title: Determination of dithiothreitol in complex protein mixtures by HPLC-MS.
abstract: 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.
authors.affiliation: Pliva Croatia Ltd., Research & Development, Zagreb, Croatia.
authors.forename: Mario
authors.initials: M
authors.lastname: Cindrić
authors.forename: Tina
authors.initials: T
authors.lastname: Cepo
authors.forename: Sabina
authors.initials: S
authors.lastname: Marinc
authors.forename: Ivan
authors.initials: I
authors.lastname: Paskvan
authors.forename: Ivana
authors.initials: I
authors.lastname: Mijić
authors.forename: Laura
authors.initials: L
authors.lastname: Bindila
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004229:Dithiothreitol / Q000032:analysis*; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011994:Recombinant Proteins; D004229:Dithiothreitol