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of the method (10 nM). The rainwater collected in Dortmund, Germany had a concentration of hydrogen peroxide ranging from 2 to 2200 ppb during daytime and 0 to 440 ppb at night. The same instrumental setup was utilized to determine gaseous hydrogen peroxide [27]. A sample was collected offline in a cryogenic trap, subsequently melted, and analyzed using the FIA system. A sampling time of approximately 2 h was required; i.e., the time resolution was relatively low. On the other hand, impressive detection limits were obtained, between 0.2 and 6 pptv depending on the relative humidity. The air in Dortmund had a daily mean concentration of hydrogen peroxide in the range 5–60 pptv and maximum levels in the range 10–220 pptv. Later, the same group measured hydrogen peroxide in Antarctic air, snow, and firn cores [28]. The air in Antarctica contained higher levels of hydrogen peroxide, 100–1050 ppt, and diurnal variations were not observed.

An online direct method for determination of gaseous hydrogen peroxide in ambient air was presented by Stigbrand et al. [29]. The analyte was collected continuously in a diffusion scrubber, in which gaseous hydrogen peroxide was captured by diffusion into a scrubber liquid. Sample plugs of aqueous hydrogen peroxide were subsequently injected into a single-line FIA system using stabilized ODI and an immobilized fluorophore. Interferences from naturally occurring organic peroxides were investigated and revealed a selective response for hydrogen peroxide, consistent with early research on POCL that investigated the effects of different organic peroxides. The detection limit for gaseous hydrogen peroxide was estimated to be 23 pptv; the linear range was limited by the operational range of the mass flow controllers rather than the chemistry, with a linear response obtained between 0.6 and 3.4 ppbv. The sample throughput was high, 120 samples per hour, resulting in a good time resolution. Van Zoonen et al. [30] determined the amount of hydrogen peroxide in rainwater using a solid-state TCPO reactor in combination with immobilized fluorophores in a single-line FIA system. Nakashima et al. [31] developed a liquid chromatographic method to determine hydrogen peroxide in cola drinks, ascorbic acid, and commercially available organic peroxides.

Recently the POCL reaction was used to study the role of hydrogen peroxide in antitumor activity [32, 33]. Addition of sodium 5,6-benzylide L-ascorbate or ascorbic acid increased the production of hydrogen peroxide, which in turn induced cell death.

3.1.2Detection of Hydrogen Peroxide Generated in a Postcolumn Reactor

Oxidases are capable of converting specific substrates and molecular oxygen into hydrogen peroxide or water, and other products [179]. By quantifying the content of hydrogen peroxide, the substrate concentration can thus be indirectly deter-

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mined. Immobilized enzyme reactors (IMERs) offer several advantages over the use of dissolved enzymes, most important being the reduction in cost and operational work. Owing to the high enzyme load in the IMERs and favorable equilibrium constants for the enzymatic reactions, 100% conversion of the substrate is commonly attained [179]. The enzymes can be either classor compound-selec- tive, and depending upon the selectivity, different instrumental configuration may be employed. A FIA system is sufficient if the enzyme is compound-selective, whereas with a class-selective enzyme a separation step is often included. This separation can be achieved with a reversed-phase LC system having a low percentage of organic modifier in the mobile phase or some kind of ion exchange separation. Higher amounts of organic solvents are in most cases incompatible with the IMER [180] owing to a loss of enzymatic activity.

Many different kinds of detection principles have been used to determine the hydrogen peroxide formed and chemiluminescent reactions are an attractive approach since the selectivity in the CL reaction combined with the highly selective enzyme results in an extremely selective and sensitive detection. The advantage of POCL compared to other CL reactions capable of determining hydrogen peroxide is its optimal pH close to 7, the same as most enzymatic reactions. On the other hand, most POCL reactions require organic solvents, which are usually incompatible with enzymes. Generally, a completely aqueous carrier/eluent transports the sample plug through the IMER, which then merges with a second carrier containing a fluorophore and the POCL reagent dissolved in a water-misci- ble organic solvent. In Table 2, applications dealing with POCL detection of enzymatically generated hydrogen peroxide are listed.

One of the most frequently studied enzymatic reaction systems involves the use of glucose oxidase:

Glucose O2 H2O → gluconic acid H2 O2

(4)

This reaction was employed by Williams et al. [34] in the first application of the POCL reaction dealing with the determination of hydrogen peroxide. Several studies of the same enzymatic reaction have been performed since then [35–39], probably because glucose oxidase is relatively inexpensive, highly stable, and well characterized. Rigin used several different enzymatic reactions coupled to the POCL chemistry; cholesterol oxidase [40], L-amino acid oxidase [41], and aldehyde oxidase [42] were all immobilized on glass supports and evaluated in a FIA system. Jansen et al. [43] also studied the stereoselective L-amino acid oxidase. Eight different L-amino acids were separated in a reversed-phase liquid chromatographic system and detected by the use of a flow cell containing immobilized 3-aminofluoranthene. Beer and urine samples were run, as well as standards. Recently, the less common amino acid isomer, the D-form, was determined in human plasma samples using FIA-IMER-POCL [44].

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Table 2 Summary of Analytes that Have Been Converted to Hydrogen Peroxide and Subsequently Quantified with the POCL Reaction

Analyte	Enzyme	Ref.
Glucose	Glucose oxidase	34–39
Cholesterol	Cholesterol oxidase	40
L-Amino acids	L-Amino acid oxidase	41, 43
Formaldehyde, formic acid	Aldehyde oxidase	42
D-Amino acids	D-Amino acid oxidase	44
Polyamines	Putrescine oxidase	45, 46
	Polyamine oxidase
Uric acid	Uricase	37, 47
Choline, acetylcholine	Choline oxidase	48–50
	Acetylcholine esterase
Choline-containing phospholipids	Phospholipase D	51
	Choline oxidase
L-Glutamic acid	Glutamate oxidase	52

Kamei et al. [45] separated spermine, spermidine, putrescine, and cadaverine in an ion-pair reversed-phase LC system and detected the hydrogen peroxide formed in the reaction catalyzed by the enzymes putrescine oxidase and polyamine oxidase with POCL. The same analytes were determined in a later study [46], together with the acetyl derivatives. The sensitive determination of uric acid, selectively converted to hydrogen peroxide by uricase, has been investigated by several authors [37, 47].

The determination of choline and acetylcholine using an IMER coupled with POCL has been performed [48–50]. Acetylcholine esterase and choline oxidase were coimmobilized in a single IMER and the analytes separated either with an ion-pair reversed-phase LC system [48] or on a cation exchanger column [49,50]. Van Zoonen et al. [49] combined these enzymes with a solid-state TCPO reactor and immobilized 3-aminofluoranthene, and one of the problems encountered was the limited water solubility of TCPO and its by-product trichlorophenol (TCP). The coupling of IMERs to ODI chemiluminescence was therefore investigated, with special attention being paid to compatibility with aqueous solutions due to a higher solubility of ODI and imidazole [50]. Two different enzyme systems were investigated: glucose oxidase and acetylcholine esterase/choline oxidase. First, different compositions (acetonitrile/water) of the carrier delivering the analyte were investigated. Surprisingly, the sensitivity was found to be essentially independent of the solvent composition. Even purely aqueous buffers can be used without loss in sensitivity, which must be regarded as a major advantage with ODI. An IMER containing glucose oxidase was incorporated in the carrier

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delivering the analyte and highly sensitive determinations of glucose were accomplished. The detection limit for glucose was 3 nM and the response was linear over the range 30 nM–10 µM. With a second IMER containing acetylcholine esterase and choline oxidase, a cation exchange separation step was included to separate the analytes. In this setup, the detection limits were higher (50 nM), mainly due to band broadening occurring in the analytical column. The response was linear from the detection limit up to 10 µM. To test the applicability of the systems, urine samples were analyzed for glucose, acetylcholine, and choline. The concentrations found in the urine were 580 20 µM β-glucose and 45 1 µM choline, while no acetylcholine was detected. Chromatograms obtained for the choline/acetylcholine system are presented in Figure 8.

Choline-containing phospholipids have been determined in human serum using an IMER consisting of coimmobilized phospholipase D and choline oxidase [51]. Recently, immobilized glutamate oxidase was used to determine L-glutamic acid in culture media [52].

Hydrogen peroxide can also be formed postcolumn by a photochemical reaction. The Birks group has generated hydrogen peroxide from good hydrogen atom donors such as aliphatic alcohols and molecular oxygen, the reaction being catalyzed by quinones. This has been utilized in two different modes. In the first, quinones were determined [53, 54] and because of their catalytic nature, up to 100 molecules of hydrogen peroxide can be formed for each quinone molecule. Consequently, detection limits in the range of tenths of femtomoles were obtained. In the other mode, the hydrogen atom donor is determined instead and a wide range of compounds lacking chromophoric groups (e.g., isopropanol, octanol, glucose, and ascorbic acid) were determined in the low-nanogram range [55].

3.2 Detection of Fluorescent Compounds

Since a relatively small number of analytes of interest have native fluorescent properties, derivatization reactions are frequently employed to enable this detection technique to be extended to a broader range of compounds. This is an excellent means of increasing the detectability for a whole range of molecules, but it is important to realize that there are certain limitations. First, it is difficult to obtain quantitative yields at low analyte concentrations. This implies that in some cases, the obtainable detection limit are not limited by the detector sensitivity, but instead by low yields in the derivatization reaction. Furthermore, to shift the equilibrium toward the product side at low analyte concentrations, as much as 104 times excess of fluorescent label may be necessary. Low concentrations of impurities in the label can be present at levels greater than the analytes of interest and as a result, numerous interfering peaks in the chromatograms may be observed. These problems are discussed in detail in Ref. 181.

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Figure 8 Chromatograms of diluted urine (I) and diluted urine spiked with 2.1 M choline and 2.3 M acetylcholine (II). Elution order: Hydrogen peroxide (0.7 min), choline (1.6 min), and acetyl choline (2.9 min). See Ref. 50 for further details.

As can be seen in Table 3, a wide range of analytes derivatized with different labels have been detected using the POCL reaction. Most of these applications have employed flow injection or liquid chromatographic techniques. An area of growing interest is the combination of capillary electrophoresis with chemiluminescence. Several strategies have been used to detect analytes with fluorescent

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Table 3 Labeled Analytes Detected by the POCL Reaction
Analyte	Label	Ref.
Amino acids	5-Dimethylaminonaphthalene-1-sulfonyl chloride	60–64
Carboxylic acids	N-(Bromoacetyl)-N′-[5-	65
	(dimethylamino)naphthalene-1-
	sulfonyl]piperazine
Carboxylic acids	3-Aminoperylene	67
Carboxylic acids	7-(Diethylamino)-3-[4-	68
	((iodoacetyl)amino)phenyl]-4-methylcoumarin
Carboxylic acids	9-Anthracenemethanol	69
Carboxylic acids	6,7-Dimethoxy-1-methyl-2(1H)-quinoxalinone-3-	70
	propionylcarboxylic acid hydrazine
Carboxylic acids	Lumarine-4	71
Aliphatic amines	5-Dimethylaminonaphthalene-1-sulfonyl chloride	72, 75
Aliphatic amines	4-Chloro-7-nitrobenzo-1,2,5-oxadiazole o-Phthal-	72
	dialdehyde
Ebiratide	4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-	73
	benzoxadiazole
Primary amines	Naphthalene-2,3-dialdehyde	74
	Anthracene-2,3-dialdehyde
Amines	Lumarine-1	75
Amphetamine-related	4-Fluoro-7-nitrobenzoxadiazole	76, 77
compounds
	Naphthalene-2,3-dicarboxaldehyde
Catecholamines	Fluorescamine	78
Nitrosamines	5-Dimethylaminonaphthalene-1-sulfonyl chloride	79, 80
Amantadine	CY5	81
Oxosteroids, oxo bile	5-Dimethylaminonaphthalene-1-sulfonyl hy-	82, 83
acids	drazine
Medroxyprogesterone	4-(N,N′-Dimethylaminosulfonyl)-7-hydrazino-	85
acetate	2,1,3 benzoxadiazole
Estradiol	5-Dimethylaminonaphthalene-1-sulfonyl chloride	87
	Lissamine rhodamine B sulfonyl chloride
Carbonyl compounds	5-Dimethylaminonaphthalene-1-sulfonyl hy-	90
	drazine
Unsaturated disaccharides	5-Dimethylaminonaphthalene-1-sulfonyl hy-	91, 92
	drazine
Aldehydes, ketones	3-Aminofluoranthene	93
Alkyl-, nitroand chloro-	5-Dimethylaminonaphthalene-1-sulfonyl chloride	94
phenols

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properties (native or labeled) with the promising fusion of capillary electrophoresis and POCL [56–59].

3.2.1Derivatization of Amino Acids

The first application describing POCL detection of fluorophores was published in 1977 and dealt with the detection of dansylated amino acids [60] separated by thin-layer chromatography. The amino acids were labeled by a reaction between dansyl chloride and the primary amine group. A few years later, Kobayashi and Imai [61] detected the same analytes with a similar detection system, but using HPLC. Gradient elution of amino acids has been successful [62–64], although significant baseline drift occurs. Miyaguchi et al. [64] extended the determination of amino acids to include N-terminal sequencing of peptides. Bradykinin was dansylated and subjected to acid hydrolysis, and the released dansylated amino acid was identified in an LC system with gradient elution.

3.2.2Derivatization of Carboxylic Acids

Kwakman et al. [65] described the synthesis of a new dansyl derivative for carboxylic acids. The label, N- (bromoacetyl)-N′-[5-(dimethylamino)naphthalene-1- sulfonyl]-piperazine, reacted with both aliphatic and aromatic carboxylic acids in less than 30 min. Excess reagent was converted to a relatively polar compound and subsequently separated from the derivatives on a silica cartridge. A separation of carboxylic acid enantiomers was performed after labeling with either of three chiral labels and the applicability of the method was demonstrated by determinations of racemic ibuprofen in rat plasma and human urine [66]. Other examples of labels used to derivatize carboxylic acids are 3-aminoperylene [67], various coumarin compounds [68], 9-anthracenemethanol [69], 6,7-dimethoxy-1-methyl- 2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide (quinoxalinone) [70], and a quinolizinocoumarin derivative termed Lumarin 4 [71].

3.2.3Derivatization of Amines

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo- 1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial- dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore

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not suitable. Lumarine 1 (a 7-aminocoumarin derivative) [75], 4-fluoro-7-nitro- benzoxadiazole (NBD-F) [76, 77], and fluorescamine [78] have also been used to derivatize amines. Nitrosamines have been determined after a denitrosation step and labeling with dansyl chloride [79,80]. Recently, Ellingson and Karnes derivatized amantadine hydrochloride with a range of different far-red dyes. Among the tested ones, a dye termed CY5 proved optimal [81].

3.2.4Derivatization of Steroids

Oxosteroids can be labeled with a fluorescent tag containing a hydrazine group. Two major products are formed, the antiand syn-isomer of the hydrazone derivative [82]. The dansyl isomers are normally separated during reversed-phase chromatographic conditions, but Imai et al. [83] discovered that if tetrahydrofuran was used in the eluent as the organic modifier, the two isomers eluted in a single peak. This nonresolving eluent in combination with removal of excess reagent improved detection limits significantly compared to a previous study [84]. Uzu et al. [85] used another hydrazine derivative, 4-(N, N-dimethylaminosulfonyl)- 7-hydrazino-2,1,3-benzoxadiazole, to determine medroxyprogesterone acetate in serum. Hydroxysteroids have been converted to ketosteroids by an enzymatic reaction using immobilized hydroxysteroid dehydrogenase, and in a second step derivatized with dansyl hydrazine [86]. Direct labeling using dansyl chloride has also been practiced, with the derivatives being separated by both reversed-phase [87] and normal-phase chromatography [88]. Appelblad et al. [89] separated 10 dansylated ketosteroids and detected them using the ODI POCL reaction. A chromatogram is depicted in Figure 9.

3.2.5Derivatization of Miscellaneous Compounds

Dansyl hydrazine has been used to label other carbonyls as well. Porous glass particles have, e.g., been impregnated with this labeling agent to derivatize carbonyl compounds in ambient air [90]. Glycosaminoglucans have been digested by specific enzymes to the corresponding unsaturated disaccharides and subsequently derivatized with dansyl hydrazine [91, 92]. The method was applied to rat peritoneal mast cells, from which glycosaminoglucans were isolated. One of the most efficient fluorophores in the POCL reaction, 3-aminofluoranthene, has been used to derivatize aldehydes and ketones [93]. Kwakman et al. [94] derivatized various phenols with dansyl chloride. Excess label was removed by allowing the organic phase to pass through an amino-bonded solid-phase extraction column. After the phenols had been separated, a photochemical reaction was allowed to take place, where the derivatives fractured into dansyl hydroxide or dansyl methoxide, and phenol. This conversion was done to prevent the efficient intramolecular quenching by phenols carrying electronegative substituents. Finally,

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Figure 9 Chromatogram showing a derivatized mixture containing 1 µM of 10 steroids: 3β-hydroxy-5-pregnen-20-one (I), 3α,21-dihydroxy-5β-pregnane-20-one (II), 4-pregnene- 3,20-dione (III), 5α-pregnane-3,20-dione (IV), 5β-pregnane-3,20-dione (V), 20α-hy- droxy-4-pregnen-3-one (VI), 3α-hydroxy-5α-pregnan-20-one (VII), 3β-hydroxy-5α-preg- nan-20-one (VIII), 3α-hydroxy-5β-pregnan-20-one (IX), 3β-hydroxy-5β-pregnan-20-one

(X).

the method was applied to analysis of Rhine river water samples for their content of phenols.

Recently the continuous-addition-of-reagent (CAR) technique [182] was applied for the determination of fluorophores by POCL chemistry [95–99]. The applicability of this technique was demonstrated by the determination of natively fluorescent acepromazine in horse plasma [95], the alkaloid harmaline in plasma [96], and other dansylated alkaloids [97]. A separation step has also been included and applied to postcolumn detection of PAHs [98] and dansylated β-carboline alkaloids [99].

Among the natively fluorescent compounds determined by the POCL reaction are PAHs in different matrices such as coal tar [100] and biomass emissions [101], and amino-PAHs in shale oil, coal oil, and coal gasifier tar [102]. NitroPAHs have no fluorescent properties, but have been reduced online (either preor postcolumn) to the corresponding amino-PAHs [103]. The two fluorescent drugs dipyridamole and benzydamine have been determined in rat plasma by

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a method developed by Nishitani et al. [104]. Urinary porphyrines have been successfully detected by the POCL chemistry in a FIA setup, in which common urinary metabolites (e.g., riboflavin and bilirubin) did not interfere [105].

3.3 Miscellaneous Applications

It was observed early that halide ions quenched the POCL reaction efficiently [183] and this behavior has been exploited analytically. The mechanism is not fully understood, but probably involves a radiationless decomposition of the charge-transfer complex; i.e., less excited fluorophores are formed [184]. It has also been observed that the quenching efficiency of a certain compound is independent of the nature and the concentration of the fluorophore and of the concentration of hydrogen peroxide, but dependent upon the nature of the oxalate [106, 183]. Van Zoonen et al. [106] used a single-line FIA setup containing a solidstate TCPO reactor and immobilized 3-aminofluoranthene. The carrier consisted of hydrogen peroxide and TRIS buffer dissolved in acetonitrile/water. A wide range of analytes was screened, and nitrite, sulfite, some anilines, and organic sulfur compounds were found to quench the CL reaction. The concentrations at the detection limits were in the low-nanogram range, and as in most indirect modes of detection, the linear range was relatively small (two to three orders of magnitude). In a more recent study [107], a separation step was included, and the solid-state TCPO reactor removed and replaced by a postcolumn solution containing 2-nitrophenyloxalate. This change of reagent increased the peak heights approximately 10-fold.

DeVasto and Grayeski [108] used the same indirect approach to determine amines in a static system. Aliphatic and aromatic amines could be determined, but in this case the linear range was even smaller (one order of magnitude). In addition, nonlinear calibration curves were obtained for the aromatic amines. Amines have been determined in a direct approach as well [109, 110]. The analytes were separated on a C8 column with an eluent containing hydrogen peroxide, but no buffer. A postcolumn flow containing TDPO and fluorophore merged with the eluent and light evolved as amines having catalytic properties eluted. The best detection limits were obtained for aliphatic amines and imidazoles (at the best 1 nM); some aromatic and heterocyclic amines were not detectable at all.

Two different approaches have been used to determine phenols without derivatization. In the first, the corresponding oxalate esters were synthesized in the traditional way (i.e., using oxalyl chloride and triethylamine) [111, 112]. Pentachlorophenol, 1-naphthol, bromofenoxim, bromoxynil, and p-cyanophenol were treated this way, after which the POCL resulting from their reaction was measured in a static system. The second approach exploits the oxidation reaction between imidazole and hydroxyl compounds at an alkaline pH, where hydrogen peroxide is formed [113]. Polyphenols, e.g., pyrogallol, pyrocatechol, and dopa-