Dwell time, Cycles in LC-MS/MS

[phamkhoa2105]

Dear,

Could you please help me explain the "dwell time" and the "cycles" in Multiple Reaction Monitoring Mode in LC-MS/MS?

And, how do they affect the analytical result?

In addition, could you show me how to determine the accurate dwell time and cycles?

Thank you so much.

[Rndirk]

Hi,

Welcome to the forum.

A peak that shows up in your chromatogram consists of data points which are connected by a line. If you have 3 data points over 1 peak, it will look like a distorted triangle. This is not enough for quantitative integration. On the other hand, 50 data points over 1 peak with give you a "noisy" peak with too much features. You see that there a optimal amount of data points over 1 peak: you want somewhere between 12-20.

With single or triple quadrupole MS detection, you typically want to look at more than 1 ion (transition) at a given moment. Your instrument can not measure them at the same time, it has to switch its filter (quad) rapidly between different masses. Here's where dwell and cycle time comes in.

Assume you want to measure 5 transitions over a certain period (also called windows).

Dwell time: the amount of time it measures 1 transition. Suppose you set this to 50 msec for each transition

Cycle time: The time it takes to aquire all transitions. This is 250 ms (50 x 5). (Note this is actually a little bit higher because it takes a few ms to switch between transitions).

What you should do with this information is optimize the amount of data points over your peak and give your instrument a minimal amount of dwell time per transition. Minimal dwell time is instrument dependent. For quantitative work, i use a minimum of 10 msec.

In the example above, it takes 250 ms for 1 cycle, so 5 transitions are measured once each during this time, then the cycle repeats. Each second, the cycle repeats 4 times (1 second/0.250 seconds). This means, if component X starts eluting at second 500 in your method , and ends eluting at second 503, it will have 3 sec x 4 = 12 data points over its peak.

The peak width is an important factor here, and depends on a lot of parameters in your method. You should check what peak widths you have and change the settings accordingly. In a given window, you want to optimize the settings for the narrowest peak. For example, for a narrow peak of 2 seconds, you want to lower the dwell time in the example above to 40 ms / transition (total dwell time 40*5 = 200ms), so you have 5 cycles per second => 10 data points over a 2 second peak.

Practice: You want to measure 8 components. Narrowest peak is 2.5sec. How much dwell time per component to get 12-20 data points per peak?

[phamkhoa2105]

Dear Rndirk,

First of all, I would like to appreciate you who gave an intensive explanation for this topic.

I am willing to give you my answer for your question.
As to 12 data points per peak, we need to set up the dwell time for each ion (transition) as follows:
- the number of cycles per second: 12 : 2.5 = 4.8 cycles/second
- cycle time for 8 components : 1 : 4.8 = 0.208 s = 208 ms
- dwell time for each transition: 208 : 8 = 26 ms
Similarly, we need to set up the dwell time of approximately 16 ms for each transition to get 20 data points per peak.

In conclusion, to measure 8 components, we need to set up the dwell time of 26 ms through 16 ms for each transition to get 12-20 data points per peak.

[Rndirk]

Dear Rndirk,

First of all, I would like to appreciate you who gave an intensive explanation for this topic.

I am willing to give you my answer for your question.
As to 12 data points per peak, we need to set up the dwell time for each ion (transition) as follows:
- the number of cycles per second: 12 : 2.5 = 4.8 cycles/second
- cycle time for 8 components : 1 : 4.8 = 0.208 s = 208 ms
- dwell time for each transition: 208 : 8 = 26 ms
Similarly, we need to set up the dwell time of approximately 16 ms for each transition to get 20 data points per peak.

In conclusion, to measure 8 components, we need to set up the dwell time of 26 ms through 16 ms for each transition to get 12-20 data points per peak.

That's right!

Note that depending on your instrument, the software calculates either the cycle time as cycles/second or as sum of dwell time, or both. Like i said it adds a couple of ms to the sum of dwell times (to switch between them). Also, the dwell times of your different components doesn't need to be identical.

To answer your original question how it affects your analytical result: too few data points are worse than too many. There's a large range where fine-tuning will have minimal impact on your quantitative results.

[phamkhoa2105]

Dear Rndirk,

I use AB SCIEX Triple Quad 3200 (ESI-QQQ). I do not know whether the software I use is which types you mentioned.

I actually want to attach the image which shows the interface in Multiple Reaction Monitoring Mode (MRM Mode) but I could not do it, although I tried several times.

Therefore, I will describe the interface to you. There are the two main regions in MRM Mode. The first region contains Q1 Mass(Da) > Q3 Mass(Da) > Time (msec), the other one called "Period Summary" includes Duration (min), Delay Time (sec) and Cycles.

Many thanks.

[Rndirk]

I'm not familiar with the software of that instrument, but i think the time (msec) should be the dwell time, and the cycles probably cycles/second. Are you not sure what to change? Try it out and see what values it gives.

[James_Ball]

Dear Rndirk,

I use AB SCIEX Triple Quad 3200 (ESI-QQQ). I do not know whether the software I use is which types you mentioned.

I actually want to attach the image which shows the interface in Multiple Reaction Monitoring Mode (MRM Mode) but I could not do it, although I tried several times.

Therefore, I will describe the interface to you. There are the two main regions in MRM Mode. The first region contains Q1 Mass(Da) > Q3 Mass(Da) > Time (msec), the other one called "Period Summary" includes Duration (min), Delay Time (sec) and Cycles.

Many thanks.

If you are using Analyst 1.6 or earlier software on the 3200 then you set the dwell time and it calculates the cycle time adding a few ms between each transition when doing MRM. (This is the instrument I have)

If you have enough time between peaks you can set up different periods with different transitions to reduce the number of transitions in each period to allow you to increase the cycles/second for better results.

If you must switch between negative and positive modes it can be done using Periods and not cost you on cycle time, but if the peaks are close enough together that you need to do it simultaneously you have to make two Experiments within the same period and the switch in polarity will cost you almost 500ms per cycle. I do that now for one test and it works because the peak widths are wide enough to still get enough scans across a peak, but if you have really narrow peaks it would not be a good idea.

[phamkhoa2105]

Dear James_Ball,

You meant when peaks were separated enough well, we could use the "Scheduled MRM".

In this mode, there are 3 variables including expected retention time (min) for each ion pair, MRM detection window (sec) and target scan time (sec) (target amount of time for each cycle during scan) which we need to set up.

For example, I have to monitor 3 transitions which have retention times of 1.2 min, 1.5 min and 1.9 min, respectively for each ion pair. Could you please show me how to set up the 3 variables above?

Many thanks.

[mhr311]

http://www.basinc.com/library/presentat ... index.html

Use of Noise Parameters to Optimize Data Collection Rates...

[James_Ball]

Dear James_Ball,

You meant when peaks were separated enough well, we could use the "Scheduled MRM".

In this mode, there are 3 variables including expected retention time (min) for each ion pair, MRM detection window (sec) and target scan time (sec) (target amount of time for each cycle during scan) which we need to set up.

For example, I have to monitor 3 transitions which have retention times of 1.2 min, 1.5 min and 1.9 min, respectively for each ion pair. Could you please show me how to set up the 3 variables above?

Many thanks.

I have never used the scheduled MRM, I have only used the Period and Experiment settings after determining retention times to break up the run into segments. Are you using Analyst or Cliquid software?

[phamkhoa2105]

Dear James_Ball,

I have used Analyst Software. And, in MRM mode, there are two options to choose Scheduled MRM Mode or MRM Mode. In Scheduled MRM, we can set up the parameters I mentioned in the previous.

There are the two main regions in MRM Mode. The first region contains Q1 Mass(Da) > Q3 Mass(Da) > Time (msec), the other one called "Period Summary" includes Duration (min), Delay Time (sec) and Cycles.

Which one did you mean?

Many thanks.