# New Challenges in (Bio)Analytical Sample Treatment Procedures for Clinical Applications
## 3 Sample Preparation Techniques for Diagnostic Purposes
### Solidphase Microextraction (SPME)  
Biological samples contain a variety of metabolites that can be used for clinical purposes in the diagnosis of various clinical conditions and many severe disorders. Among sample preparation techniques, the most widely used procedure is solid-phase microextraction (SPME), which includes sampling, extraction, and analyte preconcentration into a single step. By contact with liquid bio-matrices, analytes are transferred to the adsorbent-coated solid surface thanks to the affinity of analytes to the coating material. Direct immersion SPME (DI-SPME) and headspace SPME (HS-SPME) are the two modes of SPME operations. The first extraction technique is based on the sorption of analytes on the stationary phase immobilized on a fused silica glass rod that acts as the solid support. During the analytes' extraction, the coated fiber is directly immersed into the sample solution, while in the HS-SPME, the fiber is exposed in the vapor phase above the sample. At equilibrium, the quantity of analyte extracted by the fiber is proportional to its concentration in the sample [38]. The main advantages of this method include simplicity and speed of operations, high sensitivity, and the volume of solvent reduction; for these reasons it has been applied to the investigation of metabolites and neurotransmitters for diagnostic purposes [2]. Many metabolites and neurotransmitters are useful for the diagnosis of neurological diseases. Neurotransmitters (NTs) are basic signaling molecules used for cell communica  
Figure 1: Sources of error generated during chromatographic analysis. tions; they are involved in stress response mechanisms, the regulation of motor coordination, the control of psychomotor, gastrointestinal, and homeostatic function and neuronal communication. Many psychomotor, psychiatric, neurodegenerative, and oncological disorders are characterized by an abnormal production, release, and/or metabolism of NTs; for this reason, NTs can be used as a potential biomarker for diagnoses of [PERSON]'s and Parkinson's diseases, neuroendocrine cancers, and psychiatric illness such as schizophrenia. The most common biological fluids in the NT analysis are urine and blood, but other alternative matrices include CSF and cerebral tissue, with the related difficulty due to the presence of interfering molecules and low levels of the target analytes. Therefore, the development of validated methods of sample preparation and analytical techniques for the quantitative analysis of NT in biological samples is needed [39]. [PERSON] et al. developed a method for the determination of NTs such as dopamine (DOP), norepinephrine (NE), and serotonin (5-HT) in human urine by SPME coupled with gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS). The optimized SPME method provides a first step of extraction with polyacrylate fiber at room temperature for 45 min without the addition of NaCl; the second step was desorption at 300 \({}^{\circ}\)C, followed by a derivatization reaction carried out directly in a urine sample and SPME extraction in immersion mode in the same vial (without organic solvent or further sample treatment). The developed method has shown good linearity with correlation coefficient values of 0.9995 for DOP, 0.9999 for 5-HT, and 0.9987 for NE; the accuracy values were between 92.8% and 103.0%, and RSD% ranged between 0.67% and 4.5%. LOD values were 0.587 ug/L for DOP, 0.381 ug/L for 5-HT, and 13.5 ug/L for NE, and LOQ values were 0.81 ug/L for DOP, 0.74 ug/L for 5-HT and 21.3 ug/L for NE. The obtained satisfactory results indicate that the proposed method can be adapted for the analysis of urine samples, which contain a much lower concentration of DOP, 5-HT, and NE than those determined for healthy adults.