Combined cysteine and homocysteine quantitation in plasma by trap and release membrane introduction mass spectrometry.
Recently, a new and efficient method for total homocysteine (tHcy) quantitation in plasma using trap and release membrane introduction mass spectrometry (T&R-MIMS) with a versatile removable direct introduction membrane probe (DIMP) was described [R. Haddad, M. A. Mendes, N. F. Hoehr and M. N. Eberlin, Analyst, 2001, 126, 1212]. Herein we report on the use of the DIMP-T&R-MIMS technique for total cysteine (tCys) quantitation; hence combined tCys and tHcy quantitation in plasma or serum can be achieved. The method employs Cys and Hcy derivatization with ethyl chloroformate (after disulfide bond reduction with dithiothreitol and protein precipitation with trichloroacetic acid), preconcentration in a capillary silicone membrane, and their thermal desorption to the gas phase inside the ion source region of a mass spectrometer, at a point exactly between the two ionization filaments. Thermal desorption uses the uniform heat radiation provided by the two ionization filaments. The analytes are then ionized by electron ionization and both Cys and Hcy are quantitated by mass spectrometry using selected ion monitoring. For tCys quantitation, good linearity and reproducibility was observed for concentrations ranging from 5 to 350 microM, recovery was near 95%, and the limit of detection (LOD) was of 2 microM. This LOD is well below the mean Cys concentration in plasma, and serum samples from a large group of healthy people showed a mean tCys concentration of 132 +/- 45 microM.
Vellasco
Adriana Paula
AP
Medical Science Faculty, State University of Campinas-UNICAMP, PO Box 6111, 13083-970 Campinas, SP, Brazil.
Haddad
Renato
R
Eberlin
Marcos N
MN
Höehr
Nelci F
NF
D003545:Cysteine / Q000097:blood*; D006710:Homocysteine / Q000097:blood; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods
D006710:Homocysteine; D003545:Cysteine