Influence of data acquisition modes and data analysis approaches on non-targeted analysis of phthalate metabolites in human urine

Abstract

Humans are often exposed to phthalates and their alternatives, on account of their widespread use in PVC as plasticizers, which are associated with harmful human effects. While targeted biomonitoring provides quantitative information for exposure assessment, only a small portion of phthalate metabolites has been targeted. This results in a knowledge gap in human exposure to other unknown phthalate compounds and their metabolites. Although the non-targeted analysis (NTA) approach is capable of screening a broad spectrum of chemicals, there is a lack of harmonized workflow in NTA to generate reproducible data within and between different laboratories. The objective of this study was to compare two different NTA data acquisition modes, the data-dependent (DDA) and independent (DIA) acquisition (DDA), as well as two data analysis approaches, based on diagnostic ions and Compound Discoverer software for the prioritization of candidate precursors and identification of unknown compounds in human urine. Liquid chromatography coupled to high-resolution mass spectrometry was used for sample analysis. The combination of three-diagnostic-ion extraction and DDA data acquisition was able to improve data filtering and data analysis for prioritizing phthalate metabolites. With DIA, 25 molecular features were identified in human urine, while 32 molecular features were identified in the same urine samples using DDA data. The number of molecular features identified with level 1 confidence was 11 and 9 using DIA and DDA data, respectively. The study demonstrated that besides sample preparation, the impact of data acquisition must be taken into account when developing a NTA method and a consistent protocol for evaluating such an impact is necessary.

Keywords: Data acquisition mode; Data analysis approach; Human urine; Non-targeted analysis (NTA); Phthalate metabolites.

Fig. 1

The workflow of non-targeted analysis approach

Fig. 2

Extracted ion chromatograms (EIC). The concentration of monophthalates is 250 ppb. A EIC from DIA data, in-source full-scan, the in-source collision energy: 20 V; B EID from DDA data, full-scan ddMS2

Fig. 3

Extracted ion chromatograms (EIC). The concentration of monophthalates is 1 ppb. A EIC from DIA data, in-source full-scan, the in-source collision energy: 20 V; B EID from DDA data, full-scan ddMS2

Fig. 4

The difference of precursor ions between urine samples from infertile and fertile men. A the intensity difference of precursor ions between urine samples from infertile and fertile men; B the intensity difference of precursor ions at retention time between urine samples from infertile and fertile men

Fig. 5

Molecular features discovered by Compound Discoverer between urine from fertile and infertile men with a log-fold change > 2, p < 0.05. A Downregulated features that are present with higher intensity in samples (fertile men); B upregulated features that are present with higher intensity in samples (infertile men)