Hence, the number of samples needed to generate calibration curves for a full spectral library is (3 • (𝑛𝑛 -1) + 𝑛𝑛) • 𝑚𝑚, where 𝑛𝑛 is a number of isomers in the library, and 𝑚𝑚 is a number of biological replicates. Since calibration curves and unknown samples must be measured at the same time for accurate quantitation, the concern is that long experimental times may cause significant fluctuations in relative response factors.