## 3 Results
### Matrix Effect and Recovery  
The matrix effects and extraction recovery can be affected by the organic solvent and endogenous compounds close to the analyte elution times [31; 32]. The use of the protein precipitation method for preparation of plasma enavogliflozin samples represented the high extraction recovery but considerable matrix effects, due to the ion suppression. In order to quantify the concentration of enavogliflozin in plasma and various tissues, we tried a liquid-liquid extraction method with an isotopically labeled internal standard. For a liquid-liquid extraction, we tested an MTBE and ethyl acetate as an extraction solvent. The results showed that the liquid-liquid extraction with MTBE extracted enavogliflozin efficiently from mouse plasma and tissue samples with negligible matrix effects compared  
\begin{table}
\begin{tabular}{c c c c} \hline
**Nominal** & **Calculated** & **Accuracy (\%)** & **CV (\%)** \\
**Concentration** & **Concentration** & **Accuracy (\%)** & **CV (\%)** \\
**(ng/mL)** & **(ng/mL)** & & \\ \hline
5 & 5.073 \(\pm\) 0.069 & 101.5 & 1.351 \\
20 & 19.13 \(\pm\) 1.351 & 95.66 & 7.062 \\
50 & 48.54 \(\pm\) 3.127 & 97.08 & 6.442 \\
150 & 143.2 \(\pm\) 3.976 & 95.50 & 2.778 \\
500 & 487.2 \(\pm\) 24.43 & 97.44 & 5.015 \\
1000 & 1046 \(\pm\) 33.41 & 104.6 & 3.196 \\
3000 & 3234 \(\pm\) 101.0 & 107.8 & 3.124 \\ \hline \end{tabular} Data represented as mean \(\pm\) SD (\(n\) = 6).  
\end{table}
Table 1: Calculated concentrations of enavogliflozin in calibration standards.  
\begin{table}
\begin{tabular}{c c c c c} \hline  & **Nominal** & **Measured** & & \\
**Concentration** & **Concentration** & **Precision (CV, \%)** & **Accuracy (\%)** \\  & **(ng/mL)** & **(ng/mL)** & & \\ \hline  & 5 & 5.376 \(\pm\) 0.386 & 4.601 & 110.4 \\ Intra-day & 15 & 14.85 \(\pm\) 0.844 & 5.680 & 99.00 \\ (\(n\) = 6) & 250 & 247.2 \(\pm\) 16.19 & 6.551 & 98.88 \\  & 2000 & 2237 \(\pm\) 61.09 & 2.731 & 111.8 \\ \hline  & 5 & 5.520 \(\pm\) 0.254 & 7.186 & 107.5 \\ Inter-day & 15 & 14.15 \(\pm\) 0.831 & 5.876 & 94.31 \\ (\(n\) = 5) & 250 & 248.2 \(\pm\) 10.43 & 4.201 & 99.29 \\  & 2000 & 2117 \(\pm\) 170.3 & 8.043 & 105.8 \\ \hline \end{tabular} Data represented as mean \(\pm\) SD (\(n\) = 5 or 6).  
\end{table}
Table 2: Intra- and inter-day precision and accuracy of enavogliflozin.  
with the use of ethyl acetate. Therefore, the liquid-liquid extraction method with MTBE was chosen for the sample preparation method. Table 3 summarizes the extraction recoveries and matrix effects of enavogliflozin and IS. The extraction recoveries for enavogliflozin were calculated at three levels of QC samples and were high and reproducible, ranging from 79.75% to 88.79%, with CVs from 2.989% to 4.000%. The extraction recovery of IS was 85.76 \(\pm\) 5.879%. Therefore, the extraction process using MTBE, utilized in this study, was capable of efficiently extracting enavogliflozin and IS from mouse plasma. The matrix effects were 99.09% for Low QC, 103.6% for Mid QC, 103.2% for High QC, and 96.93% for IS, suggesting that coeluting substances did not interfere with the ionization of enavogliflozin. The low CVs (<15%) in the matrix effect indicated that there were no significant differences in the peak areas of enavogliflozin at the three concentrations across the six plasma samples. Therefore, it was possible to exclude any matrix effect on ion suppression or enhancement.