A UHPLC-MS/MS Method for the Detection of Meat Substitution by Nine Legume Species in Emulsion-Type Sausages

Abstract

Meat substitution by legume proteins in various types of meat products is a common practice. A reliable detection and quantification of these additives is required to control food specifications, especially regarding food fraud. Consequently, a UHPLC-MS/MS method for the simultaneous detection of alfalfa (Medicago sativa), broad bean (Vicia faba), chickpea (Cicer arietinum), lentil (Lens culinaris), lupine (Lupinus albus and Lupinus angustifolius), pea (Pisum sativum), peanut (Arachis hypogaea), and soy (Glycine max) proteins in meat products was developed. After protein extraction and tryptic digestion, three marker peptides for each legume species were measured by multiple reaction monitoring (MRM) using an optimized extraction protocol. To the best of our knowledge, the marker peptides for alfalfa, broad bean, chickpea, and lentil have not been reported previously. Emulsion-type sausages with 0.1, 0.4, 0.7, 1.0, 1.3, 1.6, 1.9, 2.2, and 2.5% meat substitution by each legume species, representing the concentration range between inadvertently transferred cross-contaminations and the conscious use for meat substitution, were produced for matrix calibration. No false-positive results were recorded in blank samples. In the quantification of alfalfa, broad bean, chickpea, lentil, pea, peanut, and soy, 673 of 756 measuring data of the recovery rate in unknown sausages were in the accepted range of 80–120%.

Simultaneous Mass Spectrometric Detection of Proteins of Ten Oilseed Species in Meat Products

“…The sample preparation of oilseed flours for high-resolution and homogenized emulsion-type sausages for triple-quadrupole mass spectrometry was performed as described previously [ 32 ] ( Figure 1 ). Briefly, the samples were defatted and dehydrated using acetone.…”

“…Liquid chromatography was performed with a Dionex UltiMate 3000 RS HPLC from Thermo Scientific (Waltham, MA, USA) as published previously [ 32 ]. The injection volume was 2 μL, the temperature of the column (Nucleosil 100-3 C18 HD column (125 × 2 mm; particle size: 3 μm) from Macherey–Nagel (Düren, Germany)) was set to 40 °C.…”

“…The peptide candidate markers (see Table S1 ) were synthesized and purified as described previously [ 37 ]. The identities of the peptides purified were verified as reported previously [ 32 ]. Furthermore, the synthesized peptides were used to select the five most intensive, theoretically explainable, mass transitions for each peptide marker and to optimize their MS/MS parameters (declustering potential (DP), collision energy (CE), and cell exit potential (CXP)) at the AB Sciex QTrap 5500 (Darmstadt, Germany) using syringe pump injection.…”

“…In contrast to ELISA and PCR, MS methods enable a high-throughput for the simultaneous detection of many different target proteins in food [ 29 , 30 ]. Mass spectrometric methods for the detection of oilseed species in meat products are limited to soy [ 29 , 30 ], hemp [ 31 ], or peanut and soy [ 32 , 33 ]. The Montowska working group identified species-specific peptides in oilseed cakes from coconut, evening primrose, hemp, flaxseed, milk thistle, nigella, pumpkin, rapeseed, sesame and sunflower [ 34 ].…”

“…The aim of this study was to identify new peptide markers and to develop a UHPLC-MS/MS method for the simultaneous detection of the addition of oilseed proteins from chia ( Salvia hispanica ), coconut ( Cocos nucifera ), flaxseed ( Linum usitatissimum ), hemp ( Cannabis sativa ), peanut ( Arachis hypogaea ), pumpkin ( Cucurbita ), rapeseed ( Brassica napus ), sesame ( Sesamum indicum ), soy ( Glycine max ) and sunflower ( Helianthus annuus ), with a limit of detection (LOD) of 0.01% in meat products. The peptide markers for peanut and soy were adopted from a previous study [ 32 ]. New heat-stable peptide markers require to be identified for the eight other oilseed species.…”