Methanol/chloroform/water (65:25:15) mixture was added to the ground sample at a ratio of 1:3 w/v, followed by 1 mL of internal standard amino acid solution (0.4 M Norvaline and Sarcosine in ddH2O).
Each 20[micro]L sample plus 50 [micro]L internal standard (norvaline, 0.5 [micro]M, concentration in derivatised sample: 0.25pmol [micro][L.sup.-1]) was evaporated to dryness in an LC-MS glass vial and resuspended in 20 [micro]L of 20 mM HC1, and 60 [micro]L of AccQ-Tag[TM] Ultra borate buffer was added to each sample or standard.