Are Pork Extracts Present in My Gummy Bears?

Overview

The accidental or fraudulent blending of meat and animal products from different species is highly relevant for consumers with ethical concerns against eating species such as horse or pork in particular the Jewish and Muslim communities. In this work, we present the results from the initial development of an LC-MS/MS method utilizing AB SCIEX TripleTOF^® 5600 and 4000 QTRAP^® LC/MS/MS systems for the determination of the origin of gelatin used in food products and also pharmaceutical capsules.

Introduction

Following the Food Standards Agency (FSA)’s announcement in January that horse and pig DNA had been identified in beef products sold by several supermarket chains, further testing across Europe and beyond has revealed widespread incidences of such food contamination. This intended adulteration for fanatical gain or careless false declaration of meat products is a severe problem for consumers who have ethical or religious concerns about the consumption of pork or horse, more specifically the Muslim or Jewish communities who represent about 23 % of the worldwide population. As the tolerance level for porcine and equine content in foods is 0 %, for religious reasons, the limit of detection (LOD) needs to be as low as possible and so the continued development of more sensitive methods is necessary. However, pork based products are not only used as the meat but can also be found in gelling agents in food (for example in candy, ice cream, and marshmallows) as well as in the cosmetic and pharmaceutical industry in the form of gelatin. Gelatin is made from collagen, a protein, which has been extracted from the skin, bones, and connective tissues of animals such as cows, chicken, pigs, and fish. After extraction the collagen is partially hydrolyzed to form the gelatin which is a mixture of peptides and proteins and is used in the form of sheets, granules or powder.In the production of gelatin the protein hydrolysis normally occurs with hot water or under acidic conditions. The gelatin so produced is purified and used in food manufacturing and this process again may involve elevated temperatures. Under these conditions species-specific DNA present from the original animal is often denatured or removed making the use of the polymerase chain reaction (PCR), often used in species identification, difficult^1-3 or impossible.⁴ An alternative protein-based method, ELISA (enzyme-linked immunosorbent assay), has also been used for speciation⁵ but this approach has limitations, including that it detects only one part of the protein and not multiple protein markers and so can pose a risk of producing false negatives and positives. So an LC-MS/MS approach, detecting multiple tryptic peptides as markers for confirmation offers a more accurate and reliable approach to gelatin speciation than PCR or ELISA-based techniques. Initial identification of markers was by a shotgun proteomics approach using a high-resolution mass spectrometer, AB SCIEX TripleTOF^® 5600 system, coupled to an Eksigent LC system. The method developed in this work uses the AB SCIEX 4000 QTRAP^® system where multiple reaction monitoring (MRM) was used to detect markers which then automatically trigger the acquisition of enhanced product ion (EPI) scan to provide additional sequence information to further identify the peptides and proteins and therefore the gelatin species.

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