Derivatization with heptafluorobutyric acid

Enzymatic digestion 25 with subtilisin A, ether extn, SPE cleanup, lAC cleanup, liq-liq partns, derivatization with heptafluorobutyric acid anhydride (HFB derivatives), liq-liq partns. 

Ebbighausen et al. developed a method for the assay of morphine and codeine in urine using deuterium labeled morphine and codeine. Codeine was derivatized with heptafluorobutyric acid and morphine with trifluoroacetic acid before gas chromatography on a packed column with 1 % 0V-17 on Gas Chrom Q. The LKB 9000 combination of gas chromatograph-mass spectrometer equipped with a multiple-ion detector/peak matcher was used. With a simple extraction procedure of 1 ml urine after hydrolysis a practical limit of detectability was found to be approximately 500 pg. 

Previous derivatization of the extract is necessary to improve the stability of the compounds and the sensitivity and precision of subsequent GC-MS analysis. Silyl derivatives formed for example with MSTFA [43], halogenated alkene derivatives produced with heptafluorobutyric anhydride (HFBA) [36] or pentafluoropropionic acid [58] or anhydride (PFPA), as well as acetate derivatives formed using acetic anhydride [48] have been widely employed. 

The use of 1,3-dichlorotetrafluoroacetone (DCTFA) in combination with heptafluorobutyric anhydride to form oxazohdinone derivatives provides a useful alternative method of analyzing amino acids (Husek and Felt, 1978, 1984 Husek, 1982 Husek et al., 1982). Since reaction conditions are mild. Gin and Asn can be analyzed in the presence of other free ammo acids The derivatization sequence involves four steps condensation, acylation, extraction, and additional acylation (Husek, 1982) Condensation with DCTFA proceeds smoothly within 15 min at room temperature and subsequent acylation of the side chain —NH2, —OH, or —SH groups is achieved by reaction with HFBA for 30 s Critical extraction and evaporation steps are necessary for a successful assay If quantitative analyses of Arg, Gin, and Asn are required, an additional acylation is necessary. This is achieved by heating with HFBA at 80°C for 2-3 mm. The derivatization sequence is illustrated in Fig. 4 for serine. 

Derivatization Most amino acids are nonvolatile and must be derivatized prior to separation by GC. Amino acids are both esterified and acylated to produce N(0,S)-acylamino acid esters, with the most common being (N(0,5)-trifluoroacetyl- -butylesters. Other alcohols of low relative molecular mass have also been employed for the esterification including methanol, propanol, isopropyl alcohol, butanol, or isobutyl alcohol. Several different acylating reagents have been explored as well, including acetic anhydride, trifluoroacetic anhydride, penta-fluoroproprione anhydride, or heptafluorobutyric anhydride. 

Most derivatizations for gas chromatography are esterifications or etherifications. For example, analytes containing carboxyl groups are often converted into methyl [156], [161], pentafluorobenzyl [255]-[258], or triethyl esters [259]-[261], Analytes containing acidic hydroxyl groups (phenols, chlorophenols, glycol ethers) or amino groups (e.g.. aniline) can be derivatized with such per-fluorinated compounds as heptafluorobutyric anhydride, pentafluoropropionic anhydride [169], or pentafluorobenzoyl chloride [262]. 

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