## 3 Results and Discussion
### Determination of Relative Recovery for Dilution Solutions  
To compare the dilution capacity according to the dilution factor, the RRs of four TSNA compounds that were computed by the response factor (RF) of L-WS are shown in Supplementary Figure S3 online. Comprehensive examination of two types of RR test showed that the RRs of ACN were considerably higher than those of AA. In the L-S1 phase, assessment of the average RR value of all four TSNAs for each dilution solution showed a high recovery of 137 \(\pm\) 16.6% for ACN as compared with 109 \(\pm\) 15.6 for AA. The RRs of four TSNAs in a 20-fold diluted sample with AA were considerably high (135 \(\pm\) 0.53 (NNN), 113 \(\pm\) 0.12 (NNK), 108 \(\pm\) 0.47 (NAT), and 136 \(\pm\) 0.17% (NAB); Table 3). In addition, unidentified peaks appeared due to contamination of the C18 column, which may be caused by the high contents of PG and VG in the EC liquid solution. When the sample was diluted 50-fold, the RRs of NNK and NAT were stable, while NNN and NAB exhibited relatively high recoveries (114 \(\pm\) 0.24 for NNN, 95.7 \(\pm\) 0.01 for NNK, 93.5 \(\pm\) 0.16 for NAT, and 113 \(\pm\) 0.13% for NAB). In contrast, the RRs of three TSNAs for the 100-fold diluted sample were stable with the exception of NNN (115 \(\pm\) 0.20 for NNN, 88.7 \(\pm\) 0.12 for NNK, 91.3 \(\pm\) 0.01 for NAT, and 104 \(\pm\) 0.07% for NAB). According to a previous study, the maximum concentration of TNSA detected in one of the commercial products was 60.8 ng mL\({}^{-1}\) for NNN [48]. If the E-solution is diluted 100-fold with AA, analysis of the low concentration sample is unreliable because the concentration of the diluted sample may fall outside the calibration range. In contrast, the RRs of all samples diluted with ACN were greater than 110%, regardless of the dilution factor. The 100-fold diluted sample showed the lowest RR across the three dilutions (20, 50, and 100 fold), but it was still much higher than the RR of the 100-fold diluted sample with AA (difference up to 42%). Therefore, AA was better than ACN for the dilution of the EC solution with respect to stable recovery. In the L-S2 phase, the dilution factor was fixed at 50 based on the results of experiment L-S1. The RRs of four TSNAs in spiked samples with two types of dilution solution are compared in Table 4. When diluting the samples with ACN, the RR was approximately 100% for all TSNAs at a low concentration (50 ng mL\({}^{-1}\)) of spiked samples (98.8 \(\pm\) 3.11 for NNN, 102.1 \(\pm\) 11.4 for NNK, 91.2 \(\pm\) 8.06 for NAT, and 99.3 \(\pm\) 8.77% for NAB), but increased drastically at the highest concentration of 500 ng mL\({}^{-1}\) (273 \(\pm\) 9.52 for NNN, 332 \(\pm\) 30.7 for NNK, 251 \(\pm\) 20.8 for NAT, and 261 \(\pm\) 22.0% for NAB). It is hard to explain this high recovery by only the matrix effect. In contrast, the average RRs of four TSNAs in all samples that were diluted with AA were stable at all concentrations: 83.1 \(\pm\) 4.65 for NNN, 88.0 \(\pm\) 5.16 for NNK, 81.5 \(\pm\) 3.63 for NAT, and 88.8 \(\pm\) 4.45% for NAB. Therefore, a 50-fold dilution of the E-liquid with AA was determined as a preferable pretreatment option for analysis of TSNAs in EC solution. Figure 1: Chromatograms of TSNA for the 10 ng mL\({}^{-1}\) of working standards by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (**a**) extracted ion chromatogram (EIC) of quantifier ions for each target compound and (**b**) Chromatogram in Total ion current (TIC) mode with both quantifier and qualifier ions. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Order**} & \multirow{2}{*}{**Sample Code a1**} & \multicolumn{5}{c}{**Compounds**} \\ \cline{3-6}  & & **NNN** & **NNK** & **NAT** & **NAB** \\ \hline \multicolumn{6}{l}{(A) Theoretical concentration of TSNA in the spiked sample (ng mL\({}^{-1}\)) (dilution factor: 50) b  } \\  
& 332 & 251 & 262 \\ \hline \hline \end{tabular}
\end{table}