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was extracted with C18 disposable cartridge. The extract was evaporated, and the residue was dissolved
with the mobile phase and subject to HPLC. BCPB derivative of captopril and the internal standard
(captopril derivatized with 1-benzyl-2-chloro-4-methylpyridinium bromide) were separated using RP-
HPLC within 20 min. The chromatograms are shown in Fig. 2.41. The quantitation limit at 314 nm was
10 ng, and the calibration range was 10-500 ng/ml plasma. This method enabled sensitive determination
of captopril and its disulfides in human plasma after oral administration to patients.

2.6.2—
Disulfide Reagent

The sulfhydryl-disulfide exchange reaction was used for the post column derivatization of thiols (Fig.
2.42). Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid), DTNB) and related compounds are widely
used. Yamato et al. [143, see Ellman's reagent application] reported an analytical method specific to
acetyl-coenzyme A. Acetyl-CoA separated by RP-ion pair HPLC was enzymatically converted to thio-
CoA with an immobilized enzyme reactor, and determined spectrophotometrically after post-column
derivatization with Ellman's reagent. Detection was carried out at 412 nm and the detection limit was
0.05 nmol. n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB) was also used to derivatize thiols. Faulstich et
al. [144] investigated the derivatization of protein thiols by ODNB and reported the reaction of ODNB
to be faster than Ellman's reagent. The reagent reacts with thiol groups, which are not reactive with
Ellman's reagent.

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