Determination of homocysteine thiolactone and homocysteine in cell cultures using high-performance liquid chromatography with fluorescence detection.
A sensitive and simple method utilising fluorometric detection for the simultaneous routine monitoring of homocysteine thiolactone (HTL) and homocysteine (Hcy) in biological samples has been developed. Separation relies on isocratic ion-pairing and reversed-phase chromatography while the principle of the detection is that the lactone ring in HTL molecule is cleaved with an alkali to produce Hcy, which reacts with ortho-phthalaldehyde (OPA) in the absence of an added thiol reagent to form a stable fluorescent derivative. The method has a sensitivity of 200 fmol of HTL and 100 fmol for Hcy in the sample. The present method was applied to the determination of HTL and Hcy in Hep G2 cell.
Mukai
Yoshio
Y
General Education and Research Center, Meiji Pharmaceutical University, Kiyose-Shi, Tokyo, Japan.
Togawa
Tadayasu
T
Suzuki
Toshihiro
T
Ohata
Kazuko
K
Tanabe
Shinzo
S
D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media; D006710:Homocysteine / Q000031:analogs & derivatives* / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003470:Culture Media; D006710:Homocysteine; C007957:homocysteine thiolactone