Page 266 - Modern Derivatization Methods for Separation Sciences

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resin (Duolite A-375) as catalysts. The He-Cd LIF detection method with 9-APB allows the detection of
alkylphosphonic acid at fmol levels [139].

9-APB and Br-AA are powerful reagents for phase-transfer-catalyzed fluorescence derivatization. Most
bromomethyl and bromoacetyl reagents reacted in organic solvents such as acetonitrile and acetone
with carboxylic acids. On the other hand, Br-MA, Br-AA and 9-APB react with carboxylic acids in an
aqueous matrix. Br-MA completely derivatized fatty acids within 10 min at room temperature even in
aqueous DMF and DMSO. Br-MA and Br-MMC are used in micellar-mediated labeling reactions
[111,140]. Fatty acids and valproic acid can be determined in plasma with these methods. Further, 9-
APB, a phase transfer reagent, is also advantageously used [137].

Most of this bromomethyl type reagents are easily decomposed on exposure to moisture or light to form
fluorescent by-products.

Br-DMEQ Application: Preparation of a Fluorescent Derivative of Benzoylecgonine, and Preliminary
Studies of its Application to the Analysis of Urine [122]

The pH of a urine sample (1 ml) is adjusted to about 9 with ammonia water. After 5 ml of chloroform-
isopropyl alcohol (9:1, v/v) has been added, the mixture is mechanically shaken for 10 min and
centrifuged at 1000 g for 10 min. The organic layer (4 ml) is evaporated, the residue is dissolved in 500
µl of acetonitrile. To the resulting solution is added 250 µl of 10 mM 18-crown-6 in acetonitrile and
about 5 mg of K CO and about 2 mg of Na SO , and sonicated for 1 min. After adding 250 µl of 2 mM
2
4
2
3
Br-DMEQ in acetonitrile, the mixture is heated at 80 °C for 20 min. After cooling to room temperature,
20 µl aliquots of the mixture are applied in the HPLC system (Fig. 3.29).
(2) Diazomethyl Reagents (Figs. 3.26 and 3.30)

ADAM [141], PDAM [142,143] and DAM-MC [144] have been reported. These diazomethyl reagents
offer a number of advantages as derivatization reagents. The reagents are relatively easy to synthesize
from readily available compounds and work under mild conditions without a catalysis even in the
presence of water.

In particular, ADAM has been widely used as a fluorescence labeling reagent for biologically important
carboxylic acids, and permits picomole level determination of the acids by RP-HPLC. ADAM is used
for the quantification of fatty acids including 5,8,11,14,17-eicosapentaenoic acid and arachidonic acid,
prostaglandins, oxalic acid, amino acids, carnitine, biotin, herbicides, okadaic acid, phosphoinositides,
phosphonic acid and polyether antibiotics, including monesin and laslocid [145,146]. The disadvantage
of the reagent is the fact that the reagent is very unstable even at -10 °C. In order to overcome this
drawback, a simple method for the preparation of ADAM in situ is established [147].

PDAM is more stable than ADAM, and the fluorescence intensity of PDAM esters is about five times
higher than that of ADAM derivatives (detection limits: 15-30 fmol). However, PDAM requires a
longer reaction time than ADAM to reach a constant derivatization yield. PDAM

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