If glutamine, glutamate, aspartate and asparagine are to be independently quantified, amino acids should first be separated into acidic and neutral + basic fractions prior to derivatization. This is accomplished by first applying the aqueous fraction (after MCW phase separation) to a 4 cm x 1 cm column of Dowex-1-acetate (pre-washed with 4 mL 2 M acetic acid followed by excess water until pH is neutral). Neutral + basic amino acids should be eluted with 6 mL water, and acidic amino acids with 8 mL 2 M acetic acid. The acidic amino acid fraction is dried under a stream of air in the fume hood and redissolved in 1 mL of water.

The water wash from Dowex-1-acetate containing neutral + basic amino acids should be applied to a 4 cm x 1 cm column of Dowex-50-H⁺ (pre-washed with 2 mL 2.5 M HCl, followed by excess water until pH is neutral). After applying the neutral + basic amino acids, the column is washed with 6 mL water (discarding the water wash), and neutral + basic amino acids eluted with 6 mL 6 M NH₄OH. The latter fraction should be dried under a stream of air in the fume hood.

Similarly the acidic amino acid fraction from Dowex-1-acetate, after drying and redissolving in water, should be applied to a 4 cm x 1 cm column of Dowex-50-H⁺ (pre-washed with 2 mL 2.5 M HCl, followed by excess water until pH is neutral). After applying the acidic amino acids, the column is washed with 6 mL water (discarding the water wash), and acidic amino acids eluted with 6 mL 6 M NH₄OH. The latter fraction should be dried under a stream of air in the fume hood.

The purified, dried amino acid fractions should be redissolved in 0.6 mL 60% methanol and transferred to 1 mL micro-reaction vessels for derivatization to heptafluorobutyryl isobutyl amino acid derivatives.

Where quaternary ammonium and tertiary sulfonium compounds are to be quantified, the following ion exchange sequence is recommended: Dowex-1-OH^- (to retain acidic plus neutral amino acids), Biorex-70-H⁺ (to retain bases [including SMM and choline]), and Dowex-50-H⁺ (to retain zwitterionic quaternary ammonium and tertiary sulfonium compounds [except choline-O-sulfate which is not retained and is eluted in the water wash]). Alkaline-labile quaternary ammonium and tertiary sulfonium compounds [such as beta-alanine betaine and dimethylsulfoniopropionate] can be eluted from Dowex-50-H⁺ with 6 mL 2.5 N HCl. Alkali-stable quaternary ammonium compounds can be eluted from Dowex-50-H⁺ with 6 mL 6 M NH₄OH. The bases (choline and SMM) can be eluted from Biorex-70-H⁺ with 6 mL 1 M HCl. The acidic plus neutral amino acids can be eluted from Dowex-1-OH^- with 6 mL 2.5 M HCl; however, this is not recommended when glutamine, glutamate, aspartate and asparagine are to be independently quantified, as this will result in partial hydrolysis of glutamine to glutamate and asparagine to aspartate. Amino acids eluted from Dowex-1-OH^- with 6 mL 2.5 M HCl must be further purified by Dowex-50-H⁺ ion exchange chromatography as described above, before derivatization. Choline can be extracted from the dried base fraction with acetonitrile : methanol (20 : 1 v/v). If SMM is to be quantified in the base fraction, the 6 mL 1 M HCl eluant from Biorex-70-H⁺ should be dried under a stream of air, redissolved in water, and then further purified by Dowex-50-H⁺ ion exchange chromatography as described above, eluting with 6 mL 6 M NH₄OH; choline is not eluted from the column with this reagent.