Biotechnology Techniques 12: 231–234, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 231 Analysis of carnitine, betaine, γ -butyrobetaine, and separate short-chain acylcarnitines in pigeon plasma, crop milk and tissues by HPLC coupled with UV-detection G.P.J. Janssens ∗ , H. De Rycke, M. Hesta & R.O.M. De Wilde Laboratory of Animal Nutrition, University of Ghent, Heidestraat 19, 9820 Merelbeke, Belgium ∗ Author for correspondence (Fax: +32 9 2647848; E-mail: geert.janssens@rug.ac.be) Received 22 February 1999; Accepted 1 March 1999 Key words: betaine, carnitine, chromatography, pigeon, UV-detection Abstract Carnitine, betaine, γ -butyrobetaine and separate carnitine esters are determined in blood plasma, crop milk, liver, heart and breast muscle of pigeons (Columba livia domestica) by means of HPLC with photometric detection at 245 nm. The method can be generally applied for samples of animal origin. Introduction Carnitine is a vitamin-like substance that is syn- thetized in all vertebrate animals as its laevo-isomer. Carnitine esterifies fatty acids, taken from acyl- coenzyme A, and allows them in that way to be transported into the cell mitochondria (Bremer 1983). Numerous publications have demonstrated the im- portance of the determination of carnitine in human medicine (Rebouche & Paulson 1986) and veterinary science (Fischer 1993). In most cases, only free carni- tine, total carnitine and the total of acylated carnitines are determined, and therefore useful information is lost. Moreover, for certain diseases like isovalery- lacidemia (Fries et al. 1996) the quantification of specific acylcarnitines is essential. Betaine is also considered a quasi-essential molecule in nutrition, especially related to cell mem- brane stability (Odle 1996). Several teams have al- ready developed an HPLC technique with photometric detection for carnitine and its esters (van Kempen & Odle 1992, Minkler & Hoppel 1993). This paper presents a refined HPLC method with UV-detection to quantify carnitine, betaine, γ -butyrobetaine and sev- eral acylcarnitines in plasma, liver, muscle, heart and crop milk of pigeons (Columba livia domestica), be- cause of the importance of fat metabolism in these animals. Materials and methods Chemicals L-Carnitine chloride and acetyl-L-carnitine were kindly provided by Lonza (Basel, Switzerland). Be- taine, hexanoyl-DL-carnitine chloride, octanoyl-DL- carnitine chloride and palmitoyl-DL-carnitine chloride were purchased from Sigma. Other products were pur- chased from several companies: UV grade methanol, UV grade acetonitrile and ortho-phosphoric acid from Vel (Haasrode, Belgium), triethylamine from Panreac (Montcada i Reixac, Spain), 2-bromoacetophenone from Sigma and Kieselgel 60 HF 254 from Merck. Sampling and sample preparation Blood samples were collected from leg veins in hep- arinized tubes and centrifuged. Crop milk was sam- pled by suction through a long rubber tube attached to a 1 ml pipette with a 4 mm outer diameter and a 2.5 mm inner diameter. This pipette was inserted via the oesophage into the crop while slightly stretching the animal’s throat. Breast muscle, heart and liver were