Chromatography-based methods for determining molar extinction coefficients of cytotoxic payload drugs and drug antibody ratios of antibody drug conjugates

•An HPLC method with dual detectors was developed to measure the extinction coefficients.•The MEC was solved by Beer’s law (A=εcl), with A and c obtained from DAD and CLND.•The method measures MEC accurately even when the sample is impure or in a mixture.•Compared to the only available method, our method is sample sparing and easy to implement.•The MEC has been successfully used for DAR characterization of ADCs in drug discovery. UV spectrophotometry is widely used to determine the molar extinction coefficients (MECs) of cytotoxic drugs as well as the drug antibody ratios (DARs) of antibody drug conjugates (ADCs). However, the unknown purity of a drug due to interfering impurities can lead to erroneous MECs and DARs. Hence, reliable methods to accurately determine purity and the MECs of drugs with limited quantity is urgently needed in Drug Discovery. Such a method has been developed. It achieves absolute purity and accurate MEC determination by a single automated HPLC analysis that uses less than 5μg of material. Specifically, analytical HPLC separation with online UV detection was used to resolve impurities and measure absorbance from only the compound of interest. Simultaneously, an online chemiluminescence nitrogen detector (CLND) was used to determine the concentration of the analyte. The MECs were then calculated from the absorbance and concentration results. The accuracy of the method was demonstrated using caffeine and a commercial cytotoxic drug, DM1. This approach is particularly suited to analyzing mixtures or samples with low purities. Excellent reproducibility was demonstrated by analyzing a proprietary drug with linker synthesized from different batches with very different levels of purity. In addition, the MECs of drug with linker, along with ADC peak areas measured from size exclusion chromatography (SEC), were used to calculate DARs for 21 in-house ADCs. The DAR results were consistent with those obtained by MS analysis.