**4.1 Solid-phase microextraction (SPME)**

The chemical analysis of aroma compounds in yogurt products is typically a complicated process. For example, such analysis requires an extraction stage and, despite the outstanding importance of aroma as an indicator of product quality and conformity, it is still difficult to separate aroma compounds based on common properties such as polarity or volatility. This is particularly true since most volatile organic compounds are present only in small concentrations (μg/kg to mg/kg) in yogurt [36, 37]. As a result, it is often necessary to isolate the volatiles from the complex matrix and concentrate these volatiles for analyses. Unfortunately, the extraction and concentration of volatile aroma components from yogurt products present a major analytical challenge. The most significant problems encountered during this process are:


Classical techniques for preconcentration of volatiles such as steam distillation direct extraction simultaneous steam distillation and extraction with a solvent static headspace [26, 33, 36] and dynamic headspace [30] have been applied to the extraction and concentration of volatile aromatic compounds in yogurt. In recent years, solid-phase microextraction (SPME)-based methods have been used to analyze yogurt flavors [38, 39]. Unlike conventional extraction techniques, SPME is more sensitive to experiment conditions. Any change in experiment parameters that affects the partition coefficient and adsorption rate will also affect the amount adsorbed onto the SPME fiber and the corresponding reproducibility [40].

Solid-phase microextraction (SPME) methods were developed in the 1990s by Arthur and Pawliszyn as a rapid and useful technique for volatile compound analysis. SPME coupled with GC-MS can provide high sensitivity with a small sample volume; consequently, it can be used to analyze the aroma profile of a wide variety of substances. Recently, this technique has been used to study the volatile profiles of fermented camel milk [41], grapes and wine [42], and dried fermented sausage [43].

The amount of analyte extracted on the fiber depends on the polarity and thickness of the stationary phase of the fiber, time and temperature of the extraction, agitation and pH of the sample solution, addition of salt to the sample, and the concentration of the analyte in the sample. These SPME parameters must be optimized for each analyte and matrix. Various fiber coatings are available with thicknesses from 7 to 150 μm. Although fibers coated with thicker films require longer time to reach

extraction equilibrium, they can be more sensitive because they can extract larger amounts of analyte [44].

Solid-phase microextraction onto silica fibers externally coated with a suitable stationary phase is used in combination with GC and is also directly coupled to HPLC for the analysis of low-volatile or thermally labile compounds that are not subject to GC analysis. The SPME device consists of a stand with an integrated extraction fiber inside a needle and an assembly holder. Silica fibers (1 or 2 cm long) coated on the outer surface with a thin film of an extraction phase consisting of a liquid polymer and/or a solid sorbent are commercially available. StableFlex fibers consist of a flexible condensed silicon core and are less fragile. Although SPME has maximum sensitivity to the equilibrium distribution, there is a proportional relationship between the amount of analyte adsorbed by the SPME fiber and its initial concentration in the sample prior to partition equilibrium. As a result, complete equilibrium is not necessary for quantitative analysis by SPME [44].

## **4.2 Gas chromatography with mass spectrometry (GC-MS)**

Chromatographic methods are widely used in the identification of various aromatic metabolites. In lactic acid products, these methods include Fourier transform infrared spectroscopy, electron impact ionization-mass spectrometry (EI-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectroscopy. Mass spectrometers are generally more sensitive and more selective than any other type of detector. Prior to MS analysis, metabolites must be separated, and the separated compounds must be ionized. Ionization techniques can vary, especially for GC-MS and LC-MS [45]. Each of these techniques has advantages and limitations, and no single analytical technique is yet available for the complete study of the metabolome [46].

GC-MS-based metabolome analyses have been developed and applied for metabolic profiling in plants and microorganisms. The aforementioned studies clearly demonstrated the utility of GC-MS for non-target metabolite profiling in a variety of matrices [47].

GC-MS is a combined system where volatile and thermally stable compounds are first separated by GC after which the eluted compounds are detected traditionally by mass spectrometry. In metabolomics, GC-MS has been described as the gold standard [48, 49].

Instrumental analysis of volatile compounds in yogurt is almost exclusively performed by gas chromatography (GC), although high-performance liquid chromatography (HPLC) has also been used in a limited number of cases. Various detectors, including ionization detectors (FID), thermal conductivity detectors (TCD), electron capture detectors (ECD), photoionization detectors (PID), and mass spectrometry (MS) can be used to detect volatiles [3]. In particular, GC-MS is the most popular technique used in the analysis of aromatic volatile components due to its ability to detect and quantify known compounds, identify unknown compounds, and elucidate the chemical properties of molecules. Although the sensitivity of MS depends on the nature of the analytes and the type of equipment used, the detection limits of the charged species can typically range down to picogram levels or even less. In addition to direct calibration, the quantification of volatile compounds can be performed by matrix addition of the labeled compounds or by addition of the so-called internal standard [34, 35].

The application of GC-MS has boosted research on the aroma of yogurt and other products, especially when coupled with SPME as a pretreatment method. The primary advantages of SPME are its simplicity, low cost, ease of automation and in situ sampling. SPME coupled with GC-MS has been widely used to assess the aroma chemical profiles of volatile components derived from a wide variety of matrices, including fermented milk [50], fruit and mango juice [51], grapes and wine [42], dry fermented sausage [43], and alcoholic beverages [52, 53].
