selective determination of quinones by high-performance liquid chromatography with on-line post...

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Journal of Chromatography A, 1133 (2006) 76–82 Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection Sameh Ahmed, Shuu Fujii, Naoya Kishikawa, Yoshihito Ohba, Kenichiro Nakashima, Naotaka Kuroda Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan Received 23 March 2006; received in revised form 28 July 2006; accepted 31 July 2006 Available online 22 August 2006 Abstract A new HPLC method was developed for the simultaneous determination of quinones with peroxyoxalate chemiluminescence (PO-CL) detec- tion following on-line UV irradiation. Quinones [i.e., 1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, 9,10-phenanthrenequinone] were UV irradiated (254 nm, 15 W) to generate hydrogen peroxide and a fluorescent product that were determined via PO-CL detection. Gen- eration of hydrogen peroxide from quinones with on-line UV irradiation was confirmed using flow injection analysis (FIA) system whereby incorporating an enzyme column reactor immobilized with catalase. Moreover, the structure of the produced fluorophore was confirmed using LC–MS, IR, and 1 H NMR. Afterwards, the conditions for UV irradiation and PO-CL detection were optimized. The separation of four quinones by HPLC was accomplished isocratically on an ODS column within 25 min. The detection limits (signal-to-noise ratio = 3) were 6.0 pmol/injection for 1,2-naphthoquinone, 4.4 pmol/injection for 1,4-naphthoquinone, 0.2 pmol/injection for 9,10-anthraquinone, and 0.45 pmol/injection for 9,10- phenanthrenequinone. © 2006 Elsevier B.V. All rights reserved. Keywords: Peroxyoxalate chemiluminescence detection; Quinones; UV irradiation; HPLC; Hydrogen peroxide 1. Introduction Quinones are known to play an important role in photosyn- thesis in plants and bacteria as well as for blood clotting in animals [1,2]. Quinone structure exists in some vitamins, which are necessary to maintain life as vitamins K, which take part in the blood clot action and have a naphthoquinone structure [3–5]. Some drugs have quinone structures, such as daunoru- bicin and doxorubicin, which are used as anti-tumor drugs [6,7]. So, it is important to clarify the activity and disposition of these quinones. Moreover, there is no doubt that quinones are harmful environmental pollutants. Polynuclear aromatic hydrocarbons, which are known as air pollutants, generate toxic quinones when photoxidized by sunlight [8–11]. Therefore, it is important to monitor quinones that exist in the environment. Corresponding author. Tel.: +81 95 8192894; fax: +81 95 8192444. E-mail address: [email protected] (N. Kuroda). Recently, a lot of high-performance liquid chromatographic (HPLC) methods were used as analytical tools for determination of quinones using UV detection [12–14], fluorescence detection [15,16], electrochemical detection [17], and chemiluminescence (CL) detection [18–21]. In UV detection, the sensitivity and selectivity were generally low. When the fluorescence detection was used, quinones should be converted into fluorescent hydro- quinones by reduction reaction, as the fluorescence of quinones is extremely weak. However, the efficiency of the reduction reaction was low and the fluorescence strength of the generated fluorescent compound was weak [16]. When using the electro- chemical detection method, quinones were reduced in a platinum reduction column. Despite of the high selectivity and the sen- sitivity of this detection method, the reproducibility was low [17]. Moreover, CL detection methods were reported [18–21]. The determination was based on photo-oxidation of quinones to yield hydrogen peroxide, which was monitored through CL reac- tion with peroxyoxalate chemiluminescence (PO-CL) detection using rubrene as a fluorophore [18] or through CL reaction with 0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.07.078

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Page 1: Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

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Journal of Chromatography A, 1133 (2006) 76–82

Selective determination of quinones by high-performance liquidchromatography with on-line post column ultraviolet irradiation and

peroxyoxalate chemiluminescence detection

Sameh Ahmed, Shuu Fujii, Naoya Kishikawa, Yoshihito Ohba,Kenichiro Nakashima, Naotaka Kuroda ∗

Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

Received 23 March 2006; received in revised form 28 July 2006; accepted 31 July 2006Available online 22 August 2006

bstract

A new HPLC method was developed for the simultaneous determination of quinones with peroxyoxalate chemiluminescence (PO-CL) detec-ion following on-line UV irradiation. Quinones [i.e., 1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, 9,10-phenanthrenequinone]ere UV irradiated (254 nm, 15 W) to generate hydrogen peroxide and a fluorescent product that were determined via PO-CL detection. Gen-

ration of hydrogen peroxide from quinones with on-line UV irradiation was confirmed using flow injection analysis (FIA) system wherebyncorporating an enzyme column reactor immobilized with catalase. Moreover, the structure of the produced fluorophore was confirmed usingC–MS, IR, and 1H NMR. Afterwards, the conditions for UV irradiation and PO-CL detection were optimized. The separation of four quinones

y HPLC was accomplished isocratically on an ODS column within 25 min. The detection limits (signal-to-noise ratio = 3) were 6.0 pmol/injectionor 1,2-naphthoquinone, 4.4 pmol/injection for 1,4-naphthoquinone, 0.2 pmol/injection for 9,10-anthraquinone, and 0.45 pmol/injection for 9,10-henanthrenequinone.

2006 Elsevier B.V. All rights reserved.

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eywords: Peroxyoxalate chemiluminescence detection; Quinones; UV irradia

. Introduction

Quinones are known to play an important role in photosyn-hesis in plants and bacteria as well as for blood clotting innimals [1,2]. Quinone structure exists in some vitamins, whichre necessary to maintain life as vitamins K, which take partn the blood clot action and have a naphthoquinone structure3–5]. Some drugs have quinone structures, such as daunoru-icin and doxorubicin, which are used as anti-tumor drugs [6,7].o, it is important to clarify the activity and disposition of theseuinones. Moreover, there is no doubt that quinones are harmfulnvironmental pollutants. Polynuclear aromatic hydrocarbons,

hich are known as air pollutants, generate toxic quinones whenhotoxidized by sunlight [8–11]. Therefore, it is important toonitor quinones that exist in the environment.

∗ Corresponding author. Tel.: +81 95 8192894; fax: +81 95 8192444.E-mail address: [email protected] (N. Kuroda).

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021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.07.078

HPLC; Hydrogen peroxide

Recently, a lot of high-performance liquid chromatographicHPLC) methods were used as analytical tools for determinationf quinones using UV detection [12–14], fluorescence detection15,16], electrochemical detection [17], and chemiluminescenceCL) detection [18–21]. In UV detection, the sensitivity andelectivity were generally low. When the fluorescence detectionas used, quinones should be converted into fluorescent hydro-uinones by reduction reaction, as the fluorescence of quinoness extremely weak. However, the efficiency of the reductioneaction was low and the fluorescence strength of the generateduorescent compound was weak [16]. When using the electro-hemical detection method, quinones were reduced in a platinumeduction column. Despite of the high selectivity and the sen-itivity of this detection method, the reproducibility was low17]. Moreover, CL detection methods were reported [18–21].

he determination was based on photo-oxidation of quinones toield hydrogen peroxide, which was monitored through CL reac-ion with peroxyoxalate chemiluminescence (PO-CL) detectionsing rubrene as a fluorophore [18] or through CL reaction with
Page 2: Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

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uminol [19]. However, these methods were either complicatedr suffer from sensitivity problems. GC–MS was also reportedor determination of quinones, however, it has a problem in pointf sensitivity [22]. Detection of quinones in the complex matri-es in which they are typically found presents severe challengeso most conventional techniques. Therefore, it is necessary toevelop a useful analytical method for selective determinationf quinones.

PO-CL is based on the reaction between hydrogen peroxidend diaryloxalates, which produces strong luminescence inhe presence of fluorophore through the CIEEL (chemicallynitiated electron exchange luminescence) mechanism [23].he PO-CL is easily combined with HPLC and applied to

he determination of fluorescent compounds and hydrogeneroxide [24]. Recently, we have reported the determination ofrganic peroxide by HPLC–PO-CL detection, without additionf H2O2, combined with on-line photochemical reaction.his method was based on conversion of organic peroxide toydrogen peroxide by UV irradiation, and then detected byixing with diaryloxalate and fluorophore [25]. In the course of

elated works, we found that CL was generated when quinonesere mixed with diaryloxalate after their UV irradiation andithout addition of fluorophore and hydrogen peroxide. It was

ound that there is a direct relation between the concentrationf quinone and CL intensity. Because the determined samplesn this CL method are limited to compounds that generateydrogen peroxide and a fluorescent material at the same timefter UV irradiation, it is thought that this method can detectuinones selectively. So, we aimed at the development of ann-line UV irradiation HPLC–PO-CL method for determi-ation of quinones based on this phenomenon. In this paper,our quinones (1,2-naphthoquinone, 1,4-naphthoquinone,,10-anthraquionone, and 9,10-phenanthrenequinone) werenvestigated. In addition, the generation of hydrogen perox-de and the structure of the fluorescent photoproduct wereonfirmed.

. Experimental

.1. Materials and reagents

9,10-Anthraquinone and 1,4-naphthoquinone were obtainedrom Nacalai Tesque (Kyoto, Japan), 9,10-phenanthrenequinonend 1,2-naphthoquinone were from Tokyo Chemical IndustryTokyo, Japan). Stock solutions of quinones (0.1 mM) wererepared in acetonitrile. These solutions were diluted appro-riately with the carrier solution (for flow injection analysisFIA)) or mobile phase (for HPLC) to prepare the workingolutions. Bis-(2,4,6-trichlorophenyl) oxalate (TCPO) andmidazole were obtained from Tokyo Chemical Industry;midazole was recrystallized from acetonitrile before use. Cata-ase from bovine liver (Boehringer Mannheim-Yamanouchi,okyo) was used to prepare the immobilized enzyme column

eactor (IMER, 70 mm × 2.0 mm, i.d.) [26]. Distilled wateras obtained using Simpli Lab-UV (Millipore, Bedford,A, USA) water device. Other chemicals were of extra pure

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r. A 1133 (2006) 76–82 77

.2. HPLC–PO-CL system

The HPLC system (Fig. 1) consisted of two LC 9A liquidhromatographic pumps (Shimadzu, Kyoto, Japan), a Rheo-yne 7125 injector (Cotati, CA, USA) with a 10-�l sampleoop, a Capcellpak ODS UG 120 (250 mm × 1.5 mm, i.d., Shi-eido, Tokyo), a low-pressure mercury lamp (15 W, 254 nm,igemi Standard, Tokyo), a CLD-10A chemiluminescenceetector (Shimadzu) and SIC chromatorecorder (Tokyo, Japan).TFE tubing (2.0 m × 0.25 mm i.d., GL Sciences, Tokyo) coiledround the quartz well of low-pressure mercury lamp assem-ly was used as an on-line photo reactor. Temperature ofhe photoreactor was maintained at 24 ◦C by an oven (Jasco,okyo) equipped with a circulating water system. A mixturef imidazole–HNO3 buffer (100 mM, pH 9.0) and acetonitrile60:40, v/v) was used as a mobile phase and 0.5 mM TCPO incetonitrile was used as the post column CL reagent. The mobilehase and CL reagent were degassed by vacuum degassing withonication and filtered through a 0.45 �M filter prior to use. Theow-rates of the mobile phase and the CL reagent were set at.2 and 0.35 ml/min, respectively.

.3. FIA–PO-CL system

The FIA system, in which the separation column wasemoved from the HPLC system (Fig. 1), was used for con-rmation of generation of hydrogen peroxide from quinones.MER was connected between the on-line photoreactor and theixing tee in the FIA system. A mixture of imidazole–HNO3

uffer (50 mM, pH 7.5) and acetonitrile (95:5, v/v) was used ashe carrier solution. The flow-rates of the carrier solution andhe CL reagent were set at 0.28 and 0.56 ml/min, respectively.

.4. HPLC-fluorescence system

HPLC-fluorescence system was used for identification of theuorescence characteristics of photoproducts produced fromV irradiation of quinones. The HPLC system consisted ofC 9A liquid chromatographic pump (Shimadzu), a Rheodyne125 injector (Cotati) with a 10-�l sample loop, a low-pressureercury lamp (15 W, 254 nm, Sigemi Standard), PTFE tub-

ng (2.0 m × 0.25 mm, i.d.) coiled around low-pressure mer-ury lamp assembly was used as an on-line photoreactor. Aapcellpak ODS UG 120 (250 mm × 4.6 mm, i.d., Shiseido),hich was set after UV irradiation assembly, an agilent 1100

eries fluorescence detector (Hewlett-Packard, Waldbronn, Ger-any) for multi-wavelength fluorescence detection. A mixture

f imidazole–HNO3 buffer (50 mM, pH 7.5) and acetonitrile20:80, v/v) was used as a mobile phase with a flow-rate.8 ml/min.

.5. Identification of the structure of the fluorescenthotoproduct

.5.1. Preparation of the photoproductTo a test tube containing 10 ml of 5 mM quinone solu-

ion in acetonitrile (e.g., 9,10-anthraquinone), 10 ml of 30 mM

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78 S. Ahmed et al. / J. Chromatogr. A 1133 (2006) 76–82

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etsTm9,10-anthraquinone and the fluorescent photoproduct are shownin Fig. 3. The peak of the fluorescent photoproduct newlyappeared at 3.8 min after UV irradiation. Moreover, Table 1shows the excitation maximum and emission maximum wave-

ig. 1. HPLC–PO-CL system for the determination of quinones. P1, pump 1; Pooling unit, oven equipped with cold insulator; L, low-pressure mercury lamp

ecorder.

midazole–HNO3 buffer solution (pH 7.5) was added. The reac-ion mixture was UV irradiated using a low-pressure mercuryamp (15 W, 254 nm, Sigemi Standard) for 30 min. The prepa-ation conditions were optimized for efficient conversion ofuinones to photoproduct. Acetonitrile layer containing uncon-erted quinone was removed using sub-zero temperature sepa-ation at −20 ◦C [27]. The aqueous solution was acidified withCl and extracted using ethyl acetate. The photoproduct crys-

allized from ethyl acetate layer was used for identification tests.

.5.2. LC–MSMass spectrometric analysis was performed on a MicroMass

uattro Micro tandem mass spectrometer (MicroMass, Manch-ster, UK) equipped with electrospray interface. MassLynx 4.0icro software was used. The instrument was operated in neg-

tive ion mode with the capillary voltage of 3 kV and the coneoltage of 25 V. The nebulizer gas was set at 50 l/h with desolva-ion temperature of 350 ◦C and a source temperature of 120 ◦C.he identification of the photoproduct was carried out in full-can mode and total ion current mode (TIC) by matching theetention time and mass spectrum.

Liquid chromatography was carried out on a Waters Alliance695 HPLC (Waters, Milford, USA) equipped with a columnven and a Waters 2996 photodiode array detector, an auto-ampler with 3-�l injection loop, and a reversed-phase columnCapcellpak ODS UG 120 (250 mm × 4.6 mm, i.d., Shiseido).he column was maintained at 30 ◦C and eluted under isocraticonditions at flow rate 0.2 ml/min using a mixture of acetonitrilend water (50:50, v/v) as a mobile phase.

.5.3. 1H NMR and IR1H NMR spectrum of the photoproduct was measured on a

arian Gemini 300 MHz spectrometer (Varian, CA, USA) in d6-MSO and reported in part per million (δ) relative to TMS while

R spectrum was obtained on a Shimadzu FTIR-8100A.

. Results and discussion

.1. Confirmation of the generation of hydrogen peroxide

We confirmed that CL signals obtained with the FIA systemere based on the hydrogen peroxide generated from quinones

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mp 2; I, injector; Column, Capcellpak ODS UG 120 (250 mm × 1.5 mm, i.d.);reaction coil (2.0 m × 0.25 mm, i.d.); M, mixing tee; D, CL detector; and Rec,

fter UV irradiation. To confirm this, the catalase IMER wasnserted into FIA–PO-CL system. The effect of catalase duringL was studied by detecting CL signals produced after cata-

ase treatment and without catalase treatment (Fig. 2). It wasound that the peak of each quinone was disappeared completelyith catalase treatment due to resolution of hydrogen peroxide.herefore, hydrogen peroxide generated by UV irradiation of

he quinones and its role in luminescence became clear.

.2. Fluorescence characteristics of the fluorescenthotoproduct

Quinones have extremely weak fluorescence and do not showfficient luminescence in PO-CL system. However, it was foundhat the compound produced by UV irradiation of quinone hadtrong fluorescence so it was detected via PO-CL reaction.he fluorescence intensity of the photoproduct of quinones waseasured using HPLC-fluorescence system. Chromatograms of

ig. 2. Recorder responses for 50 �M from different quinones (A) without cata-ase treatment and (B) with catalase treatment.

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S. Ahmed et al. / J. Chromatog

Fig. 3. Chromatograms of (1) 9,10-anthraquinone and (2) the fluorescent pho-taa

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oproduct using HPLC-fluorescence system. 9,10-Anthraquinone was detectedt λex 274 nm and λem 327 nm. The photoproduct was detected at λex 275 nmnd λem 334 nm after UV irradiation. Each sample concentration was 50 �M.

engths, relative fluorescent intensities (RFI), and retention timesf four quinones with and without UV irradiation. An increase ofbout 20–50 times in fluorescent intensity was observed by UVrradiation in all examined quinones. In addition, the possibilityhat the generated compounds are the same was strongly sug-ested because the retention times and the fluorescence spectraf fluorescent photoproducts were almost the same for all thetudied quinones.

.3. Confirmation of structure of the fluorescenthotoproduct

To determine the structure of the fluorescent material gener-ted by UV irradiation of quinones, LC–MS was used. Photo-roduct sample was analyzed using ESI-MS in the negative ionode. The most abundant ion peak was found at m/z 197.02 that

orresponds to [M−H]−.1H NMR and IR analysis for the fluorescent photoproduct

ere also carried out. 1H NMR (300 MHz, d6-DMSO) data con-ained: δ 7.0 (s, 2H) which corresponds to two hydrogen atomsf benzene ring, δ 10.2 (br, 2H) which corresponds to hydro-en atoms of two carboxylic groups, and δ 4.6 (br, 2H) whichorresponds to hydrogen atoms of two hydroxyl groups. Theollowing bands were obtained by IR analysis: 2500–3400/cm

trong and broad band which corresponds to carboxylic andydroxylic O H stretch. 1702.8 and 1690/cm, sharp and strongands, which correspond to C O stretch of two carboxylic

able 1luorescence characteristics and retention times of quinones and their photo-roducts using HPLC-fluorescence system

ample UV irradiation Maximumλex/λem (nm)

RFI tR (min)

,10-AnthraquinoneOff 274/327 1.9 6.0On 275/334 100.0 3.8

,4-NaphthoquinoneOff 274/333 0.3 4.7On 275/333 12.7 3.8

,10-PhenanthrequinoneOff 275/330 1.4 4.8On 276/330 28.6 3.8

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r. A 1133 (2006) 76–82 79

roups. The data obtained by 1H NMR and IR analyses showorrespondence with those obtained by 3,6-dihydroxyphthaliccid authentic sample. From the above mentioned, the fluores-ent material generated through UV irradiation of quinones wasuggested to be 3,6-dihydroxyphthalic acid.

.4. Suggested CL mechanism

The suggested mechanism for PO-CL reaction of quinoness shown in Fig. 4. When quinone receives the UV irradi-tion, it generates hydrogen peroxide and a highly fluores-ent 3,6-dihydroxyphthalic acid. Afterwards, hydrogen perox-de and the fluorescent 3,6-dihydroxyphthalic acid were mixedith TCPO via PO-CL reaction. TCPO react with the gener-

ted hydrogen peroxide to produce a high-energy intermediate,2-dioxetanedione. This intermediate transfers its energy tohe produced fluorophore, 3,6-dihydroxyphthalic acid, throughIEEL mechanism and then emission of light is observed from

he excited fluorophore when it returns to the ground state24].

.5. Optimization of HPLC–PO-CL method

Conditions for the determination of quinones with HPLC–O-CL method were optimized. Four kinds of quinones (1,2-aphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, and,10-phenanthrenequinone) were used as samples. ThePLC–PO-CL system is shown in Fig. 1.

.5.1. Optimization of UV irradiation conditionsIt is thought that the conversion efficiency of the quinones

y UV irradiation to hydrogen peroxide and a fluorescent com-ound depends on the exposure time and the temperature ofV irradiation device. Therefore, the length of the reaction coilrapped around UV irradiation lamp and the temperature of UV

rradiation device were optimized.The effect of coil length ranging from 1.0 to 4.0 m corre-

ponding to 15–60 s of UV irradiation on CL intensity wasxamined (Fig. 5). It was found that CL increased as the lengthf the reaction coil increased due to the increase in exposureime then it became constant. The reason for this phenomenons that the generation and decomposition of hydrogen peroxideave occurred simultaneously because of the UV irradiation forlong time. Moreover, the detection sensitivity (S/N ratio) waseasured against coil length for all samples. It was found that/N ratio increase by increasing coil length till maximum thenecrease because the background noise increases as the length ofhe reaction coil increases. The optimum coil length was foundo be 2 m corresponding to 30 s of UV irradiation.

Because the temperature of UV irradiation part affectedtrongly generation efficiency of hydrogen peroxide and the fluo-ophore, its effects on CL intensity and S/N ratio were examined.

here is a significant increase in CL intensity with temperature

ncrease. However, there was no significant change in S/N withhe rise in temperature because the background noise level hadisen at the same time so, 24 ◦C was selected.

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80 S. Ahmed et al. / J. Chromatogr. A 1133 (2006) 76–82

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.5.2. Optimization of CL conditionsThe concentration and pH of imidazole–HNO3 buffer solu-

ion as a mobile phase and the concentration and the flow ratef TCPO as a post column CL reagent were optimized.

Imidazole has been widely used as a catalyst for theO-CL reaction. The complex effect of imidazole on theeaction kinetics has been attributed to a combination of

ucleophilic and general-base catalysis [24]. The effect ofmidazole–HNO3 buffer solution on CL intensity was exam-ned. The highest CL intensity and S/N were obtained at 100 mMmidazole–HNO3 buffer solution. Also, the influence of pH of

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ig. 5. Effects of reaction coil length on (A) CL intensity and (B) S/N ratio. Sample,10-anthraquinone (�), and 6 �M 9,10-phenanthrenequinone (×).

te chemiluminescence reaction for quinines.

he imidazole–HNO3 buffer solution on CL was examined. Itas found that both CL intensity and S/N ratio increased with

he increase in pH, which could be attributed to faster reactioninetics at higher pH. Taking the durability of the ODS into con-ideration, pH value of 9.0 was selected for subsequent work.

Furthermore, the influence of the TCPO concentration onL intensity was examined. It was found that CL intensity and

/N ratio increase by increasing the concentration of TCPO tilloncentration 0.5 mM where it became constant. Therefore, theoncentration of 0.5 mM TCPO was selected for subsequentork. Effect of flow rate of the TCPO solution on CL inten-

s were 20 �M 1,2-naphthoquinone (♦), 10 �M 1,4-naphthoquinone (�), 4 �M

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S. Ahmed et al. / J. Chromatogr. A 1133 (2006) 76–82 81

Fig. 6. Effect of flow rate of CL reagent on (A) CL intensity and (B) S/N ratio. Samples were 20 �M 1,2-naphthoquinone (♦), 10 �M 1,4-naphthoquinone (�), 4 �M9,10-anthraquinone (�), and 6 �M 9,10-phenanthrequinone (×).

Table 2Retention times, calibration curves, and detection limits for quinones

Compound tR (min) Calibration curvea Detection limitb

(pmol/injection)Range (�M) Slopec (±SE) Interceptc (±SE) r

1,2-Naphthoquinone 7.2 10–100 0.254 (±0.015) −0.623 (±0.34) 0.998 6.01,4-Naphthoquinone 9.1 5–50 0.380 (±0.021) −0.633 (±0.21) 0.999 4.49,10-Anthraquinone 22.5 0.5–50 0.959 (±0.033) 0.088 (±0.08) 0.999 0.29,10-Phenanthrenequinone 12.6 1–50 0.685 (±0.027) 0.086 (±0.10) 0.999 0.45

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ity was also examined (Fig. 6). It was found that CL intensityncreased by the increase in flow rate and reached maximum at.35 ml/min whereas the noise remain constant then CL inten-ity decreased due to the decrease in reaction time between CLeagent, hydrogen peroxide, and fluorophore. Therefore, flowate of 0.35 ml/min was selected.

.6. Validation of the proposed method

A typical chromatogram of a standard mixture of quinonessing the proposed method and under the optimum conditionss shown in Fig. 7. Quinones were separated efficiently and all

ig. 7. Chromatogram of a standard mixture of quinones obtainedsing the proposed method. Peaks: 1—1,2-naphthoquinone (40 �M);—1,4-naphthoquinone (20 �M); 3—9,10-phenanthrenequinone (12 �M); and—9,10-anthraquinone (4 �M).

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eaks were eluted within 25 min. For each quinone calibrationurve, retention time, calibration range, correlation coefficient,nd detection limit were recorded (Table 2). The correlationoefficient of 0.998 or more was obtained in the concentrationanges of 0.5–100 �M. Also, the detection limits for quinonesbtained with the proposed method (S/N = 3) were in theange 0.2–6.0 pmol/injection. It was 3–8 times more sensitiveompared with the level obtained by GC–MS method [22]nd compared with the level obtained by other CL methods.he order of CL strength and detection sensitivity for all thetudied quinones after UV irradiation were as follows:,10-anthraquinone > 9,10-phenanthrenequinone > 1,4-aphthoquinone > 1,2-naphthoquinone. Because the differencen the detection sensitivity, it was suggested that the gener-tion efficiency of hydrogen peroxide and the fluorophore,,6-dihydroxyphthalic acid, generated by UV irradiation is anmportant factor. So, it is suggested that the overall examinationf the generation efficiency is necessary in the future.

The precision of the proposed method within- and between-ay was examined. It was found that the relative standardeviations (RSD) within-day (n = 5) and between-day (n = 3)ere 2.7–5.4% and 4.3–6.7%, respectively, so acceptable repro-ucibility was obtained.

. Conclusion

A simple, selective and sensitive HPLC–PO-CL method forimultaneous determination of quinones was developed and

our quinones were separated efficiently within 25 min. Weound that hydrogen peroxide and the highly fluorescent 3,6-ihydroxyphthalic acid were generated upon UV irradiationf quinones. Because hydrogen peroxide and the fluorescent
Page 7: Selective determination of quinones by high-performance liquid chromatography with on-line post column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

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aterial were not needed as post column reagents, only theuinones can be detected selectively. Moreover, when hydro-en peroxide is used as a post column reagent in PO-CL system,ackground noise is generated. On the other hand, because thisL method doesn’t need the addition of hydrogen peroxide, theecrease of the background noise can be expected comparedith the general PO-CL reaction. The proposed method doesot require a time-consuming labeling procedure and has sat-sfactory reproducibility and sensitivity, which is comparableo sensitive methods. The proposed method is recommended forhe determination of quinones in the fields of chemical industriesnd environmental samples.

eferences

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