(M1030-09-49) A Simple, Rapid Method for Simultaneous Quantitative Analysis of Dithiothreitol and Dihydroxy-1,2-dithiane in Cell Culture Media by LC/MS/MS

Purpose: Dithiothreitol (DTT) is a common redox reagent, which has been widely used in refolding process of recombinant proteins and protein purification. DTT in cell culture media may affect cell growth and viability, and its toxicity to different cell lines has been evaluated [1,2]; and DTT has also shown cryoprotective effect in some cell lines [3]. Therefore, it’s important to monitor the DTT concentration during cell growth, and throughout protein refolding / purification process.

Methods: DTT exists as an equilibrium mixture with Dihydroxy-1,2-dithiane (DHDT) in aqueous solution, and its stability at different pH has been studied [4]. Previously published bioanalytical method requires derivatization of DTT with copper (II) nitrate trihydrate solution to an oxidized compound [5]. Here we report a direct quantitative analysis of DTT and DHDT in acidified cell culture media using protein precipitation procedure, liquid chromatography, and tandem mass spectrometry (LC/MS/MS) without derivatization. Separation of the two forms was achieved on an XSelect HSS T3 column. DTT oxidized form (DHDT) was monitored at MRM transition of 150.9→104.9, the reduced form was monitored at MRM transition of 152.8→33.1.

Results: Two curve ranges were evaluated: 0.1-100 uM and 1-1000 uM to accommodate different concentration levels. Standards and QCs meet criteria (≥75% calibration standards/QCs within ±20% of the nominal concentrations; except ±25% at LLOQ). Good linearity was observed for both DTT and DHDT, with R² > 0.9900.

Conclusion: A robust extraction procedure was established with 20 uL sample extraction volume. DTT and DHDT were stabilized with 1% formic acid in solution. The DTT/DHDT concentration in cell culture media can be effectively quantified simultaneously by simple protein precipitation and LC/MS/MS. Baseline separation, good peak shape and column retention time were achieved for both analytes. The method was qualified in term of specificity, precision and accuracy, linearity, and limit of quantification.

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2. Yiwen Wang, Maïlys Misto, Jing Yang, Nicole Gehring, Xiaoyu Yu, Bernard Moussian*, Toxicology Reports 8 (2021) 124–130
3. Mahdokht H Aghdai, Akram Jamshidzadeh, Mahsa Nematizadeh, Mahtab Behzadiannia, Hossein Niknahad, Zahra Amirghofran, Elaheh Esfandiari, Negar Azarpira*, Hepat. Mon. 2013;13(1):e7824
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