N-Acyl Alcohol Esters

The denvatization of carboxyl groups with HFIP has limitations because the derivatives are unstable in dilute solutions and are prone to extensive fragmentation even m chemical ionization (Cl) mode. Therefore, for analyses of trace amounts of amino acids, particularly in biological fluids or perfusates, the alcohol esters are preferred. In most cases, these derivatives, even in dilute solutions, are stable for 1-2 wk at room temperature. 

The esters are prepared by an initial acid-catalyzed esterification in an anhydrous alcohol. The most useful alcohols are n-butanol and methanol. In the case of 3N HCl- -butanol, the acidified reagent can be obtained commercially from Regis Chemicals, IL. The most popular methylating reagents are 10% boron trichloride in methanol that can be obtained from Sigma Chemicals, MO 20% HCl in methanol (dropwise addition of 1 mL of acetyl chloride to 4 mL of methanol, Corradetti et al., 1983) and thio-nyl chloride in methanol (dropwise addition of 1 mL of thionyl 

The subsequent acylation step is generally performed with trifluoroacehc anhydride (TFAA), PFPA, or heptafluorobutync anhydnde (HFBA), all of which yield excellent derivatives for GC-MS. The most popular derivatives include (a) TAB (TFA/n-BuOH)- Summons et al., 1974 Schulman and Abramson, 1975 Petty et al., 1976 Kingston and Duffield, 1978 Finlayson et al., 1980 (b) PAB (PFPA/n-BuOH). Sjoquist, 1979 Lapidot and Nissim, 1980 (c) TAM (TFA/MeOH) Zaylak et al., 1977, Coutts et al., 1979 (d) PAM (PFPA/MeOH) FauU et al., 1978 Ferkany et al., 1978 Corradetti et al, 1983 (e) HAM (HFBA/MeOH) Faull and Barchas, 1983 and (f) HAB (HFPB/ -BuOH) Lapidot and Nissim, 1980 Bengtsson et al, 1981. 

Of these derivahzation procedures, the one that we have found the most sensitive for dilute samples is the PAM procedure. However, as with all the procedures utilizing alcohol esters, this one has a drawback. Dunng the esterification reaction, glutamine is hydrolyzed to glutamate and asparagine to aspartate. Therefore, these dicarboxylic acids must be isolated from the carboxamides prior to esterification. Ion exchange on Dowex AG1-X8 is extremely useful for this procedure (Corradetti et al., 1983)  

The resin is next washed twice in 2N HCL and another three times in distilled water. The resin is stored under water. 

---