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Fig. 2.42.
The reaction of Ellman's reagent and thiols.

Ellman's Reagent Application: Determination of Acetyl-CoA by Post-column Derivatization [143]

Short-chain acyl-CoA thioesters were first separated by C8 RP-HPLC using tetra-n-butylammonium
phosphate as an ion-pairing reagent. The separated thioesters were mixed with a reagent solution (30
mM Na HPO as the substrate, 15 mM (NH ) SO as the enzyme activator and 0.1 mM DTNB as the
2
4 2
4
4
color-producing reagent) and introduced to the immobilized enzyme reactor. Acetyl-CoA in the elution
mixture was converted to thio-CoA by the immobilized phosphotransacetylase and reacted with
Ellman's reagent (DTNB) in the reagent stream. The calibration curve at 412 nm was linear between 0.2
and 10 nmol, with a detection limit of 0.05 nmol.
2.6.3—
Other Reagents

Shen et al. [145] reported a simple method of determining captopril in biological fluids using p-
bromophenacyl bromide as a precolumn derivatizing reagent. The derivative was separated by C18 RP-
HPLC and detected at 260 nm. The calibration range was 20-1000 ng/ml plasma and 10-200 µg/ml
urine. Pietra et al. [146] determined glutathione and L-cysteine in pharmaceuticals after derivatization
with ethacrynic acid. Derivatives were separated by C18 RP-HPLC and monitored at 270 nm. Gotti et
al. [147] reported the determination of glutathione in pharmaceuticals and cosmetics. Glutathione was
derivatized with 4-(6-methylnaphthalene-2-yl)-4oxo-2-buteneoic acid, and the thiol adduct was
chromatographed on C8 RP-HPLC. Derivatives were detected using absorbance at 254 nm or
fluorescence (Ex. 300 nm, Em. 460 nm).

2.7—
Labelling of Other Compounds

2.7.1—
1-(2-Pyridyl)-piperazine (PYP) [148]

Chromosorb coated with PYP was evaluated for collection/derivatization of Phosgene (COCI ) gas.
2
Phosgene reacts with PYP to form a substituted urea derivative (Fig. 2.43) which is desorbed with
CH CN and determined by PR-HPLC with UV absorbance detection at either 254 or 270 nm.
3
2.7.2—
Diethyldithiocarbamate (DDTC)

The CE method is developed for the quantitative determination of the anti-cancer drug prospidin in
human tissue after its derivatization with sodium diethyldithiocarbamate (DDTC, Fig. 2.44) [149].
Detection is by UV absorption at 254 nm. The

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