Determination of Furosine in Hair by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

A sensitive and reliable analytical method for determining furosine in hair has been developed using liquid chromatography-tandem mass spectrometry. Fructose-N"-fomyl-d,-DL-lysine was synthesized and used as an internal standard. By the present method, glycated lysine levels in hair could be determined as fructose-lysine in only 0.1 mg of hair sample, ranging fhm 35.1-72.6nglmg hair among five healthy volunteers, which corresponded to 0.0635-0.153% of the total lysine contents in hair determined by amino acid analysis. Furosine, a by-product generated by acid hydrolysis of protein in various tissues, has been proposed as a marker compound for glycation of protein (Oimomi et al., 1984). Among several organic tissues, hair obtained non-invasively was expected to provide information about glycated protein levels over a period of time (Oimomi et al., 1985(Oimomi et al., , 1988Sueki et al., 1989;Kobayashi and Igimi, 1996). An HPLC method for evaluating the glycated protein as furosine was developed (Schleicher and Wieland, 198 1) and was applied to analysis of furosine in hair (Oimomi et al., 1985(Oimomi et al., , 1988Sueki et al., 1989). However, the method required a large amount of hair (10-50mg) and gave glycated protein content not as a direct value, but as the ratio of peak area of furosine to that of tyrosine.We have designed a highly sensitive, specific and accurate method for determining furosine in hair by liquid chromotography-tandem mass spectrometry (LCIMSIMS), using fructose-W-formyl-d,-DL-lysine as an internal standard. EXPERIMENTALChemicals. Furosine was obtained from Neosystem (Strasbourg, France). Ten per cent HCL:methanol was from Tokyo Kasei (Tokyo, Japan). HPLC grade acetonitrile, trifluoroacetic acid and all other solvents and reagents were from Wako Pure Chemical Industries (Osaka, Japan). Fructose-Na-formyl-L-lysine was synthesized according to the method previously reported (Sato and Imamura, 1986;Schleicher and Wieland, 1981). Fructose-W-formyl-~,-DLlysine was prepared using 4,4,5,5,-d4-DL-lysine from Isotec Inc. (Miamisburg, OH, USA) according to the same procedure as fructose-W-formyl-L-lysine. * Correspondence to: A. Takemura.Apparatus. LCIMSIMS system consisted of a 1050 HPLC system (Hewlett-Packard GmbH, Waldbronn, Germany) and a TSQ-700 tandem mass spectrometer (Finnigan MAT, Heme1 Hempstead, UK). The analytical column was a Develosil ODs HG-5 (2 rnrnx I.D. x 150 mrn, 5 pm particle size, Nomura Chemical, Seto, Aichi, Japan). Amino acid analysis was performed on a L-8500 amino acid analyser (Hitachi, Tokyo, Japan).Method. Human hair (0.1 mg) was placed in a reaction vial and fructose-Na-formyl-d4-DL-lysine (5 ng) was added. After adding 1 mL of 6 N HCl, the mixture was heated at 90°C for 24 h. The solution was concentrated to dryness with N2 gas flow and the residue was dissolved in 1 mL of 10% HC1:methanol and kept at 90°C for 30 min. After being concentrated to dryness with N, gas flow, the residue was dissolved with 200 pL of distilled water and 20 pL was applied to the LCIMSIMS. The ...

Hair as a Biological Indicator of Drug Use, Drug Abuse or Chronic Exposure to Environmental Toxicants

“…LC tandem mass spectrometry has been used for the hair analysis of furosine as a marker compound of proteins' glycation (Takemura et al 1997).…”