# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Sample Preparation
#### 5.1.3 Extraction
Ultrasonic stimulation can also increase the degree of extraction. It was shown that after four minutes of ultrasonic extraction (40 kHz, 350 W), the intensities of most of the 570 resolved peaks increased compared to two and 10 min of the traditional vortexing [79]. Moreover, by changing the pH, one can also influence the type and number of extracted metabolites. An experiment with blood components showed that the sum of unique molecular features on the chromatography-mass spectrum after extraction at pH 2, pH 7, and pH 9 is 45% more than extraction in a neutral medium [80]. Solid-phase microextraction (SPME) is based on redistributing substances between phases due to sorption or ion-exchange processes. SPME is gaining popularity as a method for sample preparation in metabolomics experiments providing significant reduction in matrix effects [81; 82]. SPME process can be fully automated, allowing for high sample throughput and improving method repeatability [83; 84]. Among the advantages of SPME, the diversity of commercially available extraction phases manufactured in standardized conditions is noteworthy [85]. For the extraction of a broad metabolome, relatively versatile silica-based C18 resins [86] and complex multicomponent and multilayer coatings, e.g., DVB/CAR/PDMS (divinylbenzene/carboxylate/polydimethylsiloxane) are used [87]. Such coatings make it possible to adapt the average pore size in different sorbent layers to different sizes of extracted analytes. The polyacrylate (PA)-coated SPME cartridge has shown excellent results for recovering volatile organic metabolites from liquid biological samples. When used, the number of well-resolved peaks and their total intensity is higher than with PDMS/DVB or CAR/PDMS coated cartridges [88]. SPME with DVB/CAR/PDMS sorbent mixture made it possible to extract and subsequently identify almost 300 unique metabolites, including hydrocarbons, amines, ethers and esters, alcohols, carboxylic acids, thiols, terpenoids, and heterocyclic compounds, etc. [89]. Moreover, when comparing various complex coatings for solid-phase extraction (PDMS/DVB, PA, DVB/CAR/PDMS, CAR/PDMS, PDMS, and PEG (carbowax-polyethylene glycol)) of volatile organic metabolites, it is also the CAR-DVB-PDMS phase which allowed to detect the largest number of metabolites (63% of the pool of identifications for the sum of all methods) [90]. However, absorbents are rarely used for panoramic profiling [89], but they show good results for extracting certain classes of substances. Thus, polar sorbents (carbowax-divinylbenzene) are effective for extracting polar (alcohols, ketones, nitroaroomatics) analytes, and nonpolar sorbents (PDMS) are effective for extracting nonpolar analytes (polycyclic aromatic hydrocarbons, benzene/toluene/ethylbenzene/xylenes) [91]. Solid-phase extraction revealed statistically significant differences between metabolomics of patients with breast cancer and healthy people for several volatile organic compounds (including benzene derivatives, terpenoids, ketones, sulfur compounds, and phenols) [92; 93].