Study of factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)-thiol derivatives by liquid chromatography.
A detailed investigation of the factors affecting the determination of total plasma 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD)-thiol derivatives (i.e. cysteine, homocysteine and cysteinylglycine) is described. Essentially, this assay entails extracting specific thiols by plasma disulphide bond reduction, protein precipitation, sulphydryl compound derivatization with the thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F), and subsequent separation with isocratic reversed-phase high-performance liquid chromatography. By improving the reliability of several analytical parameters (composition of the mobile phase, pretreatment of the sample using different reducing and protein precipitation agents, and optimization of the derivatization of thiols with SBD-F), a number of critical issues can be identified and solved.
Rizzo
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Dipartimento di Biochimica, Sezione di Analisi Chimico-Cliniche, I.R.C.C.S. Policlinico S., Matteo, Università di Pavia, Italy.
Montalbetti
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L
Valli
M
M
Bosoni
T
T
Scoglio
E
E
Moratti
R
R
D000328:Adult; D001894:Borohydrides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000097:blood* / Q000737:chemistry; D004151:Dipeptides / Q000097:blood* / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry*; D005464:Fluorobenzenes / Q000737:chemistry*; D006710:Homocysteine / Q000097:blood* / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008875:Middle Aged; D010720:Phosphines / Q000737:chemistry; D019163:Reducing Agents / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry
D001894:Borohydrides; D004151:Dipeptides; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D010720:Phosphines; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; D006710:Homocysteine; C043255:tri-n-butylphosphine; C028505:cysteinylglycine; C037802:7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate; C025364:sodium borohydride; D003545:Cysteine; D004229:Dithiothreitol