4.1 This test method provides for the qualitative and quantitative determination of denatonium benzoate in engine coolant in milligrams per litre to low percent range and requires approximately 1 mL per test, with results available in less than 10 min. Denatonium benzoate is a compound composed of a quaternary ammonium cation, denatonium and an inert anion, benzoate. In solution the denatonium benzoate exists in equilibrium between the denatonium benzoate compound, the denatonium cation and benzoate anion. By slightly adjusting the pH of the solution to be more acidic (≈ pH 4.6) the equilibrium will be shifted to the direction of forming more denatonium and benzoate ions in the solution.

1.1 This test method covers the chemical analysis of engine coolant for denatonium benzoate (DNB) by high-performance liquid chromatography (HPLC). DNB is added to potentially render engine coolant unpalatable to animals and humans. This analytical method was designed for the analysis of DNB and is not valid for any other bittering agents such as denatonium saccharide.

1.2 This test method is applicable to both new and used coolants.

1.3 Coelution of other ions may cause interferences in the detection of the denatonium cation. In the case of unfamiliar formulations, identification verification should be performed by either or both fortification and dilution of the sample matrix with denatonium ion.

1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.

1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.