# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Sample Preparation
#### 5.1.4 Derivatization
Direct silylation of keto- and aldehyde groups produces several redundant compounds, complicating chromatograms' qualitative and quantitative interpretation [99; 100]. To avoid chromatographic artifacts (e.g., keto-enol tautomerization), oximation by hydroxylamines or alkoxyamines is used before silylation. It protects keto- and aldehyde-groups, preventing cyclization of sugars and decarboxylation of \(\alpha\)-keto acids [101]. The methoxization takes 1-2 h when heated (usually up to 37-70 \({}^{\circ}\)C) or overnight at room temperature [72; 102]. It is vital to carry out silylation in an anhydrous medium, such as pyridine, since active hydrogens of water molecules react vigorously with silylating agents. Anhydrous pyridine acts as an acid scavenger and accelerates derivatization [103]. Therefore, the silylation is usually completed at room temperature or heating within an hour to 60-70 \({}^{\circ}\)C [104]. Controlled microwave radiation reduces reaction times to minutes [103; 105]. An important disadvantage against the background of all the listed advantages of TMS-derivatization is the deteriorative effect of the excess of the TMS-reagent on the sorbent of some types of chromatographic columns (e.g., carbowax (polyethylene glycol) columns) [106]. Alkylation is an alternative derivatization strategy that can be used in metabolomic profiling. Ideologically, alkylation (or aylation in the case of the reagent with aryl group) is similar to silylation, based on nucleophilic substitution of the active hydrogens from the -OH, -COOH, -SH, -NH, or -CONH groups with an aliphatic or aliphatic-aromatic group. The alkylation products are less polar and more stable than the initial molecules, which, if necessary, makes it possible to isolate and \"preserve\" derivatives [97; 107]. Popular alkylating agents are short-chain alkyl- or aryl-halides, in which the cationic moiety carries a specific property, and the anionic moiety is responsible for specific reactivity. In addition to them, dialkyl acetals, diazolalkalalanes, pentafluorobenchyl bromide (PFBBr), boron trifluoride in methanol or butanol, tetrabutylammonium hydroxide, dimethyl sulfate are used. PFBBr alkylates phenols, thiols, and carboxylic acids. Alkyl bromides are used mainly for the derivatization of carboxylic acids and tetrabutylammonium hydroxide (TMH) for low molecular weight amines and carboxylic acids [97]. Alkylation has several features, both attractive to the researcher and undesirable. The advantage of derivatization through alkylation is a wide range of reaction conditions, varying from strongly acidic to strongly basic. Another valuable property is reaction speed. For example, dialkyl acetals react so quickly with functional groups of carboxylic acids, phenols, and thiols that this reaction can be used in flash derivatization directly at the injection port. On the other hand, some alkylating reagents of this type (diazomethane and dimethyl sulfate) are extremely toxic, and some (boron trifluoride) are very unstable even at low temperatures and are extremely sensitive to moisture. Such nuances significantly complicate the process of alkyl derivatization [97]. Like the derivatization approaches described above, the nucleophilic substitution of the active hydrogen of the polar group with the RCO group of the acylating agent allows reducing the polarity of small molecules and improve their behavior in the chromatographic column, although not so effective as silylation or alkylation. Acylylation gives the ability to derivatize a wide variety of compounds for GC analysis, especially amines, amino- and organic acids in serum and plasma [108; 109]. For example, a comparison of the ethyl chloroform derivative of low molecular weight compounds in blood serum made it possible to reliably distinguish between clusters of healthy volunteers and patients with uremia [110]. Unlike the more popular silylation, acylation can be performed in an aqueous medium, which is convenient for metabolomic profiling of plasma and serum [106].