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.
- Chemically defined media
- Monolithic RPLC