Journal of Chromatography A, 1238 (2012) 54–59 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Rapid whole protein quantitation of staphylococcal enterotoxins A and B by liquid chromatography/mass spectrometry Isabel Sospedra ∗ , Carla Soler, Jordi Ma˜ nes, José Miguel Soriano Department of Preventive Medicine and Public Health, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain a r t i c l e i n f o Article history: Received 29 December 2011 Received in revised form 17 February 2012 Accepted 6 March 2012 Available online 17 March 2012 Keywords: LC–ESI/MS Staphyloccocus aureus Staphylococcal enterotoxins Milk Fruit juice a b s t r a c t Staphylococcus aureus is an important pathogen and has been indicated as the fifth causative agent of food-borne human illness throughout the world. Staphylococcal enterotoxins (SEs) are toxic compounds excreted mainly by strains of S. aureus. Among these toxins, enterotoxins A (SEA) and B (SEB) are both of the most prevalent compounds in staphylococcal food poisoning. In this work, reverse phase liquid chro- matography coupled to ESI mass spectrometry (LC–ESI/MS) has been applied for its rapid identification and quantification. Limit of detection (LOD) values were 0.5 and 0.2 ng for SEA and SEB, respectively and limit of quantification (LOQ) value was 1 ng for both enterotoxins. SEA and SEB have been analyzed as intact proteins in milk and fruit juices. Analytical methods are essential for routine monitoring purposes and safeguard public health and the proposed technique can detect and quantify successfully SEA and SEB in food samples. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Staphylococcus has been indicated by EFSA as the fifth causative agent of all reported outbreaks [1]. S. aureus is able to grow in a wide range of temperatures (from 7 to 48.5 ◦ C with an optimum from 30 to 37 ◦ C) [2], pH (from 4.2 to 9.3, with an optimum from 7 to 7.5) and sodium chloride concentrations (up to 15% NaCl) [3]. These characteristics enable S. aureus to grow in a wide variety of foods. The diverse secretion systems of bacteria provide good con- trasts between local and distant mechanisms of pathogenesis. S. aureus has an astonishing variety of both locally acting (surface- bound) and secreted, diffusible factors such as Staphylococcal enterotoxins (SEs) [4]. SEs are a family of serologically defined, low- molecular-weight basic proteins (26–30 kDa) produced by certain Staphylococcus strains in a variety of environments, including food substrates. Staphylococcal Food Poisoning (SFP) is caused by inges- tion of food containing preformed SE, is a disease where toxin is ingested in the absence of host infection. The most common symp- toms of SFP, which usually begin 2–6 h after contaminated food is consumed, are nausea, vomiting, acute prostration and abdominal cramps [5–7]. These toxins are heat stable, resistant to gut pro- teases and stable over a wide range of pH. To date, 23 enterotoxins have been identified as distinct serological entities [8]. Staphylo- coccal enterotoxin A (SEA) is the serotype most frequently involved in foodborne staphylococcal illness [9], followed by SED (37, 5%) ∗ Corresponding author. Tel.: +34 963543056; fax: +34 963544954. E-mail address: m.isabel.sospedra@uv.es (I. Sospedra). and SEB (10%) [10]. SEB is a highly heat-resistant enteric toxin and SEB-producing strains are considered as potential microbio- logical weapons of warfare and terrorism [11]. Beharka et al. [12] demonstrated that SEA and SEB toxicity was due by their binding to the 2 domain of the H-2D b molecule which induces biologi- cal activity and has physiological consequences. These toxins are commonly related to food poisoning, especially for foods requir- ing handling during processing such as milk, cheese, juices, canned meat, ham or cooked meals [2,13] because, even if the bacteria have been sterilized, the biological activity of the toxins remains unchanged. For this reason is necessary to develop a rapid, sensitive and specific method for monitoring food and beverages samples. The isolation, identification and quantification of these proteins usually require tedious steps [14–17]. Many sensitive techniques are now available to detect toxins such as SEs [8]. Immunological methods, such as immunoassays, are simple, quicker and present high sensitivity but have some drawbacks and also have impor- tant limitations. Some of them are that inactive and active SEs are nearly indistinguishable as occurs with antibody-based meth- ods [18]. Only small sample volumes can be loaded onto a gel and cross-reactive bands potentially could co-migrate with the antigen. Microslide double diffusion and ELISA have been used for test- ing food samples; however, cross-reaction with unrelated antigens [19] or endogenous peroxides may also origin false positive results [20]. In addition, heat-treated SEA (in heat processed foods) can give negative results, because heat-treated enterotoxin may aggre- gate, reducing its reactivity with antibodies [21,22]. These facts may limit the sensitivity and origin false positives or false negatives. For these reasons, the relevance of the immunological approach is 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.03.022