Measurement of very low-molecular weight metabolites by traveling wave ion mobility and its use in human urine samples

The collision cross-sections (CCS) measurement using ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) offers a great opportunity to increase confidence in metabolite identification. However, owing to the lack of sensitivity and resolution, IMS has an analytical challenge in studying the CCS values of very low-molecular-weight metabolites (VLMs ≤ 250 Da). Here, we describe an analytical method using ultrahigh-performance liquid chromatography coupled to a traveling wave ion mobility-quadrupole-time-of-flight mass spectrometer optimized for the measurement of VLMs in human urine samples. The experimental CCS values, along with mass spectral properties were reported for the 174 metabolites. The experimental data included the mass-to-charge ratio (m/z), retention time (RT), tandem MS (MS/MS) spectra, and CCS values. Among the studied metabolites, 263 traveling wave ion mobility spectrometry (TWIMS)-derived CCS values (^TWCCS_N2) were reported for the first time, and more than 70% of these were CCS values of VLMs. The ^TWCCS_N2 values were highly repeatable, with inter-day variations of < 1% RSD. The developed method revealed excellent ^TWCCS_N2 accuracy with a CCS difference (ΔCCS) within ±2% of the reported drift tube IMS (DTIMS) and TWIMS CCS values. The complexity of the urine matrix did not affect the precision of the method, as evidenced by ΔCCS within ±1.92%. According to the Metabolomics Standards Initiative, 55 urinary metabolites were identified with a confidence level of 1. Among these 55 metabolites, 53 (96%) were VLMs. The larger number of confirmed compounds found in this study was a result of the addition of ^TWCCS_N2 values, which clearly increased metabolite identification confidence.