## 1 Introduction  
Antibody-drug conjugate (ADC) is a promising biopharmaceutical consisting of three components: a monoclonal antibody, a linker, and cytotoxic drugs (payloads) [1, 2, 3, 4, 5, 6, 7, 8, 9]. ADCs undergo receptor-mediated endocytosis after reaching target cells through a specific antigen-antibody response. After endocytosis, ADCs are degraded by lysosomal enzymes, and cytotoxic drugs are released [10]. The released cytotoxic drugs cause cell death by cytotoxic effects such as mitotic arrest or the inhibition of DNA or RNA polymerization [11, 12, 13]. Because of its ability to selectively deliver these cytotoxic drugs mainly to target cells, ADCs are currently receiving a lot of interest from many pharmaceutical companies and are being developed. To date, 12 ADCs (Adcteris(r), Mylotarg(tm), Kadcyla(r), Besponsa(r), Lumoxiti(r), Polivy(tm), Padcev(r), Enherttu(r), Trodelyv(r), Blenrep(r), Zynlonta(r), and Tivdak(tm)) have been approved by U.S. Food and Drug administration (FDA) for anti-cancer therapies [14]. Inaddition, it is expected that many ADC candidates in preclinical and clinical development will be approved in the future.  
From the perspectives of the full pharmacokinetic (PK) properties of ADC, three main components including (1) total antibody (tAb), (2) antibody-conjugated drug (acDrug)/(or conjugated antibody), and (3) free payload should be quantitated from in vivo samples [15]. Among these three main analytes, acDrug demonstrates the concept that ADC exists in an active form as a conjugate [16]. The acDrug may also play an important role in the dose-response analysis of ADCs. However, it was very difficult to evaluate the acDrug of the first generation ADCs without cleavable linkers such as trastuzumab emtansine (T-DM1, brand name: Kadcyla(r)) by LC-MS, and therefore it was reported that the conjugated antibody instead of acDrug were quantitated by ELISA assay ([[https://www.accessdata.fda.gov/drugsatfda.docs/nda/2013/125427](https://www.accessdata.fda.gov/drugsatfda.docs/nda/2013/125427) Orig18000 PharmR.pdf]([https://www.accessdata.fda.gov/drugsatfda.docs/nda/2013/125427](https://www.accessdata.fda.gov/drugsatfda.docs/nda/2013/125427) Orig18000 PharmR.pdf) (accessed on 1 September 2021)) for Kadcyla. Recently, more attention is paid to the importance of the in vivo acDrug levels of ADCs regarding the efficacy, in vivo drug-antibody ratio (DAR) evaluation, and toxicity. Currently, only ADCs with cleavable linkers are easily applicable to the quantitation of acDrug by the immuno-affinity capture method followed by on-bead digestion using specific enzymes such as cathepsin B or beta-glucuronidase for the LC-MS analysis of acDrug [16; 17; 18]. Due to lack of specific sites in the linker, this method was not applicable to Kadcyla, which contains non-cleavable linkers.  
In this paper, we developed an immuno-affinity capture LC-MS/MS assay using previously reported chemical desulfuration pretreatment [19] to quantify acDrug in rat preclinical PK samples even for trastuzumab emtansine (T-DM1, brand name: Kadcyla(r)), a representative ADC with non-cleavable linkers. T-DM1 is the first FDA-approved ADC to treat metastatic human epidermal growth factor receptor 2 (HER2)-positive breast cancer and consists of the monoclonal antibody trastuzumab, maleimidomethyl cyclohexane-1-carboxylate (MCC) as a non-cleavable linker, and maytasinoid as a payload [20]. This developed assay was applied to evaluate in vitro plasma linker stability and preclinical PK studies in rats. To our best knowledge, this is the first approach to quantitatively evaluate the acDrug of T-DM1 in vivo rat PK samples.