# New Challenges in (Bio)Analytical Sample Treatment Procedures for Clinical Applications
## 3 Sample Preparation Techniques for Diagnostic Purposes
### Solidphase Microextraction (SPME)
The extraction method was developed using the biocompatible coated SPME blades that were first conditioned for 30 min with 1.5 mL of methanol/water (50:50, _v: v_) in 96-well-plates with agitation; all analytes required a time of 60 min for equilibrium extraction. To guarantee a complete immersion of the coating in the sample and to improve the method sensitivity, they have chosen a volume of saliva of 1.2 mL. After extraction, the method involves static washing with deionized water (1.5 mL) to remove matrix components from the surface of the coating and prevent subsequent desorption, no loss of analytes was detected after this step. The optimal conditions of desorption for all analytes were 90 min in acetonitrile/water 80:20 (_v: v_). The LOQ was 178, 22, 105, 185, 29, and 16 pg mL\({}^{-1}\) for cortisol, testosterone, progesterone, E3, E2, and E1, respectively. This method has shown advantages in terms of high sensitivity, reproducibility, and accuracy for the detection of endogenous steroids in saliva. These characteristics make the method better than the common immunoassay, which may lead to erroneous results due to the significant cross-reaction; the validated SPME method showed better results than liquid-liquid extraction (LLE) thanks to a cleaner extract that prevents matrix effects. Moreover, it was observed that in vivo sampling had a better efficiency than ex vivo sampling of saliva [43]. In recent years, a new special application of the SPME technique was observed that includes in vivo analysis. This method combines the advantages of the SPME technique for sample preparation that requires no, or small amount of, organic solvent and the advantages of the in vivo sampling that eliminates errors related to degradation or loss of short-life or unstable analytes; moreover, with this sampling method, it was possible to sample over time the same individual (e.g., drug monitoring). SPME was coupled to GC-MS or LC-MS/MS to separate and detect analytes or directly to MS. Usually, this extraction technique depends on the fiber and matrix characteristics. The following different SPME coating fibers have been employed to determine numerous compounds with different volatility and polarity in complex matrices: CAR/PDMS (Carboxen/polydimethylsiloxane) have been used to determine VOCs, while PDMS-DVB fibers have been used to determine the non-polar or weakly polar VOCs with GC-MS analysis, drugs and metabolites with direct MS and endogenous hydrocarbons in human breath with GC-MS. Different methods have been developed with this in vivo procedure for non-invasive and sensitive determination of different bio analytes in different types of samples in different fields of applications. Amongst complex matrices used for in vivo SPME methods, the easiest to sample is the skin with direct contact of SPME fiber placed on the skin surface; moreover, breath and saliva can be used to obtain in vivo pathophysiological information. For example, VOCs in human exhaled breath or emanating from human skin are potentially used as biomarkers for different disease diagnoses. The methodology of in vivo SPME of VOCs in human skin was used to record the VOC profile in skin cancer (e.g., basal cell carcinoma), fibrotic skin disorders, or vascular lesions. In addition, VOCs in human breath are useful in the diagnosis of lung cancer, liver disease, myocardial infarction, or diabetes [44].