Quantification of HGA and MCPG Metabolites

The quantification of HGA and MCPG plus their metabolites in serum and urine was carried out by UPLC-MS/MS as described in detail earlier (9 (link)–12 (link)). For all analyses, 25 μL of material were extracted with 300 μL of methanol containing the internal standards. Analysis was performed after butylation. For chromatographic separation 5 μL of the final extracts were injected onto an Acquity UPLC BEH C18 1.7 μm, 2.1 ×50 mm column (Waters). Gradient chromatography was performed using acetonitrile/water modified by 0.1% formic acid and 0.01% trifluoroacetic acid. Quantifications were done on a Xevo TQMS UPLC-MS/MS system (Waters, Eschborn, Germany), calculation of concentrations was conducted with single-point calibration. Concentrations of C4 to C10 acyl conjugates were also determined by this method in order to differentiate branched and unbranched C4 and C5 metabolites. The isomers of 2-MBC appeared in two separate peaks, the values of both isomers were added for quantification.