## 3 Results and Discussion
### Selection of HDEs
#### 3.1.1 HDE Characterization  
The HBD and HDA interaction results in the well-known mixture HDE. In order to determine if there is a hydrogen bond interaction between the oxygen atom of the carboxylic group located on lauric acid as HBA and the hydroxyl group of menthol as HBD, the FT-IR spectra and \({}^{1}\)H NMR spectra of the HDE (menthol-lauric acid with a molar ratio of 4:1)  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Test**} & \multicolumn{6}{c}{**Influence Factors**} & \multicolumn{6}{c}{**Analyte Response (EE\%)**} \\ \cline{2-9}  & **A: Number of** & **B: Extraction Time** & **C: Volume of** & **D: Elution** & & & \\  & **HDE-SBME Devices** & **(min)** & **Elution (\(\mu\)L)** & **Time (min)** & **BES** & **TES** & **PRO** \\ \hline
25 & 5 & 10 & 100 & 2 & 30.14 & 36.51 & 35.16 \\
26 & 3 & 30 & 200 & 5 & 73.26 & 90.39 & 87.92 \\
27 & 5 & 50 & 100 & 2 & 29.33 & 34.76 & 34.61 \\
28 & 3 & 30 & 100 & 5 & 61.89 & 75.45 & 71.70 \\
29 & 1 & 30 & 200 & 5 & 27.23 & 29.18 & 34.84 \\ \hline \hline \end{tabular}
\end{table}
Table 1: _Cont._  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multicolumn{2}{c}{**HNNDE**} & \multicolumn{3}{c}{**Peak Area**} \\ \cline{2-5}
**Ratio of Menthol–Lauric Acid** & **BES** & **TES** & **PRO** \\ \hline
2:1 & 1.081 & 5.309 & 3.864 \\
3:1 & 1.757 & 4.467 & 5.236 \\
4:1 & 1.760 & 4.608 & 5.523 \\
5:1 & 1.741 & 4.184 & 5.308 \\ \hline \hline \end{tabular} \({}^{*}\)SBME conditions were set as follows: 10.0 ug L\({}^{-1}\) of selected steroids, 20.0 mL of water as a sample solution using one SBME device stirred at 250 rpm for 30 min extraction time, and 150 \(\mu\)L of methanol as elution solvent sonicated for 5.0 min, n = 3.  
\end{table}
Table 2: The effect of using HDEs with various ratios of menthol and lauric acid in the SBME \({}^{*}\) technique.  
were recorded. As shown in Supplementary Figure (Figure S1), the FT-IR characteristic absorption peaks of lauric acid remained unchanged after the addition of menthol, while the stretching vibration of the C=O peak in lauric acid shifted from 1692 cm\({}^{-1}\) to 1739 cm\({}^{-1}\) in the HDE. The peak at 3241 cm\({}^{-1}\), corresponding to menthol's O-H stretching vibration, shifted to 3384 cm\({}^{-1}\).  
The menthol and lauric acid chemical signals show significant differences with 1:4 HDE in \({}^{1}\)H NMR spectra. The menthol spectrum (Figure 2A) shows a doublet at \(\delta\) = 3.1 ppm, while the HDE spectrum (Figure 2C) shows a singlet without significant chemical displacement. As expected, the proton bonded to the same carbon as the hydroxyl group in menthol is also a hydrogen bonding signal that appears as a multiplet with a resonance of -H-at a chemical shift of 2.0-2.2 ppm. However, in the HDE \({}^{1}\)H NMR spectrum, no detectable change is observed, and the signals are no longer well-defined multiplets, suggesting that hydrogen bond interactions between the parent molecules influence the hydrogen of menthol. The absence of the hydroxyl group of lauric acid (appears at \(\delta\) = 11.7-11.9 ppm, as shown in Figure 2B) in the HDE \({}^{1}\)H NMR spectrum supports hydrogen bonding. The physical state of the compounds further confirms this proof, as a transparent liquid is obtained after the formation of HDE solvent from initially solid compounds. Overall, the data strongly indicate the formation of hydrogen bonds between the hydroxyl groups of menthol (hydrogen bond donor) and the carboxyl group of lauric acid (hydrogen bond acceptor).