## 3 Results and Discussion  
### Method Development and Qualification  
An immuno-affinity capture LC-MS/MS assay was used for the quantification of desulfurated DM1 as acDrug of T-DM1. Desulfurated DM1 is the cleaved form of the sulfur element from DM1, and the MS/MS spectrum is shown in Figure 2. The parent [M+H]+ ion was detected at \(m/z\) 706.3, and the most abundant product ion was at \(m/z\) 547.2.  
The calibration curves with eight points in the range of 17.09-1709.44 ng/mL in duplicate were freshly prepared for all data sets. The quadratic regression using the ratios of peaks versus concentrations was weighted by 1/concentration. The acceptance of the curve was \(\geq\)0.99 for the desulfurated DM1. Figure 3 shows the calibration curve of desulfurated DM1.  
Representative chromatograms of the limit of quantification (LLOQ) and high QC are also shown in Figure 4. The peak retention time was the same in all samples.  
The intra- and inter-day runs for the accuracy and precision assays were performed for method qualification and the results are shown in Table 2. The qualification run met the acceptance criteria of \(\pm\)25% accuracy and precision for QC samples.  
Figure 3: Calibration curve for quantification of desulfurated DM1 (r = 0.99746).  
Figure 2: (a) MS/MS spectrum of desulfurated DM1 and (b) expected fragment ions of parent ion (\(m/z\) 706.3022).  
Preliminary stability tests were conducted in rat plasma under various conditions, such as short-term, long-term, and freeze-thaw, and the results are shown in Table 3. The accuracy (%) and precision (%CV) of stability samples met the acceptance criteria of \(\pm 25\%\). As a result, the desulfurated DM1 in rat plasma was stable under the different experimental conditions.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**Intra-Run Assay**} & \\ \hline
**Run No.** & **Statistics** & \begin{tabular}{c} **Low QC** \\ **(42.74 ng/mL)** \\ \end{tabular} & \begin{tabular}{c} **Medium QC** \\ **(427.36 ng/mL)** \\ \end{tabular} & \begin{tabular}{c} **High QC** \\ **(854.72 ng/mL)** \\ \end{tabular} \\ \hline Day 1 & \begin{tabular}{c} Mean concentration (ng/mL) \\ Accuracy (\%) \\ Precision (\%, CV) \\ n * \\ \end{tabular} & \begin{tabular}{c} 43.76 \\ 102.40 \\ 10.15 \\ 3 \\ \end{tabular} & \begin{tabular}{c} 419.10 \\ 98.07 \\ 8.53 \\ 3 \\ \end{tabular} & \begin{tabular}{c} 870.05 \\ 101.79 \\ 6.06 \\ 3 \\ \end{tabular} \\ \hline Day 2 & \begin{tabular}{c} Mean concentration (ng/mL) \\ Accuracy (\%) \\ Precision (\%, CV) \\ n \\ \end{tabular} & \begin{tabular}{c} 47.98 \\ 112.27 \\ 2.15 \\ 3 \\ \end{tabular} & \begin{tabular}{c} 395.68 \\ 92.59 \\ 6.54 \\ 3 \\ \end{tabular} & \begin{tabular}{c} 877.40 \\ 102.65 \\ 10.30 \\ 3 \\ \end{tabular} \\ \hline Day 3 & \begin{tabular}{c} Mean concentration (ng/mL) \\ Accuracy (\%) \\ Precision (\%, CV) \\ n \\ \end{tabular} & \begin{tabular}{c} 44.45 \\ 104.00 \\ 3 \\ \end{tabular} & \
\begin{tabular}{c} 373.00 \\ 87.28 \\ 13.75 \\ 3 \\ \end{tabular} &
\begin{tabular}{c} 745.26 \\ 87.19 \\ 12.04 \\ 3 \\ \end{tabular} \\ \hline \multicolumn{4}{c}{**Inter-Run Assay (Day 1-3)**} & \\ \hline
**Statistics** & \begin{tabular}{c} **Low QC** \\ **(42.74 ng/mL)** \\ \end{tabular} & \begin{tabular}{c} **Medium QC** \\ **(427.36 ng/mL)** \\ \end{tabular} & \begin{tabular}{c} **High QC** \\ **(854.72 ng/mL)** \\ \end{tabular} \\ \hline Mean concentration (ng/mL) & 41.06 \\ Accuracy (\%) & 96.09 \\ Precision (\%, CV) & 13.98 \\ n & 9 \\ \end{tabular} & \
\begin{tabular}{c} 99.57 \\ 5.74 \\ 9 \\ \end{tabular} &
\begin{tabular}{c} 99.47 \\ 10.12 \\ 9 \\ \end{tabular} \\ \hline \hline \end{tabular}  
* Number of samples.  
\end{table}
Table 2: The intra/inter-run assays for QC samples of desulfurated DM1.  
Figure 4: Representative chromatograms of (**a**) limit of quantification (LLOQ, 17.09 ng/mL) and (**b**) high level quality control (QC H, 854.72 ng/mL) of desulfurated DM1.