Amino acids and onium compounds can be readily extracted from plant tissues with a methanol : chloroform : water (MCW) mixture (5 : 12 : 3 v/v/v). Pre-weighed plant samples (0.2 to 1.0 gfw) should be ground to a fine powder with liquid nitrogen in a mortar and pestle and extracted with 4 x 2.5 mL aliquots of MCW.

To the total of 10 mL MCW extract add 3 mL water + 2 mL of chloroform. Mix and allow to phase-separate at 4^oC for 1 h. Remove the upper aqueous phase (containing amino acids and onium compounds), and concentrate the aqueous phase to dryness under a stream of air in the fume hood. Redissolve in 1 or 2 mL of water.

[Alternatively, samples can be extracted in 10 mL methanol and phase separated by the addition of 5 mL chloroform + 6 mL water].

For quantification of amino acids it is recommended that at least 2 internal standards (approx. 250 nmol per sample) are added to the extract prior to phase separation; alpha-amino-n-butyrate (aab) [for the neutral amino acids], and alpha-aminoadipic acid (aad) [for the acidic amino acids]. Where SMM is to be quantified in the basic amino acid fraction, deuterium-6 labeled SMM should be used as internal standard.

For quantification of glycine betaine, deuterium-9 labeled glycine betaine should be used as internal standard. For quantification of choline, deuterium-9 labeled choline should be used as internal standard.