## 4 Conclusions  
In this paper, an immuno-affinity capture LC-MS/MS method for the acDrug of T-DM1 in rat plasma was successfully developed and applied to in vitro linker stability and preclinical PK assessment in rats. The chemical desulfuration pretreatment used in this study was able to specifically cleave thioether bonds between the MCC linker and DM1 of T-DM1. The method was qualified in a dynamic range of 17.09-1709.44 ng/mL using quadratic regression with 1/concentration weighting, which was sufficient to cover all time courses for this rat PK study with a dose level of 3 mg/kg for the acDrug of T-DM1. This immuno-affinity capture LC-MS/MS method was accurate and reproducible for the determination of acDrug concentration. This method would be also helpful for the acDrug analysis of innovative ADCs with similar non-cleavable linker platforms.  
Conceptualization, S.-J.P., B.i.L. and M.-H.-P.; methodology, S.-J.P. and B.i.L. and M.-H.P.; software, S.-J.P.; validation, B.i.L., J.C., Y.P., M.-J.P. and J.-h.L.; formal analysis, S.-J.P. and B.i.L.; investigation, S.-J.P. and B.i.L.; resources, Y.G.S.; data curation, S.-J.P., B.i.L. and M.-H.P.; writing--original draft preparation, S.-J.P.; writing--review and editing, B.i.L., J.C., Y.P., M.-j.P., J.-h.L., J.L. ([PERSON]), S.H., J.L. ([PERSON]) and Y.G.S.; visualization, S.-J.P.; supervision, Y.G.S.; project administration, Y.G.S. All authors have read and agreed to the published version of the manuscript.  
Research was supported by a research fund of the Ministry of Food and Drug Safety (19172 MFDS163).  
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Chungnam National University (protocol code 202003A-CNU-024, approval date: 1 April 2020). All procedures related to animal experiments were also performed in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & \multicolumn{6}{c}{**PK Parameters**} \\ \hline  & \(\mathbf{T_{1/2}}\) & \(\mathbf{C_{max}}\) & \(\mathbf{AUC_{last}}\) & \(\mathbf{AUC_{INF}}\) & \(\mathbf{AUC_{\infty}}\) & \(\mathbf{C_{\infty}}\) & \(\mathbf{C_{\infty}}\) & \(\mathbf{V_{ss}}\) \\  & **(Day)** & **(ng/mL)** & **(Day*ng/mL)** & **(Day*ng/mL)** & **(\%)** & **(mL/Day/kg)** & **(mL/kg)** \\ \hline Mean & 4.56 & 1396.50 & 2212.16 & 2318.94 & 4.56 & 22.55 & 107.98 \\ \hline SD & 1.11 & 267.92 & 357.96 & 383.13 & 0.50 & 4.00 & 14.65 \\ \hline \hline \end{tabular}  
\(\mathbf{T_{1/2}}\), half-life; \(\mathbf{C_{max}}\), maximum plasma concentration; \(\mathbf{AUC_{last}}\), area under the curve up to last measurable concentration; \(\mathbf{AUC_{INF}}\), area under the curve from time zero to infinity; \(\mathbf{AUC_{\infty}}\), the relative ratio of \(\mathbf{AUC_{last}}\) to \(\mathbf{AUC_{INF}}\); CL, clearance; \(\mathbf{V_{ss}}\), volume of distribution.  
\end{table}
Table 4: PK parameters of desulfurated DM1 after intravenous dose of 3 mg/kg for T-DM1 in rats.  
Figure 6: Pharmacokine (PK) profile of desulfurated DM1 after intravenous dose of 3 mg/kg for T-DM1 in rats.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.  
**Conflicts of Interest:** The authors declare no conflict of interest.