Determination of hexamethylmelamine and metabolites in plasma or serum by gas—liquid chromatography with a nitrogen-sensitive detector

A gas chromatographic method for the quantitative determination of hexamethylmelamine (HMM) and five of its metabolites in plasma (or serum) is described. After adjustment of the pH of the plasma sample to about 9.5, the compounds are extracted with chloroform containing 5% of isopropanol. Amyl alcohol is added to the extract, which is then evaporated until a small volume remains. An aliquot of this solution is injected into a gas chromatograph equipped with a nitrogen—phosphorus flame ionisation detector. Separation of the methylmelamines is achieved with a 10% Carbowax 20M–2% KOH column. By programming the oven temperature unnecessarily long retention times are avoided. Using 1 ml of plasma, concentrations as low as 5 ng/ml of HMM and its metabolites can be quantitated. The method has been applied to the determination of HMM and metabolites in plasma of patients who received oral doses of HMM.