Liquid and gas-chromatography-mass spectrometry methods for exposome analysis

Alertly, the impact of exogenous chemicals on human health, and particularly the impact of their combined and interactive effects, needs to be better characterized. Most of the research exploring the effects of exogenous chemicals on human health has been focused on the impact of single groups of chemicals, even though studies revealed chemical mixtures can cause different types of combination effects, including additive and synergistic effects, and, less often, antagonistic effects [[6], [7], [8], [9]] (Fig. 1). Therefore, rather than purely targeting selected pollutants, screening a broad coverage of known and unknown compounds is of the utmost importance. It is also fundamental to explore longitudinal and retrospective studies to investigate the intricate associations between exposure, metabolism, and health outcomes. Fortunately, gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) can be designed to meet this challenge.

Journal of Chromatography A, Volume 1744, 2025, 465728: Fig. 1. Combined effects of environmental pollutants (e.g., compounds A and B) are usually additive, synergistic and, less often, antagonistic

Undoubtedly, MS-based approaches provide better coverage than other techniques applied in exposome research, such as nuclear magnetic resonance [19], but defining pre-analytical steps, separation technique(s), and post-analysis methods will vary according to the fraction of the exposome being explored. From an analytical perspective, MS-based analysis is the approach of choice in exposome research due to its ability to detect and identify a broader range of environmental substances at trace concentrations. Still, MS-based protocols face several challenges, from sampling and extraction to analysis, data processing, compound annotation, and data interpretation. Here, we provide an overview of the main steps of MS-based analytical approaches in exposome research, from sample extraction to GC- and LC-MS analysis and data processing (Fig. 2).

Journal of Chromatography A, Volume 1744, 2025, 465728: Fig. 2. Overview of sample preparation and GC- and LC-(HR)MS analysis workflows in exposomics research.

2. Mass spectrometry in exposome analysis

2.1 Sample collection and extraction

The analysis of the human (i.e., internal) exposome is conducted in a range of biological matrices, mainly using urine and blood but also feces, saliva, breath biopsy, breast milk, as well as other organs and tissues [10]. Additionally, studies may integrate other organisms (e.g., cells, rodents) to elucidate potential underlying mechanisms on complex biological systems and non-biological matrices (e.g., water, soil) to provide complementary environmental information. While many studies focused on developing methods to characterize the chemical exposome comprehensively, more effort is needed to define reporting guidelines for sampling in exposome research, including procedures for fasting (or non-fasting) conditions in human biomonitoring, sampling devices, and conditions for sample pre-processing [20]. As in any other “omics” workflow involving biological samples, sample analysis in exposomics must reflect the nature level of the chemical exposome. Thus, potential contamination during sampling should be accounted for, ensuring that the identification and quantification of exogenous compounds represent the intrinsic levels of biological matrices. This is particularly important in exposomics, as many exogenous substances posing toxicity are also found in sampling materials [21].

In addition to potential contamination sources, quenching procedures and sampling time are necessary to allow analysis of extracts reflecting endogenous levels, thereby ensuring adequate biological interpretation in highly metabolically active systems such as cells and tissues. One way to improve sampling routines for exposome is to employ pre-analytical procedures universally applicable for non-target metabolomics whenever possible [[22], [23], [24], [25], [26]].

Sample preparation methods in exposomics have been explored more thoroughly than sampling strategies and were also similar to those described for non-target metabolomics analysis. As recent and comprehensive reviews on exposomics sample preparation are available in the literature [27,28], we will focus on overarching concepts rather than defining specific methods. In metabolomics, smaller volumes are typically sufficient because most endogenous analytes are present at relatively high concentrations. However, this is not the case for some exogenous substances, which might be detected near the detection limit of MS technologies. Consequently, different sample preparation methods, such as liquid extraction and solid-phase extraction, have been developed to explore the exposome considering different matrices and chemical space covered.

2.2. Mass spectrometric-based analysis

GC or LC combined with high-resolution mass spectrometry (HRMS) are the most widely employed methods in exposome analysis. Despite the appreciated sensitivity and peak resolution capabilities of GC, LC can cover a broader chemical space and, therefore, has been applied more frequently in exposome studies. A recent systematic review revealed that more than 60 % of exposome studies employ LC-HRMS while remaining studies mostly rely on GC-based methods [32]. Other authors indicate that the fraction of LC-HRMS-based studies in exposomics is even higher, up to 80 % [33]. Targeted quantification using low-resolution (triple quadrupole) mass spectrometry (QqQ-MS) remains in use to some extent due to its sensitivity, but primarily as a complementary technique. Other MS-based techniques, including capillary electrophoresis-MS (CE-MS) [34], selected ion flow tube MS (SIFT-MS) [35], and MS imaging (MSI) [36], also have been employed for selected applications.