Acrylamide by HPLC-MS/MS

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

8 posts Page 1 of 1
Dear all,
I need your ideas and help with the analysis of acrylamide in potato chips by HPLC-MS/MS. I managed to have an acceptable chromatogram with an Zorbax RX-C8 employing an isocratic method with 98% of acidified water (1% of acetic acid). However, I am not able of finding a suitable extraction protocol which allow me to achive limit of detection of 300 ug/kg set up by teh European legislation. I have tried diferent extraction methods, with cartridged without cartridges, .... :roll: :roll: :roll:
I have also derivitized the acrilimadide, this method was very succefull as it increase considerably de signal however I detected acrilamide everwhere :twisted: :twisted: :twisted: .... I don't know if it is because i used plastic bottles and plastic pipettes.


Lookign forward for your ideas and suggestions :idea: :idea:
Which instrument do you use? I would advice not to use 1% acid in your mobile phase for LCMS. As a rule of thumb stay below 0.1%.

We report acrylamide from 30 µg/kg on dry food (potato (chips), fries,..). I can give you some general tips and keep an eye on this topic if you have more specific questions.

- We found APCI(+) to give a better signal than ESI(+)
- Use an internal standard. Deuterated acrylamide is cheap!
- We use a simple extraction with water followed by protein precipitation and filtering
- Optionally you can do an LLE cleanup with dichloromethane/hexane to remove fat (we do this for fries)
Thank you very much for your reply Rndirk,

The instrument I use is an HPLC 1100 from agilent and a API Qtrap 2000 from Applied Biosystems. The ionization mode is ESI, the mobile phase is based on EU method, I can changed to 0.1% with no problem, this is a simple solution.

Which internal standard do you recomend? The one I found it cost around 300 €/5 mg, it is a bitte expensive for us.

I have tried a simple extraction with water but the signal intensity is very low to be able to achive a good LOD.

If you prefer you can send me a private email with all your recomendations, I will be more than happy to try everything.

Thank again,
Carol
Product 72834 from Sigma aldrich: acrylamide-d3, 500ppm, 5mL for 62.5€ is the one we use. An internal standard will not solve your sensitivity problem however...

Are you sure the peak is low because the sample prep is not OK? Is it possible that you're just not detecting it properly? You need to be able to detect, let's say, a standard solution of 10-50 µg/L acrylamide in water before you start worrying about sample prep reaching 300µg/kg.

You should start by infusing an acrylamide solution of about 0.1-1 ppm in the MS and determine the parent, daughters and collision energy among other parameters. Preferably use a T-piece for infusion.

Next step is to develop a chromatographic method. You can achieve decent peak shape and retention on reversed phase. Check application notes/literature.
Hi again,
Thank you for your interest,

With my instrument and my HPLC-MS/Ms method I am able to detect approximately 25 ng/ml. However, in the potato extract I don't see anything.


Carol
carol wrote:
Hi again,
Thank you for your interest,

With my instrument and my HPLC-MS/Ms method I am able to detect approximately 25 ng/ml. However, in the potato extract I don't see anything.


Carol


With that instrument sensitivity you can do a 1g sample extracted with 10ml water and have final detection limit of 250ug/Kg theoretically.

Do you add 25ng Acrylamide to 1g potato chips then extract with 10ml water to verify detection limit?
The past is there to guide us into the future, not to dwell in.
Hi and thanks for your help,

I prepare a matrix calibration curve, to 2 g of potato chips I spike acrylamide to the final concentracion of 0, 250,500, 750 and 1000 ug/kg, then I start the extraction 20 ml of water, shaked for one hour, centrifuged and filtered twice. This is one of the most comun protocol of extraction. I have also used other extraction protocol which purified the extract with different SPE columns

Carol
This looks indeed like a normal protocol although the extraction time is rather long. I would advice to double check the calculations for spiking.

The next thing you can try is to spike the acrylamide on an extract after the sample preparation. Compare this chromatogram with a chromatogram obtained for acrylamide at the same concentration in water.

If you see the analyte by spiking post-extraction compared to pre-extraction, you have a problem with recovery.

If not, you have a problem with matrix effects.
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