Talanta 80 (2009) 294–306 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products Ana Juan-García ∗ , Guillermina Font, Cristina Juan, Yolanda Picó Laboratori de Bromatologia i Toxicologia, Facultat de Farmàcia, Universitat de València, Av. Vicent Andrés Estellés s/n, 46100 Burjassot, València, Spain article info Article history: Received 26 March 2009 Received in revised form 14 June 2009 Accepted 29 June 2009 Available online 4 July 2009 Keywords: b-CMs NanoMate QIT MS Nanoelectrospray abstract beta-Casomorphins (b-CMs) are bioactive peptides derived from casein with opioid agonist effects simi- lar to morphine. The use of electrospray (ESI) with quadrupole ion-trap mass spectrometry (QIT-MS) for these compounds in two matrices, cheese and milk, was examined. It was compared to a liquid chro- matography (LC) coupled to mass spectrometry (LC–MS), and a “soft” ionisation technique, NanoMate, with selected ion monitoring (SIM), which are unreliable for the determination of trace casomorphins in derived milk products. b-CM mass fragmentation pathways were done for the four most common b-CMs: -casomorphin (1-5) bovine (b-CM-5), -casomorphin (1-7) bovine (b-CM-7), [D-Ala2, D-Pro4,Tyr5]- -casomorphin (1-5) amide (b-CM-10) and -casomorphin (1-5) amide [D-Ala2,Hyp4,Tyr5] (b-CM-11). The major product ions obtained in QIT-MS were used to construct fragmentation pathways for b-CMs. The different collision energies using automated nanoelectrospray ion source NanoMate and conven- tional LC in QIT-MS were studied. Calibration data for b-CMs, using spiked milk or cheese samples (10g or 10mL), were: NanoMate/MS (25–1000 g/L), r 2 = 0.998; NanoMate/MS 2 (5–1000 g/L), r 2 = 0.9992; NanoMate/MS 3 (2.5–1000 g/L), r 2 = 0.9998. Reproducibility data (% RSD, N = 5) for NanoMate/MS n mode ranged between 2.0 at 500 g/L and 7.0 at 10 g/L. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Peptides yielded by the digestion of beta-casein have opioid agonist effects similar to morphine. They are named beta- casomorphins (b-CMs) [1]. These bioactive peptides are bound to opioid receptors and exhibit morphine-like effects. They have high content on proline residues which it is related with effects as opioid (casomorphins) antithrombotic, antihypertensive, immunomodu- lators or mineral carriers [6,5]. Although their effect in adult human is limited to the gastroin- testinal tract, as they are not absorbed or are subject to enzymatic degradation in the intestinal wall, in newborns and young infants, the effects presented are more evident, as the widespread hypothe- sis used to explain autistic symptoms in autism spectrum disorders (ASD) as shown in some studies [2,3]. It has been observed that many children with ASD present gastrointestinal symptoms such as inflammation, diarrhea, gastroesophageal reflux, abdominal pain and increased intestinal permeability [4,5]. It has also been observed that b-CMs induce alteration of the intestinal electrolyte transport, affect the human mucosal immune system, possibly via opioid receptors, and an important function in the digestive pro- cess may be served [6]. Investigations have shown that b-CMs ∗ Corresponding author. E-mail address: ana.juan@uv.es (A. Juan-García). significantly inhibited stimulated lamina propria lymphocyte pro- liferation (LPL) [5,7]. Continued developments in food technology and nutritional sci- entific interest in food peptides have grown considerably in the last years and in consequence it has given rise to the synthesis or production of potential bioactive peptides. This interest has been increased due to the presence of food component necessaries for organism such as amino acids, nitrogen and nutritional and func- tional values. In fact in the food industry, peptides derived from whey proteins, sometimes are used as skim milk replacers in ice cream, as fat replacers, as whole eggs replacers, as nondiary dessert products and in enteral, infant and sport nutrition [8,9]. And in consequence, detrimental components are sometimes presented in natural forms in food, but others, some effects on food components could cause effects and provide bioactive peptides because of the development of modern food processing and storage techniques. Methods used in food processing such as hydrolysis, oxidation, fermentation, pH changes, heat and microbial enzymes can cause structural and chemical changes and synthesis or production of potential bioactive peptides. In the case that it concerns us the bio- logically active peptides produced from milk proteins are generated in natural way like in fermentation process of milk products. Milk is of great importance in the early years of human life and adult age [6,8], it is one of the basic foods well studied over the years, and its major proportion of nutrient components are proteins, the main source of natural biologically active peptides (b-CMs). 0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2009.06.062