A CE method was developed and validated for the assessment of the chiral purity of the drug tenofovir applying a quality by design approach. Following selection of a quaternary ammonium β‐CD as chiral selector, a fractional factorial resolution V+ design was employed for identification of the critical process parameters, while a central composite design served for method optimization. The final method used a 40/50.2 cm, 50 μm id fused‐silica capillary, a BGE composed of a 100 mM sodium phosphate buffer, pH 6.4, containing 45 mg/mL quaternary ammonium β‐CD, an applied voltage of 18 kV, and a capillary temperature of 22°C. Robustness was assessed by a Plackett–Burman design. The method was validated according to guideline Q2(R1) of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use and enabled the determination of the (S)‐enantiomer of tenofovir at the 0.1% level.

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the identification and quantification of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to internal or external perturbations in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe µPhos, an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96–well cell culture experiments in < 8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to < 200 µL, we demonstrate increased sensitivity, over 90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify more than 20,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize > 5,000 phosphorylation sites from 5 µg. This depth covers key intracellular signaling pathways, rendering sample-limited applications and extensive perturbation experiments with hundreds of samples viable.