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Selective Determinationof Harmol by Room-Temperature Phosphorimetry:A Comparative Performancewith Micellar ElectrokineticCapillary ChromatographyFlávia F. C. Marques a , Cabrini F. de Souza a , FláviaS. Figueiredo a & Ricardo Q. Aucélio aa Departamento de Química , Pontifícia UniversidadeCatólica do Rio de Janeiro , Rio de Janeiro, BrazilPublished online: 17 Jul 2008.

To cite this article: Flávia F. C. Marques , Cabrini F. de Souza , Flávia S.Figueiredo & Ricardo Q. Aucélio (2008) Selective Determination of Harmol byRoom-Temperature Phosphorimetry: A Comparative Performance with MicellarElectrokinetic Capillary Chromatography, Analytical Letters, 41:9, 1648-1657, DOI:10.1080/00032710802122339

To link to this article: http://dx.doi.org/10.1080/00032710802122339

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LUMINESCENCE

Selective Determination of Harmol byRoom-Temperature Phosphorimetry: A

Comparative Performance with MicellarElectrokinetic Capillary Chromatography

Fl�aavia F. C. Marques, Cabrini F. de Souza, Fl�aavia S. Figueiredo,and Ricardo Q. Aucelio

Departamento de Quımica, Pontifıcia Universidade Cat�oolica doRio de Janeiro, Rio de Janeiro, Brazil

Abstract: Selective determination of harmol in the presence of other b-carbolinealkaloids without the need for previous separation of components was achievedby Solid Surface Room-Temperature Phosphorimetry (SSRTP) using HgCl2.Detection of harmol at concentrations as low as 5.2� 10�7 mol L�1 can be madein urine samples. Recovery of 100� 12% was achieved. The analyticalperformance of SSRTP was compared to Micellar Electrokinetic CapillaryChromatography (MECC).

Keywords: Harmol, micellar electrokinetic chromatography, selective determi-nation of beta-carbolines, solid surface room-temperature phosphorimetry

Received 10 October 2007; accepted 15 February 2008.Financial support from the Brazilian agencies FAPERJ and FINEP is

gratefully acknowledged. Aucelio, Figueiredo, and de Souza thank CNPq, andMarques thanks CAPES and FAPERJ for scholarships.

Address correspondence to Ricardo Q. Aucelio, Departamento de Quımica,Pontifıcia Universidade Cat�oolica do Rio de Janeiro, Rio de Janeiro, Brazil.Fax: þ 5521 3527-1637; E-mail: aucelior@puc-rio.br

Analytical Letters, 41: 1648–1657, 2008Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print/1532-236X onlineDOI: 10.1080/00032710802122339

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INTRODUCTION

b-carboline alkaloids occur naturally in plants such as Peganum harmalaand in other plant species throughout the world. They are also found asproducts of pyrolysis of indoles and proteins and thus occur in cigarettesmoke, charcoal grilled meats, and teas (Adachi et al. 1991; Nishigataet al. 1980). Harmane and norharmane are the main and most simpleb-carbolines, being endogenously produced in human and animal tissueas a product of secondary metabolism (Bidder et al. 1979); however, mostgenerally, the presence of harmane and norharmane in the human body isfrom exogenous sources (Pfau and Skog 2004). The metabolic routeof harmane leads to the formation of harmine, a more liposolubleb-carboline, which is therefore more favorably distributed in the bodytissues. Harmol is the result of the hydroxylation of harmane, and it isthe intermediary reactive species that will undergo methylation to formharmine. The ability to detect amounts of harmol in body fluids mayserve as an indication of the metabolism of b-carbolines, showing howthe liver is dealing with these substances in the organism.

Solid Surface Room-Temperature Phosphorimetry (SSRTP) hasbeen established as a selective analytical technique, allowing ultra-tracedetermination of many organic substances of clinical and biologicalinterest, especially alkaloids (Arruda and Aucelio 2002; Aucelio andCampıglia 1994). Phosphorescence may be selectively induced from a spe-cific substance by choosing the right experimental conditions—in parti-cular, the use of a selective heavy atom enhancer (Vo Dinh andHooyman 1979). The presence of heavy atoms in the vicinity of a mol-ecule may induce or enhance its phosphorescence due to the increase ofthe spin-orbit coupling interaction. This phenomenon can affect sing-let-triplet transitions and either radiative or nonradiative decay fromthe excited triplet state; therefore, the increasing in phosphorescencequantum yield does not always occur, because of the selective nature ofthe heavy atom effect based on these different interactions between ana-lytes and heavy atoms (Vo Dinh 1984).

Capillary Electrophoresis (CE) is a powerful way to separate chargedchemical species before performing selective detection, in general byUV-vis absorption photometry. Micellar Electrokinetic CapillaryChromatography (MECC) is a mode of CE that is used to separateuncharged compounds by the difference in their water-charged organizedassembly partition coefficients (Tavares 1996). In MECC, chargedmicelles are the most commonly used pseudostationary phase.

In the present work, SSRTP is evaluated as a tool to enable the selec-tive determination of harmol in the presence of other b-carbolines with-out the need for previous separation of components. The performance of

Determination of Harmol by SSRTP 1649

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the SSRTP method was compared to an improved method based onMECC. The method based on MECC was adapted from other workdescribed in the literature (Cheng and Mitchelson 1997), aiming for theachievement of more robust conditions to separate and detect b-carbolines.

EXPERIMENTAL

Instrumentation

Phosphorescence was measured on an LS-55 Perkin-Elmer spectrofluori-meter (Perkin-Elmer, Kent, UK) using 3 ms delay and 3 ms gate times inorder to minimize secondorder scattering and fluorescence from samples.Data was acquired using FLWinlab software (Perkin-Elmer) and spectralbandpass was at 10 nm. A front surface accessory (Perkin-Elmer) wasused to measure phosphorescence from cellulose substrates. This appar-atus was modified to enable nitrogen purging directly onto the sampleduring measurements. MECC was performed on an Agilent CE capillaryelectrophoresis system (Agilent, California, USA) equipped with a diodearray UV-vis absorption photometric detector and data acquisition andtreatment software supplied by the manufacturer. Photochemical treat-ment to reduce cellulose background was carried out in a laboratory-made photochemical reactor loaded with six 6 W mercury sterilizationlamps (each one with most intense emission wavelength at 253 and inthe 296–313 nm range).

Reagents

Filter paper (Whatman No. 42-Whatman, Kent, UK) was employed as asubstrate. Deionized water (resistivity of 18.2 MXcm) was from a waterultra-purifier master system 1000 (Gehaka, Sao Paulo, Brazil). Nitrogen(99.996%) was from Lynde Gases (Rio de Janeiro, Brazil), and it wasfurther purified by passing it through an ammonium metavanadate sol-ution and drying it in a silica gel bed. Methanol, acetonitrile, mercury(II) chloride, Sodium Dodecil Sulfate (SDS), boric acid, and sodiumhydroxide were purchased from Merck (Darmstadt, Germany). Harmol,harmane, harmine, harmaline, and norharman were from Across Organ-ics (New Jersey, USA). Analytical grade reagents were employed.

Procedure

Paper substrates were washed in hot boiling water (2 h in a Sohxletextractor), dried with an infrared lamp, and cut in circles of 18 mm of

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diameter. These substrates were then treated with UV radiation (2 h) in aphotochemical reactor. Methanol=water 25=75%, v=v was employed toprepare stock solutions of b-carbolines from which the working solutionswere prepared by sequential dilutions using ultra-purified water.Deionized water was used to prepare the HgCl2 0.25 mol L�1 and SDSsolutions. Urine samples fortified with b-carbolines were used to testthe methods. The pH of the samples was adjusted to around 7.0, bythe addition of NaOH 0.1 mol L�1, and centrifuged for 15 min at2.500 rpm, in order to perform a cleanup.

Five mL of analyte standard solutions or sample solutions were depositedin the center of the substrate, using an adjustable micropipette. The center ofthe substrates was previously spotted with three sequential 5 mL aliquots ofHgCl2 0.20 mol L�1. These substrates were then dried (for 2 h) at room tem-perature in a vacuum desiccator. Two minutes prior to and during the phos-phorescence measurement, a dry nitrogen flow was passed over the substratesurface to minimize quenching effects from oxygen and air moisture.

The following conditions were used for MECC: uncoated fused silicacapillary of 57 cm (effective length of 50 cm) and 50mm i.d.; applied potentialof 25 kV, temperature held constant at 30�C, and injection made at 50 mbarfor 10 s. The working electrolyte solution was freshly prepared and consistedof 20 mmol L�1 borate buffer (pH 9.0) containing SDS (50 mmol L�1) andacetonitrile 15%, in volume. The absorption of the analyte peak was detectedat 254 nm. For new capillary tubes, the following conditioning procedure wasused: (i) flushing with NaOH 1.0 mol L�1for 15 min, (ii) waiting for 2 min, (iii)flushing with water for 10 min, (iv) flushing with acetonitrile=water=boratebuffer (14=29=57%, v=v=v) for 10 min, and (v) flushing with working electro-lyte solution for 2 min. Before each sample, standard or blank injection, thefollowing conditioning was used: (i) flushing with acetonitrile for 1 min, (ii)flushing with water for 1 min, (iii) flushing with NaOH 1 mol L�1 for 2 min,(iv) flushing with water for 1 min, and (v) flushing with working electrolytesolution for 1 min. After five runs, a cleaning procedure was used to restorethe separation performance: (i) flushing with acetonitrile for 5 min, (ii) flush-ing with NaOH 1 mol L�1 for 3 min, (iii) flushing with water for 5 min, and (iv)flushing with working electrolyte solution for 1 min.

RESULTS AND DISCUSSION

Experimental Conditions for the Selective Determination of

Harmol Using SSRTP

The b-carbolines presented intense fluorescence when dissolved inaqueous solution and natural phosphorescence when placed in solid

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substrates. Such natural phosphorescence is probably favored bythe endocyclic heteroatoms (nitrogen) present in the structure of themolecule. Although signal intensities are different from each of theb-carbolines studied (harmol, harmane, harmine, norharman, andharmaline), the maximum wavelengths of excitation (kexc at 250 and336 nm) and emission (kemat 486 nm) are common among theseb-carbolines. In order to find spectral conditions to perform phosphori-metric determination of harmol, the effects of several heavy atom saltshave been tested, aiming to find one that would selectively enhance phos-phorescence from harmol while decreasing the signal from the otherb-carbolines. While no success was achieved by using Iodine, Tl(I),Ag(I), and Pb(II), the presence of 0.27 mg of Hg(II) in the substrateincreased almost three times the phosphorescence from harmol whiledecreasing significantly the signals from the other four b-carbolines.Although Hg(II) is an unusually heavy atom enhancer, it has been usedfor the determination of thalidomide by SSRTP (Aucelio and Campıglia1994). The presence of Hg(II) caused a small blue shift on the kem of har-mol to 485 nm. Substrates containing different amounts of HgCl2 wereused in order to evaluate the effect on the phosphorescence for each ofthe b-carbolines (2 nmol deposited on the substrate). Since maximumconcentration of the HgCl2 solution was 0.20 mol L�1 (limited by thesolubility of the salt), multiple additions of 5 mL were used in order toget larger amounts of HgCl2 in the center of the substrate. Up to fivesequential additions were used (1.35 mg of HgCl2), as can be seen inFig. 1a. For harmol, stronger phosphorescence (about seven times itsnatural phosphorescence) was achieved with three additions of HgCl20.20 mol L�1 (0.81 mg). In these conditions, phosphorescence from 2 nmolof harmane, harmaline, or norharman was almost eliminated. For har-mine, the signal magnitude that was observed in the presence of0.27 mg of HgCl2 remained the same. Although relatively small whencompared to the harmol signal, phosphorescence from the other b-carbo-lines could cause spectral interferences in the determination of harmol ifthey are present in higher amounts in the sample. For this reason, asecond-order derivative technique was applied in order to explore thesmall difference between the kem (485 nm) of harmol in contrast to thekem (495 nm) observed for the other b-carbolines. In this case, isodifferen-tial points (kiso) were identified, where harmol signal can still be measuredin a region where the signals from the other compounds were at theirbaselines. The kiso were identified by the comparison of the signal profilesand from interference tests using synthetic mixtures of b-carbolines(Fig. 1b and Table 1). In the presence of harmane, norharman, or harma-line, the kiso at 434 nm was chosen because it enabled the measurement ofintense signal from harmol. For mixtures also containing harmine, the

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Figure 1. (a) Effect of the amount of HgCl2 on the phosphorescence ofb-carbolines; (b) Second derivative spectra of b-carbolines in the presence of0.81 mg of HgCl2. SDS modified substrates containing 5 mL of b-carbolinesolutions at 1� 10�4 mol L�1. The spectrum of harmol is attenuated (� 0.1 meansthat displayed signal is 10% of the original one).

Determination of Harmol by SSRTP 1653

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most indicated kiso value was at 564 nm, where phosphorescence of theharmol standard was statistically similar to the one measured from mix-tures containing the other four b-carbolines in quantities ten times higherthan harmol. In Table 1, an interference study made with harmane andharmine indicated noninterferences in the harmol phosphorescence, ascan be seen by the ratio values close to one. Results with harmalineand norharman were similar to those observed with the harmol=harmanearmane mixture.

Experimental Conditions for the Selective Determination of

Harmol Using MECC

The literature (Cheng and Mitchelson 1997) describes experimentalconditions to perform separation and photometric determination ofsix b-carbolines using the anionic surfactant SDS as the pseudostation-ary phase. In their work, borate buffer (pH 9.0) was used in a mediumcontaining 15% of acetonitrile in volume and urea as the organicmodifier. In the present work, adaptations of these conditions weremade, in order to allow more efficient and reproducible separation ofb-carbolines, because the use of the reported conditions implicate norobust conditions for separation with larger changes in migration timesand analyte peak areas. In addition, shortcut in the electrode was com-mon, due to the deposition of urea, requiring frequent disassemblingand cleaning of the system. In the adapted experimental conditionsset in this work, the use of urea was eliminated, and two conditioningprocedures were applied: a faster one in between runs and a longerone, applied from time to time, in order to restore the efficiency ofseparation and minimize dispersion of peaks. Under these conditions,separation was achieved within 21 min (Fig. 2).

Table 1. Evaluation of the kiso at 564 nm for the determination of harmol(2 nmol) by second derivative SSRTP using Hg(II)

Composition of the b-carboline mixture (relativeproportion in mol L�1)

Iharmol in

mixture=Iharmol

harmol:harmine:harmane:harmaline:norharman (1:1:1:1:1) 0,92� 0,10harmol:harmine:harmane:harmaline:norharman(1:10:10:10:10)

1,12� 0,16,

Iharmol in mixture=Iharmol: Ratio between RTP measured from 2 nmol of harmolfrom the b-carboline mixture and the RTP measured from 2 nmol of harmol froma standard solution.

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Analytical Performance in the Determination of Harmol

Analytical figures of merit for the developed SSRTP method and for theadapted MECC method were estimated and listed in Table 2, togetherwith other important parameters for these methods. Three analyticalcurves were constructed, using harmol standards (from 1� 10�6 to5� 10�5 mol L�1) and plotting the analytical signal in function of the con-centration (mol L�1) of the standard solution injected into the capillary ordeposited on the substrate. Detection power of the methods was evalu-ated as the concentration or the amount of harmol that enabled an ana-lytical signal equal to three times the blank signal. In other words, this

Figure 2. (a) Electropherograms of a sequential of harmol (tm ¼ 8.25 min)standards: (i) 1� 10�5, (ii) 2� 10�5 e, and (iii) 5� 10�5mol L�1. (b) Electro-pherograms of (i) harmol (1� 10�5 mol L�1) in urine, (ii) urina, and (iii) urinecontaining: a) harmaline (1� 10�4 mol L�1 ; tm ¼ 20.5 min), b) harmine(1� 10�4 mol L�1; tm ¼ 17.1 min), c) harmane (1� 10�4 mol L�1; tm ¼ 15.1 min),d) norharman (1� 10�4 mol L�1; tm ¼ 13.8 min), and e) harmol (1� 10�5 mol L�1;

tm ¼ 11.2 min).

Determination of Harmol by SSRTP 1655

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parameter indicated the minimum quantity of harmol that can be effec-tively detected. For SSRTP this value was 5.2� 10�7 mol L�1 or 0.59 ngof harmol while for MECC the value achieved was 5.0� 10�6 mol L�1.Repeatability was estimated by the standard deviation of consecutivesignal measurements from the 1� 10�5 mol L�1 harmol standard(11.7 ng of harmol in the solid substrate). Repeatability was about2.5% (n ¼ 10) for SSRTP and about 4.2% (n ¼ 5) for MECC.

The selective methods were tested using urine samples fortifiedwith the analyte of interest (harmol) and the two potential interferentb-carbolines (harmane and harmine). Previously, tests in nonfortifiedurine samples indicated no signals from the matrix when using conditionsset for the harmol determinations using SSRTP. For samples containingharmol (1� 10�5 mol L�1), harmane (1� 10�4 mol L�1), and harmine(1� 10�4 mol L�1), the average recovery for harmol was 100� 12%(n ¼ 4) for SSRTP and 91� 2% (n ¼ 3) for MECC. These resultsindicated the suitability of the SSRTP method for such application. AStudent’s t-test indicated no difference in the results achieved by thetwo methods (tcalculated ¼ 0.64 < tcritical ¼ 2.23 for p ¼ 0.05; nSSRTP ¼ 4;nMECC ¼ 3).

CONCLUSIONS

In this work, selective determination of harmol was achieved using SSRTPwithout the need for previous separation of components. The SSRTPmethod was compared to the method based on MECC, and the analyticalperformance, including the recovery of harmol in urine samples fortified

Table 2. Conditions for selective determination of harmol using SSRTP andMECC

SSRTP MECC

Measurement conditions kiso ¼ 564 nm(kexc ¼ 336 nm)0.81 mg of HgCl2

kabs ¼ 254 nmand tm ¼ 21 min

r2 0.9914 0.9999Repeatability at 2� 10�5 mol L�1

(number of replicates)2.5% (n ¼ 10) 5% (n ¼ 5)

Detection powera 5.2� 10�7 mol L�1

(0.59 ng in absolutevalue)

5.0� 10�6mol L�1

aConcentration or mass of harmol producing analytical signal is equal to 3times the blank signal.

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with harmine and harmane, was very satisfactory. In practical terms,determinations using SSRTP were made in a more sensitive, simpler,and cheaper way when compared to the ones made using MECC.However, the experimental conditions adjusted for MECC enable thequantification of harmol in addition to the other four b-carbolines.

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Adachi, J., Mizoi, Y., Naito, T., Yamamoto, K., Fujiwara, S., and Nimomiya, I.1991. Determination of beta-carbolines in foodstuffs by high-performanceliquid-chromatography and high-performance liquid-chromatographymass-spectrometry. J. Chromatogr., 538: 331–339.

Arruda, A.F. and Aucelio, R.Q. 2002. Room temperature phosphorimetry for theselective determination of yohimbine in the presence of reserpine-like indolicalkaloids. Anal. Sci., 18: 831–834.

Aucelio, R.Q. and Campıglia, A.D. 1994. Solid surface room temperature phos-phorimetry analysis of reserpine in pharmaceutical formulations. Talanta,41: 2131–2136.

Aucelio, R.Q. and Campıglia, A.D. 1994. Pharmaceutical formulation analysis ofthalidomide by solid surface room temperature phosphorimetry. Mikrochim.Acta 117: 75–85.

Bidder, T.G., Schomaker, D.W., Boettger, H.G., Evans, H.E., and Cummins, J.T.1979. Harman in human-platelets. Life Sci., 25: 157–164.

Cheng, J. and Mitchelson, K.R. 1997. Improved separation of six harmanealkaloids by high-performance capillary electrophoresis. J. ChromatographyA., 761: 297–305.

Micke, G.A., Fujita, N.M., Tonin, F.G., Costa, A.C., and Tavares, M.F.M. 2006.Method development and validation for isoflavones in soy germ pharmaceu-tical capsules using micellar electrokinetic chromatography. J.Pharm.Biomed. Anal., 41: 1625–1632.

Nishigata, H., Yoshida, D., and Matsumoto, T. 1980. Determination of the yieldof norharman and harman in the pyrolitic products of proteins. Agric. Biol.Chem., 44: 209–210.

Pfau, W. and Skog, K. 2004. Exposure to beta-carbolines norharman andharman. J. Chromatogr. B, 802: 115–126.

Tavares, M.F.M. 1996. Capillary electrophoresis: Basic concepts. Quimica Nova,19: 173–181.

Vo Dinh, T. 1984. Room Temperature Phosphorimetry for Chemical Analysis,Chemical analysis series, Vol. 101. New York: J. Wiley & Sons.

Vo Dinh, T. and Hooyman, J.R. 1979. Selective heavy-atom perturbation foranalysis of complex mixtures by room temperature phosphorimetry. Anal.Chem., 51: 1915–1921.

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