Chemiluminescence in Analytical Chemistry

and unsaturated fatty acids [35, 36], and central nervous stimulants [37, 38] could be detected at fmol levels using N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as a labeling reagent. For labeling of primary and secondary amines, ABEI is used with N, N′-disuccinimidyl carbodiimide (DSC) as a condensing reagent. 2-Chloro-1-methylpyridinium iodide (CMPI) and 3,4-dihydro-2H-pyrido[1,2- α]pyrimidin-2-one are employed as condensing reagents to label carboxylic acids with ABEI (Fig. 8). 4-Isothiocyanatophthalhydrazide (ILITC) [39] and 6-isothio- cyanatobenzo[g]phthalazine-1,4 (2H,3H)-dione (IPO) have been developed for the labeling of amines [40]. Femtomol levels of amino acids and amines could be detected by use of these reagents. IPO has also been applied to analysis of maprotiline, a widely used antidepressant, in human plasma [41].

Figure 8 Labeling reaction of ABEI with (A) primary and secondary amines, and

(B) carboxylic acids.

Hydrogen peroxide and peroxides can also be detected utilizing the CL from luminol derivatives. Determination of peroxides is very important to obtain information on human diseases such as a atherosclerosis and aging, and also on food spoilage. Lipid hydroperoxides such as phosphatidylcholine hydroperoxide in rat tissues separated by HPLC were successfully determined by using the CL generated by the reaction of luminol with cytochrome c or microperoxidase [42]. An HPLC determination of hydrogen peroxide with a cation-exchange gel column was examined and a detection limit of 4 pmol was obtained. This method was successfully applied to determination of hydrogen peroxide in coffee drinks [43].

An IMER immobilizing 3 α-hydroxysteroid dehydrogenase was employed for HPLC determination of cholic acid and a detection limit of 2 pmol was achieved [44]. This approach has the advantage of permitting a repeatable use of the enzyme.

Methods measuring CL via the luminol-type reaction products from analytes have been proposed. Derivatization reactions of analytes with luminol-type reagents are shown in Figure 7B. α-Keto acids including phenylpyruvic acid and α-dicarbonyl compounds were reacted with 4,5-diaminophthalhydrazide (DPH) under different conditions to give corresponding chemiluminescent luminol-type compounds. The detection limits of eight biologically important α-keto acids and five α-dicarbonyl compounds (phenylglyoxal, diacetyl, 2,3-pentanedione, 2,3- hexanedione, and 3,4-hexanedione) were in the range of 4–50 fmol [45] and 1.1–300 fmol per injection [46], respectively. N-Acetylneuraminic acid (NANA) derivatized with DPH was determined with a detection limit of 9 fmol [47]. DPH has also been extended to the sensitive determination of 3α, 5β-tetrahydroaldoste- rone [48] and dexamethasone in human plasma [49]. A new luminol-type reagent, 6-aminomethylphthalhydrazide (6-AMP), was synthesized as a CL derivatization reagent for 5-hydroxyindoles (Fig. 7B) [50]. The detection limits for 5-hydroxy- indoles were in the range of 0.7–4 fmol (S/N 3) (Fig. 9). The advantages of these methods using DPH and 6-AMP are high sensitivity and selectivity due to the unique derivatization reaction.

6.2 HPLC-CL Detection Using Lucigenin Derivatives

Reducing sugars can be determined using lucigenin; oxidized products of reducing sugars with sodium periodate react with lucigenin to generate an intense CL. This phenomenon is based on the reaction of lucigenin with the α-hydroxycarbo- nyl group. Therefore, compounds such as glyceraldehyde, cortisol, phenacylalcohols, and phenacylesters, which contain an α-hydroxycarbonyl group in their structure, are determined sensitively by this system. Detection limits of corticosteroids and p-nitrophenacyl esters were reported to be ca. 0.5 pmol per injection [51].

Figure 9 Chromatogram of 5-hydroxyindoles derivatized with 6-AMP. Peaks (2.5 pmol each on column): 1 5-hydroxytryptophan; 2 serotonin; 3 5-hydroxyindole-3-acetic acid. (From Ref. 50.)

An HPLC-CL determination of environmentally important chlorophenols was reported by using 10-methyl-9-acridinium carboxylate as a CL label. A twostep derivatization was used to produce the CL derivatives (Fig. 10). Following the separation under reversed-phase conditions, the CL reaction was performed by the base-catalyzed postcolumn oxidation. The quantum efficiency was dependent on the species of analytes. The detection limit of chlorophenols (S/N 3) ranged from 300 amol to 1.25 fmol per injection (Fig. 11) [52].

6.3 HPLC-PO-CL Detection

The nature of the aryloxalate used in a PO-CL system is an important factor in view of the produced CL intensity. Several aryloxalates, e.g., bis(2,4,6-trichloro- phenyl)oxalate (TCPO), bis(2,4-dinitrophenyl)oxalate (DNPO), bis(2,6-diflu- orophenyl)oxalate (DFPO), bis(pentafluorophenyl)oxalate (PFPO), and bis[2-

Figure 10 CL derivative of chlorophenol and its CL reaction.

Figure 11 Chromatogram of chlorophenols. Peaks (40 fmol each on column): 1 chlorophenol; 2 4-chlorophenol; 3 2,4-dichlorophenol; 4 2,4,6-trichlorophenol. (From Ref. 52.)

Figure 12 Structure of ODI.

(3,6,9-trioxadecyloxycarbonyl)-4-phenyl]oxalate (TDPO), were evaluated on their properties [53–55]. Among them, TCPO, TDPO, and DNPO are the most frequently used. Recently, 1,1′-oxalyldiimidazole (ODI) was applied to determine hydrogen peroxide, and the results showed that ODI was about 10 times more sensitive than TCPO (Fig. 12) [56].

In the PO-CL system, the compounds showing native fluorescence or that fluoresce after chemical derivatization can be detected. As examples of the POCL detection of native fluorescence compounds, dipyridamole and benzydamine in rat plasma [57] and fluphenazine [58] have been reported; in the former method, the detection limits of dipyridamole and benzydamine were 345 pM and 147 nM in plasma, respectively. Diaminoand aminopyrenes were sensitively determined using TCPO and their detection limits were in the sub-fmol range [59]. Carcinogenic compounds such as 1- nitropyrene and its metabolites, can also be determined by the HPLC-PO-CL system. Nonfluorescent nitropyrenes were converted into the corresponding fluorescent aminopyrenes by online reduction on a Zn column followed by detection; 2–50-fmol detection limits were achieved in the determination of ethanol extracts from airborne particulates (Fig. 13) [60].

Figure 13 Chromatogram of an airborne particulate sample. Peaks: 1 1,6-dinitropyr- ene; 2 1,8-dinitropyrene; 3 1,3-dinitropyrene; 4 2-fluoro-7-nitrofluorene; 5 1- nitropyrene. (From Ref. 60.)

Figure 14 Representative fluorescence derivatization reagents used for PO-CL detection. DNS-Cl, dansyl chloride; DBD-F, 4-(N,N-dimethylaminosulphonyl-7-fluoro-2,1, 3-benzoxadiazole; NDA, naphthalene-2,3-dicarboxaldehyde; DNS-H, dansyl hydrazine; DBD-H, 4-(N,N-dimethylaminosulphonyl-7-hydrazino-2,1,3-benzoxadiazole).

For analysis of nonfluorescent compounds, many fluorescence derivatization reagents can be used for HPLC with PO-CL detection (Table 2). Representative fluorescence derivatization reagents are shown in Figure 14. Dansyl derivatives such as dansyl chloride (DNS-Cl) and dansyl hydrazine (DNS-H) are often utilized as fluorescence labeling reagents. DNS-Cl has been utilized for PO-CL detection of amino acids [7, 63–66]. A highly sensitive microbore HPLC-PO- CL detection technique for DNS amino acids was also investigated and the detection limits obtained were 0.8–1.7 fmol [91]. Determination of methamphetamine and its metabolites was investigated and determination limits as low as 10–30 fmol were obtained [71]. For labeling of the carbonyl group, DNS-H is widely used. Oxosteroids and oxobile acid ethyl esters [81], hyaluronic acid (HA) in blood plasma [82], HA, chondroitin sulfate, and dermatan sulfate [83] were successfully determined using DNS-H.

Femtomol levels of detection limits were also achieved in the determination of stimulant amines with the benzofurazan derivative 4-(N,N-dimethylaminosul- phonyl)-7-fluoro-2,1,3-benzoxadizole (DBD-F) [73]. DBD-F was successfully applied to the PO-CL detection of amino acids and epinephrine [74] and a β- blocker, metoprolol [75]. 4-(N,N-Dimethylaminosulphonyl)-7-hydrazino-2,1,3- benzoxadizole (DBD-H) has also been used for PO-CL determination of a neuronal cell protective compound, propentofylline. The method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus [85].

Figure 15 Chromatograms of catecholamines obtained from (a) human and (b) Sprague-Dawley rat plasma. Peaks: NE norepinephrine; E epinephrine; I 3,4- dihydroxybenzylamine; DA dopamine. (From Ref. 88.)

Naphthalene-2,3-dicarboxaldehyde (NDA), which reacts with primary amines to give highly fluorescent cyanobenz[ f ]isoindole (CBI) derivatives, has also been used for determination of amphetamine-related compounds [70], dopamine, and norepinephrine [72]; sub-fmol to fmol amounts of these were detected by HPLC with the PO-CL detection.

A highly sensitive method was developed for determination of plasma catecholamines. These analytes were derivatized online with ethylenediamine to give the corresponding intensely fluorescent compounds, and then detected by the POCL reaction [88]; the detection limit for all the catecholamines obtained was 1 fmol (S/N 2) (Fig. 15). Detection limits of amol levels (S/N 3) for catecholamines were obtained by the precolumn derivatization method using 1,2-diaryl- ethylenediamine derivatives as fluorescence derivatization reagents [89, 90]. In these methods, TDPO permitting a highly sensitive determination of catecholamines was used as the aryloxalate.

Hydrogen peroxide was determined with fmol detection limits by using rhodamine 6G and pyrimidopyrimidine derivatives as fluorescent enhancers. The method employing the latter reagent was applied to cola drinks [92]. Sensitive