Table 2. A typical analytical sequence for two samples.

Sample run	HPLC run	comment
Asp RMs	no gradient	linearity check
Glu RMs	no gradient	calibration check
15AAmix	short then long	precision check
sample #1	short then long	replicate analysis
sample #1	short then long
sample #1	short then long
15AAmix	short then long	precision check
sample #2	short then long	replicate analysis
sample #2	short then long
sample #2	short then long
Asp RMs	no gradient	linearity check
15AAmix	short then long	precision check

During the linearity and calibration runs, Asp and Glu RMs were injected into an isocratic flow of 99.7% A / 0.3% B that had a stable baseline. Samples and the 15AAmix were analysed using a short run followed by a long run. Both short runs and long runs included RMs, injected before the sample, as additional checks of δ¹³C linearity and calibration; these short and long RMs eluted in regions where baselines were not entirely stable.