Page 257 - Modern Derivatization Methods for Separation Sciences

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Urine is diluted 20 times with water and passed through a disposable filter (0.45 µm). To a 200µl
portion of the diluted urine sample, 200 µl of 0.15 M benzylamine in aqueous 90% methanol, 100 µl of
a mixture of 0.1 M CAPS buffer (pH 11.0) and methanol (3:7, v/v) and 100µl of 50 mM potassium
hexacyanoferrate (III) in aqueous 50% methanol are added successively. The mixture is allowed to
stand at 50 °C for 20 min. A 50-µl portion of the final reaction mixture is applied onto RP-HPLC. Fig.
3.24 shows a typical chromatogram obtained with a standard mixture and a human urine sample.

3.2.2.4—
Guanidino Compounds

Guanidino compounds and arginine can be selectively converted into fluorescent derivatives by reaction
with PQ [97] or benzoin [98,99] (Fig. 3.25).

PQ reacts with guanidino compounds under alkaline and subsequent acidic conditions to give
fluorescent derivatives. The reagent has been employed only for the post-column HPLC.

On the other hand, benzoin yields a single fluorescent derivative for guanidino compound. Thus, the
reaction with benzoin is used for both pre and post-column derivatization in HPLC.

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