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Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media

Citation

Alwael, H and Connolly, D and Barron, L and Paull, B, Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media, The Journal of Chromatography A, 1217, (24) pp. 3863-3870. ISSN 0021-9673 (2010) [Refereed Article]

Copyright Statement

2010 Elsevier

DOI: doi:10.1016/j.chroma.2010.04.036

Abstract

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC-MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50mm×2mm I.D. monolithic C18 column with UV detection at 355nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5mL/min with an elevated column temperature. For RPLC-MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R2>0.999 for 0.25-500μM for cysteine and 0.25-250μM for cystine using the LC-UV method, sensitive, with detection limit of 36nM for cysteine, and precise, with ≤1.1% RSD for both retention time and peak area (n=6). Samples (n=31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26μg/mL and cystine from 1062.02 to 1348.13μg/mL. © 2010 Elsevier B.V.

Item Details

Item Type:Refereed Article
Research Division:Chemical Sciences
Research Group:Analytical Chemistry
Research Field:Separation Science
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Chemical Sciences
Author:Paull, B (Professor Brett Paull)
ID Code:72038
Year Published:2010
Web of Science® Times Cited:14
Deposited By:Research Division
Deposited On:2011-08-18
Last Modified:2015-08-04
Downloads:0

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