## 3 Results and Discussion
### Method Validation  
The validation in terms of linearity, selectivity, accuracy (R\({}_{\text{M}}\)), precision (intra- and inter-day variability), sensitivity (LOD and LOQ) and carry over was conducted according to the guidelines. The linearity was tested by a calibration curve including eight levels of 0.2, 0.5, 1, 2, 5, 10, 20 and 50 ng mL\({}^{-1}\). The calibration curve was built by plotting the peak area ratio of each analyte to IS versus the concentration ratio of analyte to IS with a linear regression weighting factor of 1/concentration. Regression coefficients (R\({}^{2}\)) of the calibration curves were greater than 0.9978 with deviations less than 12% for all concentrations, indicating a good linearity within the ranges defined. The mothed should be able to differentiate the target analyte and endogenous components in matrices. The selectivity was proved using 8 individual blank serum samples, which were analyzed before and after fortified with target analytes at levels close to the LOQs. No obvious interferences from endogenous components were observed at the retention time of each analyte, as shown in Figure 3. In addition, the complete chromatographic separation of all the target compounds provided further evidence for the high specificity of the method. Figure 2: Optimization of enzyme concentration. The Sensitivity (LOD/LOQ) of the assay were obtained by analyzing spiked serum samples at low levels. The LOD and LOQ for each analyte were determined as signal to noise ratio (S/N) greater than 3 and 10, which ranged 0.02-0.06 ng mL\({}^{-1}\) and 0.1 and 0.2 ng mL\({}^{-1}\) respectively for all target compounds, as given in Table 2. The values demonstrated a high improvement in sensitivity [24, 27, 28, 29, 30]. The accuracy and precision were investigated from six replicates of fortified serum samples at three levels (0.5, 1.0 and 5.0 ng/ mL) once a day on three consecutive days. Method recovery (R\({}_{\text{M}}\)) which had been corrected by IS were from 91.6% to 123.6% for all the analytes at three concentrations (Table 2). The relative standard deviation (RSD) of intra-day and inter-day, representing the precision of the method, were ranged 2.53%-5.99% and 3.62%-8.22%, respectively. Besides, extraction recovery (R\({}_{\text{E}}\)) of sample preparation step and the matrix effect (signal suppression/enhancement, SSE) of instrument analysis step showed great results, meaning that acceptable accuracy can also be achieved even without IS compensation. No carry-over was observed within the assay which was performed by injecting three blank solvents after injection of a high concentration standard at 100 ng mL\({}^{-1}\). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \multirow{2}{*}{**Analytic**} & **R\({}_{\text{E}}\) (Extraction recovery, \%)** & **Matrix Effect** & **Precision \%** & **Spiked Value** & **R\({}_{\text{M}}\) (Method** & **LOQ (ng mL\({}^{-1}\))** & **LOQ (ng mL\({}^{-1}\))** \\  & **recovery \%)** & **Effect** & **Intra-day** & **Inter-day** & **(ng mL\({}^{-1}\))** & **recovery \%)** & **LOQ (ng mL\({}^{-1}\))** & **LOQ (ng mL\({}^{-1}\))** \\ \hline ZEN & 94.06 & 85.2 & 2.53 & 3.62 & 1 & 110.1 & 0.02 & \\  & & & & & 5 & 102.2 & & (fmol) & (0.6 fmol) \\  & & & & & 0.5 & 100.4 & 0 & 2 & 0.04 \\ a-ZOL & 99.95 & 78.09 & 3.12 & 4.29 & 1 & 103.6 & (6 fmol) & (1 fmol) \\  & & & & & 5 & 109.0 & 0 & 9.1 \\ g-ZOL & 111.8 & 84.17 & 3.81 & 4.96 & 1 & 92.9 & (6 fmol) & (2 fmol) \\  & & & & & 5 & 92.4 & 0 & 105.6 & 0 & 0.2 \\ a-ZAL & 106.5 & 76.83 & 4.32 & 8.22 & 1 & 110.0 & (6 fmol) & (1 fmol) \\  & & & & & 5 & 106.5 & & & \\  & & & & & 0.5 & 100.5 & & & \\ g-ZAL & 116.1 & 77.24 & 4.25 & 5.98 & 1 & 106.1 & 0.1 & 0.02 \\  & & & & & 5 & 103.2 & & (3 fmol) & (0.6 fmol) \\  & & & & & 0.5 & 123.6 & & & \\ ZAN & 100.4 & 81.77 & 5.99 & 7.04 & 1 & 110.7 & 0.1 & 0.03 \\  & & & & & 5 & 100.1 & & (3 fmol) & (1 fmol) \\ \hline \end{tabular}
\end{table} 
Table 2: Extraction recovery, matrix effect, accuracy, precision and sensitivity of the method.