# Application of Thin-Film Microextraction to Analyze Volatile Metabolites in A549 Cancer Cells
## 1 Introduction  
Non-small cell lung cancer (NSCLC) is still one of the most common cancer diseases, with a five year survival rate varying from 92 to 0% [1] depending on the cancer stage at initial diagnosis. Due to the low specificity of symptoms (e.g., cough) NSCLC is often diagnosed at a late stage of the disease, with approximately 38% of patients being diagnosed at stage IV [2]. Thus, an early, easy to handle, and relatively cheap screening test is desired to improve current methods for cancer detection. Nowadays, volatile organic compounds (VOCs) are receiving increasing interest as potential biomarkers related to lung cancer [3], but their trace concentrations require a preconcentration step prior to offline instrumental analysis. In this regard, solid-phase microextraction (SPME) is a widely used preparation technique that relies on obtaining equilibrium between analyte concentration in a sample and the amount extracted by the device [4]. Various formats of SPME devices are commonly applied for analysis of liquid samples in clinical and pharmacological research, whereas for preconcentration of volatile metabolites from gaseous samples, small fibers made of fused silica (or nitinol) coated with a chosen sorptive phase are the gold standard [5]. SPME fibers have been proven to extract VOCs from diverse human body fluids including exhaled breath samples, tissues from stomach cancer patients [6] and from lung cancer patients [7, 8]. For in vitro models of lung cancer, the A549 adenocarcinoma cell line is often employed, and SPME fibers have been successfully applied to VOCs extraction from a cell culture headspace [9], even during their growth phase [10]. A pharmacometabolic study on cisplatin influence on volatilome of A549 cells has been investigated utilizing SPME fibers, with particular VOCs shown to be related to cell apoptosis [11]. Due to limited extraction efficiency resulting in relatively low sensitivity of fiber-based SPME devices, thin film microextraction (TFME) has been developed. TFME can have diverse geometry, for instance, a metal blade covered with solid sorbent immersed in polyacrylamide glue, which is commonly used in Liquid-Phase Microextraction including Direct Immersion Single Drop Microextraction (DI-SDME) [12]. Another option is a mesh-like geometry, where the polymer particles of the extraction phase are immersed in biocompatible glue made of Poly(dimethylsiloxane) (PDMS) and a prepared slurry is sprayed to form a thin layer on carbon mesh. This structure is highly resistant to temperature, allowing thermal desorption of extracted analytes, making this variant suitable for analysis of volatile compounds by means of gas chromatography [13]. Different chemical compositions of the extraction phase may be used to adjust sample preparation protocol, either broadening the range of analytes for untargeted analysis or narrowing it to the target group, considering the analytes polarity, volatility and its concentration in a sample, as well as the complexity of the sample matrix [13]. In this regard, strong adsorbents such as Carboxen are recommended for extraction of trace levels of highly volatile analytes of small molecular weight (<150). However, larger molecules, especially those containing functional groups responsible for \(\pi\)-\(\pi\) interactions (e.g., conjugated double bonds), bind to the microporous carbon surface too strongly and may not allow sufficient desorption [14]. For this reason, mixed-phases are used in the SPME technique, where fibers composed of a layer of divinylbenzene (DVB) suspended in polydimethylsiloxane (PDMS) and coated with a layer of Carboxen (CAR) are the most common choice for extraction of volatiles with a wider molecular weight range. However, the limitation of this approach is a reduced sample capacity due to the thinner coating of each sorbent, hindering the analysis of trace-VOCs in a complex matrix (containing far more abundant interfering substances).