Development of a liquid chromatographic method for picomole determination of S-sulfocysteine in trifluoroacetic acid extracts of neonatal rat brain.
Neonatal Sprague Dawley rat brain tissue was extracted with methanol, acetonitrile, acetic acid and trifluoroacetic acids (TFA). Among the extractants tested, 0.1 M TFA gave the highest recovery, 73.4 +/- 5.2% (slope of regression of 'added' vs. 'found' and standard error of the slope) of S-sulfocysteine (SSC). The poorest recovery of SSC was found with acetonitrile and 90% methanol extractions (less than 10%). Possible reasons for the low recoveries have been explored. The recovery of SSC from aqueous standards in 0.1 M TFA is 92 +/- 5%. Detection of picomole quantities of SSC has been demonstrated with a combination of the optimized extraction procedures and our previously developed detection system. Supernatant of rat brain homogenate (0.10 M TFA as extractant) was evaporated to dryness in a vacuum centrifuge. Residues were reconstituted with deionized water. Samples were separated on a reversed phase column. The mobile phase was 20 mM aqueous acetate buffer (pH 5.2) containing 0.40 mM cetyl trimethylammonium p-toluene sulfonate and 2 vol.% methanol. Electrochemical detection used dual series gold-mercury amalgam electrodes. For the first time, S-sulfocysteine was detected in normal neonatal rat brain. Its concentration is 0.99 +/- 0.25 pmol/mg brain tissue. The results indicate that TFA, rarely reported an an extractant, efficiently recovers SSC from rat brain tissues.
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Department of Chemistry, Chevron Science Center, University of Pittsburgh, PA 15260, USA. sweber@pitt.edu
Sandberg
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Weber
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SG
D000818:Animals; D000831:Animals, Newborn / Q000378:metabolism*; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis; D004563:Electrochemistry; D005374:Filtration; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D012997:Solvents; D014269:Trifluoroacetic Acid; D014465:Ultrasonics
D012997:Solvents; C011119:S-sulphocysteine; D014269:Trifluoroacetic Acid; D003545:Cysteine