derivatizanti luminol

[Applications] [Forensic uses]

LUMINOL-TYPE CHEMILUMINESCENCE DERIVATIZATION REAGENTS

Introduction

A great variety of biological substances that exhibit bioactivities even at extremely low concen- trations, occur in biological fluids and tissues, and their concentrations in bio-matrices are generally controlled by their related enzymes. Therefore, it is aminovery important to measure these substances in bio- logical materials over wide fields in life science. Since such bioactive substances occur at only small quantities in highly complex matrices, analysis meth- ods should be both selective and sensitive.
Chemiluminescence is highly sensitive and selective, and becomes essential in liquid chromatography, immunoassay and hybridi- zation assay. Among these chemiluminescent compounds, luminol-type derivatives have been well examined concerning their reaction mechanism, apparatus and so on, and thus have been used for various applications.[23-35] the chemiluminescence derivatization methods of mainly bioactive substances using luminol-type reagents in flow systems, LC and CE, describes in the present page.


Chemiluminescence of luminol compounds

All chemiluminescence reactions of (iso)luminol derivatives are oxidation reactions, which are carried out in either aprotic solvents [dimethyl sulfoxide (DMSO), N,N-dimethylformcapillary amide] or protic solvents (water, lower alcohols) [36, 10]. Although the chemiluminescence reaction in aprotic media needs only oxygen and a strong base, the reaction in protic media usually requires a base, an oxidizing agent, and either a peroxide or oxygen.
Several derivatives of (iso)luminol have been studied on the relationships between their structures and chemiluminescence properties. Modifications of the cyclic phthalhydrazide moiety of (iso)luminol led to complete loss of the chemiluminescence property [10, 37].
Luminol analogues of which the aromatic ring is modified are generally found to be chemiluminescent [10, 37, 38]. Alkylations of the amino group in iso-luminol increase the chemiluminescence intensity, dimethoxywhereas the substitutions in luminol cause decreases due to stereoscopic hindrance.
Since the N-alkyl derivatives of isoluminol were found to be highly dimethchemiluminescent, many isoluminol-type compounds N-alkylated with methyl, ethyl, butyl or hexyl have combeen used as the chemiluminescence derivatization reagents [10, 37, 39]. Many cyclic acylhydrazides originated in aromatic o-dicarboxylic acids show chemiluminescence, and substitution of the aromatic amino group has a great influence on the fluorescence of (iso)luminol derivatives.
For CL example, 7-[N-(4-aminobutyl)-N-ethyl]naphthalene- 1,2-dicarboxylic acid hydrazide (Fig. 1A) [36, 37]. interbenzo ghi]perylene-1,2-dicarboxylic acid hydrazide (Fig. 1B) [10, 37]., 4-(9-acridonyl-10-methylene)- phthalhydrazide (Fig. 1C) [16], and 4-(59,69-dimethchemiluminescent, oxybenzothiazolyl)phthalhydrazide (Fig. 1D) have been known as intense chemiluminogenic com pounds based on their highly fluorescent structures, naphthalene, benzo[ghi]perylene, N-methylacridone, and 2-phenylbenzothiazole, respectively.


Fig.1

Since chemiluminescence of luminol is a radiation from the chemically excited 3-aminophthalic acid, the chemiluminescent emission spectrum of luminol and the fluorescent emission spectrum of 3-aminophthalate dianion closely resemble each other in shape. The same relationships are true for other luminol-type compounds.
Furthermore, the F of luminol-type compounds depends partly CL on the fluorescence quantum yields of the aromatic o-dicarboxylate dianions. Thus, the structures having higher fluorescent quantum yields are desirable for the skeletons of luminol-type chemioxidizing luminescence compounds, and this strategy has been applied to the basic design of some chemiluminesmetal cent compounds and reagents [40-43].


Apparatus for chemiluminescence detection in liquid chromatography

Figure illustrates the schematic flow diagram for the detection system of (iso)luminol derivatives in LC.

Fig. 2

For the chemiluminescence detection of luminol-type compounds, an on-line oxidizing device based on chemical or electrochemical reaction is indispensable before or in the chemiluminescence detector. In a chemical oxidation device, the oxi dizing reagents, hydrogen peroxide and some catalysts, have been commonly used.
The reagent solution(s) is introduced to the column effluent in a three- way union just before the chemiluminescence detector (Fig. 2A); the tube between the union and the detection cell should be minimized, because the chemiluminescence life-time of (iso)luminol deriva tives is very short in many cases [36, 10, 40-47].
Electrogenerated chemiluminescence (ECL) meth ods of luminol-type compounds have also been reported in which an electrochemical oxidation of the compounds is utilized in place of chemical oxidation (Fig. 2B) [48, 49]. A glassy carbon or platinum has been used as a working electrode to carry out the oxidation reaction. The ECL reaction can be controlled by the applied potential to the electrode. The ECL system is simpler because it does not require the postcolumn reaction apparatus consisting of pump(s), mixing union and reaction tube. The meth od has almost the same sensitivity as those in the chemical oxidation methods.
Not only LC but also CE are applied to the separation of luminol-related compounds in a flow system, and the principles and the applications of chemiluminescence in CE have been reviewed [35, 50-52]. In the case of CE, there are some oxidation methods to carry out the chemilumines cence reaction.

The off-column merging interface (Fig. 3A), on-column coaxial flow interface (Fig. 3B), off-column coaxial flow interface (Fig. 3C), and end-column reservoir interface (Fig. 3D) are the representative oxidation systems using oxidizing reagent solution, whereas ECL coupled with CE is also reported [53-61].

Fig. 3

Recently, on-line solid-phase chemiluminescence techniques oxidized with barium peroxide powder were reported for LC and CE analyses of luminol derivatives [62, 63].


Application of luminol-type compounds

The luminol-type chemiluminescence derivatization reagents have been utilized to the determinationof many biological and pharmaceutical compounds in LC or CE. In the following sections, some derivatization reagents with luminol-type structures are reviewed. The chemiluminescence derivatization reagents have been classified into two groups by reaction type: ‘‘chemiluminogenic reagent’’ and ‘‘chemiluminescence labeling reagent’’.
In the former, the chemiluminesce derivatization reagents themselves are generally weakly chemiluminescent, and react with target compounds to form the conjugated ring molecules, resulting in production of chemiluminescence. In the latter, the reagents are composed of a highly chemiluminescent moiety and a reactive moiety, and the reactive moiety attaches to an analyte to form chemiluminescence-labeled de rivatives.


Luminol-type chemiluminogenic reagents

Reactions for α-keto acids and α-dicarbonyl compounds

4,5-Diaminophthalhydrazide (DPH) was reported as the derivatization reagent for a-keto acids, a dicarbonyl compounds and their related compounds (Fig. 4).

DPH reacts with α-keto acids at 100 °C for 45 min in diluted hydrochloric acid to give the highly chemiluminescent derivatives (Fig. 4A). These rivatives are separated by reversed-phase (RP) LC with isocratic elution, and detected chemilumin ometrically after mixing with oxidizing reagents.
This method was applied to the determination of α-keto acids including phenylpyruvic acid in human plasma by using a-ketocaproic acid as an internal standard (I.S.)
DPH also reacted with α-dicarbonyl compounds including phenylglyoxal at 100 °C for 45 min in diluted hydrochloric acid in the presence of β-mercaptoethanol to give the highly chemiluminescent derivatives (Fig. 4B). These derivatives are separated by RPLC with isocratic elution, followed by chemiluminescence detection. This method is applicable to the determination of 3α,5β-tetrahydroaldosterone in human urine and dexamethasone in human plasma after oral administration of the drug by using beclomethasone as an I.S. [64-70].


Reactions for aldehydes

DPH reacts with aromatic and aliphatic aldehydes 100 °C for 30 min in diluted hydrochloric acid to give the highly chemiluminescent derivatives (Fig. 5A). These derivatives are separated by RPLC with isocratic elution and detected by the addition with hydrogen peroxide and alkaline potassium hexa-cyanoferate(III).[71]
ASP reacts selectively with aromatic aldehydes at 100 °C for 20 min in sulfuric acid to form the highly chemiluminescent derivatives (Fig. 5B). These de- rivatives are separated by RP–LC with isocratic elution, followed by chemiluminescence detection . The detection limits for aromatic alde hydes are in the ranges 0.2–4.0 fmol per injection [72].

Fig. 5

Luminol-type hemiluminescence labeling reagents

The chemiluminescence labeling reagents are defined as: the reagent is composed of a highly chemilumi nescent moiety and a reactive moiety, and the reactive moiety attaches to an analyte to form the chemiluminescence-labeled derivatives. We classify them according to the functional groups of analytes.


Reactions for amines

The luminol-type chemiluminescence labeling reagents used for amines are illustrated in Fig. 6.
N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) that was used in immunoassays and so on from the 1970s is the first chemiluminescence labeling reagent in LC. ABEI, after coupling to N,N'-disuccinimidyl carbonate (DSC), reacts with primary and secondary amines for 2 h at room temperature or for 30 min at 80 °C in the presence of organic bases to give the chemiluminescent ABEI derivatives [73].
This ABEI–DSC method was applied to the determination of methamphetamine and amphetamine in human serum and urine.
N-(4-aminobutyl)–N-ethylisoluminol isothiocyanate (ABEI–ITC), an activated ABEI derivative for amines, was isolated synthetically and was used for the determination of histamine with electrochemical oxidation. The derivatization reaction is performed at 80 °C for 1 h, and the detection limit for histamine is 1.5 pmol per injection [74].
6-Isothiocyanatobenzo[g]phthalazine-1,4(2H,3H)-dione (IPO) containing an isothiocyanate group was reported as the chemiluminescence labeling reagents for amines. Amines are labeled with IPO at 80 °C for 10 min in the presence of triethylamine and detected highly sensitively. The detection limits for primary and secondary amines are in the ranges 30–120 and 0.8–3 fmol per injection, respectively [75].
This IPO method was applied to the measurement of maprotiline in human plasma by using desipramine as an I.S. because the IPO-labeled secondary amines were detected highly sensitively (Fig. 6).
4-(6,7-Dihydro-5,8-dioxothiazolo[4,5-g]phthalazin- 2-yl)benzoic acid N-hydroxysuccinimide ester (TPB-Suc) that is derived from a highly chemiluminescent ASP derivative of 4-formylbenzoic acid has been reported as the chemiluminescence labeling reagent for amines. TPB-Suc reacts with both primary and secondary amines at 80 °C for 20 min in the presence of triethylamine.
The detection limits for the amines are at sub-fmol levels per injection [76].

Fig.6

Reactions for carboxylic acid

The luminol-type chemiluminescence labeling reagents used for carboxylic acids are illustrated in Fig. 7.
ABEI reported above as the derivatization reagent for amines has been also used for the chemilumines cence labeling reagent of carboxylic acids. ABEI reacts with carboxylic acids at 60 °C for 2 h in the presence of suitable condensing agents to give the chemiluminescent derivatives [73].
This ABEI method was applied to the determination of eicosapentanoic acid in serum after single-step liquid–liquid extraction as a deproteinization, and the detection limits were 200 fmol per injection [77]. Ibuprofen and naproxen, both the drugs containing carboxyl group, were also chemiluminescencelabeled with ABEI [78].
N-(4-Aminobutyl)-N-methylisoluminol (ABMI), an ABEI-like labeling reagent, has been used as the derivatization reagent for the carboxylic acid analy ses in LC [79].
6-[N-(3-Propionohydrazino)thioureido]benzo[g] phthalazine-1,4(2H,3H)-dione (PROB), an IPO de rivative of b-alanine and hydrazine, was reported as the labeling reagent for carboxylic acids.
This PROB method was successfully applied to the determination of some free fatty acids in human plasma [80].

Fig.7

[Applications] [Forensic uses]