770 (2002) 237–241 Journal of Chromatography B, www.elsevier.com / locate / chromb Short communication Monitoring of dithiothreitol clearance by means of micellar electrokinetic chromatography ˇ * ˇ ˇ´ ´ Zdenek Glatz , Petra Sevcıkova ´ˇ ´ Department of Biochemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic Abstract A new method for specific determination of dithiothreitol (DTT) using micellar electrokinetic chromatography and on-column reaction with reactive disulfide–2,29-dipyridyldisulfide is described. DTT in this reaction is quantitatively transformed into a mixed disulfide concomitantly with formation of equimolar amount of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of DTT is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 2.5 mM (correlation coefficient 0.993) with a detection limit of 2.5 mM. The inter-day reproducibility of the peak area was 1.35% and the inter-day reproducibility of the migration time 0.56%. The method can be applied for DTT monitoring both in chemical and biological systems.  2002 Elsevier Science B.V. All rights reserved. Keywords: Dithiothreitol 1. Introduction as a cancer treatment modality [10]. DTT is also routinely used to solubilize recombinant proteins Dithiothreitol (DTT) synthesised in 1949 by from inclusion bodies [11]. Evans et al. [1] is a well known sulfhydryl reagent. In view of the fact that DTT readily undergoes Since 1964 when Cleland described its function [2] it autooxidation in solution [12,13] the monitoring of was used to protect the sulfhydryl groups of small the DTT state during biochemical and chemical molecular compounds as well as proteins in a variety experiments is an important task. Previous methods of biochemical applications. The radioprotective used to determine DTT in solutions have included action of the compound has received significant spectrophotometry [14] and HPLC with UV spectro- attention for a number of years and its mechanisms photometric [15,16] and fluorescence [17] detection. have been examined in chemical [3–5] and bio- Recently a method describing the usage of the on- logical [6–9] systems. Moreover DTT has been column reaction of 2,29-dipyridyldisulfide (DPDS) applied in studies on heat cell killing in hyperthermia for the specific detection of thiols (RSH) during micellar electrokinetic chromatography (MEKC) (Fig. 1) has been published [18]. The purpose of this *Corresponding author. Tel.: 1420-541-129-401; fax: 1420- paper is to demonstrate its applicability for moni- 541-211-214. E-mail address: glatz@chemi.muni.cz (Z. Glatz). toring of DTT clearance during different types of 1570-0232 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0378-4347(01)00526-6