(T1530-01-06) Development of an Ultra-sensitive and Highly Selective Method for Quantification of Monomethyl auristatin E (MMAE) in Human Plasma Using HPLC‑MS/MS

Purpose: An Antibody-Drug Conjugate (ADC) is an innovative approach to targeted cancer therapy that links a monoclonal antibody (mAb) with a potent cytotoxic drug (payload) through a chemical linker. Monomethyl auristatin E (MMAE) is one such synthetic antineoplastic agent, noted for its high toxicity. Due to its toxicity, MMAE is typically conjugated to a monoclonal antibody to facilitate targeted delivery to cancer cells. As a result, the plasma concentrations of MMAE are extremely low, posing a challenge for accurate quantification. An ultra-sensitive assay will be critical for pharmacokinetic studies, efficacy assessments, and safety evaluations in ADC-based therapies. In this paper, we aim to develop an ultra-sensitive and highly selective ion-exchange method for quantifying MMAE in human plasma.

Methods: A 200-µL sample of human K₂EDTA plasma was spiked with an isotope-labeled internal standard (IS), MMAE-d₈, pretreated with formic acid (FA), and then loaded onto a conditioned cation exchange solid‑phase extraction (SPE) cartridge. After washing, the samples were eluted using a mixture of ammonium hydroxide (NH₄OH), acetonitrile (MeCN), and water. The eluted extracts were dried under nitrogen flow and subsequently reconstituted with a mixture of MeCN and water. The reconstituted samples were injected into a pentafluorophenyl (PFP) column, with mobile phases composed of FA, water, ammonium acetate (NH₄OAc), and MeCN. Detection was conducted using a SCIEX Triple Quad 6500 MS/MS system in positive ion mode, monitoring MMAE and its IS at ion transitions of 718.6→686.6 and 726.6→694.6, respectively.

Results: The extraction method was evaluated using the internal AMOEBA method development procedure, in which the protein precipitation extraction (PPE) and solid phase extraction (SPE) were systematically compared, indicating that SPE achieved the highest sensitivity and the cleanest baseline. Further optimization following AMOEBA procedures revealed that the cation exchange SPE yielded the highest recovery and selectivity, allowing a strong organic wash without analyte loss. One of the biggest challenges in quantifying ADC payloads is the potential release of the payload during sample extraction and extract storage. A commercially available ADC drug using MMAE as its payload was tested and demonstrated sufficient stability during both the extraction procedures and the storage of sample extracts. After incubation of the plasma sample with 50µg/mL ADC for approximately 4 hours at room temperature, we observed MMAE release at a molar ratio of 0.02%. To reduce chromatographic interference at lower concentrations, the Halo PFP 2.7µm 2.1×50mm column was selected after assessments demonstrated its superior selectivity compared to alkyl chain columns. This selectivity is attributed to its combination of electronegative and hydrophobic interactions. Using mobile phases composed of FA, NH₄OAc, MeCN, and water, a symmetrical peak was obtained using isocratic elution with 80% MeCN. However, due to observed co-elution interference, a more selective gradient was developed. The sample was initially loaded onto the column with a low percentage of MeCN, which then quickly increased to 80% MeCN for the isocratic elution stage. The gradient utilized the U-shape retention characteristic of the PFP column, which exhibits increased retention under high-organic or high-aqueous conditions. The method demonstrated excellent accuracy and precision over the range of 1.00 to 1000pg/mL. The inter-assay accuracy ranged from 89.1% to 102.4% with precision ranging from 2.8% to 13.2%. Sufficient sensitivity was achieved at 1.00pg/mL, with no significant interference or carryover observed.

Conclusion: An ultra-sensitive, highly selective, and robust assay for MMAE using HPLC-MS/MS was seccessfully developed.