Addition of an appropriate solvent to the sample is the simplest way for sample preparation prior to GC analysis. For example, hexane was used as dilution solvent in the determination of methyleugenol in tea tree oils. To 5 µL of oil in 2 mL GC vials, 1 mL of n-hexane and 50 mg/L internal standard (IS) (n-tetradecane) were added and mixed thoroughly prior to measurement [25]. Meanwhile in another study, hexane was used as extraction solvent in the determination of methyleugenol, eugenol and isoeugenol in fish fillets [30]. However, due to the complexity of the extracts, further sample preparation (clean-up) by solid phase extraction (SPE) was implemented.

The use of ultrasound is known to be useful in diminishing the cost of processing and handling time in the food and chemistry industry. Ultrasound is also a key player in achieving the objective of green chemistry especially in extractions [34]. In ultrasound assisted solvent extraction, the ultrasonic energy passes through a liquid solvent containing the sample in the form of waves. When the waves hit the sample’s surface, it generates a perpendicular or parallel force to the surface. Sonic energy is converted to mechanical energy and presented in the form of shock waves equivalent to several thousand atmospheric pressure. The rapid localised surge in pressure and temperature contributes improved migration of the solvent into the sample. This results in the improved extraction of the analytes [35].

In a recent study for the determination of eight alkenylbenzenes (eugenol, methyleugenol, acetyl eugenol, trans-isoeugenol, safrole, estragole, myristicin and trans-anethole) in pepper and its varieties using GC, a solvent extraction aided by ultrasound was developed. Ethyl acetate (AcOEt) was chosen as extraction solvent because it was suitable for the extraction of non-polar alkenylbenzenes. The procedure was very straightforward, a weighed sample (1 g), AcOEt (20 mL) and dicyclohexylmethanol (10 µL, internal standard) was sonicated at 37 kHz for 45 min at room temperature in an ultrasonic bath. After centrifugation at 3700 rpm (5 min), an aliquot of the AcOEt supernatant was subjected to GC analysis. The mean recovery reported was acceptable in the range of 70% to 120% [20].

LLE is widely used to transfer an analyte from an aqueous matrix into an extraction solvent. The aqueous matrix and extraction solvent should be immiscible or partially miscible with each other so that the two solvents can be separated easily. Moreover, the analytes from the aqueous matrix should be soluble in the extraction solvent and ideally exhibit high partition coefficients in the solvent. The common extraction solvents used are dichloromethane, chloroform, n-butyl chloride, hexane and toluene [36]. An example of an LLE procedure was in the quantification study of methyleugenol in 120 food samples including basil, laurel, tarragon, allspice, nutmeg, lemongrass, cinnamon and anise [32]. In this work, 60% ethanol (that showed highest extraction efficiency) was used as aqueous matrix and methyl tert-butyl ether (MTBE) was used as extraction solvent. Three extraction solvents used for evaluation were 1,1,2-trichloro-1,2,2-trifluoroethane, n-hexane and MTBE. MTBE and 1,1,2-trichloro-1,2,2-trifluoroethane showed a similar extraction behaviour (about 90% at first extraction), whereas n-hexane had a lower yield (about 65% at first extraction). 1,1,2-trichloro-1,2,2-trifluoroethane was an ozone-depleting chemical; therefore, MTBE was used for further experiments in the study. Cyclodecaneone was used as internal standard (IS) in the experiment to improve the accuracy of quantification. For solid samples, ethanolic homogenate was made by mixing with 100 mL of 60% ethanol. Beverage solution or 1 mL of the ethanolic homogenate was spiked with 200 µL of 40 mg/L cyclodecanone (IS), 5 mL of water and 1 mL of extraction solvent. After shaking and centrifugation, the organic phase was subjected to GC analysis. The recovery for this sample preparation was notable in the range from 95% to 105%.

LPME which is a miniaturized version of LLE was introduced by Jeannot can Cantwell in the early 1990s [37]. It is designed to be a simple, low cost, high recovery, low solvent consumption and rapid extraction method. LPME is a three-phase extraction method where analytes are extracted from a liquid matrix using a small amount (e.g., tens of microliters) of immiscible extraction solvent with the aid of another solvent. There are three main types of LPME including dispersive liquid-liquid microextraction (DLLME), single-drop microextraction and hollow-fiber LPME [38].

An innovative dual DLLME [39] was used for the enrichment of six phenylpropenes, including anethole, estragole, methyleugenol, eugenol, safrole and myristicin in oil samples [26]. In DLLME, a dispersant is used to help the extractant to form fine droplets in the sample solution. The dispersant and extractant are typically organic solvents. The formation of fine droplets increases the extraction efficiency by increasing the contact between extractant and analytes [39]. The procedure for the dual DLLME (forward and back DLLME) method for the alkenylbenzenes is shown in Figure 3. In the first (forward) DLLME, a biodegradable surfactant (TX-100) was used to reduce the surface tension of oil by adsorption at the liquid–liquid interface, increasing the dispersion of extraction phase (n-hexane) into the sample oil phase. The organic phase was then subjected to the second (backward) DLLME in order to remove the surfactant which was detrimental during GC-MS analysis. In the backward DLLME, water and ethyl acetate (dispersant) were added to the hexane extract, and the surfactants were removed into the water phase. The efficiency of the dual DLLME method was determined by calculating the enrichment factor which were 4.5, 9.1, 9.8, 16.7 and 37.1× for estragole, eugenol, methyleugenol, safrole and myristicin, respectively [26].

QuEChERS was initially established to detect pesticide residues in fruits and vegetables but had now rapidly gained attention in the extraction of analytes from different matrices. It is a very popular technique with thousands of papers published according to PubMed [40]. QuEChERS involves two simple steps. The first step is sample preparation and extraction. During this step, the sample is homogenised and an internal standard is added to enhance quantification accuracy. Various acids, salts and buffer are also added to improve the extraction efficiency. Second step is sample extract clean-up. The solvent extract from the first step is cleaned-up using dispersive solid-phase extraction to eliminate possible interfering compounds especially from food extracts [41].

QuEChERS was recently used in the determination of methyleugenol in food samples [31]. For both solid and semi-solid food samples, the sample (2 g) was weighed and placed in a 15 mL centrifuge tube. Methyleugenol-D₃ (100 µL) and AcOEt (10 mL) was added, and the mixture was vortexed (1 min). Anhydrous magnesium sulphate (2 g) and sodium chloride (0.5 g) was added and the resulting mixture was sonicated for 10 min. After centrifugation, 1 mL of supernatant was collected for clean-up. This was by adding anhydrous magnesium sulphate (500 mg) and primary/secondary amine PSA (100 mg). PSA is a solid phase extraction adsorbent used for sample clean-up, which is available commercially. After the vortex and centrifugation steps, the sample was filtered through a 0.22 µm polytetrafluoroethylene (PTFE) syringe filter and analysed by GC. Satisfactory recoveries were obtained in the range of 94.3–100.3% by this method [31]. In another study aimed to determine flavouring substances in food, a similar extraction method was developed but was less time consuming, straightforward and matrix-independent. The samples (2.5 g) were weighed in a plastic centrifuge tube and spiked with dicyclohexylmethanol (25 µL, IS). AcOEt (5 mL) was added, and the mixture was shaken at room temperature for 30 min. Subsequently, magnesium sulfate (2 g) and sodium chloride (0.5 g) were added. The mixture was then shaken by hand and centrifuged (3500 rpm) for 10 min. 0.5 mL of supernatant were collected and filtered with a mini-uniprep PTFE filter. The recoveries for this extraction method were considered acceptable within the range of 70–120% [28], which was a little poorer than the first extraction method described.

SPE uses a liquid phase and a solid phase to separate analytes from a solution. It is often used to clean-up the sample before final chemical analysis. In general, the procedure starts by loading a sample solution into a SPE column, and undesired components are removed by flushing with a non-eluting solvent. An appropriate (eluting) solvent is loaded into the column to wash off the desired analytes that were retained in SPE material during the initial steps.

A phenyl-containing group SPE material was used to clean-up the hexane extract of fish fillets that potentially contained methyleugenol, eugenol and isoeugenol [30] (see Section 2.2.1). The SPE column was preconditioned with hexane and AcOEt. It was then flushed with hexane (clean-up) and then AcOEt was used to elute the targeted analytes for GC analysis. The authors examined the efficiency of hexane, AcOEt, acetonitrile (ACN) and dichloromethane (DCM) to elute targets from phenyl SPE columns. They found that hexane was not able to elute eugenol, isoeugenol and methyleugenol but was able to wash off the interferences from the fish fillet samples during SPE. In contrast, AcOEt, ACN and DCM were able to elute the analytes from the phenyl SPE columns. However, AcOEt was chosen to elute the analytes because ACN and DCM can produce adverse effects on human health [42]. Overall, the proposed solvent extraction and SPE clean-up methodology produced acceptable recovery values for methyleugenol, eugenol, isoeugenol from 76.4% to 99.9%. The methodology is also free from rotary evaporation and nitrogen blowing.

According to the type of interaction between the water and/or steam, there are two main variants for extraction using distillation: steam distillation and hydro-distillation. Steam distillation has been used for a long time in the analysis of volatiles from high-water-content foods, beverages and essential oils [43,44]. It employs steam and/or water as extracting agent to vaporise the volatile analytes from the raw material. The volatile analytes are vaporised by absorbing heat from the steam. The vapor phase resulted from the process is cooled and condensed. Based on their immiscibility, water separates from the organic phase. As a result, two products are formed which are called the volatile oil (upper phase) and the hydrosol (bottom phase). In dry steam distillation, the steam is forced to flow through a supported matrix. This variant enables the steam to be heated above the boiling point and thus becomes superheated steam. In direct steam distillation, the matrix is inserted above the water in the boiling flask, supported by perforated grid or screen [45]. Steam distillation had been conducted by Muráriková, et al. [27] on basil essential oil which contains alkenylbenzenes.

Hydro-distillation is a common extraction method used in GC analysis. In hydro-distillation, there are three main physicochemical processes involved: hydro-diffusion, hydrolysis and decomposition by heat [46]. Clevenger apparatus is the equipment employed in hydro-distillation as shown in Figure 4. The name was titled after its inventor, Joseph Franklin Clevenger in 1928. The round-bottomed flask at the bottom contains the mixture of material and water. As the steam rises, the steam assembles in the condenser and the condensate falls into a burette. In the burette, oil floats (Oi) on the water. After few hours of extraction, the oil can be collected for further use [47].

Since 2010, eight articles described hydro-distillation for the extraction of alkenylbenzenes in various types of food related samples. In the analysis of methyleugenol present in Cymbopogon khasianus Hack., Gogoi, et al. [24] collected fresh plant materials that were shade dried for 24 h. Shade drying results in significant increase in essential oil contents [48], most likely by the significant reduction of the water content. In the extraction of alkenylbenzenes in essential oil, the amount of sample used ranged from 50 g to 300 g and the distillation time were carried out between 2 to 3.5 h. The extracted oils were collected and treated with anhydrous sodium sulfate to remove the water in the oil. Subsequently, the samples were stored at 4 °C until GC analysis [17,18,21,22,23,24,29]. Joy, Berle, Affolter and Pegg [18] showed the average recovery was 85.3 ± 3.2% using hydrodistillation technique in their study. An interesting study by Niculau, et al. [49] used the Clevenger type apparatus for the hydro-distillation (at 110 °C for 2 h) of Pimenta pseudocaryophyllus essential oil which contained chavibetol and methyleugenol. Semi-preparative HPLC was then used to purify chavibetol and methyleugenol from the crude essential oil. GC with FID was used to determine the purity, with chavibetol (98.7%) having a higher purity compared to methyleugenol (85.3%). The purification method of hydrodistillation method coupled with semi-preparative HPLC can provide chavibetol with high purity and recovery. HPLC will be discussed in the next section, but for analysis.

Many studies evaluated the use of different solvents such as hexane, methanol and ethanol for the solvent extraction of secondary metabolites in various plant parts (e.g., leaves and seeds) [59]. They have found that highly polar solvents like methanol gave better recoveries. This was the most likely reason leading to the authors’ decision to use methanol as extraction solvent during the determination of alkenylbenzenes using HPLC. In fact, during this review period, only solvent extraction was used in conjunction with HPLC. First, Gursale, et al. [51] used methanol in the solvent extraction of alkenylbenzenes in Cinnamomum zeylanicum Blume. Recovery of methyleugenol was 99% after solvent extraction with sonication followed by simple filtration. The recovery value was considered acceptable because the recovery value was within 80% to 110% (see FDA Guidelines for the Validation of Chemical Methods) [60]. This study was an inspiration for later studies. For example, Alajlouni, et al. [58] used methanol to extract alkenylbenzenes from plant food supplements. The recoveries found for estragole, myristicin, apiol was 85.3% ± 2.9%, 101.1% ± 5.4% and 94.5 ± 7.6%, respectively. Suparmi, et al. [13] determined alkenylbenzenes in Indonesian jamu. The recoveries found for methyleugenol, elemicin, safrole, myristicin and estragole were 103.8% ± 0.1%, 108.1% ± 1.8%, 105.6% ± 23.0%, 96.1% ± 1.3%, 99.7% ± 3.4, respectively. However, solvent extraction with methanol cannot extract all alkenylbenzenes with good repeatability (e.g., safrole with ~20% error).

In the determination of myristicin in pesto sauce, Al-Malahmeh and co-workers [55] used the methanol extraction method of Ávila and co-workers [61] with few modifications. Originally, Ávila and team macerated dry samples (e.g., basil leaves) with only 15 mL methanol for 12 h at 50 °C. The sample was filtered and wash with water and methanol. The filtrates were collected and quantitatively diluted and then stored at 5 °C [61]. In the Al-Malahmed and team extraction method, a larger volume of methanol (i.e., 80 mL) was used for maceration. The resulting samples were centrifuged for 5 min and an aliquot of the supernatant was stored at −20 °C [55]. They used the margin of exposure (MOE) approach to evaluate the potential risk of myristicin from intake of pesto sauce. Based on the levels of myristicin found in the pesto sauces studied, >30 g consumption of pesto sauce on a daily basis will achieve MOE levels that will warrant priority for risk management.

Meanwhile, the study by Lung et al. [53] was interested to know the effect of microwave irradiation on the levels of linalool and myristicin in plants. The plants were exposed to microwave irradiation prior to extraction. Similar to methanol extraction, the sample was soaked in the solvent, sonicated and then filtered prior to HPLC analysis. No explanation or systematic studies on other solvents were conducted, however very high recovery for myristicin was achieved between 98%–98.48%. Interestingly, they found that the concentration of myristicin in plants was significantly affected by microwave irradiation, but not linalool.