Ambrosia artemisiifolia L. (common ragweed) is an invasive plant whose allelopathic properties have been suggested by its field behaviour and demonstrated through phytotoxicity bioassays. However, the nature of the molecules responsible for the allelopathic activity of common ragweed has not been explored. The main objective of this study was to identify the phytotoxic molecules produced by A. artemisiifolia. A preliminary investigation has indicated that a methanol extract of A. artemisiifolia completely inhibited the germination of cress and radish. Semi-preparative fractionation of the methanol extract allowed separating of phytotoxic fraction which contained a single compound. The structure of this compound was elucidated by liquid chromatography–mass spectrometry (LC-MS)/MS, high-resolution mass spectral, nuclear magnetic resonance, and Fourier transform infrared spectra as sesquiterpene lactone isabelin (C₁₅H₁₆O₄). The effect of pure isabelin was tested on four different weed species, confirming the inhibitory activity of molecule. The results indicate directions for the future studies about herbicidal specific activity of isabelin, as pure molecule or in the crude extract, as a potential candidate for biological weed control.

Bioanalytical methods to quantitate conjugated payloads are essential for assessing antibody-drug conjugate (ADC) stability and pharmacokinetics (PK). Dual-payload ADCs present analytical challenges; different linker chemistries can require complex digestion conditions to perform the cleavage. Developing separate methods for each linker combination can be time and resource demanding. Rat tritosomes—purified lysosomal fractions from Triton-treated rat liver—provide a comprehensive enzymatic mixture that mimics the lysosomal environment. The presented bioanalytical method combines immunoaffinity purification with tritosome-mediated digestion for simultaneous quantitation of dual-conjugated payloads. The method was applied to a model dual-payload ADC containing two different cytotoxic payloads, conjugated using different enzymatically cleavable linkers, with an unrelated DAR (drug-to-antibody ratio). Method validation in mouse plasma demonstrated excellent accuracy (bias ± 20%, LLOQ and ULOQ ± 25%) and precision (coefficient of variation CV% ≤ 20%, LLOQ and ULOQ ± 25%) across all concentration levels (lower to upper limit of quantitation, LLOQ to ULOQ) for both payloads, with 100% of quality control samples (QCs) meeting acceptance criteria for hybrid LC-MS/MS quantitation methods. This tritosome-based approach provides a unified, efficient platform for multi-payload ADC bioanalysis, eliminates linker-specific method optimization, and enables robust support for preclinical studies. The method has been tested for accuracy and precision on 4 different model ADCs and employed to quantify the conjugated payloads in in vivo samples from a homozygous hFcRn transgenic mouse model (Tg32) PK study, resulting in reliable data in accordance with total antibody measurements.

Bioanalytical methods to quantitate conjugated payloads are essential for assessing antibody-drug conjugate (ADC) stability and pharmacokinetics (PK). Dual-payload ADCs present analytical challenges; different linker chemistries can require complex digestion conditions and to perform the cleavage. Developing separate methods for each linker combination can be time and resource demanding. Rat tritosomes—purified lysosomal fractions from Triton-treated rat liver—provide a comprehensive enzymatic mixture that mimics the lysosomal environment. The presented bioanalytical method combines immunoaffinity purification with tritosome-mediated digestion for simultaneous quantitation of dual conjugated payloads. The method was applied to a model dual-payload ADC containing two different cytotoxic payloads, conjugated using different enzymatically cleavable linkers, with not related Dar (drug-to-antibody-ratio). Method validation in mouse plasma demonstrated excellent accuracy (bias ± 20%, LLOQ and ULOQ ± 25%) and precision (coefficient of variation CV% ≤ 20%, LLOQ and ULOQ ± 25%) across all concentration levels (lower to upper limit of quantitation ,LLOQ to ULOQ) for both payloads, with 100% of quality control samples (QCs) meeting acceptance criteria for hybrid LC-MS/MS quantitation methods. This tritosome-based approach provides a unified, efficient platform for multi-payload ADC bioanalysis, eliminates linker-specific method optimization and enables robust support for preclinical studies. The method has been tested for accuracy and precision on 4 different model ADCs and employed to quantify the conjugated payloads in in-vivo samples from a homozygous hFcRn transgenic mouse model (Tg32) PK study, resulting in reliable data in accordance with total antibody measurements.