Accounting for Matrix Effects

Discussions about GC and other "gas phase" separation techniques.

17 posts Page 1 of 2
I seem to have some type of matrix effect or something going on in the GC-FID analysis of a formulation I am looking at that seems to give me a consistent 75% recovery.

Using in-house methodology for HPLC UV-Vis, we don't see this matrix effect, so it is specifically related to GC-FID methodology.

I am looking to analyze Prallethrin in a liquid formula that contains some other compounds and a solvent system that is propylene carbonate, peg 400 and pluronic.

I guess my main question is what types of phonemena may be causing me to see a reduced area in both the active peak of interest as well as the internal standard (although the decrease in ISTD area is not nearly as great as the active of interest)? Especially phenomena that would be specific to GC-FID that I do not observe in HPLC UV-Vis?

My overall experience with analyzing more complex samples on GC-FID is significantly less than HPLC, so I'm not necessarily sure what I should look at first.

I have great resolution between Prallethrin and the ISTD (Triphenyl Phosphate), and all of the other components in the formulation elute well before the peaks of interest as well. I can share a chromatogram if necessary but it doesn't look like I'm seeing a lower area because of an issue with co-eluting peaks or a discrepancy between integrating nearby peaks properly.

Sorry this was a bit long-winded.
Like HPLC, GC can suffer from similar problems. In my case (HPLC) the analyte was only being partially retained until it reached a limit and was dumped in a blank.

In most cases Helium is the carrier case and you cannot manipulate its composition. It is the thermal conditions that ensure complete elution of your analyte. Try raising the oven temperature until you are assured it is completely eluted.

Also give us the exact chromatographic conditions? What is the boiling point of your analyte?
Your not the only one who has had issues! The EPA was not able to meet its own acceptance criteria DB5-MS column (30 m x 250 µm i.d., 0.25 µm thickness) and MS (Agilent 7890).

Maybe you are better off with an HPLC method!
HPLC chemist wrote:
Like HPLC, GC can suffer from similar problems. In my case (HPLC) the analyte was only being partially retained until it reached a limit and was dumped in a blank.

In most cases Helium is the carrier case and you cannot manipulate its composition. It is the thermal conditions that ensure complete elution of your analyte. Try raising the oven temperature until you are assured it is completely eluted.

Also give us the exact chromatographic conditions? What is the boiling point of your analyte?


Column: DB-1701 30x.25x.25 (Cannot change this parameter)
Inj vol: 1uL
Split: 100ml/min
Linear Vel. 35cm/s (helium)
Oven: 150ºC 0.5min
5ºC/min to 220ºC and hold 5min
3ºC/min to 275 and hold 13min
Injecor: 280ºC
Detector: 320ºC

Boiling point of prallethrin: 315.5ºC per pubchem.

I realize that the injector is well below the boiling point but I would imagine I should be getting a similar area count as the standard if the concentration in the standard and sample solutions are identical. I will be increasing the injector temperature as one of my troubleshooting ideas but I would just like to see what other's think.

Here is a chromatogram of the sample and standard overlayed. Black is the sample, blue is the standard.Everything separates and the active and ISTD are eluting with the same RT in the sample as the standard.

Image
HPLC chemist wrote:
Your not the only one who has had issues! The EPA was not able to meet its own acceptance criteria DB5-MS column (30 m x 250 µm i.d., 0.25 µm thickness) and MS (Agilent 7890).

Maybe you are better off with an HPLC method!



Unfortunately for some international reasons, I am forced to follow GC-FID analysis. As I mentioned we use an internal HPLC method for US and internal analysis.

The basis of my method is the Prallethrin GC analysis by

CIPAC.http://www.cipac.org/index.php/p21/460-prallethrin-743

So my hands are tied in regards to being forced to do a GC-FID analysis as well as limiting the parameters I can change before I have to do a full blown inter-laboratory collaborative study (which I am trying to avoid due to the cost involved and our timeline won't allow it).
I can't seem to get actual method details from the link that you supplied

Is your sample prep an extraction of the sample or a complete dissolution of the sample in your solvent?

In the former case the Pluronic surfactant may be affecting the partition into the solvent compared to your external standard.

Have you tried the "standard addition" method of adding target analyte to your sample to overcome possible matrix effects?
Regards

Ralph
This is almost certainly due to a differenc ein matrix between samples and standards. Do you standards have all the carriers and excipients etc that are in the samples ?

Peter
Peter Apps
Peter Apps wrote:
This is almost certainly due to a differenc ein matrix between samples and standards. Do you standards have all the carriers and excipients etc that are in the samples ?

Peter


No, but if this product is analyzed internationally in international labs, those labs will not have access to all of the inerts and excipients and other formulation ingredients to prepare their standards in the matrix. My concern is that I want to be able to produce results that can be reproducible by other labs.

With this in mind, I do like GOM's idea of standard addition. This is not something that I have thought of doing and it might solve my problems.
GOM wrote:
I can't seem to get actual method details from the link that you supplied

Is your sample prep an extraction of the sample or a complete dissolution of the sample in your solvent?

In the former case the Pluronic surfactant may be affecting the partition into the solvent compared to your external standard.

Have you tried the "standard addition" method of adding target analyte to your sample to overcome possible matrix effects?


If you would like to see the exact method I can e-mail it to you if you want to provide me with an e-mail.

With that in mind, the sample preparation is a simple dilution of the sample in Acetone. The formulation is liquid, so it's 13g of sample diluted to 100mL in acetone.

I have not tried standard addition, I will need to check with our contractor that deals with CIPAC and discuss with him whether this type of change would be acceptable without the changes being considered a "major modification" to the methodology.
That is useful information.

I understand your concerns with regards to matrix matching your external standard

I would like to see the full method out of interest.. Thank you

Try tailingpeak@gmail.com --- only used for details that cannot be easily posted on the forum

I would prefer to keep discussions confined to the forum for all to share, learn from and to contribute.

Let us know how you get on with the standard addition approach :-)
Regards

Ralph
GOM wrote:
That is useful information

I would like to see the full method out of interest.. Thank you

Try tailingpeak@gmail.com

However, I would prefer to keep discussions confined to the forum for all to share, learn from and to contribute.

Let us know how you get on with the standard addition approach :-)


Unfortunately while the standard addition approach would probably work well, I had a discussion with our consultant that deals with all of our CIPAC work, and he suggests doing that type of change to the methodology might not be accepted as easily. My first action is going to crank up the injector to 325ºC and see if that helps with my recovery.

I have sent you the methodology but so the rest of the forum is clear on the accepted preparation:

Standard preparation: Make a 1mg/mL standard in acetone.
Sample preparation: Weigh out enough sample that contains 1mg/mL target analyte and dilute in acetone.

The standard and sample preparation in the accepted method are very simple and straightforward. Unfortunately this means that even something fairly straightforward, such as standard addition, could be considered a "major modification" and thus would require an inter-laboratory collaborative study while something as simple as increasing injector temperature would be considered an acceptable "minor modification" and would allow me to perform the analysis without any further trouble.

It's more so that I need to try to work around the methodology that I need to try to already adhere to with as little alteration as possible, while still getting acceptable results. In many cases here my hands are going to be tied, and with my lack of dealing with matrix effects in GC-FID, I turn to the forums to discuss possible solutions.

Another idea I have is to screen different solvents to see if any of them have an impact on the matrix effect.
Why not determine the recovery and correct for it ? - as long as it is repeatable the results will still be accurate and precise.

Peter
Peter Apps
Peter Apps wrote:
Why not determine the recovery and correct for it ? - as long as it is repeatable the results will still be accurate and precise.

Peter


That's another option. My recovery must be 90 - 110% based on the guidelines for the concentration I'm looking at. I don't know if this recovery is "as is" or if I can apply some type of correction factor. I have a meeting with my consultant on Wednesday to discuss this sort of thing. That's obviously another route as my repeatability is well below acceptable limits.
To provide an update: I have switched to a DB-WAX column (much different than the DB-1701 I was using previously) and now my active peak has very consistent areas that are in line with my standard injections.

Now I have a new problem that, every injection (whether it's from the same vial or from the next vial) has a continuously increased ISTD area, about a count of 10000 per injection - which is clearly affecting my recovery.

I am using triphenyl phosphate for my ISTD.

Oven is isothermal at 245C and injector is 325C.

Aside from possible carryover from previous injections, what could the reason be for a constant increase in area count of the ISTD? My standard injections had consistent areas for the ISTD - so it seems to be sample related.
You will have to select a different ISTD since the boiling point of is far above 245 Celsius. Thus, you are not getting complete volatization and elution. See attached;

https://www.google.ca/search?source=hp& ... dmwMIJnPlA

In addition check website applications for what you are doing. Like www.restek.com , www.agilent.com .
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