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 TWCCSN2
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 TWCCSN2
database was built for 87 steroids
(142 ions). The cross-laboratory database consists of average TWCCSN2
values obtained by the four TWIMS
instruments in triplicate measurements. In general, lower deviations
were observed between TWCCSN2
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 TWCCSN2
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 TWCCSN2
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.
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