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The gradient used was as follows (at a flow rate of 0.5 ml min 1 ): 0% solvent B for 1.6 min, followed by an increase to 30% solvent B over 2.4 min, a further increase to 90% solvent B over 0.5 min, a hold at 90% solvent B for 0.5 min, a return to 0% solvent B over 0.1 min and equilibration at 0% solvent B for 1.4 min, for a total run time of 6.5 min per sample

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doi: 10.1046/j.1523-5378.2003.00146.x

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