Determination of underivatized amino acid d 13 C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non-essential amino acid profile on the isotopic signature of individual amino acids in fish James McCullagh 1 , Julia Gaye-Siessegger 2y and Ulfert Focken 2 * 1 Chemistry Research Laboratory, Oxford University, Mansfield Road, Oxford OX1 3TA, UK 2 Department of Aquaculture Systems and Animal Nutrition in the Tropics and Subtropics, University of Hohenheim, Stuttgart, Germany Received 14 November 2007; Revised 20 March 2008; Accepted 21 March 2008 This study provides data for the effect of dietary non-essential amino acid composition on the d 13 C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of d 13 C values of individual amino acids from diets and tissues without derivatization. Diet affected the d 13 C of individual amino acids in fish. For fish on Diets 1–3 amino acid d 13 C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in 13 C. Improvements are necessary before all amino acid d 13 C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists. Copyright # 2008 John Wiley & Sons, Ltd. The relatively new method of liquid chromatography/ isotope ratio mass spectrometry (LC/IRMS), allowing the determination of isotopic signatures of individual amino acids from proteins without prior derivatization, 1 was used here for the first time in a study on protein metabolism in fish. The importance of the dietary non-essential/conditionally essential amino acid (hereafter referred to as non-essential amino acids) composition has received little attention in fish nutrition. Only a few studies have examined the effect of the dietary non-essential amino acid composition on growth performance. 2–4 The steadily increasing demand for aqua- culture products 5 has caused a corresponding demand for feeds used in aquaculture, which must contain less fish meal and more plant protein since the supply of fish meal is limited. A frequent observation is that the growth of fish on diets based on plant proteins is significantly lower than on fishmeal-based diets, although the essential amino acids are balanced. 6,7 Differences in the composition of non-essential amino acids might be one of the reasons for this observation. 4 In the present experiment, rainbow trout (Oncorhynchus mykiss) were fed a commercial trout diet (control) or three purified diets which differed in their non-essential amino acid composition. All three diets did not contain protein, only synthetic amino acids. Diet 1 resembled the amino acid composition of fish meal. While the essential amino acids must be provided with the food, the non-essential amino acids can be synthesized by the animal from other amino acids, e.g. from glutamate, aspartate, serine and phenyl- alanine (which are further on referred to as precursor amino acids). In Diet 2 some non-essential amino acids were RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2008; 22: 1817–1822 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3554 *Correspondence to: U. Focken, Department of Aquaculture Sys- tems and Animal Nutrition in the Tropics and Subtropics, University of Hohenheim (480B), Fruwirthstr. 12, 70599 Stutt- gart, Germany. E-mail: focken@uni-hohenheim.de y Present address: Fisheries Research Station Baden-Wu ¨ rttemberg, Untere Seestraße 81, 88085 Langenargen, Germany. Contract/grant sponsor: DFG Grant; contract/grant number: GA 1068/2-1. Copyright # 2008 John Wiley & Sons, Ltd.