~! Because I know I must learn something here!!
I love this forum and am glad to be one here.

Re: Your mobile phases, when you say it's mainly around 85% ACN:water "with buffer" and acetic acid... what is the buffer? What is your injection volume?

What is the solvent for your standards? Diazinon looks great (quant 305.1>169.1, qual 305.1>153.1) when I calibrate it from 1-125 ppb in ACN with 0.1% formic acid (yeah you shouldn't inject a strong solvent in RPLC, but the autosampler adds some 0.1% FA/water before injection). However we once received a water sample to analyze for a few including diazinon, so I prepared some calibrations in 0.1% FA - I found the diazinon degraded significantly over the timescale of the LC run.

Quinalphos I have not had any trouble with, from 4-500 ppb.

Fenitrothion elutes as three peaks for me, probably because tech-grade fenitrothion includes proper fenitrothion ROP(=S)(OMe)2, as well as isomers ROP(=O)(SMe)OMe and RSP(=O)(OMe)2. And apparently AccuStandard's fenitrothion is the same. We calibrate it from 10-1250 ppb and we have to set the integration parameters to integrate all three peaks as one or it'll end up inconsistent.

Re: Signal not varying much with concentration - what happens when you inject blanks? Do they still look like blanks, or do you still appear to get a strong signal for diazinon?

[catlover]

The buffer I used is ammonium acetate, injection volume is 5 microlitre.
I used ACN as solvent for all standards.
BTW, I would like to know if you means that diazinon will degrade in water sample???

I tried to use lower CE for MRM of diazinon, then the calibration is done well.
My adviser said it is because the signal is really high and MS is "saturated" on detecting the analytes.
I think it is the same problem for Quinalphos.

For Fenithrothion, I will tried to analyze it once more with new integration setting because it is now set as auto-integration and I don't know if it affect the results.

Anyways, thank you for your response!!
I'm sure it will help me a lot~!

[Camisotro]

The buffer I used is ammonium acetate, injection volume is 5 microlitre.
I used ACN as solvent for all standards.
BTW, I would like to know if you means that diazinon will degrade in water sample???

I tried to use lower CE for MRM of diazinon, then the calibration is done well.
My adviser said it is because the signal is really high and MS is "saturated" on detecting the analytes.
I think it is the same problem for Quinalphos.

For Fenithrothion, I will tried to analyze it once more with new integration setting because it is now set as auto-integration and I don't know if it affect the results.

Anyways, thank you for your response!!
I'm sure it will help me a lot~!

My experience is that in 0.1% formic acid (aqueous), diazinon degrades rapidly enough that you can't do a calibration curve. It is stable in 0.1% formic acid with acetonitrile though. I don't know if the same happens in neutral water.

Saturated signal may indeed be a problem as well. Detuning the collision energy to reduce signal is a reasonable strategy, as long as you still get enough sensitivity to see all the levels you want to see.

[catlover]

Again, thank you so much for your reply.
In fact, I still have questions on Quinalphos case, I tried reducing CE, Cone voltage, resolution but still the signal drop in 150 ppb standard.
However, interestingly, calibration is done perfectly with R^2 =0.99 after preparing new standard sets from 1 ppm stock.
It is the same with Fenithrothion.
I am still figuring out why....
Anyways, I will try mixing all standard to check if the separation (LC) done well and may play with gradient setting.
Hope everything will be alright. =]

[BHolmes]

Saturated signal may indeed be a problem as well. Detuning the collision energy to reduce signal is a reasonable strategy, as long as you still get enough sensitivity to see all the levels you want to see.

How reasonable is it to detune a compound so that signal is not at saturation level? Why not just make a new mix with lower concentrations?