MMAE-d8 (Synonyms: Monomethyl auristatin E-d8; Deuterated labeled MMAE)

This scheme is from the literature, please modify it according to your specific needs. 
1. Cells treated with ADC for 20 hours were collected, counted, and resuspended in 1.67 ng/ml deuterium-labeled MMAE to a cell density of 8.3×10⁵ cells/ml.
2. The samples were lysed at -20°C for 16 hours and then centrifuged at 21,100 RCF for 15 minutes at 4°C. The supernatant was transferred to a 12×75 mm borosilicate glass tube and dried under a stream of nitrogen.The residue was resuspended in 20% methanol and 0.1% (v/v) formic acid (To generate a standard curve, different concentrations of 0.017-3.33 ng/ml MMAE were mixed with 1.67 ng/ml MMAE-d8 in untreated tumor cell lysate and treated as described above).
3. LC-MS/MS Analysis: Chromatographic separation was performed on a column (30°C, 5 μm, 50 mm×3 mm) using a solvent gradient method.Solvent A was H₂O containing 0.1% (v/v) formic acid, and solvent B was acetonitrile containing 0.1% (v/v) formic acid.The gradient used was 0-1 min (20% B to 60% B), 1-1.5 min (60% B to 95% B), 1.5-3 min (95% B), 3-4.1 min (95% B to 20% B), 4.1-5 min (20% B) at a flow rate of 0.5 ml/min.The sheath, auxiliary and sweeping gasses were set to 50, 15, and 1 arbitrary units, respectively.The spray voltage was set to 3.5 kV and the vaporizer and capillary temperatures were maintained at 375°C and 350°C, respectively.The transitions of 718.8 m/z−134.1 m/z and 727.6 m/z–134.1 m/z were used for the quantification of MMAE and MMAE-d8, respectively.
4. Sample collection and data analysis.
References:
[1] Kang J C, Sun W, Khare P, et al. Engineering a HER2-specific antibody–drug conjugate to increase lysosomal delivery and therapeutic efficacy[J]. Nature biotechnology, 2019, 37(5): 523-526.