# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Sample Preparation
#### 5.1.4 Derivatization  
Derivatization can be applied before or after chromatographic separation (Figure 3, stage 4). In GC, pre-column derivatization is much more common. Post-column derivatization is quite an exotic procedure enhancing the detectability of the analytes through rapid physical-chemical conversion (pyrolysis, catalytic hydrogenation, etc.). In this case, the chromatogram of the initial mixture is recorded, and then the substance is modified for more accurate mass spectrometric analysis. For example, for post-column derivatization, a dehydrogenation microreactor installed between the GC and MS was used. Since only six-membered rings without quaternary carbon atoms undergo aromatization, this post-column dehydration makes it possible to differentiate between cyclopentane and cyclohexane hydrocarbons [94]. As part of this review, we will focus on predominant pre-column methods. During GC-MS analysis, volatile low molecular weight compounds pass through a chromatographic column heated to about 300 \({}^{\circ}\)C and higher. However, most metabolites (e.g., glucose, lactate, pyruvate, palmitate, etc.) have higher boiling points due to their polar functional groups. They, therefore, are not volatile at the highest temperature allowed for a GC system. Pre-column chemical derivatization protects polar functional groups, improves the volatility and thermal stability of the molecule [95]. The replacement of acidic protons of amino-, hydroxyl-, carboxyl-, and thiol-groups with other groups (silyl-, alkyl formate-, etc.) weakens intermolecular interactions, decreasing the boiling point and polarity of the metabolite, and increasing its stability [96; 97]. The choice of the optimal derivatization reaction for GC-MS-based metabolomics is not always a simple task because there are a plethora of various compromises, more or less suitable for specific molecular targets. Most popular and universal derivatization protocols use trimethylsilylaton strategy and its variants [55]. Alternative variants (alkylation and acylation complete the list of the top three most abundant techniques) expand the \"toolbox\" of derivatization and will also be described below. Silyulating agents replace the active proton in many functional groups, including OH, COOH, SH, NH, CONH, POH, and SOH with trimethylsilyl (TMS) group [97]. The good volatility and stability characteristics make silylated derivatives highly suitable for GC-MS analysis (Figure 4). The most commonly used silylation agents are N,O-bis-trimethylsilyltrifluoroacetamide (BSTFA), N-methyl-trimethylsilyltrifluoroacetamide (MSTFA), and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Among popular trimethylsilylacetamides, MSTFA is the most volatile, mild, and versatile reagent for the complex profiling of metabolites of blood components [56]. The silylation potential of BSTFA is close to that of MSTFA, but the volatility is slightly lower, which may positively affect the ease of use of this derivatizing agent. Moreover, the stability of BSTFA derivatives ensures low noise and moderate detector fouling [97] (Figure 4). Figure 4: The typical scheme of trimethylsilylation. Bimolecular nucleophilic substitution (SN2) replaces the active hydrogen (-OH, -COOH, -NH, -NH2, and -SH) by the trimethylsilyl group. Compared to the initial compounds, silylated derivatives generally are more volatile and thermally stable, thus yielding advanced separation of symmetric and narrow chromatographic peaks. The molecular weight of the initial molecule plays an essential role in the derivatization. If the molecule has a low molecular weight, there are no fundamental differences between the TMS agents. For relatively large metabolites (dicarboxylic acids, syrenes, monohydrooxy-polycyclic aromatic hydrocarbons) without hindered functional groups (9-hydroxyfluorene, sugars), MTBSTFA is an excellent solution. A significant advantage in using MTBSTFA is the absence of highly volatile by-products that interfere with early eluting peaks [98].