# HS-SPME Gas Chromatography Approach for Underivatized Acrylamide Determination in Biscuits
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the reference method for AA determination in food [9; 10; 11; 12; 13; 14; 15]. This methodology has the advantage of not requiring AA derivatization, but an extensive clean-up methodology for sample defatting and deproteinization is necessary [9]. Due to the complexity of food matrices, which account for a large number of water-soluble compounds that affect chromatographic separation and interfere with detection method, LC requires isotope-labeled internal standards such as d3-acrylamide [16; 17; 18] and \({}^{13}\)C-3-acrylamide [16]. The gas chromatography technique can also be used for AA quantification [19; 20]. However, an AA derivatization is always required to promote its volatility and thermal stability [9; 21]. Furthermore, the direct injection of AA results in a high background noise that limits the possibility to achieve a low detection limit [9] and the possibility to generate in situ AA in the heated GC injection port in the presence of AA precursors (sugars and asparagine) co-extracted from the food matrix [9; 22; 23]. Some methods proposed in literature are compiled in Table 1. The use of solid-phase microextraction (SPME) in headspace mode (HS) [24; 25; 26; 27] minimizes the purification requirements. Due to AA high-water solubility, it can be easily extracted from food matrices with water at room temperature [23]. However, this AA high-water solubility compromises its transfer to the headspace [25], representing a drawback for its implementation. Consequently, a methodology that allowed the simultaneous extraction and quantification of AA, without derivatization and implementation of complex steps, is a major need. The aim of this study was to develop a simple and reliable methodology to quantify AA using HS-SPME followed by gas chromatography coupled with quadruple mass spectrometry detection (GC-qMS) in ion extraction chromatography (IEC) mode (\(m/z\) 71 and 55). AA extraction conditions including the type of fiber coating, the extraction temperature and time, and the amount of organic solvent in the aqueous solution were optimized. The applicability of this methodology was further tested in biscuit samples, including the ones with low AA content being produced using mitigation strategies (asparaginase and \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Extraction Method** & **Detection Method** & **Clean Up** &
\begin{tabular}{c} **AA Deriva-** \\ **itization** \\ \end{tabular} & **LoD/LoQ** & **Food Product** & **Reference** \\ \hline SLE & LC-HRMS & Yes & No & 2.65/5 ppb & Several foods * & [10] \\ DLIME & UPLC-MS/MS & Yes & No & 0.9/3.0 ug/L & Brewed coffee & [11] \\ SLE & LC-MS/MS & Yes & No & 0.3/1 ug/kg & Bread & [13] \\ SLE & LC-QQ-MS/MS & Yes & No & LoQ:1.3–1.4 mg/kg & Insect-based products ** & [14] \\ SLE & LC-MS/MS & Yes & No & 0.62/1.89 ug/kg & Corn snacks & [15] \\ SLE & LC-MS & Yes & Yes & 6.6/19.6 ug/kg & Potato chips, crisppread, & [16] \\ SLE & GC-MS & Yes & No & 4.0/11.8 ug/kg & butter, and cookies & [16; 28] \\ SLE & GC-MS & Yes & No & 5.6/16.7 ug/kg & & [16; 28] \\ SLE & LC-MS/MS & Yes & No & 9.2/12.5 ug/kg & & [17] \\ SPE & LC-MS & Yes & No & 3.55/11.8 ug/kg & Biscuits & [18] \\ DSPE & GC-MS & Yes & No & 6.7/20.3 ug/kg & Dark Chocolate & [19] \\ DLIME & GC-MS & Yes & No & 0.6/2 ug/kg & Roasted nuts and seeds & [20] \\ HS-SPME & GC-MS & No & Yes *** & 1/3 ug/kg & Coffee Beans & [21] \\ DSPE & GC-MS & Yes & No & LoQ:15-40 ug/kg & Food products & [22] \\ \hline \end{tabular}
\end{table} 
Table 1: Literature survey on methods used to extract and quantify acrylamide in food products.