Liquid chromatography analysis of N-(2-mercaptopropionyl)-glycine in biological samples by ThioGlo 3 derivatization.
N-(2-Mercaptopropionyl)-glycine (MPG) is a synthetic aminothiol antioxidant that is used in the treatment of cystinuria, rheumatoid arthritis, liver and skin disorders. Recent studies have shown that MPG can function as a chelating, cardioprotecting and a radioprotecting agent. Several other studies have shown that it may also act as a free radical scavenger because of its thiol group. Thiol-containing compounds have been detected in biological samples by various analytical methods such as spectrophotometric and colorimetric methods. However, these methods require several milliliters of a sample, time-consuming procedures and complicated derivatization steps, as well as having high detection limits. The present study describes a rapid, sensitive and relatively simple method for detecting MPG in biological tissues by using reverse-phase HPLC. With ThioGlo 3 [3H-Naphto[2,1-b] pyran, 9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) phenyl-3-oxo-)] as the reagent, highly fluorescent derivatives of thiols can be obtained that are suitable for HPLC. MPG is derivatized with ThioGlo 3 and is then detected flourimetrically by reverse phase HPLC using a C18 column as the stationary phase. Acetonitrile: Water (75:25) with acetic acid and phosphoric acid (1 mL/L) is used as the mobile phase (excitation wavelength, 365 nm; emission wavelength, 445 nm). The calibration curve for MPG is linear over a range of 10-2500 nM (r=0.999) and the coefficients of the variation of within-run and between-run precision were found to be 0.3 and 2.1%, respectively. The detection limit was 5.07 nM per 20 microL injection volume. Quantitative relative recovery of MPG in the biological samples (plasma, lung, liver, kidney and brain) ranged from 90+/-5.3 to 106.7+/-9.3 %. Based on these results, we have concluded that this method is suitable for determining MPG in biological samples.
Penugonda
Suman
S
Department of Chemistry, University of Missouri-Rolla, 1870 Miner Circle, Rolla, MO 65409, USA.
Wu
Wei
W
Mare
Suneetha
S
Ercal
Nuran
N
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007668:Kidney / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011714:Pyrans / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D008625:Tiopronin / Q000097:blood / Q000378:metabolism*
C434651:9-acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho(2,1-b)pyran; D005456:Fluorescent Dyes; D011714:Pyrans; D011758:Pyrroles; D008625:Tiopronin