Interlaboratory and Interplatform Study of Steroids Collision Cross Section by Traveling Wave Ion Mobility...

Collision cross section (CCS) databases based on single-laboratory measurements must be cross-validated to extend their use in peak annotation. This work addresses the validation of the first comprehensive ^TWCCS_N database for steroids. First, its long-term robustness was evaluated (i.e., a year and a half after database generation; Synapt G2-S instrument; bias within ±1.0% for 157 ions, 95.7% of the total ions). It was further cross-validated by three external laboratories, including two different TWIMS platforms (i.e., Synapt G2-Si and two Vion IMS QToF; bias within the threshold of ±2.0% for 98.8, 79.9, and 94.0% of the total ions detected by each instrument, respectively). Finally, a cross-laboratory ^TWCCS_N database was built for 87 steroids (142 ions). The cross-laboratory database consists of average ^TWCCS_N values obtained by the four TWIMS instruments in triplicate measurements. In general, lower deviations were observed between ^TWCCS_N measurements and reference values when the cross-laboratory database was applied as a reference instead of the single-laboratory database. Relative standard deviations below 1.5% were observed for interlaboratory measurements (<1.0% for 85.2% of ions) and bias between average values and ^TWCCS_N measurements was within the range of ±1.5% for 96.8% of all cases. In the context of this interlaboratory study, this threshold was also suitable for ^TWCCS_N measurements of steroid metabolites in calf urine. Greater deviations were observed for steroid sulfates in complex urine samples of adult bovines, showing a slight matrix effect. The implementation of a scoring system for the application of the CCS descriptor in peak annotation is also discussed.