# Recent Applications of Solid Phase Microextraction Coupled to Liquid Chromatography
## 2 Applications
### Biological
#### 2.1.3 Metabolomic  
SPME, including the in vivo format, can be also a very suitable means for metabolomic studies. Metabolomics, which is defined as the comprehensive analysis of metabolites in a biological specimen, is currently one of the most powerful analytical approaches for studying the functional status of biological systems in numerous research fields. As metabolites are the end-products of anabolic and catabolic pathways within living organisms, their comprehensive profiling may considerably help to understand many molecular mechanisms. As a matter of fact, metabolomics is deeply influenced by the sample pre-treatment procedure; it is crucial to maintain the metabolic integrity of biological samples so that metabolomic profiles can really be the direct expression of the biochemical status of a given organism. In vivo solid-phase microextraction was proposed by [PERSON] and co-workers [PERSON] and co-workers [PERSON] (2018) as a sampling method for metabolomics studies of living systems using liquid chromatography-mass spectrometry and mice as a model system. The results showed that it was possible to extract more than 1500 untargeted metabolites with different polarities using a single mixed-mode coated fiber. This sampling approach is potentially able to capture unique metabolites and detect changes in metabolic profiles associated with any kind of modification occurring in the organism under investigation. For instance, two recent different in vivo metabolomic studies were carried out in the muscle tissue of living trout by [PERSON] et al. [PERSON] _et al._ (2019); [PERSON] _et al._ (2020) using SPME fibers with biocompatible coatings and LC coupled to high resolution mass spectrometry (HRMS). Lipidic profiles were obtained [PERSON] _et al._ (2019) and compared to those obtained with an ex vivo SPME study of samples stored for a prolonged period, to determine the influence of sample storage, and to those obtained by solid liquid extraction. The great potential of the new approach was evident, since it enabled the characterization of a variegated lipidome, even detecting compounds not determinable with ex vivo SPME or SLE. Furthermore, the changes in the metabolic profile associated to short- and long-term exposition to sublethal doses of benzo[a]pyrene (BaP) [PERSON] _et al._ (2019) were determined in the muscle tissue of living fish by SPME-LC-HRMS, focusing on interesting dose and time-dependent alterations observed in the exposure by case-control model. The results indicated that BaP exposition was able to decrease the number of detected compounds. In vivo SPME, using a mixed-mode (C18 with benzenesulfonic acid) coating, coupled to LC-MS allowed the detection of more than 50 oxylipins, brain mediators involved in key processes, under normal physiological conditions in awake moving rats [PERSON] _et al._ (2019). Using the same well established experimental set-up as in vivo microdialysis, the approach permitted to obtain detailed oxylipin profiles in real time, eliminating post-mortem changes randomly observed in oxylipin concentrations. SPME-LC metabolomic studies were also performed ex vivo in biological fluids. A comprehensive study [PERSON] _et al._ (2019) reported the extraction of 36 different metabolites with a wide range of polarities in biological fluids by comparing the performances of more than 40 fiber coatings. Mixed-mode coatings, polar-enhanced polystyrene\(-\)divinylbenzene, and phenylboronic acid resulted to be the most suitable for the simultaneous extraction of the largest number of heterogeneous compounds and, consequently, for untargeted metabolomic profiling applications. The mixed-mode SPME coated fiber coupled to LC-HRMS was then employed to develop a fast method for the metabolomic profiling of human plasma. A comparison with ultrafiltration and solvent precipitation clearly showed the numerous advantages of the new approach in terms of metabolite coverage, method precision, and ESI ionization suppression.