Possessing high protein and fibre content combined with low carbohydrate and fat levels, lupin has gained considerable attention for its nutritional advantages over other legumes. Lupin is used in some food products but primarily as animal feed. The consumption of lupin products can be constrained by the presence of the toxic secondary metabolites, the quinolizidine alkaloids. The industrial threshold set by Australia and some European countries for alkaloid content in lupin is 200 mg/kg for animal feed and 100 mg/kg for human intake.
To monitor alkaloids, we developed and validated a rapid LC-MS/MS method for the determination of six quinolizidine alkaloids and one indole alkaloid in lupin. The final method involves extraction by water/acetonitrile, ultrasonication, separation using NaCl and MgSO4, alkalinisation by NaOH and analysis using HPLC-MS/MS. The separation was achieved by HPLC in 6 min directly coupled to MS/MS on a 6500 QTRAP. The analytes were detected in ESI positive mode and MRM parameters were optimised to obtain the highest sensitivity.
The method showed acceptable recovery for all tested compounds at a wide range from 1.09 to 109 mg/kg, with mean recovery of 95% for the total alkaloid content. The LOQs of α-isolupanine, 13-α-hydroxylupanine, (+)-lupanine, and sparteine was 0.01 mg/kg, 0.05 mg/kg for angustifoline, and 0.5 for both of gramine and (-) Lupinine. The total alkaloid LOQ was 1.09 mg/kg. Low CVs were achieved for alkaloids (<3.6%) among six replicates at three concentration levels. The method was tested on a commercial lupin flour sample yielding an alkaloid concentration of 443.5 ± 11.7 mg/kg (CV 2.6%).
The simple, rapid, robust approach with low detection limits was used to confirm that lupin different phenotypes fulfil the industry thresholds. This will be benefit lupin breeders in selecting cultivars with the lowest QA content and ensuring the safety of food and feed.