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Determination of dithiothreitol in complex protein mixtures by HPLC–MS
Author(s) -
Cindrić Mario,
Čepo Tina,
Marinc Sabina,
Paškvan Ivan,
Mijić Ivana,
Bindila Laura,
PeterKatalinić Jasna
Publication year - 2008
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200800207
Subject(s) - dithiothreitol , chemistry , chromatography , derivatization , detection limit , aqueous solution , atmospheric pressure chemical ionization , chemical ionization , mass spectrometry , tandem mass spectrometry , ionization , ion , organic chemistry , enzyme
Dithiothreitol (DTT) and other reducing agents are typically used in refolding processes of recombinant human proteins during their purification from inclusion bodies. Due to its toxicity, it is essential to monitor the clearance of DTT throughout the analytical flow from the refolding phase to the final formulated product. Here we report a direct, simple, and fast liquid chromatography method using UV and tandem mass spectrometry (MS/MS) detection for DTT evaluation in complex protein mixtures. In aqueous solution DTT exists as an equilibrium mixture of the oxidized and the reduced form (H 2 DTT → DTT ox ) and the quantitation tools should therefore be applicable to both forms in a single step or in multiple steps. Oxidation of DTT with aqueous copper(II) nitrate trihydrate solution was introduced to determine a single oxidized compound, i. e. DTT ox . Proteins and other components of high molecular masses were separated from DTT ox by ultrafiltration. Consequently, efficient separation of the DTT ox from other flow‐through mixture components (sugars, polymers, salts, protein stabilizers) was achieved on an Atlantis dC 18 column. After chromatographic separation, DTT ox was selectively identified by UV absorbance at 285 nm or by selected reaction monitoring, measuring signal transition between m/z 151 → 105. The method was validated in terms of specificity, accuracy, precision, linearity, and limit of quantification and detection. A reversed‐phase HPLC separation method with atmospheric pressure chemical ionization and MS/MS detection in negative ion mode is highlighted as a viable alternative to currently existing quantitation methods involving DTT derivatization and HPLC fluorescence detection. The described approach offers simple, straightforward, selective, and high‐throughput DTT quantitation in protein mixtures.