Effortless acrylamide extraction: Key sample prep tips & tricks

If you have experience testing food processing biproducts such as acrylamide, then you already know all the challenges that come with the territory. Acrylamide, a probable carcinogen formed during high-temperature cooking, poses significant health risks. Accurate detection is crucial for food safety and public health. With any sample preparation technique, there can be difficulties, but extracting acrylamide from food matrices poses special complications for several reasons. This text delves into these complexities and explores advanced techniques for acrylamide extraction.

Understanding the complex matrix:

Food matrices are complex in nature due to the various substances found within the food itself, such as fiber, fats, oils, proteins, minerals, vitamins, etc. These substances can interfere with acrylamide analysis, making extract clean-up an absolute must prior to analysis. A great way to remove these unwanted matrix components is to utilize specialized solid phase extraction (SPE) chemistry such as ISOLUTE^® Multimode. This SPE sorbent contains non-polar (C18), strong cation exchange (SO3-) and strong anion exchange (-NR3+) functional groups. These groups allow for the removal of acidic, basic and non-polar interferences components from the matrix while allowing acrylamide to pass through due to its small and polar properties.

Navigating chemical structure challenges:

Acrylamide is very small and highly polar analyte, making it extremely water soluble. Since there are no acidic or basic groups in the acrylamide structure, it does not ionize under certain pH conditions, making anion or cation exchange solid phase extraction (SPE) impossible. It’s difficult to retain using reversed phase SPE, and C18 media does not retain acrylamide at all. Therefore, specialized sorbents are required in order to extract the analyte from your sample. ISOLUTE^® ENV+ is a hyper crosslinked hydroxylated polystyrene-divinylbenzene copolymer with a very high surface area, making it ideal for extracting polar analytes. For this reason, ISOLUTE^® ENV+ is one of the few sorbents that can effectively retain acrylamide from aqueous solutions.

Effective sample pre-treatment:

Acrylamide is extracted with water, and isotopic labelled acrylamide is added. The extract is then centrifuged, and the supernatant is cleaned up using two solid phase extraction (SPE) columns. In 2002, the European Standard Method for Acrylamide (EN16618: 2015), was developed as one of the most robust acrylamide analytical protocols. This technique utilizes a two-step SPE approach:

     Step 1) Remove unwanted matrix interferences

     Step 2) Retain acrylamide, wash away additional interferences, and then elute and collect the acrylamide for analysis.

To achieve this, the following steps can be performed using Biotage products:

Step 1: ISOLUTE^® Multimode Clean-Up

The first SPE column contains silica based C18 groups as well as anion and cation exchangers (ISOLUTE^® Multimode, 1 g/6 mL, p/n 904-0100-C). Since acrylamide is not retained by the column, the extract is just passed through and collected. The purpose of using this column is to retain as many matrix components as possible (non-polar compounds as well as anions and cations) without retaining acrylamide. Essentially, the first SPE column acts as a chemical filter (or matrix scavenging column).

Step 2: ISOLUTE^® ENV+ SPE Clean-Up

The second SPE column contains a polymer-based phase with a relatively high capacity to bind acrylamide (ISOLUTE^® ENV+ 500 mg/6 mL p/n 915-0050-C). The extract is loaded onto the column, which is then washed with water and finally eluted with a mixture of 60 parts methanol and 40 parts water by volume. The purpose of this step, apart from further cleaning of the extract, is to concentrate the extract and obtain low limits of quantification. After evaporation of the methanol, the extract is analyzed by LC-MS/MS.

Managing volatility:

Working with volatile analytes like acrylamide can present unique challenges, particularly in preventing evaporation loss after extraction from the sample matrix. If you analyze a sample and find no acrylamide, it’s important to ensure that your technique is robust enough to consistently protect the analyte and capture the target. When measuring low level acrylamide (<1ppb), concentrating the sample extract using solvent evaporation is essential to meet the methods sensitivity requirements. Using a keeper solvent such as ethylene glycol allows you to concentrate down to 10uL, without evaporative loss of acrylamide. The keeper solvent needs to have a higher boiling point than the primary solvent being evaporated to ensure stability and prevent evaporative loss during the concentration process.

Scaling the method:

Although the EN method uses relatively large column formats, the acrylamide extraction and clean up method can be successfully scaled down to use smaller columns or even 96-well plate formats. Reduced bed masses offer the advantages of lower sample volumes and subsequently reduced final extract volumes. This can lead to higher throughput due to reduced processing time and reduced post-extraction evaporation.

The table below suggests appropriate volumes for use with reduced bed masses.

*Note these are based on theoretical considerations and should be optimized by users.

Summary

Extracting acrylamide from food matrices is challenging due to complex food components, acrylamide's volatility, and its polar, non-ionizable structure, necessitating specialized solid phase extraction (SPE) techniques. The European Standard Method (EN16618:2015) utilizes a two-step SPE process: ISOLUTE^® Multimode removes matrix interferences, while ISOLUTE^® ENV+ retains and elutes acrylamide for LC-MS/MS analysis. To prevent evaporative loss during concentration, a keeper solvent like ethylene glycol is used, enabling detection at low levels. The method is scalable to smaller columns or 96-well plates, reducing sample volumes and processing time for higher throughput.

To learn more about this technique, download our application note!