The Importance of Derivatizing Reagent in Chromatography Applications for Biogenic Amine Detection in Food and Beverages

Table 3

Various derivatizing agents for gas chromatography purposes and their characteristics [65–67].

No.	Reagent	Chemical structure	Characteristics
1	Acylation
	α-Methoxy-α-trifluoromethylphenylacetic acid (MTPA)		(i) It is acidic and called Mosher’s acid
			(ii) Very effective in generating ions of analytes
	Acetic anhydride		(i) Improves selectivity in GC, MS, and FID detectors, especially negative chemical ionization
			(ii) Generally used by base compounds such as biogenic amines
	4-Carbethoxyhexafluorobutyryl chloride		(i) Very stable for secondary amines because it can remove the excess agent by adding protic solvents
	Heptafluorobutyrylimidizole (HFBI)		(i) Fast reaction and suitable for amine and alcohol
			(ii) Byproduct is not acidic
	N-Methyl-N-bis (trifluoroacetamide) (MBFTA)		(i) Acceptable and fast for primary and secondary amines
			(ii) Byproduct is volatile
	N-(Trifluoroacetyl)-prolyl chloride (TPC)		(i) Generally applied for amine compounds
			(ii) With a proton at the chiral center in the α position to the carbonyl group in order to avoid racemization through keto-enol tautomerization and thus reaction and storage conditions must be controlled
	Pentafluorobenzoyl chloride (PFBCI)		(i) Very reactive, obtaining the most sensitive ECD derivatives of amine
	Propyl chloroformate		(i) Derivative analyte is water soluble; thus, the byproducts can be removed using water
2	Alkylation
	BF3/methanol (n-butanol)		(i) Obtaining methyl (butyl) ester with acid
			(ii) Fast and quantitative esterification/transesterification
			(iii) No side reactions with volatile byproducts
	Tetrabutylammonium hydroxide (TBH)		(i) Very reactive to low molecular weight amines
	Trimethylanilinium hydroxide (TMAH)		(i) Knows as a flash alkylation reagent
			(ii) Quantitative derivatization of nitrogen-bearing molecules
3	Silylation
	Hexamethyldisilazane (HMDS)		(i) A weak (trimethylsilyl) TMS donor
			(ii) It applies without solvent, yet the reagent capacity can be increased by the acidic catalyst
			(iii) Byproduct can leave the reaction mixture as the reaction goes to completion
	N, O-Bis (trimethylsilyl) acetamide (BSA)		(i) Product derivatives are stable
			(ii) Byproduct elutes with the analyte
			(iii) Reactions are fast and quantitative
			(iv) BSA and its byproducts are more volatile than other silylating reagents causing less chromatographic interference
	N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA)		(i) Faster than BSA and more complete
			(ii) Reacts with a range of polar organic compounds by replacing active hydrogens with a TMS group
			(iii) TMCS addition is recommended in order to control hydroxyl presence and other functionalities
			(iv) Reduces the sensitivity of FID detector
	N-Methyl-N-(t-butyldimethylsilyl) trifluoroacetamide		(i) Very reactive and stable
			(ii) To hinder alcohol and amine groups and add a catalyst such as t-butyldimethylchlorosilane
			(iii) Does not release HCl
			(iv) Very suitable for mass spectrometry detector as it provides high-mass ions
	Trimethylchlorosilane (TMCS)		(i) A catalyst and forms HCl as a byproduct
			(ii) Completes the derivatization step after secondary amines derivatized by BSTFA by adding 1–20% TMCS to BSTFA
	Trimethylsilyldiethylamine (TMS-DEA)		(i) Suitable for amino and carboxylic acids
	N-Trimethylsilylimidazole (TMSI)		(i) Reacts with -OH and cannot react with aliphatic amines
			(ii) It is stable thermally yet more susceptible to hydrolysis