ORIGINAL PAPER

A new luminol chemiluminescence reaction usinga tetravalent nickel-periodate complex as the oxidant

Chunyan Yang & Zhujun Zhang & Jinli Wang

Received: 13 January 2009 /Accepted: 25 March 2009 /Published online: 9 October 2009# Springer-Verlag 2009

Abstract We report on a new chemiluminescence (CL)reaction that occurs between luminol and di-periodatonickelatein alkaline medium. The possible mechanism of thissystem is discussed based on the kinetics of the reaction,its CL and UV spectra. The system exhibits a sensitivitythat is higher than any other system based on luminol. Itwas successfully applied to the determination of isoniazidin serum.

Keywords Chemiluminescence . Luminol .

Diperiodatonickelate . Isoniazid

Introduction

In recent years, extremely sensitive analytical techniquesbased on CL and bioluminescence systems have receivedconsiderable attention. CL analysis has many advantagessuch as simplicity of instrument, low detection limit, largecalibration range and short analysis time that make themethod attractive [1, 2]. So CL analysis has been widelyused in many fields such as pharmaceutical science,environmental science and life science.

The oxidation of luminol (3-aminophthalhydrazide) inalkaline medium is one of the most efficient CL reactions

[3–7]. Luminol undergoes CL reactions with a range ofoxidants. The oxidants including hydrogen peroxide,oxygen, potassium permanganate, ferricyanide, tetravalentcerium ion, lead dioxide and oxygen-free radicals such assuper oxide anion (•O2

−), hydroxy radical (•OH) and nitricoxide (NO) were often used in this CL system. Sometransition metals in their highest oxidation states, such astetravalent nickel, can be stabilized by chelating withsuitable polydentate ligand. Although there are manyreports concerning the use of diperiodatonickelate (DPN)as oxidant in alkaline medium, the use of DPN in CLanalysis was never studied. In this paper, the CL phenom-ena were found be accompanied by a reaction betweenDPN and luminol in alkaline medium. The chemilumines-cence can be detected with low concentrations of luminol,but chemiluminescence can hardly be observed when thesame concentration of luminol reacts with other oxidants. Inthe experiments, it was also found that some compoundssuch as crimetidine, mezlocillin sodium, ganciclovir,lincomycin, ampicillin sodium, cefuroxime sodium, azi-thromycin, metoclopramide, isoniazid, adrenaline, furose-mide, and dopamine can greatly enhance the CL intensityof the luminol-DPN reaction. Isoniazid (isonicotinic acidhydrazide) is a tuberculostatic agent effective againstmycobacterium strains. Finding a sensitive and rapidmethod for the determination of isoniazid in biologicalfluids is therefore of great importance. The luminol-DPNsystem was studied using low concentrations of luminoland isoniazid as a model. In the same concentration ofluminol, the CL intensity of the luminol-DPN-isoniazidsystem is much stronger than that of the luminol-H2O2-metal ion system, which is one of the most well-knownluminol CL reactions [8–11]. Especially when the concen-tration of luminol is below 10−8 mol L−1, the CL of theluminol-DPN-isoniazid system was clearly to be observed,

Electronic supplementary material The online version of this article(doi:10.1007/s00604-009-0223-6) contains supplementary material,which is available to authorized users.

C. Yang : Z. Zhang (*) : J. WangCollege of Chemistry and Materials Science,Shaanxi Normal University,Xi’an 710062, Chinae-mail: zhangzhujun18@yahoo.com.cn

Microchim Acta (2009) 167:91–96DOI 10.1007/s00604-009-0223-6

while the CL of the luminol-H2O2-metal ion system washardly observed. Here a new CL system with highsensitivity was designed. In order to validate the applica-bility of the present CL system in real samples, it wasapplied to determine isoniazid in serum.

Experimental

Material

Luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione, 99%,Shanxi Normal University, Xi’an, China, http://www.snnu.edu.cn/); isoniazid (Drug and Biological Products Examina-tion Bureau of China, Beijing, http://www.nicpbp.org.cn/CL0001/); Potassium persulfate (Shanghai Aijian ChemicalReagent Company, http://www.ajchem.com); potassium hy-droxide, hydrogen peroxide, sodium nitrate, sodium period-ate, nickel sulfate, calcium chloride (Shanghai ChemicalReagent Research Institute, http://www.scrri.com/). All thereagents were of analytical grade. Deionized and doublydistilled water was used throughout the work.

The 0.01 mol L−1 luminol stock solution was preparedby dissolving 1.772 g luminol in 1 L 0.1 mol L−1 carbonatebuffer and then leaving to stand for approximately 24 hbefore use. The luminol solution was stable for at least1 month when stored in the dark.

Apparatus

The CL-FIA system used in this work is shown in Fig. 1.Two peristaltic pumps (HL-2, Shanghai Huxi, China, http://www.canytec.com.cn/) were used to deliver all the chem-icals at a flow rate of 1.5 mL min−1. Polytetrafluoroethylene(PTFE) flow tubes (0.8 mm i.d.) were used to connect all thecomponents in the flow system. 50 μL of sample solutionwas injected into the water stream by an eight-way injectionvalve and then mixed with the reagent streams. The CLsignal was monitored by a IFFM-A multifunction chemilu-minescence analyzer (Remex Analytical Instrument Co. Ltd.,Xian, China, http://www.xaremex.com/). The UVabsorbancewas detected with the TU1901 UV-Vis spectrophotometer(Beijing Purkinje General Instrument Co., Ltd. China, http://www.pgeneral.com/). The chemiluminescence spectrum wasmonitored using a F-4600 fluorescence spectrophotometer(Shimadzu, Japan, http://www.shimadzu.com.cn/).

Synthesis of diperiodatonickelate (IV)

Metal complexes containing nickel in the tetravalent stateshave been the subject of considerable interest and study.Murthy et al. [12] once brought forward two separate one-electron reduction steps, but the process is not considered

owing to the absence of induced polymerization ofacrylamide which indicates the presence of free radicals inthe system, a general feature of one-electron-transferreactions. A two-step single-electron-transfer mechanismwas proposed by Liu [13] to explain the creation of radicalsand the initiation.

The nickelate (IV)-periodate complex (KNiIO6· (1/2)H2O) was prepared using the method suggested by Murthy[12]. A portion of the KNiO6·(1/2)H2O crystals was treatedwith 1.0 mol L−1 KOH and a small amount of KIO4 at 50degrees centigrade under stirring in order to prepare thestock solution. The undissolved material was filteredthrough a G3 sintered crucible to get a clear solution whichwas found to be sufficiently stable. The UV-vis absorptionspectrum of DPN is shown in Fig. S1 (supportinginformation). The concentration of DPN solution wasdetermined gravimetrically after reducing nickel (IV) tonickel (II) as the dimethyl glyoxime complex.

Procedures

The flow injection system used in this study is shown inFig. 1. The distilled water delivered the standard isoniazidor the samples in the sample loop solution to react with themixture of luminol solution and DPN solution in the flowcell to produce strong CL. The CL signal was detected withan IFFS-A multifunction chemiluminescence analyzer.

Result and discussion

The comparison of CL intensity of luminol with differentoxidants or catalyzers

The oxidation of luminol in alkaline medium is one of themost important CL reactions. Luminol undergoes CLreactions with a range of oxidants, including oxygen,

Fig. 1 Schematic diagram of the CL-FIA system. a isoniazid solutionor samples; b distilled water; c luminol solution; d DPN solution; V:injection valve; F: spiral glass flow cell; PMT: photomultiplier tube;pump1, pump2: peristaltic pump

92 C. Yang et al.

hydrogen peroxide, potassium permanganate, ferricyanide,tetravalent cerium ion, lead dioxide and oxygen freeradical. For some oxidants, e.g. hydrogen peroxide, one ofthe most well-known CL reactions, a catalyst, such as Cu2+,Cr3+, Co2+, is also required for CL. In this paper, we foundthat DPN produces a strong redox reaction with luminol,emitting CL. The CL was detected when the concentrationof luminol was only up to 10−8 mol L−1, but the CL ofluminol with other oxidants of the same concentration asluminol could hardly be observed (Fig. S2, supportinginformation). Our experiments also showed that somecompounds greatly enhanced the CL intensity of theluminol-DPN reaction. In this paper, the CL system wasstudied using isoniazid as a model. In the same conditions,the CL of the luminol-H2O2-metal ion system is muchlower than that of the luminol-DPN-isoniazid system. Forexample, in the luminol-H2O2-metal ion (Cu2+, Cr3+, Co2+)CL system, the CL can be clearly observed when theconcentration of luminol is up to 1.0×10−5 mol L−1, whilein the luminol-DPN-isoniazid system, the experimentalresult is excellent when the concentration of luminol isonly 2.0×10−7 mol L−1(Fig. S3, supporting information).So this method possesses the advantages of low reagentconsumption as well as high sensitivity and selectivity.

Kinetics curve of the CL reaction

Before performing the flow-injection method, the kineticcharacteristics of the proposed CL reaction were studiedusing the batch method. In the batch mode, the experimen-tal parameters were kept constant, and the typical responsecurve (intensity versus times) of the luminol (3.0×10−7 mol L−1) CL reaction catalyzed by isoniazid (4.0×10−7 g mL−1) in the presence of DPN (5.0×10−5 mol L−1)was recorded to study the kinetic characteristic of the CLreaction. Figure 2 demonstrates that the CL reaction was avery quick reaction. The CL intensity peak appeared within

2 s after injecting the isoniazid solution. The CL signalsdecreased to the baseline within 30 s. The kinetic curveindicates that the CL method is rapid and sensitive enoughto perform the determination of isoniazid.

Mechanism discussion

In order to explore the possible mechanism of this CLenhancing phenomenon, the following experiments wereperformed. Firstly, the CL emission spectrum was obtainedusing the modified F-4600 fluorescence spectrophotometer(Shimadzu, Japan) combined with the flow-injection sys-tem, with the light source taken off. The obtained CLspectrum is shown in Fig. 3. As can be seen, the maximumemission wavelength of the luminol-DPN or luminol-DPNCL system catalyzed by isoniazid was located at about425 nm. It is well known that aminophthalate is theluminophor, and the maximum emission of the CL reactionis at 425 nm. So the CL emitter in the both CL reactionsbetween luminol and DPN with and without isoniazid is thesame species, which is the oxidation product of luminol.

Series experiments were performed to find out moredetails about the isoniazid-enhanced luminol-DPN CLreaction. Firstly, the UV-vis absorption spectra of DPN,luminol, isoniazid and their mixtures were studied in thebasic medium (Fig. S4, supporting information). UV-visspectra of luminol and isoniazid alone and a mixture did notreveal the appearance of any extra peak after mixing bothsolutions. The absorption of DPN and isoniazid almostdisappeared as they were mixed. Secondly, when DPN andisoniazid were mixed in alkaline medium, the color of DPNfaded. However, when strong oxidant (K2S2O8 or HNO3)was added to this nearly colorless solution, the primarycolor of DPN recovered. Considering the above experi-ments, it is obvious that a redox reaction takes placebetween isoniazid and DPN. What is more, when themixture of DPN and isoniazid was injected into luminol,

Fig. 2 Kinetics curves of luminol-DPN catalyzed by isoniazid. luminol:3.0×10-7 mol L−1; DPN: 5.0×10−5 mol L−1; isoniazid: 4.0×10−7 g mL−1. 1: luminol + DPN; 2: luminol + DPN + isoniazid

Fig. 3 CL spectrum of luminol and DPN in the presence (1) and (2)absence of isoniazid;. luminol: 1.0×10−5 mol L−1; DPN: 2.4×10−5 mol L−1; isoniazid: 4×10−4 g mL−1

A new luminol chemiluminescence reaction 93

the CL enhancement decreased with the mixing time, untilit was hardly observed. The observation implies theexistence of a medium product which may be a free radicalin isoniazid oxidation [13], although more evidences arenot available.

We performed more experiments to evaluate the existenceof a free radical. When isoniazid standard solution was firstlymixed with DPN flow instead of luminol in the present flowsystem (Fig. 1), the CL intensity was much lower than thatobtained without the above flow system change. By varyingthe tube length from DPN-isoniazid confluence point toluminol confluence point, it was found that the longer thetube length, the weaker the CL observed. These two

experiment results coincide with the suggested existence ofa free radical. Especially according to the reported study[14], isoniazid can be oxidized by diperiodatocuprate (III) toproduce a free radical. So it can be concluded that the CLdifference observed in the above experiments is attributed tothe instability of the free radical.

Fig. 4 Proposed CL reactionmechanism a Proposed CLreaction mechanism ofluminol-DPN; b Proposed CLreaction mechanism ofluminol-DPN-isoniazid

Table 1 Results of recovery tests on human serum

Sample Found(ng mL−1)

Added(ng mL−1)

Total found(ng mL−1)

Recovery/ % RSD/ %

No.1 7.1 8.0 14.5 92.5 2.7

10.0 17.8 107 2.3

20.0 27.0 99.5 0.5

20.0 32.9 103 2.3

No.2 12.3 40.0 51.2 97.3 3.2

60.0 72.9 101 1.7

80.0 127.0 102 1.3

No.3 45.5 100.0 143.7 98.2 1.0

200.0 245.8 100 0.2

Table 2 Figures of merit of comparable methods of CL determinationof isoniazid

CL system Analytical range; Application Referencesdetection limit

Luminol-DPN 4–600 ng mL−1; Human serum This work1.2 ng mL−1

ElectrogeneratedManganese(III)

0.1–10 μg mL−1; Pharmaceutical(tablet)

[15]32 ng mL−1

Luminol-NBS 8–600 ng mL−1; Pharmaceutical(tablet)

[16]4.0 ng mL−1

Luminol-NCS 6–200 ng mL−1 Pharmaceutical(tablet)

[16]4.0 ng mL−1

Luminol-SDCC 4–100 ng mL−1; Pharmaceutical(tablet)

[17]2 ng mL−1

Luminol-TCCA 6–200 ng mL−1; Pharmaceutical(tablet)

[17]3 ng mL−1

Ru(phen)32+-Ce(IV) 70–6,500 ng mL−1; Pharmaceutical

(injection)[18]

25 ng mL−1

Luminol-potassiumhexacyanoferrate(III)

0–1 μg mL−1; Pharmaceutical(tablet;injection)

[19]0.03 ng mL−1

ECL 0.3–1,000 ng mL−1; Urine sample [20]0.1 ng mL−1

94 C. Yang et al.

Based on the above discussion, the possible enhance-ment mechanism of isoniazid with respect to the CLreaction between DPN and luminol may be that isoniazidas reducing agent is able to reduce DPN in an alkalinesolution, creating a free radical. The produced free radicalreacts with luminol, yielding an unstable endoperoxide andthen leads to an electronically excited aminophthalate. Thiscan be expressed as shown in Fig. 4, although moreevidence is not available.

Optimization of CL reaction conditions

Stability of DPN

Quadrivalence nickel should be stabilized by chelating withsuitable polydentate ligand due to its limited stability inaqueous medium. The stability of DPN in aqueous solutionwas studied based on the variety of CL intensity within 2 h(Fig. S5, supporting information). As can be seen, the CLintensity varied little within 2 h when the DPN concentra-tion was up to 2.0×10−5 mol L−1.

Optimization of the CL reaction conditions was carriedout according to the schematic diagram shown in Fig. 1. Toachieve the highest enhancement of the CL intensity ofluminol-DPN-isoniazid, the optimal conditions for the CLreaction system were investigated using the ratio of the CLsignal (1.0×10−7 g mL−1) to noise influence (S/N) atdifferent concentration levels of luminol, DPN, potassiumhydroxide. Thus, the chosen conditions for the CL systemare 2.0×10−7 mol L−1 luminol and 1.0×10−3 mol L−1

potassium hydroxide mixed solution, 2.4×10−5 mol L−1

DPN and 2.0×10−2 mol L−1 potassium hydroxide mixedsolution.

Analytical parameters

Under the optimum experimental conditions, the CL intensitywas linear to the isoniazid concentration from 4.0×10−9–6.0×10−7 g mL−1, and the detection limit was 1.2×10−9 g mL−1

(3σ) with a relative standard deviation for 5.0×10−8 g mL−1

isoniazid solution of 4.1% (n=11). The regressionequation was ΔI=13.495c–26.71 (c being the isoniazidconcentration (ng mL−1), and ΔI being the relative CLintensity) with R2=0.9922 (n=11).

Interference study

The effect of foreign substances was tested by analyzing astandard solution of isoniazid (1.0×10–8 g mL−1) to whichincreasing amounts of interfering substances were added.The tolerable concentration ratios with respect to 1.0×10–8 g mL−1 isoniazid at an interference level of 5% were over1,000 for K+, Na+, Ca2+, Cl−, SO42−, NO3−, starch,

carbamide, lysine, glutamic acid, leucine, serine; 500 for Fe3+,Cu2+, Cr3+, Co2+, lactic acid; 200 for lactose, glucose, uricacid, asparagine; 100 for citrate, oxalate; 50 for ascorbic acid.The interference of protein in human serum could be ignoredwhen human serum was ultrafiltered, thus implying that thepresent method may be directly applied to the determination ofisoniazid in human serum.

Applications

The proposed method has been utilized for the determina-tion of isoniazid as a model in human serum. A 2 mL blankserum sample was collected and transferred to a centrifugalfilter unit, then centrifuged at 10,000 rpm for 10 min at 4 °C.Then 1 mL filtrate was transferred to a 100 mL volumetricflask and diluted to the mark with doubly distilled water. Theblank serum was injected into the CL system, and the blanksignal recorded. The amount of isoniazid in human serumand the recovery obtained are shown in Table 1.

Comparison with other CL systems

Based on this experiment, a novel CL system wasdeveloped, which was successfully applied to determineisoniazid in serum. Although several CL methods havebeen reported for isoniazid analysis [15–20], some methodswere only applied to the determination of isoniazid inpharmaceutical preparations by reason of their poorselectivity and sensitivity, but the method can be appliedto the determination of isoniazid in biological samples(human serum) (Table 2).

Conclusions

A novel CL method combined with the flow injectionsystem has been proposed. The characteristics of theluminol-DPN CL system were investigated by usingisoniazid as a model. Using a low luminol concentrationcan produce excellent result with a higher selectivitycompared to other luminol systems. The possible CLmechanism is proposed briefly.

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