Page 28 - Modern Derivatization Methods for Separation Sciences

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pravastatin sodium. HPLC with UV detection was not sensitive enough to measure reliably the low
ng/mL level of quantification needed for biological samples.

Dumousseaux et al. [3] developed a method for the determination of pravastatin in plasma by using an
immobilized antibody column extraction followed by HPLC with a laserinduced fluorescence detector
after fluorogenic derivatization. For the extraction of pravastatin in plasma samples, in which 100
µg/mL levels of organic acids and/or fatty acids exist, a simple yet specific immobilized-antibody-
mediated cleanup was performed. The immobilized-antibodymediated extraction method was first
introduced by Glencross et al. [4] for the determination of 17β-estradiol. A plasma sample was applied
to the column and washed with water, and the drug was eluted with methanol.

After evaporation of methanol, pravastatin was derivatized with N-dansyl-ethylenediamine (DNSED) at
the carboxyl end in the presence of diethyl phosphorocyanidate (DEPC) and triethylamine (TEA) in
dioxane (Fig. 1.1.1). The optimal DNS-ED derivatization condition was extensively examined. The best
solvent for the derivatization was found to be dioxane, amongst DMF, tetrahydrofurane, ethyl acetate,
and dioxane. Pravastatin sodium was first dissolved in a small amount of DMF and then diluted with
dioxane (2 to 4-fold, v/v) because of its solubility. Fig. 1.1.2 shows the effect of DNSED concentration
on the derivatization. The peak area of the pravastatin-DNS-ED adduct was increased with an increase
of DNS-ED and became constant in the range of DNS-ED concentrations of more than 100 µg/mL. The
effect of all the reagent concentrations on the derivatization and HPLC determination was studied, as
shown in Fig. 1.1.3. The reagents were prepared as dioxane solutions. The highest peak area was
obtained when pravastatin was derivatized under molar concentrations of DNS-ED:DEPC:TEA =
0.001:1:1. A concentration of 100 µg/mL for DNSED was chosen as the best compromise between the
reaction yield and the chromatographic noise due to a high concentration of the reagent.

Fig. 1.1.2.
Effect of N-dansyl-ethylenediamine concentration
on the derivatization. [Reproduced from ref.
3, p. 1633, Fig. 2.].

From these results, the concentration of reagents was fixed as follows: DNS-ED, 100 µg/ml (= 0.34
mM); DEPC, 51.8 µlml (0.34 M); and TEA, 47.6 µl/ml (0.34 M). For this condition, the derivatization
process was completed within 5 min at room temperature to yield a maximum and a constant peak area.

The DNS-ED derivatization method was designed to make good use of a highly sensitive He-Cd laser-

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