Page 243 - Modern Derivatization Methods for Separation Sciences

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DMEQ-COCI Application: Determination of Amantadine in Human Plasma by HPLC

A 50-µl aliquot of plasma is pipetted into a 10ml screw-capped test tube together with 10 µl of 1-(1-
adamantyl)ethylamine (IS: 1.0 µg/ml) solution, 50 µl of 1 M NaOH and 2 ml of toluene. After vortex-
mixing for about 2 min, the mixture is centrifuged at 1000 g for 10 min. The supernatant (1.5 ml) is
transferred to another tube and is evaporated to dryness under a nitrogen stream. To the residue are
added 1.0 mM DMEQ-COCI solution (100 µl) and 5.0 mM triethylamine solution (50µl). The resulting
mixture is allowed to stand at room temperature (15-40 °C) for 5 min. A 20 µl portion of the final
reaction mixture is applied onto RP-HPLC. Figs. 3.11A and B are typical chromatograms obtained with
drug-free plasma and plasma spiked with amantadine, respectively [49].

(3) Isothiocyanates (Edman 's Reagent)

Isothiocyanates and isocyanates react with primary amines to give thiourea (thiohydantoin) and urea
derivatives, respectively (Fig. 3.5C). However, isocyanates react quite readily with water and alchohols
to give urethanes. For this reason they are generally replaced by the less reactive isothiocyanates. Hence
bolt the former and latter groups are used as reactive groups for the derivatization of amines and
alcohols, respectively.

Fig. 3.11.
Chromatograms obtained from: (A) drug-free plasma;
and (B) plasma spiked with amantadine. Peaks:
1 = amantadine; 2 = (l-adamantyl)ethylamine (IS).
Column: YMCPackC8 (5 µm; 250 × 4.6 mmI.D.).
Mobile phase: methanol-acetorntrile-water (35:35:30,
v/v). Flow-rate: 1 ml/min. [Reproduced from ref.
49, p. 470, Fig. 4.].

Edman's reagents are used not only in the precolumn determination of amino acids, but also in the
microanalysis of peptide sequences. The following reagents have been developed: FITC [50], DANITC
[51], DNSITC [52], 3- and 4-POPITCs [53], BHBT-NCS [54], CIPITC [55] and DTDITC [56] (Fig.
3.12). These reagents do not show great differences in sensitivity and reactivity.

(4) Succinimidyls

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