A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Changed isoflavone levels in red clover (Trifolium pratense L.) leaves with disturbed root nodulation in response to waterlogging.

The effect of disturbed root nodulation on the quantitative and qualitative composition of the main isoflavonoid glucoside malonates, glucosides, and aglycones in the leaves of Trifolium pratense L. grown under waterlogging conditions was investigated. Isoflavonoids are involved in the regulation of root nodule activity and the establishment of the mycorrhizal association. Isoflavonoid determination was performed using reversed-phase liquid chromatography coupled to mass spectrometric and UV absorbance detection. In response to waterlogging, the concentrations of biochanin A and biochanin A-7-O-glucoside malonate, biochanin A-7-O-glucoside, and genistein-7-O-glucoside in the leaves increased two- to threefold after a lag period of 3 wk because of disturbed root nodulation. The other isoflavones detected formononetin, formononetin-7-O-glucoside malonate, and formononetin-7-O-glucoside-did not show any significant changes related to waterlogging. After restoring normal soil water conditions, the concentrations of biochanin A and its glucoside and glucoside-malonate rapidly returned to the initial values, whereas the concentration of genistein-7-O-glucoside remained high.