To achieve the intended system pressures when coupling to MS, the volume of post-column tubing is kept to an absolute minimum (33 cm or 38 cm of 0.10 mm ID tubing when used with incorporated in the MS system to facilitate automated switching of inputs) to ensure highest chromatographic performance and reliability of the system. These methods were initially used with Q-ToF instruments (Waters Xevo G2-S) utilising Z-spray configured electrospray sources but have been compatibility tested with mass spectrometers from other instrument manufacturers. Any high-resolution MS instrument capable of adequately desolvating a 0.6 mL/min flow (challenging at 99% aqueous effluent) and a rapid scanning rate capable of producing more than 10 points per peak for accurate quantitation (excluding any duty cycle consumed for alternative purposes including structural characterisation by MS/MS or dynamic range extension by interleaving scans with an attenuated beam) should be suitable for utilising these methods. When paired with Waters Xevo G2-S instruments, we observed a propensity towards in source fragmentation (ISF) owing to the nature of the ion optics. Consequently, acquisition of routine profiling data with simple MS1 is sufficient to generate rich spectra inclusive of diagnostic fragmentation patterns. This acquisition strategy is complemented with data-dependent analysis (DDA) MS/MS analyses on pooled QC samples (QC materials are discussed below in greater detail) and may be further augmented with targeted MS/MS analyses on specific samples of interest as needed. Finally, it should be noted that the timeof-flight MS instrument is regularly tuned to produce the highest resolution achievable at or near maximal sensitivity. By prioritising sensitivity over other instrument attributes (i.e. resolution in orthogonal acceleration time-of-flight mass spectrometry), the amount of sample required for each analysis can be minimised. In turn, larger scale analysis (batches of one to two thousand study samples) can be more practically achieved without stopping and disassembling for cleaning and maintenance. This is a central tenet of our "continuous analysis" approach to delivering high quality LC-MS raw data.