research article estimation of cutoff values of cotinine...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 386784 11 pageshttpdxdoiorg1011552013386784

Research ArticleEstimation of Cutoff Values of Cotinine in Urine and Saliva forPregnant Women in Poland

Joanna Stragierowicz1 Karolina MikoBajewska1 Marta Zawadzka-Stolarz2

Kinga PolaNska3 and Danuta Ligocka1

1 Department of Toxicology and Carcinogenesis Nofer Institute of Occupational Medicine 8 Teresy Street 91-348 Lodz Poland2Nofer Institute of Occupational Medicine 8 Teresy Street 91-348 Lodz Poland3Department of Environmental Epidemiology Nofer Institute of Occupational Medicine 8 Teresy Street 91-348 Lodz Poland

Correspondence should be addressed to Danuta Ligocka ligockaimplodzpl

Received 26 April 2013 Accepted 16 September 2013

Academic Editor Peter P Egeghy

Copyright copy 2013 Joanna Stragierowicz et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Setting appropriate cutoff values and the use of a highly sensitive analytical method allow for correct classification of the smokingstatus Urine-saliva pairs samples of pregnant women in the second and third trimester and saliva only in the first trimester werecollected Offline SPE and LC-ESI-MSMS method was developed in the broad concentration range (saliva 04ndash1000 ngmL urine08ndash4000 ngmL)The mean recoveries were 37 plusmn 76 for urine and 991 plusmn 26 for saliva LOD for saliva was 012 ngmL and forurine 005 ngmL LOQ was 04 ngmL and 08 ngmL respectively Intraday and interday precision equaled respectively 12 and34 for urine and 23 and 64 for salivaThere was a strong correlation between salivary cotinine and the uncorrected cotinineconcentration in urine in the second and third trimesters of pregnancyThe cutoff values were established for saliva 129 ngmL andurine 423 ngmL or 531 120583gg creatinine with the ROC curve analysisThe developed analytical method was successfully applied toquantify cotinine and a significant correlation between the urinary and salivary cotinine levels was found The presented cut-offvalues for salivary and urinary cotinine ensure a categorization of the smoking status among pregnant women that is more accuratethan self-reporting

1 Introduction

The most commonly used biomarker of exposure to tobaccosmoke is cotinine as a metabolite of nicotine The measure-ment of the cotinine concentration in various biological fluidsis directly proportional to the degree of exposure to nicotine[1] The determination of cotinine is recommended for theassessment of active tobacco smoking monitoring of envi-ronmental tobacco smoke (ETS) exposure and impact evalu-ation of smoking cessation programs [2]Themost importantadvantage of using cotinine as a biomarker of tobacco smokeand ETS is the fact that about 72 of nicotine is convertedto cotinine [3] and the half-life of cotinine averages about17 hours in comparison to the one averaging 2-3 h in caseof nicotine [4] The total nicotine content in tobacco (by

weight of tobacco) averaged 102mg [5] while the nicotineintake per cigarette averaged 104mg [6] representing about10

According to the Global Adult Tobacco Survey (GATS)in Poland in 2009 approximately 24 of women aged 15ndash49years were smokers (out of whom 21 were daily cigarettesmokers) [7] Based on the Pregnancy-related AssessmentMonitoring Survey (PrAMS) the most recent results showthat evenmore than 12 of pregnant women in Poland smoke[8]

The effect of tobacco smoking is not limited to the one ofnicotine which is responsible for addiction to smokingbut also involves the influence of various toxic substancesreleased from burning cigarettes like carbon monoxidePAHs heterocyclic compounds N-nitrosoamines aromatic

2 BioMed Research International

amines N-heterocyclic amines aldehydes or volatile hydro-carbons among which 69 are known carcinogens [9ndash11] Themost important adverse health effects associated with mater-nal cigarette smoking are premature rupture of membranesplacental abruption or preeclampsia [12] uteroplacentalinsufficiency and reducing the blood flow to the fetusMaternal smokingmay also result in lower birth weight of thenewborns [11] Based on the analysis performed in Poland inthe newborns prenatally exposed to ETS the birthweight waslower by 335 g plusmn 903 than that in the case of the nonexposednewborns (119875 lt 0001) after adjustment for maternal edu-cational level marital status prepregnancy weight childgender and gestational age [13] Maternal nicotine exposuremay cause changes in the development and maturing of theoffspringrsquos lungs which can result in the organ being moresusceptible to disease and likely to manifest reduced lungfunction [14] Smoking during pregnancy may have long-term consequences on the neurobehavioral development ofchildren [8]

Due to numerous highly adverse effects of smoking dur-ing pregnancy there is a need to monitor the extent of expo-sure spread the knowledge of these effects to the fetus andpromote smoking cessation Evaluation of the smoking statusamong pregnant women is based mainly on a self-reportedquestionnaire However only a confirmation by a laboratoryanalysis may lead to correct and reliable classification sincepregnant women (and not only they) are reluctant to admitthat they smoke

Interindividual variability in the metabolism of nicotineis due to the gender and ethnic differences in the activity ofenzymes (CYP2A6 and UGT1A) and to some extent geneticpolymorphisms of the CYP2A6 gene [15] Like many otherphysiological processes also the metabolism of nicotinechanges during pregnancy The observed variability in themetabolic clearance of cotinine may markedly increase by140 during pregnancy resulting in a half-life shorter bynearly 50 than the one in the nonpregnant state [16]

The explanation of these changes could be the influenceof a higher concentration of estradiol during pregnancy [17]which induces the activity of CYP2A6 responsible for themetabolism of nicotine [18]

As reported by Rebagliato et al [19] the salivary cotininelevel was significantly lower during pregnancy comparedwith the postpartum oneTherefore it is necessary to identifya cutoff value to avoid misclassification of smoking and non-smoking pregnant women

The ROC analysis is increasingly used to determine thecut-off values for biomarkers of exposure to tobacco smoke[20ndash23]

The primary aim of this study was to establish the optimalcut-off value for cotinine in saliva and urine of pregnantwomen in Poland and to compare the diagnostic effectivenessof three smoking tests cotinine in saliva and in urine and inurine with creatinine correction The secondary one was todevelop a sensitive and specific method for determining thecotinine level in urine and saliva in a broad range of con-centrations Finally our aim was also to estimate the utilityof these matrices for both rapid screening used in order toidentify potential smokers and more accurate determination

of the degree of exposure to tobacco smoke especially thatconcerning pregnant women

2 Methods

21 Population From the biobank of the Polish Motherand Child Cohort Study (REPRO PL) saliva-urine samplescollected in trimester II and III of 69 women were selected aswell as the survey data on the smoking status of the pregnantwoman the smoking habit of her husbandpartner and a con-sent to smoking in the apartment In addition each of thesewomen had a saliva sample taken during the first trimesterof pregnancy The complete description of the cohort waspublished elsewhere [24] In short the inclusion criteria weresingle pregnancy up to 12 weeks of gestation no assistedconception no pregnancy complications and no chronicdiseases as specified in the study protocol [24]Themean ageof 69 women was 2641 plusmn 497 years Based on the surveydata it was found that in the first trimester 1969 womenwere smokers and in the second and third trimesters thisratio equaled 1769 Smoking was permitted in 52 of theapartments in the first trimester of pregnancy but in the thirdtrimester such permission to smoke at home decreased byapproximately 9

To find the correlations between the matrices we ana-lyzed saliva and urine samples collected at the same time

22 Standards and Reagents Cotinine (98) internal stand-ard-cotinine-d

3(98) and ammonium acetate (98) were

obtained from Sigma Aldrich Acetonitrile and MethanolUltra Gradient HPLC Grade were supplied by Baker Aceticacid (gt99) was purchased from Fluka Ultrapure water wasobtained fromMilli-Q-Plus Ultra-PureWater System (Milli-pore USA) All working standards of cotinine and cotinine-d3were prepared in acetonitrile and stored at minus20∘C Solid

phase extraction manifold was maintained on Supelco andOASIS HLB LP 96-Well Plate 60120583m (60mg) was supplied byWaters (USA) Control urine lyophilized ClinCheckControlfor toxic organic compounds was purchased from RecipeChemicals (Germany)

23 Biological Samples Collection Saliva was collected frompregnant women into a Salivette with citric acid (SarstedtGermany)The amount of approximately 1-2mL of saliva waseasily obtained by having the women chew a cotton swab atleast 30min after eating or drinking A clear fluid sample wasobtained by centrifuging the Salivette and used for analysisA 50mL volume of morning urine was collected frompregnant women to a 100mL polypropylene container (BenePoland) All saliva and urine samples were transported to thelaboratory in a cool box and stored at minus20∘C until analysis

24 Samples Preparation Urine and saliva samples werethawed before the analysis thoroughly mixed and trans-ferred into 20mL polypropylene tubes The samples werecentrifuged for 10min at 11000 rcf (MIKRO 120 Hettich Zen-trifugen TuttlingenGermany) To 025mLof urine or 05mLof saliva water and 20120583L of internal standard (cotinine-d

3)

were added and mixed vigorously Each well of the Oasis

BioMed Research International 3

Table 1 Validation parameters of the LC-MSMS method of determination of cotinine in saliva and urine

LOD[ngmL]

LOQ[ngmL]

Range of linearity[ngmL]

Correlation coefficient1199032

Uncertainty(119896 = 2) []

Accuracy[] Recoverylowast []

Urine 005 08 08ndash4000 09997 93 427 9372 plusmn 76

Saliva 005 04 04ndash1000 09993 60 233 9910 plusmn 26

lowastAll values are means plusmn SD

HLB extraction plate was prewashedwith 2mL of acetonitrilefollowed by 2mL of water Then the samples were placedon the plate and washed with 10mL of water (in case ofsaliva samples) and 10mL of 20 methanol in water (incase of urine samples) The analytes were eluted with 1mLof acetonitrile and 20120583L of the extract was injected into thechromatographic system

25 Calibration The working solutions were prepared byappropriate dilutions of the standard stock solutions Thestandard stock solutions of 1mg cotinine or cotinine-d

3mL

were further diluted with acetonitrile to obtain the workingsolutions of cotinine (10 120583gmL 400 ngmL and 20 ngmL)and cotinine-d

3(25 120583gmL) Calibration standards were

prepared for saliva at the following concentrations 02 0510 50 10 50 100 200 500 ngmL and for urine 02 05 1050 10 50 100 200 500 1000 ngmL All working solutionswere stored at minus20∘C Calibration curves were generatedusing linear regression with 1X weighting

26 Liquid Chromatography-Mass Spectrometry The chro-matographic separation was performed using the Waters2695 Alliance LC System (Waters USA) on the analyticalcolumn X-Terra MS C 18 35120583m 21 times 150mm (Waters)

The following mobile phase was used A 7 (watercontaining 004 of ammonium acetate with 005 of aceticacid) and B 93 (acetonitrile) with an isocratic mode andflow rate of 02mLmin

The Micromass Quattro Micro API tandem mass spec-trometer (Waters USA) was coupled to the HPLC Alliancesystem The mass spectrometer was operated in the electro-spray positive mode the capillary was kept at 10 kV and thesource temperature was maintained at 130∘C the desolvationgas flow was 600 Lh and the desolvation temperature waskept at 350∘C and the cone energy was 33V and the collisionenergy was 21 eV for both cotinine and cotinine-d

3 The

specific ion transitions for cotinine and cotinine-d3were

monitored in a multiple reaction monitoring mode (MRM)with a dwell time of 03 s

27 Creatinine Correction The creatinine level was deter-mined according to the Jaffe automated method Urinesamples with a creatinine concentration lower than 03 gL orhigher than 30 gL were excluded [25]

28 Statistical Analysis Receiver operating characteristic(ROC) curve analysis was used for three purposes to testthe performance of the diagnostic system (how close to 1 isAUC) to compare the performance of the three tests and toestablish the optimal cut-off values for cotinine in saliva and

in urine and creatinine-corrected concentration in urineStatistical analysis was performed based on the measurementof cotinine in the biological material taking into accountthe self-reported information on smoking and the data fromthe questionnaires concerning the ETS exposure at home ofsamples donorsThe level of statistical significancewas kept at119875 lt 005TheROCanalysismodule of the IBMprogramSPSSver 200 (IBM SPSS) was used for the ROC curves analysisand comparison of the area under the curve (AUC)

The correlation between the cotinine concentrations insaliva and urine was analyzed with the IBM SPSS Statistics200

3 Results

31 Optimization of the HPLC-MSMS Conditions Themassspectrometer conditions were optimized by monitoring coti-nine and cotinine-d

3ion pairs for quantification in theMRM

mode The best results were achieved with mz transitions ofquantification traces 1772 gt 802 1802 gt 802 and con-firmation 1772 gt 982 1802 gt 1012 for cotinine andcotinine-d

3 respectively

The retention time of cotinine and cotinine-d3was

approx-imately 25min and the total run time equaled6min The use of the structurally identical internal standard(cotinine-d

3) eliminated most of the quantification errors

32 Linearity The relationship between the response andconcentration of cotinine in a range of 04ndash1000 ngmL (forsaliva samples) and 08ndash4000 ngmL (for urine samples) waslinear with the correlation coefficient (119903) of the calibrationcurve 119903 gt 0998 or higher Detailed parameters of themethodvalidation are presented in Table 1

33 Sensitivity The limit of detection (LOD) defined as theconcentration of an analyte that gives at least a 3 1 signal to-noise ratio was 012 ngmL (for saliva) and 005 ngmL (forurine) and the limit of quantification (LOQ) defined as theconcentration of an analyte that gives at least a 10 1 signal-to-noise ratio was 04 ngmL (for saliva) and 08 ngmL (forurine)

34 Precision and Recovery The intraday and interday preci-sions of the method were estimated by the analysis of controlurine (ClinCheck Level 1) and saliva samples (saliva fromsmokers) at the concentration of 248 ngmL of urine (controlrange 198ndash298 ngmL) and about 250 ngmL of saliva on thesame day and on four consecutive days (Table 2)

To determine the recovery of the extraction from theurine and saliva samples seven different cotinine levels were


Table 2 Recovery of cotinine in urine and saliva samples

Expected value[ngmL]

Measuredlowast[ngmL] Recovery []

UrineBefore spiking0884 plusmn 0055 ngmL

08 063 plusmn 001 78540 388 plusmn 34 9780 764 plusmn 3 956120 1131 plusmn 46 9432000 1977 plusmn 963 9883800 3728 plusmn 766 981

SalivaBefore spiking0701 plusmn 0041 ngmL

04 040 plusmn 0037 100520 2006 plusmn 056 100340 384 plusmn 42 95980 80 plusmn 29 1005120 1136 plusmn 22 947500 5061 plusmn 736 1011900 907 plusmn 105 1007


Table 3 Intraday and interday precision of cotinine in urine andsaliva samples

Sample Intraday precision Interday precision

119899Mean

[ngmL] RSD [] 119899Mean

[ngmL] RSD []

Urine 3 2575 12 12 2590 34Saliva 4 2433 23 16 2449 64

added Three repetitions of each concentration level wereanalyzed and the ratio of themeasured amounts to the addedamounts was calculated (Table 3)

35 Tests of Usability Smoking and Nonsmoking Women Totest the usability of the method in total 138 pairs of urine-saliva samples were analyzed (69 pairs in trimester II and 69pairs in trimester III) as well as 69 saliva samples collected intrimester I In all urine and saliva samples the concentrationsof cotinine were above LOD (Table 4)

To assess the correlations between thematrices the linearregression and Pearsonrsquos correlations were calculated Theresults are shown in Figure 1 The correlations between thesaliva and the uncorrected cotinine concentration in urine intrimester II (119903 = 0932) and III (119903 = 0925) were higher thanthe aggregated data of trimester II and III (119903 = 0851) bothbeing statistically significant (119875 lt 001) Similar trends wereobserved for the correlation between saliva and creatinine-corrected cotinine in urine but the corresponding 119903 valueswere lower that is 0720 0865 and 0780 for trimester IIIII and the total period of pregnancy respectively

To verify the smoking status various questionnaires areusually taken into account In this study we took into accountinformation on the smoking status obtained in the survey

The research model was used to compare different matri-ces (saliva urine) and the creatinine correction of cotinineconcentrations of urine Also an analysis was conducted ofthe effect of the period in the pregnancy on the cut-off value

Statistical analysis was performed to compare the cotinineconcentrations in the samples taken from women at differentperiods of pregnancy Saliva samples were collected duringperiodic medical examinations in trimester I II and III ofpregnancy and urine samples in trimester II and III

Receiver operating characteristic (ROC) curve analysis isa graphical and quantitative technique used for determina-tion of the optimal cut-off value Selecting the optimal cut-offvalue for individual biomarkers of smoking was conductedusing the Youdenrsquos index J which is defined as the maximumsumof sensitivity and specificity decreased by 1 that is 119869max =(sensitivity + specificity) minus 1 [26] The cut-off value is ldquoopti-malrdquo when the index has the maximum value

On the basis of the content of cotinine in saliva thecut-off values for the corresponding uncorrected cotinine inurine and creatinine-corrected urine were calculated Theoptimized cut-off values and parameters of ROC curvesanalysis of cotinine in saliva and urine for pregnant womenare shown in Table 5

When choosing the optimal cut-off value it is importantto take into account both sensitivity and specificity and themaximum value of Youdenrsquos index was the decisive factor

ROC analysis showed that the optimal cut-off value sepa-rating smokers and nonsmokers for saliva varies dependingon the period of pregnancy and equals 319 ngmL for thefirst trimester 181 ngmL for the second one and 1147 ngmLfor the last one with the sensitivity of 100 and specificity of96 for all tests In addition the optimum cut-off value wasdetermined on the level of 188 ngmL (983 896 119899 = 241)for cotinine in saliva throughout pregnancy (trimesters I IIand III)

Theoptimal cut-off value for cotinine in the urine samplescollected in trimester II was higher than in trimester III bothfor the urine-53 ngmL versus 3444 ngmL and the crea-tinine corrected concentration-6835120583gg creatinine versus4853 120583gg creatinine (Table 5)

The ROC analysis for the combined samples taken duringthe period from the second to the third trimester of preg-nancy showed lower cut-off values for the cotinine level in alltested matrices 1245 ngmL for saliva and 423 ngmL and5309 120583gg creatinine for urine with the sensitivity equaling100 and specificity of 95 (Table 5)

The determined cut-off for cotinine in saliva samplescollected at the beginning of the pregnancy was 319 ngmL(sensitivity 100specificity 96)

These values were established to distinguish betweenpregnant smokers and nonsmokers

4 Discussion

We developed and validated the method for rapid sensitiveand specific determination of cotinine in urine and salivaBy achieving the low limit of quantification (04 ngmL forsaliva and 08 ngmL for urine) the presented method can beuseful to assess the exposure to tobacco smoke as ETS andactive smoking Using the noninvasive methods of samplingbiological material like urine or saliva the smoking statuscan be more easily assessed especially with lower volume ofbiological fluids than in other studies


2500

2000

1500

1000

500

0

6000

4000

2000

0

0 100 200 300 400

Trimester II Trimester II-creatinine corrected

Trimester III Trimester III-creatinine corrected

Trimesters II and III Trimesters II and III-creatinine corrected

Urin

e (ng

mL)

2500

2000

1500

1000

500

0

Urin

e (ng

mL)

Urin

e (ng

mL)

R = 0932

P lt 001

R = 0923

P lt 001

R = 0865

P lt 001

R = 0780

P lt 001

R = 0851

P lt 001

R = 0720

P lt 001

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400

Saliva (ngmL)

Urin

e (120583

gg

crea

tinin

e)U

rine (120583

gg

crea

tinin

e)1200

1000

800

600

400

200

0

8000

6000

4000

2000

0

Urin

e (120583

gg

crea

tinin

e)

8000

6000

4000

2000

0

Figure 1 Comparison of the urinary and salivary cotinine concentrations depending on the trimester of pregnancy and creatinine correctedconcentration


Table 4 Cotinine in saliva and urine classification based on self-reporting smoking status

119873 AM SD GM 95 CINonsmoking

SalivaTrimester I 50 599 688 4437 4376ndash4498Trimester II 52 486 842 3190 3116ndash3263Trimester III 52 386 414 3060 3024ndash3096

Urine Trimester II 52 1078 2931 4497 4242ndash4751Trimester III 52 1027 3250 3070 2787ndash3352

Urine corrected Trimester II 52 1433 3561 5531 5221ndash5841Trimester III 52 1443 3014 5546 5267ndash5826

Smoking




Table 5 Optimized cutoff values of cotinine in saliva and urine for pregnant women (Youdenrsquos index 0956 plusmn 00084)

Smoking AUClowast SE 95 CI Cutoff Sensitivity SpecificityYes No

Saliva [ngmL]Trimester I 19 50 0998 0003 0992ndash1000 3190 100 96Trimester II 17 52 0997 0004 0989ndash1000 1810 100 96Trimester III 17 52 0997 0004 0989ndash1000 1147 100 96Trimesters II amp III 34 104 0997 0003 0992ndash1000 1285 100 96

Urine [ngmL]Trimester II 17 52 0997 0004 0989ndash1000 5300 100 96Trimester III 17 52 0991 0008 0976ndash1000 3444 100 94Trimesters II amp III 34 104 0994 0004 0997ndash1000 4231 100 95

Urine corrected [120583gg]Trimester II 17 52 0995 0005 0986ndash1000 6835 100 96Trimester III 17 52 0990 0008 0974ndash1000 4853 100 94Trimesters II amp III 34 104 0993 0004 0984ndash1000 5309 100 95

lowast119875 lt 00001

A number of other analytical methods have been appliedfor the measurement of cotinine in urine or saliva includ-ing immunological methods for example enzyme linkedimmunosorbent assay (ELISA LOD = 13 ngmL) Howevercross-reactivity is still the major concern for immunologicalmethods especially with nicotine metabolites 31015840-hydroxyc-otinine and 31015840-hydroxycotinine-glucuronide [27] The mostcommonly used analytical techniques are gas chromatog-raphy with flame-ionization detection (GC-FID LOQ =500 ngmL) [28] mass spectrometry (GC-MS LOQ =10 ngmL) [29] and liquid chromatography with mass spec-trometry (UPLC-MSMS LOQ = 11 ngmL) [30 31] Thesemethods have been used to distinguish smokers from non-smokers Many gas chromatographic methods are character-ized by relatively high limits of quantification which makesit more complicated to access the ETS exposure In this case

the method of choice should make it possible to achieve alimit of detection lower than 1 ngmL [32] In order to evalu-ate such low concentrations it is very important to minimizeion suppression in particular in the chromatographymethodcoupled with mass spectrometry or tandemmass spectrome-tryMany analytical methods do not use internal standards oruse structurally different internal standards than radiolabeledcompounds such as milrinone [33] diphenylamine [34] andacetaminophen [35] which may contribute to the increase ofion suppression [36]

A current state-of-the-art method for determination ofcotinine in biological material is liquid chromatography withmass spectrometry (LC-MSMS) and atmospheric pressureor electrospray ionization which gives high sensitivity Theresults of our study offered a method characterized byimproved sensitivity selectivity and high throughput in


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva-trimester I Trimester II

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine correctedTrimesters II and III

Saliva Urine

Urine corrected

1 minus specificity 1 minus specificity

1 minus specificity1 minus specificity

Figure 2 ROC curves analysis of smoking status based on questionnaires and measurement of cotinine in saliva and in urine and creatininecorrected cotinine in urine are shown in Figure 2

comparison to other conventional techniques The struc-turally identical deuterated internal standard (cotinine-d

3)

increases the selectivity of themethod eliminates the effect ofion suppression and results in higher precision and accuracyof the measurement The advantage of the use of the 96-wellextraction plate is the possibility to cleanupmultiple samplesblank and quality control samples in parallel under the

same conditions This sample preparation procedure makesit possible to achieve a lower limit of detection (LOD =005 ngmL) by minimizing the matrix effect with only 025mL of urine or 05 mL of saliva needed for the analysis

So far many studies have been published concerningcotinine cutoff points yet only a few included pregnantwomen In 1998 Klebanoff et al [37] established a serum


cotinine cut-off value 10 ngmL for pregnant women whichcorresponds to the concentration in saliva of 125 ngmLThisestimation is based on the study of Jarvis who discovered thatthe concentration of cotinine in saliva is 125 times higherthan in serum [38] For the nonpregnant women-14 ngmLserum [39] and 12 ngmL [40] In Japan for a large validationstudy (119899 = 5128) on tobacco smoke exposure duringpregnancy the cutoff for serum cotinine was established at1148 ngmL which corresponds to 1435 ngmL in saliva [41]As for saliva the values obtained for pregnant women wereon the level of 13 ngmL [42] and 24 ngmL [43 44]

Interindividual variabilities in themetabolism of nicotineare due to the gender- and ethnicity-dependent differences inthe activity of enzymes (CYP2A6 and UGT1A) or to someextent genetic polymorphisms of the CYP2A6 gene [15]

As for urine the results recorded among pregnant womenwere 100 ngmL [45] and among the nonpregnant ones-50 ngmL [39] 200 ngmL [46] and 550 ngmL [21]

From the physiological point of view pregnancy is adynamic process during which there increases inter alia thevolume of blood (30ndash40) [47] distribution volume and rateof nicotine metabolism [16]

Probably these are the main reasons for which the cut-off values for cotinine in saliva and urine at different stages ofpregnancymay differmaterially from those set for the generalpopulation In our study we compared changes in the con-centration of cotinine in the samples taken from women atdifferent times of pregnancy during periodic medical exami-nationsThe cut-off values determined for trimester II and IIIfor both saliva and urine indicate a slight change (reduction)However the difference between the values determined forthe first trimester for saliva that is 319 ngmL was signifi-cantly higher for those determined for the second trimesterand third trimester respectively 181 ngmL and 1147 ngmLIn view of the relatively small changes in the metabolism ofnicotine in early pregnancy the cut-off value of 319 ngmL forthat period may be taken as a defining value for nonpregnantwomen

The correct determination of the smoking status is par-ticularly important in epidemiological studies assessing theeffects of exposure to environmental factors on the outcomeof pregnancy where smoking is an important confoundingfactor that should be taken into account in the analysis andinterpretation of results

Minimizing the number of misclassified cases we esti-mated the optimal cutoff point for pregnant women as1285 ngmL for salivary cotinine and 423 ngmL or 531 120583ggcreatinine for urinary cotinine The observed slight decreaseof cotinine cut-off values both for saliva andurine in trimesterIII as compared with II confirms the previous findingsThese findings correspond to those obtained in a Nakayimaand Yokoirsquos study where a large interindividual variability incotinine N-glucuronidation (ca 89-fold) in human micro-somes in vitro was reported [14] The hypothesis concerninga decrease of the free cotinine concentration in the bodyfluids of pregnantwomenmay clarify our observation but stillneeds verification in the further research

In our study cotinine concentrations in urine sampleswere compared with those in saliva samples to estimate the

differences and correlations between the matrices For theurinary and salivary cotinine levels in the second and thirdtrimesters high correlations were observed better for saliva-urine than for saliva-creatinine-corrected urine That con-firms the findings of a previous study on the lesser use ofcreatinine correction [48 49] The mean urinary creatinineconcentration in our study was 089 gL (with the resultslt03 gL themeanwas 082 gL) which is amuch lower resultcomparedwith the nonpregnant women aged 20ndash49 with themean being 12 gL [50] A more reliable parameter for theassessment of diuresis would be obtained if the collection of24-hour urine samples was used However in epidemiologicstudies spot samples are more practical than 24 hr urinesamples [50]

For themonitoring of exposure to tobacco smoke usuallyonly one type of biological samples is collectedThe choice ofthe sample type depends on the purpose of the study and theanalytical technique that is available in the laboratory Thisis particularly important in case of nonsmokers where theexpected concentrations are very low Our study shows thatthe levels of cotinine in urine are about 4ndash55 times higherthan in saliva depending on the use of creatinine correctionOn the other hand saliva is a matrix that is easier to cleanup than urine All the advantages and disadvantages of thosematrices must be taken into account while selecting themethod of sample preparation and analytical technique Inthis study we used saliva samples for rapid screening of thesmoking status Urinary cotinine can be used to estimatemore precisely the level of exposure to tobacco smoke espe-cially in nonsmokers and to differentiate the nonexposednonsmokers and exposed nonsmokers In our analysis wetook into account only the survey data as a criterion forclassification as smokers-nonsmokers but a widespread lackof acceptance for smoking during pregnancy may be a causeof untrue answers

In 2007 Zielinska-Danch et al set a cut-off value for coti-nine in urine of 500 ngmL for the general population [21]In later studies Zielinska-Danch et al [22] using ROC curveanalysis obtained the value of 327 microgramsg creatinine

Exposure to the second-hand smoke is region specificand in some countries a low level of exposure results in amuch lower cutoff cotinine value Until 2009 many studiesreferred to the cut-off value established by Jarvis et al [39]that equaled 14 ngmL serum for the general populationwhile the new value of 447 ngmL (corresponding to559 ngmL saliva) for women was established as a result ofthe NHANES survey [20] However the authors proposeda general cut-off point of 3 ngmL or ethnic-specific valuesbetween 1 and 6 ngmL serum [20] Benowitz et al [20]established also a urine cut-off value of 15 ngmL usingan extrapolation of the above results and average ratios ofcotinine in urine and plasma

The cut-off value for cotinine in urine (determined byGoniewicz et al [23]) equaling 315 ngmL (119899 = 637) repre-sents the multiethnic population including the Poles that islikely to be exposed to tobacco smoke in the same way as thegroup of pregnant women studied in our research

The optimal saliva cut-off value of 129 ngmL is com-parable to that equaling 1435 ngmL saliva (derived form


1148 ngmL plasma) obtained for Japanese pregnant women[41]Most likely the high cutoff point concerning Polish preg-nant women represents a similar second-hand exposure totobacco smoke Taking into account the smoke-free law thesubstantial reduction of the ETS exposure can be expected aswell as a decrease of tobacco smoke biomarkers (cotinine insaliva and urine) As a consequence also lower a cut-off valuemight be established

5 Conclusion

The results of the analytical method validation indicate thatthe developed procedure can be applied for routine deter-mination of cotinine in urine and saliva samples Achievinga low limit of quantification (04 ngmL for saliva and08 ngmL for urine) allows not only distinguishing betweensmokers and nonsmokers but also quantifing the exposure toenvironmental tobacco smoke and degree of active smokingOur results showed a significant correlation between theurinary and salivary cotinine levels Our study presents forthe first time results of ROC curve analysis used to determinethe cut-off values for the assay of cotinine in saliva and urineand in creatinine-corrected urine as a marker of exposureto tobacco smoke in women during different periods ofpregnancy in order to distinguish their status of smokingThis is the first such study of pregnant women in Poland

The ROC analysis with the application of the Youdenrsquosindex helped to determine the optimal cut-off value forcotinine in saliva (189 ngmL) and in urine (423 ngmL and531 120583gg creatinine) for the first time for pregnant women inPoland

The analysis of the diagnostic usefulness of cotininedetermination in saliva and urine and the evaluation of theusefulness of the creatinine correction of the cotinine levelshowed that all three proposed cut-off values are character-ized by high sensitivity and specificity

Our results suggest that during the interpretation ofthe analysis of cotinine the period of pregnancy when thesamples of urine or saliva are taken for the assessment ofexposure to tobacco smoke may have some significance

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The research was supported by the Grant PNRF-218-AI-107from Norway through the Norwegian Financial Mechanismwithin the Polish-Norwegian Research Fund

References

[1] N L Benowitz ldquoBiomarkers of environmental tobacco smokeexposurerdquo Environmental Health Perspectives vol 107 no 2 pp349ndash355 1999

[2] SRNT Subcommittee on Biochemical Verification ldquoBiochem-ical verification of tobacco use and cessationrdquo Nicotine andTobacco Research vol 4 no 2 pp 149ndash159 2002

[3] N L Benowitz and P Jacob III ldquoMetabolism of nicotine to coti-nine studied by a dual stable isotope methodrdquo Clinical Pharma-cology andTherapeutics vol 56 no 5 pp 483ndash493 1994

[4] N L Benowitz ldquoCotinine as a biomarker of environmentaltobacco smoke exposurerdquo Epidemiologic Reviews vol 18 no 2pp 188ndash204 1996

[5] L T Kozlowski N YMehta C T Sweeney et al ldquoFilter ventila-tion and nicotine content of tobacco in cigarettes from Canadathe United Kingdom and the United Statesrdquo Tobacco Controlvol 7 no 4 pp 369ndash375 1998

[6] N L Benowitz and P Jacob III ldquoDaily intake of nicotine duringcigarette smokingrdquo Clinical Pharmacology and Therapeuticsvol 35 no 4 pp 499ndash504 1984

[7] Global Adult Tobacco Survey (GATS) ldquoFact Sheet Poland2009-2010rdquo httpnccdcdcgovGTSSDataAncillaryDataRe-portsaspxCAID=1

[8] A Wojtyła M Gozdziewska P Paprzycki and P BilinskildquoTobacco-related foetal origin of adult diseases hypothesismdashpopulation studies in Polandrdquo Annals of Agricultural and Envi-ronmental Medicine vol 19 no 1 pp 117ndash128 2012

[9] V Haufroid and D Lison ldquoUrinary cotinine as a tobacco-smoke exposure index A minireviewrdquo International Archives ofOccupational and Environmental Health vol 71 no 3 pp 162ndash168 1998

[10] IARC IARCMonographs on Evaluation of Carcinogenic Risks toHumans Volume 83 Tobacco Smoke and Involuntary SmokingWHO International Agency for Research on Cancer LyonFrance 2002

[11] N L Benowitz and D A Dempsey ldquoPharmacotherapy forsmoking cessation during pregnancyrdquo Nicotine and TobaccoResearch vol 6 supplement 2 pp S189ndashS202 2004

[12] A Castles E K Adams C LMelvin C Kelsch andM L Boul-ton ldquoEffects of smoking during pregnancy five meta-analysesrdquoThe American Journal of Preventive Medicine vol 16 no 3 pp208ndash215 1999

[13] K Polanska W Hanke W Sobala and D Ligocka ldquoImpact ofprenatal environmental tobacco smoke exposure on birthparametersrdquo Przegld Lekarski vol 67 no 10 pp 835ndash837 2010

[14] G S Maritz and R Harding ldquoLife-long programming impli-cations of exposure to tobacco smoking and nicotine beforeand soon after birth evidence for altered lung developmentrdquoInternational Journal of Environmental Research and PublicHealth vol 8 no 3 pp 875ndash898 2011

[15] M Nakajima and T Yokoi ldquoInterindividual variability in nico-tine metabolism C-oxidation and glucuronidationrdquo DrugMetabolism and Pharmacokinetics vol 20 no 4 pp 227ndash2352005

[16] D Dempsey P Jacob III and N L Benowitz ldquoAcceleratedmetabolismof nicotine and cotinine in pregnant smokersrdquo Jour-nal of Pharmacology andExperimentalTherapeutics vol 301 no2 pp 594ndash598 2002

[17] E Higashi T Fukami M Itoh et al ldquoHuman CYP2A6 isinduced by estrogen via estrogen receptorrdquo Drug Metabolismand Disposition vol 35 no 10 pp 1935ndash1941 2007

[18] E S Messina R F Tyndale and E M Sellers ldquoA major role forCYP2A6 in nicotine C-oxidation by human liver microsomesrdquoJournal of Pharmacology and Experimental Therapeutics vol282 no 3 pp 1608ndash1614 1997

[19] M Rebagliato F Bolumar C D V Florey et al ldquoVariations incotinine levels in smokers during and after pregnancyrdquo TheAmerican Journal of Obstetrics and Gynecology vol 178 no 3pp 568ndash571 1998


[20] N L Benowitz J T Bernert R S Caraballo D B Holidayand J Wang ldquoOptimal serum cotinine levels for distinguishingcigarette smokers and nonsmokers within different racialethnic groups in the United States between 1999 and 2004rdquoTheAmerican Journal of Epidemiology vol 169 no 2 pp 236ndash2482009

[21] W Zielinska-Danch W Wardas A Sobczak and I Szołtysek-Bołdys ldquoEstimation of urinary cotinine cut-off points distin-guishing non-smokers passive and active smokersrdquo Biomark-ers vol 12 no 5 pp 484ndash496 2007

[22] W Zielinska-Danch M L Goniewicz I Szołtysek-Bołdys etal ldquoEstimation of optimal levels of tobacco biomarkers todistinguish active and passive smokers using ROC analysisrdquoPrzegld Lekarski vol 66 no 10 pp 636ndash640 2009

[23] M L Goniewicz M D Eisner E Lazcano-ponce et al ldquoCom-parison of urine cotinine and the tobacco-specific nitrosam-ine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL) and their ratio to discriminate active from passivesmokingrdquoNicotine and Tobacco Research vol 13 no 3 pp 202ndash208 2011

[24] K Polanska W Hanke J Gromadzinska et al ldquoPolish motherand child cohort studymdashdefining the problem the aim of thestudy and methodological assumptionsrdquo International Journalof OccupationalMedicine and Environmental Health vol 22 no4 pp 383ndash391 2009

[25] WHO Biological Monitoring of Chemicals Exposure in theWorkplace vol 1 WHO Geneva Switzerland 1996

[26] N J Perkins and E F Schisterman ldquoThe inconsistency of mdashoptimalrdquo cutpoints obtained using two criteria based on thereceiver operating characteristic curverdquo The American Journalof Epidemiology vol 163 no 7 pp 670ndash675 2006

[27] A Matsumoto T Ino M Ohta et al ldquoEnzyme-linked immun-osorbent assay of nicotine metabolitesrdquo Environmental Healthand Preventive Medicine vol 15 no 4 pp 211ndash216 2010

[28] F Kardani A Daneshfar and R Sahrai ldquoDetermination ofnicotine anabasine and cotinine in urine and saliva samplesusing single-drop microextractionrdquo Journal of ChromatographyB vol 878 no 28 pp 2857ndash2862 2010

[29] J S Torano and H J M van Kan ldquoSimultaneous determinationof the tobacco smoke uptake parameters nicotine cotinine andthiocyanate in urine saliva and hair using gas chromatography-mass spectrometry for characterisation of smoking status ofrecently exposed subjectsrdquo Analyst vol 128 no 7 pp 838ndash8432003

[30] J Kuhn T Vollmer C Martin D Hendig and C Knabbe ldquoFastand sample cleanup-free measurement of nicotine and cotinineby stable isotope dilution ultra-performance liquid chromatog-raphy-tandem mass spectrometryrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 67-68 pp 137ndash143 2012

[31] P Jacob III L Yu M Duan L Ramos O Yturralde and N LBenowitz ldquoDetermination of the nicotine metabolites cotinineand trans-31015840-hydroxycotinine in biologic fluids of smokersand non-smokers using liquid chromatography-tandem massspectrometry biomarkers for tobacco smoke exposure andfor phenotyping cytochrome P450 2A6 activityrdquo Journal ofChromatography B vol 879 no 3-4 pp 267ndash276 2011

[32] L Song W Davis S M Abrams et al ldquoSensitive and rapidmethod for the determination of urinary cotinine in non-smokers an application for studies assessing exposures to sec-ond hand smoke (SHS)rdquo Analytica Chimica Acta vol 545 no2 pp 200ndash208 2005

[33] R Q Gabr M E Elsherbiny V Somayaji P T Pollak and D RBrocks ldquoA liquid chromatography-mass spectrometry methodfor nicotine and cotinine utility in screening tobacco exposurein patients taking amiodaronerdquo Biomedical Chromatographyvol 25 no 10 pp 1124ndash1131 2011

[34] H S Shin J G Kim Y J Shin and S H Jee ldquoSensitive andsimple method for the determination of nicotine and cotininein humanurine plasma and saliva by gas chromatography-massspectrometryrdquo Journal of Chromatography B vol 769 no 1 pp177ndash183 2002

[35] R Apinan A Choemung and K Na-Bangchang ldquoA sensitiveHPLC-ESI-MS-MSmethod for the determination of cotinineinurinerdquo Journal of Chromatographic Science vol 48 no 6 pp460ndash465 2010

[36] L L Jessome and D A Volmer ldquoIon suppression a major con-cern in mass spectrometryrdquo LC-GC North America vol 24 no5 pp 498ndash510 2006

[37] M A Klebanoff R J Levine J D Clemens R Dersimonianand D G Wilkins ldquoSerum cotinine concentration and self-reported smoking during pregnancyrdquo The American Journal ofEpidemiology vol 148 no 3 pp 259ndash262 1998

[38] M J Jarvis P Primatesta B Erens C Feyerabend and ABryant ldquoMeasuring nicotine intake in population surveyscomparability of saliva cotinine and plasma cotinine estimatesrdquoNicotine and Tobacco Research vol 5 no 3 pp 349ndash355 2003

[39] M J Jarvis H Tunstall-Pedoe C Feyerabend C Vesey and YSaloojee ldquoComparison of tests used to distinguish smokersfrom nonsmokersrdquoThe American Journal of Public Health vol77 no 11 pp 1435ndash1438 1987

[40] M J Jarvis J Fidler J Mindell C Feyerabend and R WestldquoAssessing smoking status in children adolescents and adultscotinine cut-points revisitedrdquo Addiction vol 103 no 9 pp1553ndash1561 2008

[41] S Sasaki T S Braimoh T A Yila E Yoshioka and R KishildquoSelf-reported tobacco smoke exposure and plasma cotininelevels during pregnancymdasha validation study inNorthern JapanrdquoScience of the Total Environment vol 412-413 pp 114ndash118 2011

[42] H K Hegaard H Kjaergaard L F Moslashller H Wachmann andB Ottesen ldquoDetermination of a saliva cotinine cut-off to dis-tinguish pregnant smokers from pregnant non-smokersrdquo ActaObstetricia et Gynecologica Scandinavica vol 86 no 4 pp 401ndash406 2007

[43] L Owen and A McNeill ldquoSaliva cotinine as indicator of ciga-rette smoking in pregnant womenrdquo Addiction vol 96 no 7 pp1001ndash1006 2001

[44] N R Boyd R AWindsor L L Perkins and J B Lowe ldquoQualityof measurement of smoking status by self-report and salivacotinine among pregnant womenrdquo Maternal and Child HealthJournal vol 2 no 2 pp 77ndash83 1998

[45] H J Jhun H G Seo D H Lee et al ldquoSelf-reported smokingand urinary cotinine levels among pregnant women in Koreaand factors associated with smoking during pregnancyrdquo Journalof Korean Medical Science vol 25 no 5 pp 752ndash757 2010

[46] S L Bramer and B A Kallungal ldquoClinical considerations instudy designs that use cotinine as a biomarkerrdquo Biomarkers vol8 no 4 pp 187ndash203 2003

[47] W J McGanity E B Dawson and A Fogelman ldquoNutrition inpregnancy and lactationrdquo in Modern Nutrition in Health andDisease M E Shils J A Olson and M Shike Eds pp 705ndash726 Lea and Febiger London UK 8th edition 1994


[48] P Jatlow SMcKee and S SOrsquoMalley ldquoCorrection of urine coti-nine concentrations for creatinine excretion is it usefulrdquo Clin-ical Chemistry vol 49 no 11 pp 1932ndash1934 2003

[49] G O Petersen C E Leite J M Chatkin and F V ThiesenldquoCotinine as a biomarker of tobacco exposure developmentof a HPLC method and comparison of matricesrdquo Journal ofSeparation Science vol 33 no 4-5 pp 516ndash521 2010

[50] D B Barr L C Wilder S P Caudill A J Gonzalez L L Need-ham and J L Pirkle ldquoUrinary creatinine concentrations in theUS population implications for urinary biologic monitoringmeasurementsrdquo Environmental Health Perspectives vol 113 no2 pp 192ndash200 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


amines N-heterocyclic amines aldehydes or volatile hydro-carbons among which 69 are known carcinogens [9ndash11] Themost important adverse health effects associated with mater-nal cigarette smoking are premature rupture of membranesplacental abruption or preeclampsia [12] uteroplacentalinsufficiency and reducing the blood flow to the fetusMaternal smokingmay also result in lower birth weight of thenewborns [11] Based on the analysis performed in Poland inthe newborns prenatally exposed to ETS the birthweight waslower by 335 g plusmn 903 than that in the case of the nonexposednewborns (119875 lt 0001) after adjustment for maternal edu-cational level marital status prepregnancy weight childgender and gestational age [13] Maternal nicotine exposuremay cause changes in the development and maturing of theoffspringrsquos lungs which can result in the organ being moresusceptible to disease and likely to manifest reduced lungfunction [14] Smoking during pregnancy may have long-term consequences on the neurobehavioral development ofchildren [8]

Due to numerous highly adverse effects of smoking dur-ing pregnancy there is a need to monitor the extent of expo-sure spread the knowledge of these effects to the fetus andpromote smoking cessation Evaluation of the smoking statusamong pregnant women is based mainly on a self-reportedquestionnaire However only a confirmation by a laboratoryanalysis may lead to correct and reliable classification sincepregnant women (and not only they) are reluctant to admitthat they smoke

Interindividual variability in the metabolism of nicotineis due to the gender and ethnic differences in the activity ofenzymes (CYP2A6 and UGT1A) and to some extent geneticpolymorphisms of the CYP2A6 gene [15] Like many otherphysiological processes also the metabolism of nicotinechanges during pregnancy The observed variability in themetabolic clearance of cotinine may markedly increase by140 during pregnancy resulting in a half-life shorter bynearly 50 than the one in the nonpregnant state [16]

The explanation of these changes could be the influenceof a higher concentration of estradiol during pregnancy [17]which induces the activity of CYP2A6 responsible for themetabolism of nicotine [18]

As reported by Rebagliato et al [19] the salivary cotininelevel was significantly lower during pregnancy comparedwith the postpartum oneTherefore it is necessary to identifya cutoff value to avoid misclassification of smoking and non-smoking pregnant women

The ROC analysis is increasingly used to determine thecut-off values for biomarkers of exposure to tobacco smoke[20ndash23]

The primary aim of this study was to establish the optimalcut-off value for cotinine in saliva and urine of pregnantwomen in Poland and to compare the diagnostic effectivenessof three smoking tests cotinine in saliva and in urine and inurine with creatinine correction The secondary one was todevelop a sensitive and specific method for determining thecotinine level in urine and saliva in a broad range of con-centrations Finally our aim was also to estimate the utilityof these matrices for both rapid screening used in order toidentify potential smokers and more accurate determination

of the degree of exposure to tobacco smoke especially thatconcerning pregnant women

2 Methods

21 Population From the biobank of the Polish Motherand Child Cohort Study (REPRO PL) saliva-urine samplescollected in trimester II and III of 69 women were selected aswell as the survey data on the smoking status of the pregnantwoman the smoking habit of her husbandpartner and a con-sent to smoking in the apartment In addition each of thesewomen had a saliva sample taken during the first trimesterof pregnancy The complete description of the cohort waspublished elsewhere [24] In short the inclusion criteria weresingle pregnancy up to 12 weeks of gestation no assistedconception no pregnancy complications and no chronicdiseases as specified in the study protocol [24]Themean ageof 69 women was 2641 plusmn 497 years Based on the surveydata it was found that in the first trimester 1969 womenwere smokers and in the second and third trimesters thisratio equaled 1769 Smoking was permitted in 52 of theapartments in the first trimester of pregnancy but in the thirdtrimester such permission to smoke at home decreased byapproximately 9

To find the correlations between the matrices we ana-lyzed saliva and urine samples collected at the same time

22 Standards and Reagents Cotinine (98) internal stand-ard-cotinine-d

3(98) and ammonium acetate (98) were

obtained from Sigma Aldrich Acetonitrile and MethanolUltra Gradient HPLC Grade were supplied by Baker Aceticacid (gt99) was purchased from Fluka Ultrapure water wasobtained fromMilli-Q-Plus Ultra-PureWater System (Milli-pore USA) All working standards of cotinine and cotinine-d3were prepared in acetonitrile and stored at minus20∘C Solid

phase extraction manifold was maintained on Supelco andOASIS HLB LP 96-Well Plate 60120583m (60mg) was supplied byWaters (USA) Control urine lyophilized ClinCheckControlfor toxic organic compounds was purchased from RecipeChemicals (Germany)

23 Biological Samples Collection Saliva was collected frompregnant women into a Salivette with citric acid (SarstedtGermany)The amount of approximately 1-2mL of saliva waseasily obtained by having the women chew a cotton swab atleast 30min after eating or drinking A clear fluid sample wasobtained by centrifuging the Salivette and used for analysisA 50mL volume of morning urine was collected frompregnant women to a 100mL polypropylene container (BenePoland) All saliva and urine samples were transported to thelaboratory in a cool box and stored at minus20∘C until analysis

24 Samples Preparation Urine and saliva samples werethawed before the analysis thoroughly mixed and trans-ferred into 20mL polypropylene tubes The samples werecentrifuged for 10min at 11000 rcf (MIKRO 120 Hettich Zen-trifugen TuttlingenGermany) To 025mLof urine or 05mLof saliva water and 20120583L of internal standard (cotinine-d

3)

were added and mixed vigorously Each well of the Oasis



LOD[ngmL]

LOQ[ngmL]










3mL







3 The







3 Results




3 respectively



















119899Mean


[ngmL] RSD []

Urine 3 2575 12 12 2590 34Saliva 4 2433 23 16 2449 64















4 Discussion



2500

2000

1500

1000

500

0

6000

4000

2000

0

0 100 200 300 400




Urin

e (ng

mL)

2500

2000

1500

1000

500

0

Urin

e (ng

mL)

Urin

e (ng

mL)

R = 0932

P lt 001

R = 0923

P lt 001

R = 0865

P lt 001

R = 0780

P lt 001

R = 0851

P lt 001

R = 0720

P lt 001

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400

Saliva (ngmL)

Urin

e (120583

gg

crea

tinin

e)U

rine (120583

gg

crea

tinin

e)1200

1000

800

600

400

200

0

8000

6000

4000

2000

0

Urin

e (120583

gg

crea

tinin

e)

8000

6000

4000

2000

0








Smoking









lowast119875 lt 00001





00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















LOD[ngmL]

LOQ[ngmL]










3mL







3 The







3 Results




3 respectively



















119899Mean


[ngmL] RSD []

Urine 3 2575 12 12 2590 34Saliva 4 2433 23 16 2449 64















4 Discussion



2500

2000

1500

1000

500

0

6000

4000

2000

0

0 100 200 300 400




Urin

e (ng

mL)

2500

2000

1500

1000

500

0

Urin

e (ng

mL)

Urin

e (ng

mL)

R = 0932

P lt 001

R = 0923

P lt 001

R = 0865

P lt 001

R = 0780

P lt 001

R = 0851

P lt 001

R = 0720

P lt 001

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400

Saliva (ngmL)

Urin

e (120583

gg

crea

tinin

e)U

rine (120583

gg

crea

tinin

e)1200

1000

800

600

400

200

0

8000

6000

4000

2000

0

Urin

e (120583

gg

crea

tinin

e)

8000

6000

4000

2000

0








Smoking









lowast119875 lt 00001





00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























119899Mean


[ngmL] RSD []

Urine 3 2575 12 12 2590 34Saliva 4 2433 23 16 2449 64















4 Discussion



2500

2000

1500

1000

500

0

6000

4000

2000

0

0 100 200 300 400




Urin

e (ng

mL)

2500

2000

1500

1000

500

0

Urin

e (ng

mL)

Urin

e (ng

mL)

R = 0932

P lt 001

R = 0923

P lt 001

R = 0865

P lt 001

R = 0780

P lt 001

R = 0851

P lt 001

R = 0720

P lt 001

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400

Saliva (ngmL)

Urin

e (120583

gg

crea

tinin

e)U

rine (120583

gg

crea

tinin

e)1200

1000

800

600

400

200

0

8000

6000

4000

2000

0

Urin

e (120583

gg

crea

tinin

e)

8000

6000

4000

2000

0








Smoking









lowast119875 lt 00001





00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2500

2000

1500

1000

500

0

6000

4000

2000

0

0 100 200 300 400




Urin

e (ng

mL)

2500

2000

1500

1000

500

0

Urin

e (ng

mL)

Urin

e (ng

mL)

R = 0932

P lt 001

R = 0923

P lt 001

R = 0865

P lt 001

R = 0780

P lt 001

R = 0851

P lt 001

R = 0720

P lt 001

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400 500 600

Saliva (ngmL)0 100 200 300 400 500 600

Saliva (ngmL)

0 100 200 300 400

Saliva (ngmL)

Urin

e (120583

gg

crea

tinin

e)U

rine (120583

gg

crea

tinin

e)1200

1000

800

600

400

200

0

8000

6000

4000

2000

0

Urin

e (120583

gg

crea

tinin

e)

8000

6000

4000

2000

0








Smoking









lowast119875 lt 00001





00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Smoking









lowast119875 lt 00001





00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10


00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine

Urine corrected

Trimester III

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

Saliva Urine


Saliva Urine

Urine corrected





3)





















5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































5 Conclusion







Acknowledgment


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article estimation of cutoff values of cotinine...

Documents