Convenient Synthesis of Stable Deuterium-Labeled Alkylpyrazines for Use in Stable Isotope Dilution Assays

Stable isotope dilution assays (SIDA) provide for accurate and precise quantitation of aroma components, such as alkylpyrazines, which are often present in low concentrations in complex food matrices. The unavailability of labeled standards is the main limitation to the widespread use of SIDA. This study describes the chlorination of several alkylpyrazines to form the corresponding chloroalkylpyrazine compounds, which are efficient starting materials for the synthesis of deuterium-labeled alkylpyrazines, namely [H-2(3)]-2-methylpyrazine (d-1), [H-2(5)]-2-ethylpyrazine (d-2), [H-2(3)]-2,3(or 6)-dimethylpyrazine (d-3(A), d-3(B)), [H-2(3)]-2,[H-2(3)]-6-dimethylpyrazine (d-3(C)), [H-2(5)]-2,[H-2(5)]-6-diethylpyrazine (d-4), [H-2(5)]-2-ethyl-3(or 6)-methylpyrazine (d-5(A), d-5(B)), 2,[H-2(3)]-3,5-trimethylpyrazine (d-6), [H-2(5)]-2-ethyl-3,6-dimethylpyrazine (d-7), [H-2(5)]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[H-2(3)]-5-methylpyrazine (d-9), which were obtained in good yields (57-100%) and high purities (86-98%). These stable isotopes were used as internal standards in SIDA to accurately and precisely determine selected alkylpyrazines in commercial peanut butter, cocoa powder, and instant coffee. 2,3-Diethyl-5-methylpyrazine (p-9) and 2-ethyl-3,5-dimethylpyrazine (p-8), despite their low abundance, had the highest odor-active values among the 13 pyrazines quantified in all products due to their very low odor thresholds.