# Recent Applications of Solid Phase Microextraction Coupled to Liquid Chromatography
## 2 Applications
### Biological
#### 2.1.2 In Vivo Sampling  
Another crucial branch of biological applications is represented by in vivo sampling procedures. They should allow the preservation of the sample to ensure that the information eventually obtained are effectively representative of the situation of the living organism under investigation. Fiber SPME perfectly fits this requirement thanks to its syringe-like format that allows a faster and simpler sampling, compared to alternative methods (i.e., microdialysis or ultrafiltration), followed by retraction into the holder for storage, protection, and transportation of the extracted compounds. [PERSON] et al. developed three original SPME-LC-MS/MS applications for the analysis of target pharmaceuticals (carbamazepine, diazepam, nordiazepam, and others) in fish samples. In the first study [38], a method for the determination of the bioconcentration of trace contaminants in fish bile was first validated in rainbow trout after laboratory exposures and then applied to fathead minnow caged upstream and downstream of a local wastewater treatment plant. The method performances in field were demonstrated by the detection of some analytes detected in fish bile at the downstream location. In the second one [39], the same approach was applied to the in vitro and in vivo study of kinetic desorption rate constants of the analytes in agarose gel model used to simulate tissue matrix and fish muscle (dorsal muscle), respectively, using a triple quadrupole-linear ion trap (Qtrap) mass spectrometer as LC detector. The third work [40] was focused on the development and evaluation of a new improved in vivo SPME device (PDMS coating), that enabled a simplified sampling procedure without the need of fish anesthetization, as demonstrated by the in vivo determination of the target drugs in the muscle tissue of wild river fishes. In situ SPME (mixed-mode fibers, octadecyl and benzenesulfonic acid groups) with LC-MS/MS detection was proposed as a green in vivo method for the direct underwater sampling of a very wide range of different small compounds from corals, so that the organisms can remain in their natural habitat [41]. More than 280 species were identified, mostly eicosanoids, steroids, prenol lipids, fatty acids, glycerolipids, and glycerophospholipids, likely because of the sampling location, near tentacles, biological membranes of corals. Three SPME (reverse phase amide fiber, RPA) calibration methods for in vivo application, i.e., on-fiber standardization, dominant pre-equilibrium desorption, and diffusion-based interface model were compared by [PERSON] et al. [42] using fenoterol as a drug model and LC-MS/MS detection. The methods were also validated against SPME equilibrium extraction external calibration and protein precipitation. The study demonstrates that the diffusion-based model was the most convenient for future in-vein blood SPME investigations, to sample the circulating blood of animals without the need to withdraw representative samples. In fact, the same research group subsequently developed, using the same RPA fiber, a new in vivo SPME-LC-MS/MS procedure [43] to determine the pharmacokinetics of fenoterol and methoxyfenoerol in rat blood using the diffusion-based calibration interface model. The results obtained were in good agreement with those obtained by a traditional approach of blood withdrawal followed by protein precipitation, indicating the utility of the proposed method. A protocol for the in vivo determination of benzodiazepines and metabolites in beagles by SPME-LC-MS/MS was validated by [PERSON] and co-workers [44], using a polypyrrole wire coating exposed to the bloodstream through a SPME probe directly inserted into a peripheral vein by means of a standard medical catheter.