## 3 Results and Discussion  
### Calibration and Quality Assurance/Quality Control  
Table 2 shows the calibration results and summary of the quality assurance/quality control (QA/QC) information of L-WS using a LC-MS/MS system. Figure 1 shows the retention times for individual TSNA compounds under these instrumental conditions. The standard calibration curve was examined by computing a regression line of peak area ratio for weight (pg) of TSNAs in L-WS using the least squares method. All of the calibration curves of four TSNA compounds showed \begin{table}
\begin{tabular}{c c c c c c c c} \hline \multicolumn{1}{c}{**(A) High-Performance Liquid Chromatography (LC-20 AD, Shimadzu, Japan)**} \\ \hline \multicolumn{1}{c}{**Column: ZORBAX Eclipse Plus C18 (Agilent, USA), 3 \(\times\) 150 mm, Particle size: 3.5 μm**} \\ \hline \multicolumn{1}{c}{Oven temp:} & 55 & \multicolumn{2}{c}{\({}^{\circ}\)C} \\ Injection volume: & 1 & & \multicolumn{2}{c}{\(\mu\)L} \\ Flow rate: & 0.4 & & \multicolumn{2}{c}{mL min\({}^{-1}\)} \\ Pump mode: & \multicolumn{2}{c}{Binary gradient} \\ Mobile phase A: & \multicolumn{2}{c}{0.1\% Acetic acid in water} \\ Mobile phase B: & \multicolumn{2}{c}{0.1\% Acetic acid in methanol} \\ Gradient: & Time (min) & 0 & 4 & 7 & 8 & 20 & 25 \\ Solvent B (\%) & 55 & 98 & 98 & 2 & 2 & 55 \\ Total analysis time: & 25 & & & \multicolumn{2}{c}{min} \\ \hline \multicolumn{1}{c}{**(B) Tandem Mass Spectrometry (LCMS-8040, Shimadzu, Japan)**} \\ \hline \multicolumn{1}{c}{Acquisition mode:} & MRM & & & & \\ Electrospray ionization: & ESI & & & & \\ Polarity: & Positive & & & & \\ Nebulizing gas flow (N\({}_{2}\)): & 3.0 & & & \multicolumn{2}{c}{mL min\({}^{-1}\)} \\ Drying gas (N\({}_{2}\)): & 15.0 & & & \multicolumn{2}{c}{mL min\({}^{-1}\)} \\ CID gas (Ar): & 230 & & & \multicolumn{2}{c}{kPa} \\ Interface voltage: & 4.5 & & & \multicolumn{2}{c}{kV} \\ Loop time: & 0.348 & & & \multicolumn{2}{c}{s} \\ Dwell time: & 26 & & & \multicolumn{2}{c}{msec} \\ \hline \multicolumn{1}{c}{**(C) Mass spectrum parameters for MRM condition**} \\ \hline \multicolumn{1}{c}{**Compounds**} & \multicolumn{2}{c}{**Precursor**} & \multicolumn{2}{c}{**Product Ion**} & \multicolumn{2}{c}{**CE * (V)**} & \multicolumn{2}{c}{**Dwell time (m s)**} \\ \cline{3-6} \multicolumn{1}{c}{} & \multicolumn{1}{c}{**Ion (_un/z_)**} & \multicolumn{1}{c}{**Quantifier (_un/z_)**} & \multicolumn{1}{c}{**Qualifier (_un/z_)**} & \\ \hline \multicolumn{1}{c}{**NNN**} & 178 & 148.1 & & 12 & 26 \\  & & & 120.1 & 23 & 26 \\  & & & 119.1 & 33 & 26 \\
**NNK** & 208 & 122.1 & & 11 & 26 \\  & & & 79.1 & 30 & 26 \\  & & & 106.1 & 18 & 26 \\
**NAT** & 190 & 160 & & 13 & 26 \\  & & & 79.1 & 39 & 26 \\  & & & 106.1 & 22 & 26 \\ \hline \end{tabular} \({}^{*}\) Collision Energy.  
\end{table} 
Table 1: Operational condition of high-performance liquid chromatography (HPLC)–tandem mass spectrometry (MS/MS) system and multiple reaction monitoring (MRM) conditions for the analysis of tobacco-specific nitrosamines (TSNAs). excellent linearity (R\({}^{2}\) > 0.999) in the concentration range of 0.5 to 100 ng mL\({}^{-1}\) (Figure S2). The MDL for TSNA was calculated as the product of the standard deviation of seven replicate analyses based on the Student's t-value (t = 3.14 for degrees of freedom = 6) at 99% confidence level according to US EPA guidelines. The MDL of NNN, NNK, NAT, and NAB were estimated as 4.40, 4.47, 3.71, and 3.28 ng mL\({}^{-1}\), respectively. The relative standard error (RSE) was stable at \(\sim\)2% at a L-WS concentration of 5 ng mL\({}^{-1}\). \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Order** & **Compound** & **RF 1 ** & **RSE (\%) 3 ** & **MDL (ng mL\({}^{-1}\)) 3 ** \\ \hline
1 & NNN & 40,707 & 0.9999 & 0.74 & 4.40 & 4.40 \\
2 & NNK & 79,308 & 0.9999 & 0.49 & 4.47 & 4.47 \\
3 & NAT & 82,614 & 0.9999 & 1.17 & 3.71 & 3.71 \\
4 & NAB & 53,392 & 0.9997 & 0.59 & 3.28 & 3.28 \\ \hline \end{tabular}
\end{table} 
Table 2: Results of calibration analysis and quality assurance (QA) information obtained using liquid phase working standards (L-WS).