Validation of the LC/MS method. (A) Principal analysis score plots (PCA) for RBC extracts obtained from 5 healthy individual donors who were subjected to 3 consecutive freeze-thaw cycles before LC/MS analysis. The chromatograms were processed by automatic peak detection software. The variables (ie, couples of m/z ratio, retention time) contained in the resulting data matrix were mean-centered and scaled to unit variance before PCA. Data for each person are circled, and the samples corresponding to the first, second, and third freeze-thaw cycles are red, green, and blue, respectively. (B) Levels of the 3 indicated metabolites as a function of the number (F1 = 1, F2 = 2, and F3 = 3) of freeze-thaw cycles. A.U. indicates arbitrary units. (C) More than 1700 signals among 3000 detected in the positive and negative ionization mode exhibited coefficient correlations more than 0.7 with the dilution factor. (Inset) An example of correlation between UHPLC/MS areas and dilution of RBCs samples. C1 to C5 indicate control persons.