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Published OnlineFirst August 16, 2010; DOI: 10.1158/1055-9965.EPI-10-0483

Cancer
Epidemiology,
iomarkersrevention

arch Article

h-Throughput Simultaneous Analysis of Five Urinaryabolites of Areca Nut and Tobacco Alkaloids byope-Dilution Liquid Chromatography-Tandem Mass

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ctrometry with On-Line Solid-Phase Extraction

g-Wen Hu1,4, Yan-Zin Chang3, Hsiao-Wen Wang1, and Mu-Rong Chao2

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kground: Areca nut and tobacco are commonly used drugs worldwide and have been frequently used innation.Wedescribe the use of on-line solid-phase extraction and isotope-dilution liquid chromatography-

mass spectrometry for the simultaneous measurement of five major urinary metabolites of bothnut and tobacco alkaloids, namely, arecoline, arecaidine, N-methylnipecotic acid, nicotine, ande.thods: Automated purification of urine was accomplished with a column-switching device. After theon of deuterium-labeled internal standards, urine samples were directly analyzed within 13 minutes.ethod was applied to measure urinary metabolites in 90 healthy subjects to assess areca nut/tobaccore. Urinary time course of arecoline, arecaidine, and N-methylnipecotic acid was investigated in fivey nonchewers after oral administration of areca nut water extracts.ults: The limits of detection were 0.016 to 0.553 ng/mL. Interday and intraday imprecision were <10%.recoveries of five metabolites in urine were 97% to 114%. Mean urinary concentrations of arecoline,dine, N-methylnipecotic acid, nicotine, and cotinine in regular areca nut chewers also smokers were,816, 1,298, 2,635, and 1,406 ng/mg creatinine, respectively. Time course study revealed that after ad-ration of areca nuts extracts, the major urinary metabolite was arecaidine with a half-life of 4.3 hours,ed by N-methylnipecotic acid with a half-life of 7.9 hours, and very low levels of arecoline with a half-0.97 hour.clusions: This on-line solid-phase extraction liquid chromatography-tandem mass spectrometry meth-tly provides high-throughput direct analysis of five urinary metabolites of areca nut/tobacco alkaloids.
od firs
Impact: This method may facilitate the research into the oncogenic effects of areca nut/tobacco exposure.Cancer Epidemiol Biomarkers Prev; 19(10); 2570–81. ©2010 AACR.

that dcured(calcibetelIndia,In Ta

duction

areca nut (fruit of the Areca catechu tree) is themost commonly used psychoactive substance in

orld after tobacco, alcohol, and caffeine (1). It isonly consumed by Asian populations and migrated

ving in Africa, Europe, and North Americaan be chewed alone or in a variety of ways

with bnut hformsa flavcatechout to1985 bhad fois cartion gwithois alsoThe

arecol

ns: Departments of 1Public Health, 2Occupational, and 3Institute of Medicine, Chung Shan MedicalDepartment of Family and Community Medicine,al University Hospital, Taichung 402, Taiwan.

ary data for this article are available at Cancer Epide-rs & Prevention Online (http://cebp.aacrjournals.org/).

uthor: Mu-Rong Chao, Department of Occupational, Chung Shan Medical University, No.110, Sec.1,d, Taichung 402, Taiwan. Phone: 886-4-2473-00226-4-23248194. E-mail: [email protected]

9965.EPI-10-0483

ssociation for Cancer Research.

l Biomarkers Prev; 19(10) October 2010

on August 18, 2018. © 20cebp.aacrjournals.org from

iffer by region. Betel quid contains fresh, dried, orareca nut, catechu (Acacia catechu), and slaked limeum oxide and calcium hydroxide) wrapped in aleaf (Piper betle). In some countries, particularly inmost habitual chewers of betel quid add tobacco.

iwan, the green unripe areca nut is often chewedetel inflorescence, but tobacco is not added. Arecaas also been available in commercially preparedin the last few decades. The product is basicallyored and sweetened dry mixture of areca nut,u, and slaked lime with tobacco (gutkha) or with-bacco (pan masala; ref. 3). A previous evaluation iny the International Agency for Research on Cancerund that chewing betel quid with tobacco (group 1)cinogenic to humans (4). Recently, the new evalua-oes further to conclude that chewing betel quidut tobacco (group 1) and areca nut itself (group 1)carcinogenic to humans (5).
re are four main areca alkaloids in areca nut:ine, arecaidine, guvacine, andguvacoline.Arecoline,
10 American Association for Cancer Research.

http://cebp.aacrjournals.org/

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ain alkaloid present at up to 1% of dry weight, isht to be responsible for a central cholinergiclation and monoamine transmission, which thentes both sympathetic and parasympathetic effectsArecoline has been further shown to be implicatedpathogenesis of oral diseases because of its geno-mutagenic, and carcinogenic potential (3, 8). How-relatively little is known about the metabolism ofline as well as other alkaloids. Until recently, Giri9) reported a metabolic map of arecoline and arecai-n the mouse and found that the major metabolite ofrecoline and arecaidine wasN-methylnipecotic acid.theless, except for arecoline, none of the areca nutid metabolites has ever been identified in humans.acco smoking has long been recognized as a majorof death and disease in many countries (10). Nico-the major alkaloid of tobacco and is responsible foro addiction. When tobacco is smoked, nicotine isntly absorbed into the bloodstream through theand rapidly metabolized to many different com-s (e.g., cotinine, trans-3-hydroxycotinine, nornico-nd cotinine-N-oxide; ref. 11). These nicotineolites have been recently measured in urine to pro-a better estimate of exposure to tobacco smoke.g these metabolites, cotinine with a longer half-lifehours (12) is by far the best documented and mostntly utilized maker (13, 14).eral chromatographic-based techniques have beenoped for the measurement of tobacco alkaloidsreca nut alkaloids (mostly arecoline only) in biolog-mples, such as high performance liquid chromatog-(HPLC) with UV detection (HPLC-UV), gasatography-mass spectrometry (GC-MS), and liquidatography-tandem MS (LC-MS/MS; refs. 15-18).ormer two methods, however, can be difficult toout in the clinical laboratory and are labor inten-require time-consuming sample preparation, orit inadequate specificity when used to test urine.S/MS is a relatively new and powerful technologyan overcome the sensitivity and selectivity issues inalysis of urinarymetabolites. Accurate quantificationets at extremely low levels in matrix has frequentlyon the use of stable isotope-labeled standards toensate for the loss of analyte during sample prepara-hich has been the most critical step to eliminate thex effect for analysis by mass spectrometry (19).ermore, the on-line sample extraction using an-switching device is an extremely useful techniquepare biological samples automatically for LC-MSds (20, 21). Its advantages include less ion suppres-nd relatively short run times, as well as higher sensi-and selectivity, especially forurine samples containingiderable amount of coeluting interferences.ause of the serious health consequences of areca nutbacco and the fact that most areca nut users alsobacco either in the form of chewing or smoking
, methods for a simultaneous determination of uri-etabolites of both areca nut and tobacco alkaloids
time pafter

acrjournals.org

on August 18, 2018. © 20cebp.aacrjournals.org wnloaded from

tremely important. We report here a new, highlyive isotope dilution LC-MS/MS method coupledn-line solid phase extraction (SPE) for simultaneousion of five urinary metabolites of areca nut and to-alkaloids (arecoline, arecaidine, N-methylnipecoticnicotine and cotinine). This method was applied toigate the urinary concentrations of these five meta-s in non–areca nut chewers who are also non-ers, non–areca nut chewers but regular cigaretteers, and regular areca nut chewers who are also cig-smokers. Furthermore, the urinary time course ofline, arecaidine, and N-methylnipecotic acid wasinvestigated in five healthy males after oral admin-on of areca nut water extracts.

rials and Methods

icalsvents and salts were of analytical grade. Reagentspurchased from the indicated sources: arecoline,dine, nicotine, and cotinine from Sigma-Aldrich;thylnipecotic acid from Oakwood Product; them-made arecoline-d3, arecaidine-d3, and N-lnipecotic acid-d3 from Ryss Lab; and nicotine-d4

otinine-d3 from Cerilliant.

ration of water extract of areca nutsmercial fresh and unripe areca nuts (about the size

olive) were purchased from a local shop in Taiwan.al of 12 areca nuts (∼36 g) were ground and sus-d in 60 mL of deionized water. The mixture wasd for 1 hour at room temperature and the extractollected by centrifugation. This extraction procedureepeated once more by adding 60 mL of deionizedto the residue. Both extracts were pooled andmixedrepresenting 12 areca nuts (∼36 g) of extracted ma-in 120 mL deionized water, for further use in theourse study as described in a later section.

cipants and urine sampless study was approved by the Institutional Reviewof Chung Shan Medical University Hospital.ss-sectional study. Single spot urine samples werened from 90 apparently healthy individuals (31reca nut chewers also nonsmokers, 26 non–arecaewers but regular cigarette smokers, and 33 regularnut chewers also cigarette smokers). A question-was used to obtain data on subject age, body mass(BMI), and the areca nut chewing or smoking

s (self-reported daily consumption of areca nutr cigarettes).e course study. Five healthy male volunteers whoot chew areca nut and smoke cigarettes in the pastwere each administered 20 mL of water extracts ofnut (representing two areca nuts) orally. Urine sam-ere collected immediately before and at different
oints (2, 4, 6, 8, 10, 12, 14, 17, 24, 27, and 33 hours)administration of the extracts. Each subject was
Cancer Epidemiol Biomarkers Prev; 19(10) October 2010 2571



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ed to drink at least 150 mL of water after each voidure an adequate volume of urine. Urine volumeeasured at each void in a calibrated container0 mL fractions were saved. Urine samples fromhe cross-sectional study and the time course studykept at 4°C during sampling, and stored at −20°Cto analysis. Urinary creatinine was also measuredch sample using a HPLC-UV method describedng (23).

ltaneous analysis of five urinary metabolites ofnut/tobacco alkaloids using on-lineC-MS/MSparation of urine samples. The urine samplesthawed, vortexed, and then heated to 37°C fornutes to release possible alkaloid metabolites fromitate. After centrifugation at 5,000 g, 20 μL of urinediluted 10 times with a solution containing 2 ngof arecoline-d3, arecaidine-d3, N-methylnipecotic3, and cotinine-d3, and 4 ng of nicotine-d4 asal standards in 2% (v/v) methanol containing(v/v) trifluoroacetic acid (TFA).rimary standard stock solution mixture of five ana-1,000 μg/mL) was prepared by dissolving the sament of each analyte in 10% (v/v) methanol and thenr diluting with 2% (v/v) methanol/0.1% (v/v) TFAld appropriate working solutions. Calibrators werein drug-free pooled urine and prepared by spiking
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r Epidemiol Biomarkers Prev; 19(10) October 2010


ower calibrators. Calibrators were then processednalyzed as urine samples. Two linear ranges wereined for arecoline, arecaidine, N-methylnipecotic

and cotinine, from 0.006 to 0.375 ng (low range:, 0.012, 0.023, 0.047, 0.094, 0.188, and 0.375 ng)rom 0.375 to 24 ng (high range: 0.375, 0.75, 1.5,.0, 12, 24 ng), whereas the ranges from 0.094 toand from 1.5 to 24 ng were applied for nicotine.omated on-line SPE. The column-switching systemin this study was as described in detail elsewhereIt consisted of a switching valve (two-positionelectric actuator from Valco) and a C18 trap column2.1 mm i.d., 5 μm, ODS-3, Inertsil). The switchingfunction was controlled by PE-SCIEX controlare (Analyst, Applied Biosystems). The column-hing operation, including the LC gradients usedg the on-line cleanup and the analytical procedures,marized in detail in Table 1. When the switchingwas at position A, 50 μL of prepared urine sampleloaded on the trap column by an autosampler (Agi-100 series, Agilent Technology), and a binary pumpnt 1100 series, Agilent Technology) delivered the/v) methanol/0.1% (v/v) TFA at a flow rate ofL/minute as the loading and washing buffernt Ia). After the column was flushed with the load-ffer for 1.8 minutes, the valve switched to the injec-osition (position B) to inject the sample into the LC. At 5 minutes after injection (Table 1), the valve
witched back to position A, and the trap columnashed with a mobile phase (eluent I) with a gradient
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100% solvent Ia to 100% solvent Ib (75% methanol/TFA; see Table 1), followed by 100% solvent Ia forinutes for equilibration of the column andration for the next analysis. The total run time3 minutes.uid chromatography. After automatic sample clean-e Table 1 at the 1.8-minute time point), the sampleutomatically transferred onto a C18 column (250 ×i.d., 5 μm, ODS-3, Inertsil). The mobile phase was

/v) methanol containing 0.1% (v/v) formicFA; solvent IIa) and was delivered at a flow rateμL/minute. At 7.0 minutes after injection, the mo-hase was varied to 83% solvent IIa for 2 minutes,ed by 100% solvent IIb (50% methanol/0.1% FA)minutes and rapidly back to 100% solvent IIa.

ctrospray ionization MS/MS. The sample elutingthe HPLC system was introduced into a TurboIon-source installed on an API 3000 triple-quadrupolespectrometer (Applied Biosystems), operated inve mode with a needle voltage of 5.5 kV, nitrogen

Figure 1. Chemical structure and tandem mass spectrometry paramete

nebulizing gas, and turbogas temperature set at. Data acquisition and quantitative processing were

statisttest w

acrjournals.org


plished with Analyst software, ver. 1.4 (Appliedstems). For all analytes, the [M+H]+ ion was select-the first mass filter. After collisional activation, twoent ions were selected: the most abundant fragmentas used for quantification (quantifier ion), and thed most abundant ion was used for qualificationfier ion). For the stable isotope-labeled internal stan-only one fragment ion was selected. The chemicalure and optimization results for each analyte inle reaction monitoring scan mode are given in Fig. 1.e parameters were as follows: nebulizer gas flow, 10;in gas flow, 10; collision-assisted-dissociationow, 12; turbo gas flow, 8. Peak full-width at half-um was set to 0.7 Th (Thomson = 1 amu per unit

e) for both Q1 and Q3.

tical methodsan and SD were used to describe the distributionsinary metabolites and the demographic data forsubjects. The data were analyzed using the SAS

five urinary metabolites of areca nut/tobacco alkaloids.

ical package (SAS, version 9.1). Mann-Whitney Uas used to compare the continuous variables




Hu et al.

Cancer Epidemiol Biomarkers Prev; 19(10) October 2010 Cancer Epidemiology, Biomarkers & Prevention2574

on August 18, 2018. © 2010 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from



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g groups. Spearman correlation coefficients wereto study the relationship of urinary metabolitesntrations to self-reported daily areca nut (or ciga-consumption. Multiple linear regression modelsused to investigate the relationship of urinaryolites concentrations to self-reported daily arecar cigarette) consumption after adjusting for otherles (i.e., age and BMI).

lts

ne SPE LC-MS/MS analysis of five urinaryolites of areca nut/tobacco alkaloids inn urineomatography and mass spectra. A typical on-lineC-MS/MS chromatogram for five urinary metabo-nd their isotope internal standards of an areca nutr also cigarette smoker is shown in Fig. 2. Theion time of arecoline was 9.9 minutes. The positivespray ionization (ESI) mass spectrum of arecolinened a [M+H]+ precursor ion at m/z 156 and prod-ns at m/z 44 (quantifier ion, Fig. 2A) and m/z 113fier ion, Fig. 2B) due to loss of C6H8O2 or C2H5N;cursor ion at m/z 159 and product ion at m/z 47cterized the arecoline-d3 (Fig. 2C). For arecaidine,tention time was 7.2 minutes. The [M+H]+ precur-n of arecaidine was at m/z 142 and product ionsred at m/z 44 (quantifier ion, Fig. 2D) and m/z 99fier ion, Fig. 2E), resulting from the loss of C5H6O2

5N; a precursor ion at m/z 145 and product ion at7 characterized the arecaidine-d3 (Fig. 2F). Mean-, the retention time for N-methylnipecotic acid.4 minutes. Its [M+H]+ precursor ion was at4 and product ions appeared at m/z 98 (quantifierig. 2G) and m/z 126 (qualifier ion, Fig. 2H)d by the loss of CH2O2 or H2O; a precursor ion147 and product ion at m/z 73 characterized the

thylnipecotic acid-d3 (Fig. 2I). Because seriousting interference was observed at the first andd most abundant fragment ions (m/z 101 anda third abundant fragment ion (m/z 73) was used-methylnipecotic acid-d3. In terms of nicotine, theion time was 9.1 minutes. However, a slight differ-n retention time between the analyte (9.1 minutes)ts deuterated internal standard (8.9 minutes) wased. The retention time difference could be attrib-to the altered hydrophilic nature of the nicotineal standard labeled with four deuterium atoms,was known as “deuterium isotope effect” duringed phase LC separation (24). The [M+H]+ precur-
n of nicotine was at m/z 163 and gave product ions not d
-d4; m/z 177→80 (M) and m/z 177→98 (N) for cotinine and m/z 180→80 (O) for co

acrjournals.org


ig. 2K) corresponding to loss of the CH5N or; a precursor ion at m/z 167 and product ion at

36 characterized the nicotine-d4 (Fig. 2L). For coti-the retention time was 10.4 minutes. The [M+H]+

rsor ion of cotinine was at m/z 177 and its productere at m/z 80 (quantifier ion, Fig. 2M) and m/z 98

ifier ion, Fig. 2N) corresponding to loss of theNO or C5H5N; a precursor ion at m/z 180 andct ion at m/z 80 characterized the cotinine-d3

2O). The transitions obtained for the ESI/MS-MSsis of nicotine and cotinine were in agreement withpreviously reported (17, 25, 26).it of quantification and limit of detection. The limitntification was defined as the lowest concentrationne that could be reliably and reproducibly mea-with values for accuracy, intraday imprecision,terday imprecision <20%. Using the present meth-e limits of quantification were determined to be0.30, 0.09, 1.80, and 0.17 ng/mL on column (2.5,5, 90 and 8.5 pg in an injection volume of 50 μL)ecoline, arecaidine, N-methylnipecotic acid, nico-nd cotinine, respectively, based on direct measure-of diluted calibration solutions. The limits ofion (LOD) in urine, defined as the lowest concen-n that gave a signal-to-noise ratio of at least 3,0.016, 0.078, 0.037, 0.553, and 0.028 ng/mL on col-(0.8, 3.9, 1.85, 27.7, and 1.4 pg) for arecoline, arecai-N-methylnipecotic acid, nicotine, and cotinine,tively.earity, precision, accuracy, and recovery. Two linearation curves covering the low concentration range-0.375 ng for arecoline, arecaidine,N-methylnipecoticand cotinine, and 0.094-1.5 ng for nicotine) and theoncentration range (0.375-24 ng for arecoline, arecai-N-methylnipecotic acid, and cotinine, and 1.5-24 ngotine) were obtained by serial dilution of calibratorsdrug-free urine. Each calibrator contained 2 ngof arecoline-d3, arecaidine-d3, N-methylnipecotic3, and cotinine-d3 and 4 ng of nicotine-d4. Linearsion was calculated with nonweighting and non-forced, and the linear equations of each analytemmarized in Table 2. The correlation coefficientsbtained were >0.99 in all cases. Over the entirentration range of the calibration curves, the meanved percentage deviation of back-calculated concen-ns was between −15.2% and +9.6% with an impreci-CV) <15%. For each metabolite in urine, the peakty was also confirmed by comparing the peak area(quantifier/qualifier) with those of the calibrators.acceptance criterion, ratios in urine samples should
eviate by more than ± 25% from the mean ratios in
132 (quantifier ion, Fig. 2J) and m/z 106 (qualifier the calibrators.

2. Chromatograms of five urinary metabolites of areca nut/tobacco alkaloids of a regular areca nut chewer who is also a smoker using/MS coupled with on-line SPE. Multiple reaction monitoring transitions of m/z 156→44 (A) and m/z 156→113 (B) for arecoline and m/z 159→47 (C) fore-d3; m/z 142→44 (D) and m/z 142→99 (E) for arecaidine and m/z 145→47 (F) for arecaidine-d3; m/z 144→98 (G) and m/z 144→126 (H) forylnipecotic acid and m/z 147→73 (I) for N-methylnipecotic acid-d3; m/z 163→132 (J) and m/z 163→106 (K) for nicotine and m/z 167→136 (L) for

tinine-d3.




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Lin r2 Intra

Urine Urin Urin

Arecol L: y = 0.9996 26.8 (1 102.0 512.4H: y = 0.9992 4.1‡ 1.4 1.

Arecai L: y = 0.9991 26.1 (1 109.2 491.0H: y = 0.9999 6.1 1.9 1.

N-met L: y = 0.9987 23.7 (1 100.1 481.2H: y = 0.9994 7.2 4.6 3.

Nicotin L: y = 0.9994 27.0 (2 106.0 500.0H: y = 0.9980 8.5 2.8 1.

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precision and accuracy of the present methodevaluated by spiking drug-free urine sample witheled standard mixture at three different concentra-(25 ng/mL for urine 1, 100 ng/mL for urine 2,00 ng/mL for urine 3; each standard mixturened equal amounts of each analyte) and repeated-asuring the five analytes in these three urineles. The intraday and interday CVs were 1.2%% and 1.0% to 8.1%, respectively. Intraday anday accuracy were 95% to 109% and 94% torespectively.overy was evaluated by adding unlabeled stan-mixture at five concentrations (6.25, 12.5, 50, 100,g/mL) to a urine sample that initially contained/mL of each analyte (urine 1), and measuringreplicates of these samples. As shown in Table 2,ean recoveries were 97% to 114% as estimatedthe increase in the measured concentration afterion of the analyte divided by the concentrationas added, whereas the recoveries as calculatedthe slope of the regression were 95% to 104%.99).trix effects. Matrix effects were calculated fromeak areas of the internal standard added to theard mixture solutions (prepared in 2% methanolining 0.1% TFA) and compared with the peakof the internal standard that was added to eachry sample. The relative change in peak area ofternal standard was attributed to matrix effects,reflect both on-line extraction losses and ion

ession due to the urinary matrix. In this study,atrix effects for five metabolites were <30%urine samples. Although the use of stable iso-abeled internal standards could have compensat-r different matrix effects, a low matrix effect

(Continued on the fo

ved in this study ensures a high sensitivity ofethod (27).

of thrmulti



ry excretion of five metabolites of arecaobacco alkaloidsotal of 90 healthy subjects were recruited into the-sectional study, including 31 non–areca nuters also nonsmokers, 26 non–areca nut chewersgular cigarette smokers, and 33 regular areca nuters also cigarette smokers. The characteristics ofrticipants and the five urinary metabolites concen-ns are summarized in Table 3. The three groupssimilar in age and BMI (P > 0.05, by Mann-Whitneyt). As for the urinary metabolites adjusted for uri-reatinine, the group of non–areca nut chewers alsookers had mean urinary nicotine and cotinine con-tions of 0.9 and 3.1 ng/mg creatinine, respectively,ondetectable concentrations of areca nut alkaloids.roup of smokers but nonchewers had a self-ted mean consumption of 17.6 cigarettes/day andurinary nicotine and cotinine of 1,514 and

g/mg creatinine, respectively, and nondetectablentrations of areca nut alkaloids. For the group ofnut chewers also smokers, they reported meanconsumptions of 23.0 cigarettes and 28.0 arecand had mean urinary concentrations of arecoline,dine, N-methylnipecotic acid, nicotine, and cotinine9, 5,816, 1,298, 2,635, and 1,406 ng/mg creatinine,tively. The association between self-reported dailynut (or cigarette) consumption and the correspondingry metabolites was further analyzed using Spear-orrelation coefficients. The urinary concentrationscoline, arecaidine, and N-methylnipecotic acid ass the sum of these three metabolites were foundassociated with the self-reported number of arecaewed per day (n = 33; r = 0.71, P < 0.01 for areco-= 0.59, P < 0.01 for arecaidine; r = 0.56, P < 0.01 forthylnipecotic acid; and r = 0.60, P < 0.01 for the sum

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y metabolites of areca nut alkaloids and daily con-tion of areca nut were not confounded by otherbles, including age and BMI (P < 0.01). For thery metabolites of tobacco alkaloids, there was noation between urinary nicotine or cotinine (or thef nicotine and cotinine) and the self-reported dailytte consumption for the smokers (n = 59; r = 0.05,.73 for nicotine; r = 0.24, P = 0.07 for cotinine; and08, P = 0.53 for the sum of the two metabolites).ple linear regression analysis also revealed no sig-nt correlation between urinary nicotine metabolitesaily cigarette consumption after adjustment for ageMI (P = 0.67).

course of three metabolites of arecalkaloids in human urinee male volunteers were each orally administeredof water extract of areca nuts, and urine samples

collected at 0 (predose), 2, 4, 6, 8, 10, 12, 14, 17, 24,d 33 hours after dosing. The results of LC-MS/MSsis revealed that 20 mL of water extract of arecarepresenting two areca nuts) contained 1,838 μgμmole) of arecoline, 968 μg (6.87 μmole) of are-e, and 82 μg (0.57 μmole) of N-methylnipecoticSurprisingly, areca nut itself also contained N-ylnipecotic acid, which has not been reportedously. Figure 3 shows rapid formations of areca

covery was estimated from the slope of the regression of the me

kaloids in urine after the administration of water ex-f areca nuts. The levels of arecoline (Fig. 3A) and

regreCorre

acrjournals.org


dine (Fig. 3B) were dramatically increased from 0highest concentrations of 1.84 and 1,306 ng/mg cre-e, respectively, within 2 to 4 hours and decreasedally. By 12 hours after administration, the levels ofine were nondetectable in all urine samples whereasean level of arecaidine decreased by almost 93%ng/mg creatinine at 12 hours) and remained de-d throughout the experiment (8.49 ng/mg creati-at 33 hours). In terms of N-methylnipecotic acidC), the mean level was significantly increased fromhe highest concentration of 304.5 ng/mg creatinine6 hours. The mean level of N-methylnipecotic acid

hen decreased by 41% (179.2 ng/mg creatinine) atrs and slightly increased again at 10 hours andased gradually thereafter. By 33 hours, the meanof N-methylnipecotic acid was decreased by 92%ng/mg creatinine). Furthermore, by the end of theiment, it was found that the major urinary metabo-as arecaidine with a total excretion of 4.3 toole, followed by N-methylnipecotic acid of 1.3-ole and arecoline of 0.004-0.008 μmole for five

volunteers.the urinary half-life of each metabolite, semiloga-ic mean urinary excretion concentrations versusurves were constructed for each metabolite excre-data not shown). The urinary elimination rateant of each metabolite was calculated by linear

d concentration versus the added concentration.

2. Linearity, precision and recovery (Cont'd)

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Cancer Epidemiol Biomarkers Prev; 19(10) Oc

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reviations: L, low; H, high.g-free pooled urine samples were individually spiked with unlabeled standard mixture (containing equal amounts of eachlyte) at three different concentrations (25 ng/mL for urine 1, 100 ng/mL for urine 2, and 500 ng/mL for urine 3). Each urinelysis was repeated five times for the intraday and interday tests; the interday test was carried out over a period of 50 days.covery of the analytes in urine was estimated by the addition of unlabeled standard mixture at five different concentrations5, 12.5, 50, 100, 200 ng/mL) to a urine sample (urine 1). The recovery was estimated from the increase in measured concen-on after addition of the analyte divided by the concentration that was added., %.

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metabolites ranged from 0.96 to 0.99, indicating afit of the first-order reactions. The correspondingry half-life of elimination (t1/2 of elimination)alculated from the elimination rate constant (k) ac-g to the equation: t1/2 of elimination = 0.693/k. The
ves of arecoline, arecaidine, and N-methylnipecotic (manu
detectrespecet al.limitsrespeof arefocuseet al.LC-Mextrac

ere found to be 0.97, 4.3, and 7.9 hours, respectively.

ssion

have developed a rapid, specific, and sensitivepe-dilution LC-MS/MS method incorporatinge SPE and isotopic internal standards that canltaneously detect five urinary metabolites ofnut/tobacco alkaloids with the LODs of 0.016 to
ng/mL on column (0.8-27.7 pg) and a total analysiser sample as short as 13 minutes.
reportively.



-MS/MS has received a great deal of attentionyears because it can provide a sensitive andive means for comprehensive measurement ofple metabolites. Tuomi et al. (29) and Xu et al. (30)ibed LC-MS/MS methods involving an off-lineal) SPE cleanup for nicotine and cotinine and hadion limits of 1 to 10 ng/mL and 0.1 to 1 ng/mL,tively. Similarly, the method described by Heavner(31) involved also an off-line SPE and had detectionof 4.4 and 3.7 ng/mL for nicotine and cotinine,ctively. In terms of the metabolite measurementca nut alkaloids, previous methods were mostlyd on the quantification of arecoline alone. Pichini(18) and Zhu et al. (32) developed the ion trapS methods following a two-step liquid-liquidtion purification or off-line SPE purification, and

3. Overall
teristics of the study parti
ted LODs of 0.4 and 8 ng/mL fApparently, the method establi

Cancer Epidemiology, Bioma

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reca nut chewers alsononsmokers
reca nut chewers butigarette smokers

nut chewers alsoarette smokers
c cig
31
26 33 ears n (SD) 43.0 (13.2) 43.6 (11.1) 37.9 (9.6) ge 21-63 25-62 24-59 g/m2
n (SD) 25.1 (3.4) 23.4 (3.2) 24.5 (4.1)
ge 17.4-31.6 18.6-29.4 17.4-29.3 ttes/day n (SD) 0 17.6 (9.3) 23.0 (11.0) ge 5-40 5-60 nuts/day n (SD) 0 0 28.0 (23.8) ge 3-100 ine, ng/mg cre atininen (SD) ND ND 23.9 (39.3) ge ND-141.8* dine, ng/mg cr eatininen (SD) ND ND 5,816 (12,541) ge 7.2-66,053 hylnipecotic ac creatinine id ng/mgn (SD) ND ND 1,298 (2,580) ge 0.8-13,833 e, ng/mg crea tininen (SD) 0.9 (1.8) 1,514 (1,330) 2,635 (3,078) ge ND-7.8† 173-5,514 0.6-12,563 e, ng/mg crea inin tinine
ean (SD) 3.1 (3.0) 820 (460) 1,406 (1,496)ange ND-10† 314-2,406 41.9-6,423

reviation: ND, not detectable.e out of 33 (3%) urine samples had a nondetectable level of arecoline.

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which involves on-line sample cleanup/purifica-oupled to isotope dilution LC-MS/MS, has lower(0.553 ng/mL for nicotine, 0.028 ng/mL for

ne, and 0.016 ng/mL for arecoline) than these tool toin resAlt

arecaaboubest orodenline incomp1,000havetaboliGiri emetab6 of tAmonfoundarecaiInterearecaiurineand FIn t

sampamoumL) aconceworthlow tchewenuts (relativtectabFig. 3tweenthe coarecathreelites asessinthe uthe mtionssimilaof >30atininmetabcant csumpsum opossibof succigare(e.g., 0the caaidine. C, N-methylnipecotic acid. Points, mean; bars, SE.

acrjournals.org


d provides direct and simultaneous determinationjor urinary metabolites of both areca nut and tobac-aloids, which would be used as a high-throughputmonitor the subjects with either one or both habits

earch and clinical practice.hough there are as many as 600 million chewers ofnut products worldwide, relatively little is knownt the metabolism of areca nut alkaloids. To thef our knowledge, in the past 40 years, only severalt studies (9, 33, 34) and one clinical trial with areco-Alzheimer patients (16) have been reported. This

ares very poorly with nicotine, for which more thanpapers have been published and 24 metabolitesbeen identified (11). One of the most significant me-sm studies of areca nut alkaloids in rodent is that oft al. (9). With the use of a metabolomic approach, 11olites of arecoline were identified in mice urine andhese metabolites were shared with arecaidine.g these metabolites, N-methylnipecotic acid wasto be the major metabolite of both arecoline anddine after arecoline (or arecaidine) administration.stingly, in this study, N-methylnipecotic acid anddine were for the first time identified in the humanof areca nut chewers in addition to arecoline (Table 3ig. 3).he cross-sectional study (Table 3), all of the urineles from areca nut chewers presented quantifiablents of N-methylnipecotic acid (range, 1.9-21,534 ng/s well as arecaidine (4.6-102,800 ng/mL) with thentrations well above the LODs of our method. It isnoting that the urine samples contained relatively

o nondetectable levels of arecoline for areca nutrs although arecoline is the major alkaloid in areca6). This may be due to the fact that arecoline has aely short half-life in urine and could be barely de-le after 12 hours of oral administration, as shown in. Moreover, there were significant correlations be-self-reported daily areca nut consumption andrresponding urinary metabolites (i.e., arecoline,idine, and N-methylnipecotic acid or the sum ofmetabolites), suggesting that these three metabo-re quantitatively representative biomarkers for as-g the exposure to areca nut alkaloids. In terms ofrinary nicotine/cotinine measurement (Table 3),ean values of the nicotine and cotinine concentra-for the groups of nonsmokers and smokers arer with previously reported ranges (e.g., nicotineng/mg creatinine and cotinine of >100 ng/mg cre-e for smokers; refs. 13, 35). However, unlike urinaryolites of areca nut alkaloids, there was no signifi-orrelation between self-reported daily cigarette con-tion and urinary nicotine or cotinine as well as thef these two metabolites for smokers. Despite theility of recall bias in questionnaire studies, the lackh a correlation might be because nicotine found intte smoke varies substantially from brand to brand
.1-2.3 mg/cigarette; ref. 36); however, it may not be
ously reported methods. More importantly, our

3. Time course of three urinary metabolites of areca nut alkaloidsministration of water extract of areca nuts. A, arecoline.

se for areca nuts because the alkaloids contents




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quite similar in the commercial-size unripe arecae.g., ∼7 mg/g of arecoline; ref. 37). Moreover, avariability in smoking behavior (e.g., puffing vol-nd duration and/or inhalation depth) may haveed the nicotine intake per cigarette smoked (38,nd this most likely could have interfered withrrelation analysis between self-reported cigarettemption and urinary nicotine or cotinine.vious studies have shown that arecoline readilys the blood-brain barrier and metabolizes rapidly.95 minute in plasma) after its i.v. administrationjects with Alzheimer's disease (16). In this study,sults of time course measurement also revealed ametabolism of this compound after oral adminis-n of areca nut water extracts with an estimatedife of 0.97 hour in urine. Meanwhile, relativelyr half-lives were obtained for arecaidine (t1/2urs) and N-methylnipecotic acid (t1/2 7.9 hours).ncreasing half-lives of arecoline < arecaidine <thylnipecotic acid estimated in this study couldr support the existence of a metabolic pathwayfied in a mouse model by Giri et al. (9), in whichine is firstly hydrolyzed to arecaidine and then un-es carbon-carbon double-bond reduction to yieldthylnipecotic acid.thermore, the results of the time course study alsoed that arecaidine was the major metabolite inn urine after administration of areca nut water ex-, composing 23% to 33%, of the dose administratedlculated by total excreted arecaidine divided bymount of arecoline and arecaidine in the water ex-f areca nuts), followed by N-methylnipecotic acidto 15% (total excreted N-methylnipecotic acid di-

by total amount of three areca nut alkaloids in theextract) and arecoline at 0.03% to 0.07% (total ex-arecoline divided by total arecoline in the watert). Our finding was slightly different from the pre-study in mouse (9), showing thatN-methylnipecoticas the predominant metabolite of arecoline or

dine (composing up to 30-38% of the dose). Suchsistency could be partially explained by the inter-s differences in enzyme activity (e.g., reducingn-carbon double bonds of arecaidine to formthylnipecotic acid) between mouse and human.onclusion, this study describes a simple, rapid, and
le LC-MS/MS method for direct determination of Rece
iew of agents and causative mechanisms. Mutagenesis 2004;251–62.ernational Agency for Research on Cancer. Tobacco habits other

thanitrisk

5. Intaremo85

6. Loan



ids. When combined with on-line SPE and isotopeon, this method could allow for high-throughputsis of urinary metabolites without compromisingy and validation criteria. Three urinary metabolitesca nut alkaloids, namely, arecoline, arecaidine, andthylnipecotic acid, were firstly shown in the urinesular chewers and were found to be highly correlatedthe self-reported daily consumption of areca nuts.correlations may further illustrate the adequacythree metabolites as biomarkers for assessing ex-e to areca nuts. Interestingly, however, our resultse course study showed that for arecoline, being theabundant alkaloid in areca nut, very little was ex-unchanged (0.03-0.07% of the dose) with a shortfe of 0.97 hour after administration of areca nutsextracts. This finding indicates that arecoline aloneot be a suitable biomarker and the information ofst may have been missed owing to its rapid elimi-. Therefore, from the point of view of practical

cation, simultaneous determination of arecoline,idine, and N-methylnipecotic acid (together com-g 29-47% of the dose administrated) could providere comprehensive evaluation of areca nuts expo-Epidemiologic studies have shown that most arecahewers also have tobacco-chewing or cigarette-ing habits. The combination of both habits has beenn to dramatically increase the risk of developingancer up to ∼90 times (40). Our method for deter-ion of three metabolites of areca nut alkaloids to-r with nicotine and cotinine may facilitate therch into the oncogenic effects of both areca nutobacco exposure.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

thank the National Science Council, Taiwan, for financial supportNSC 97-2314-B-040-017-MY2 and NSC 97-2314-B-040-015-MY3),ih-Ming Chen and Wei-Lin Cheng for help in sample preparation.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 05/07/2010; revised 06/18/2010; accepted 07/06/2010;
ajor urinary metabolites of areca nut and tobacco published OnlineFirst 09/14/2010.
rencesnstock A. Areca nut-abuse liability, dependence and public health.dict Biol 2002;7:133–8.pta PC, Ray CS. Epidemiology of betel quid usage. Ann Acadd 2004;33:31–6.ir U, Bartsch H, Nair J. Alert for an epidemic of oral cancer dueuse of the betel quid substitutes gutkha and pan masala: a

n smoking; betel-quid and areca-nut chewing; and some relatedrosamines. IARC monographs on the evaluation of carcinogenics to humans. Vol. 37. Lyon: IARC; 1985.ernational Agency for Research on Cancer. Betel-quid andca-nut chewing and some areca-nut-derived nitrosamines. IARCnographs on the evaluation of carcinogenic risks to humans. Vol.
. Lyon: IARC; 2004.rd GA, Lim CK, Warnakulasuriya S, Peters TJ. Chemical andalytical aspects of areca nut. Addict Biol 2002;7:99–102.
Cancer Epidemiology, Biomarkers & Prevention



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u NS. Neurological aspects of areca and betel chewing. Addictl 2002;7:111–4.i YS, Lee KW, Huang JL, et al. Arecoline, a major alkaloid ofca nut, inhibits p53, represses DNA repair, and triggers DNAmage response in human epithelial cells. Toxicology 2008;249:–7.i S, Idle JR, Chen C, Zabriskie TM, Krausz KW, Gonzalez FJ. Atabolomic approach to the metabolism of the areca nut alkaloidscoline and arecaidine in the mouse. Chem Res Toxicol 2006;19:8–27.ctor RN. The global smoking epidemic: a history and statusort. Clin Lung Cancer 2004;5:371–6.kkanen J, Jacob P III, Benowitz NL. Metabolism and dispositionetics of nicotine. Pharmacol Rev 2005;57:79–115.tsuki H, Hashimoto K, Arashidani K, et al. Studies on a simulta-us analytical method of urinary nicotine and its metabolites, andir half-lives in urine. J UOEH 2008;30:235–52.inrich J, Hölscher B, Seiwert M, Carty CL, Merkel G, Schulz C.otine and cotinine in adults' urine: The German Environmentalrvey 1998. J Expo Anal Environ Epidemiol 2005;15:74–80.tersen GO, Leite CE, Chatkin JM, Thiesen FV. Cotinine as a bio-rker of tobacco exposure: development of a HPLC method andparison of matrices. J Sep Sci 2010;33:516–21.

riharan M, VanNoord T. Liquid-chromatographic determination ofotine and cotinine in urine from passive smokers: comparison withs chromatography with a nitrogen-specific detector. Clin Chem1;37:1276–80.

thana S, Greig NH, Holloway HW, et al. Clinical pharmacokineticsarecoline in subjects with Alzheimer's disease. Clin Pharmacoler 1996;60:276–82.ofnagle AN, Laha TJ, Rainey PM, Sadrzadeh SM. Specific detec-of anabasine, nicotine, and nicotine metabolites in urine by liquidomatography-tandem mass spectrometry. Am J Clin Pathol 2006;:880–7.hini S, Pellegrini M, Pacifici R, et al. Quantification of arecoline (are-nut alkaloid) in neonatal biological matrices by high-performanceid chromatography/electrospray quadrupole mass spectrometry.pid Commun Mass Spectrom 2003;17:1958–64.gh R, Farmer PB. Liquid chromatography–electrospray ionization–ss spectrometry: the future of DNA adduct detection. Carcinogen-s 2006;27:178–96.CW, Wang CJ, Chang LW, Chao MR. Clinical-scale high-throughputlysis of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-tion liquid chromatography–tandem mass spectrometry with on-lineid-phase extraction. Clin Chem 2006;52:1381–8.CW, Chao MR, Sie CH. Urinary analysis of 8-oxo-7,8-ydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine bytope-dilution LC-MS/MS with automated solid-phase extraction:udy of 8-oxo-7,8-dihydroguanine stability. Free Radic Biol Med10;48:89–97.CH, Ko YC, Huang HL, et al. The precancer risk of betel quidwing, tobacco use and alcohol consumption in oral leukoplakiaoral submucous fibrosis in southern Taiwan. Br J Cancer 2003;

:366–72.ng YD. Simultaneous determination of creatine, uric acid, creati-
e and hippuric acid in urine by high performance liquid chroma-raphy. Biomed Chromatogr 1998;12:47–9.ng S, Cyronak M, Yang E. Does a stable isotopically labeled
40. Kochca

acrjournals.org


ernal standard always correct analyte response? A matrix effectdy on a LC/MS/MS method for the determination of carvedilolantiomers in human plasma. J Pharm Biomed Anal 2007;43:701–7.

I, Huestis MA. A validated method for the determination ofotine, cotinine, trans-3′-hydroxycotinine, and norcotinine in humansma using solid-phase extraction and liquid chromatography-ospheric pressure chemical ionization-mass spectrometry.ass Spectrom 2006;41:815–21.llegrini M, Marchei E, Rossi S, et al. Liquid chromatography/ctrospray ionization tandem mass spectrometry assay for deter-nation of nicotine and metabolites, caffeine and arecoline inast milk. Rapid Commun Mass Spectrom 2007;21:2693–703.kvis E, Rosing H, Beijnen JH. Stable isotopically labeled internalndards in quantitative bioanalysis using liquid chromatography/ss spectrometry: necessity or not? Rapid Commun Mass Spec-m 2005;19:401–7.RooijBM,BoogaardPJ,RijksenDA,CommandeurJN,VermeulenNP.naryexcretionofN-acetyl-S-allyl-L-cysteineupongarlicconsumptionhuman volunteers. Arch Toxicol 1996;70:635–9.omi T, Johnsson T, Reijula K. Analysis of nicotine, 3-hydroxycotinine,tinine, and caffeine in urine of passive smokers by HPLC-tandemss spectrometry. Clin Chem 1999;45:2164–72.X, Iba MM, Weisel CP. Simultaneous and sensitive measurementanabasine, nicotine, and nicotine metabolites in human urine byuid chromatography-tandem mass spectrometry. Clin Chem04;50:2323–30.avner DL, Richardson JD, Morgan WT, Ogden MW. Validation andplication of a method for the determination of nicotine and fivejor metabolites in smokers' urine by solid-phase extraction andid chromatography-tandem mass spectrometry. Biomed Chro-togr 2005;19:312–28.u MM, Chen HX, Han FM, Chen Y. Analysis of arecoline in rat urine,d. identification of its metabolites by liquid chromatography–dem mass spectrometry. Chromatographia 2006;64:705–8.ry R. The metabolic interconversion of arecoline and arecolinexide in the rat. Biochem J 1971;122:503–8.ri S, Krausz KW, Idle JR, Gonzalez FJ. The metabolomics of−)-arecoline 1-oxide in the mouse and its formation by humanvin-containing monooxygenases. Biochem Pharmacol 2007;73:1–73.Weerd S, Thomas CM, Kuster JE, Cikot RJ, Steegers EA. Varia-n of serum and urine cotinine in passive and active smokers andplicability in preconceptional smoking cessation counseling. Envi-Res 2002;90:119–24.Y, Morimoto K. Exposure level to cigarette tar or nicotine is asso-ted with leukocyte DNA damage in male Japanese smokers.tagenesis 2008;23:451–5.ng CK, Lee WH, Peng CH. Contents of phenolics and alkaloids inca. catechu Linn. during maturation. J Agric Food Chem 1997;45:85–8.nowitz NL, Jacob P III, Kozlowski LT, Yu L. Influence of smokinger cigarettes on exposure to tar, nicotine, and carbon monoxide.ngl J Med 1986;315:1310–3.herer G. Smoking behaviour and compensation: a review of therature. Psychopharmacology (Berl) 1999;145:1–20.YC, Huang YL, Lee CH, Chen MJ, Lin LM, Tsai CC. Betel quid

ewing, cigarette smoking and alcohol consumption related to oralncer in Taiwan. J Oral Pathol Med 1995;24:450–3.




2010;19:2570-2581. Published OnlineFirst August 16, 2010.Cancer Epidemiol Biomarkers Prev Chiung-Wen Hu, Yan-Zin Chang, Hsiao-Wen Wang, et al. Spectrometry with On-Line Solid-Phase ExtractionIsotope-Dilution Liquid Chromatography-Tandem MassMetabolites of Areca Nut and Tobacco Alkaloids by High-Throughput Simultaneous Analysis of Five Urinary

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