Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Chromatogr B Analyt Technol Biomed Life Sci. Author manuscript; available in PMC 2012 February 1.
Published in final edited form as:
PMCID: PMC3050598

Determination of the Nicotine Metabolites Cotinine and Trans-3′-Hydroxycotinine in Biologic fluids of Smokers and Non-Smokers using Liquid Chromatography - Tandem Mass Spectrometry: Biomarkers for Tobacco Smoke Exposure and for Phenotyping Cytochrome P450 2A6 Activity


The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3′-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by Cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography – tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3′-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1 ng/ mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.

Keywords: Nicotine, Cotinine, trans-3′-hydroxycotinine, Cytochrome P450 2A6 (CYP2A6), tobacco, secondhand smoke

1. Introduction

Worldwide, tobacco-related diseases cause about 5 million premature deaths per year.[1] Most of these deaths occur in smokers, but smokeless tobacco use[2] and exposure to secondhand smoke in non-smokers also poses a significant health risk.[3; 4] Most smokers in the United States say they want to quit, but the majority of them are unable to do so, in large part because of nicotine addiction.[5]

Determining exposure to nicotine is of interest to resear chers studying the effects of tobacco use on health, to clinicians who need an objective outcome measure for tobacco dependence treatment programs, to scientists studying exposure to secondhand smoke and its effects, and for numerous other areas of inquiry into the pharmacology and toxicology of nicotine and tobacco. A widely used approach for measuring exposure is determination of tobacco-derived biomarkers in biologic fluids.[6; 7; 8] In this regard, the nicotine metabolite cotinine is the most widely used, and has excellent specificity for both active use of tobacco and for secondhand smoke exposure,[6; 9; 10]except in individuals using nicotine-containing medications.[7] Cotinine concentrations have been determined in a variety of biological matrices, including plasma, serum, urine, saliva, hair, and nails.[6; 9; 11; 12; 13; 14; 15] Saliva concentrations are highly correlated with plasma concentrations,[16; 17] and since obtaining saliva does not require venipuncture, saliva is the preferred biofluid for many studies. Urine concentrations are generally much higher than those in plasma or saliva [18], and for this reason urine analyses can provide greater sensitivity for assessing low level exposure.

Trans-3′-hydroxycotinine (3HC) is, in most individuals, the major metabolite of cotinine.[19; 20] Its concentrations in urine generally exceed cotinine concentrations by 3-4 fold,[19; 20] but in plasma or saliva, cotinine concentrations are generally higher than those of 3HC.[21; 22] Consequently, determination of 3HC, as well as cotinine, might provide a more sensitive measure of exposure, especially when urine is used. The conversion of cotinine to 3HC, as well as the conversion of nicotine to cotinine in humans is largely mediated by the liver enzyme cytochrome P450 2A6 (CYP2A6) (Figure 1).[23; 24] Recently, we reported a high correlation between the ratio of 3HC to cotinine concentration in plasma and nicotine oral clearance. This ratio provides a convenient measure to phenotype individuals for CYP2A6 activity.[25; 26] This method is being used for large-scale pharmacogenetic studies.

Figure 1
Metabolism of nicotine to cotinine and trans-3′-hydroxycotinine

Numerous methods for determination of cotinine in biologic fluids have been reported, including gas chromatography (GC),[11; 27] high performance liquid chromatography (HPLC) [28], gas chromatography - mass spectrometry (GC-MS)[11; 29; 30], liquid chromatography - mass spectrometry (LC-MS)[22; 31; 32; 33; 34; 35], and immunoassay procedures.[36; 37; 38]. Chromatographic and chromatographic-mass spectrometric methods have been used for determination of 3HC as well,[11; 39] but, to our knowledge, immunoassay methods have not. Liquid chromatography - tandem mass spectrometry (LC-MS/ MS) with triple-stage quadrupole instruments is widely used for low-level quantitation of basic drugs, their metabolites, and various endogenous substances in biologic fluids.[40; 41] During the past few years, LC-MS/ MS methods for determination of sub-nanogram per milliliter concentrations of nicotine and its metabolites have been reported.[22; 31; 32; 33; 42].

As part of our studies of the pharmacology and toxicology of nicotine and tobacco, we required methods for determination of both cotinine and 3HC in various biological matrices. For studies of low-level secondhand smoke (SHS) exposure, methods with very high sensitivity were required, in order to achieve limits of quantitation of 0.1 ng/ mL or lower. In addition, the methods had to be practical for analysis of large numbers of samples. This paper describes LC-MS/ MS methods for simultaneous determination of low concentrations of cotinine and 3-HC in human biofluids. Ad vantages of the methods include: 1.] straight-forward extraction procedures that are convenient for large batches of samples; 2.] excellent precision, accuracy, and sensitivity with lower limits of quantitation (LLOQ) ranging from 0.02 to 0.1 ng/ mL for 1 ml volume samples; and 3.] the methods have been applied to and validated for plasma, urine and saliva samples, the major biofluids that are used for tobacco smoke exposure assessment.

2. Materials and Methods

2.1. Biofluid Samples

Plasma, saliva, and urine samples were collected and analyzed for studies that have been reported elsewhere.[43; 44; 45] All studies received approval of the appropriate institutional review boards. Typically, 7 mL of blood is collected in vacutainer tubes containing 100 USP units of lithium heparin, then centrifuged to ob tain plasma. Plasma is transferred to polypropylene cryogenic vials. Saliva is collected into 20 mL polypropylene vials. Prior to collection, subjects are asked to wash their mouths with water. If necessary to collect sufficient volume, subjects may ch ew on a piece of paraffin. However, it should be noted that stimulation of saliva flow may affect cotinine concentrations.[46] Urine is acidified to a pH of 2-3 with solid sodium bisulfate. Biofluid samples are stored at −20° C until analysis.

2.2. Reagents and Standards

Cotinine perchlorate and trans-3′-hydroxycotinine perchlorate were synthesized as previously described.[47; 48] The internal standards, cotinine-d9 and trans-3′- hydroxycotinine-d9 (Figure 2) were synthesized by modification of published procedures.[47; 48; 49] The HPLC mobile phase was prepared from HPLC grade water and HPLC grade methanol from Burdick-Jackson. These were buffered using formic acid (ACS reagent grade) and ammonium formate (Certified) from Fisher Chemical Company (Pittsburgh, PA). Optima grade methylene chloride used for extractions, ACS reagent grade 60-62% perchloric acid, ACS reagent grade hydrochloric acid, and CP tripotassium phosphate monohydrate (Acros) were from Fisher.

Figure 2
Chemical structures of internal standards

2.3. Instrumentation

LC-MS/ MS analyses were carried out with a Thermo Surveyor or Agilent 1200 HPLC interfaced to a Thermo-Finnigan TSQ Quantum Ultra triple-stage quadrupole mass spectrometer for analyses requiring maximum sensitivity, or using a Hewlett - Packard 1090 HPLC interfaced with a Finnigan TSQ 7000 triple-stage quadrupole mass spectrometer with an API2 ion source. Solvent evaporation was carried out using a Savant Automatic Environmental SpeedVac Model AES 2000.

2.4. Working Standards and Controls

Standards were prepared from the perchlorate salts of cotinine and 3HC in 0.01 M HCl/ HPLC grade water, and concentrations were corrected to those of the free bases. Dilutions were made in 0.01 M HCl to prepare working standards ranging from 0.01 to 20 ng/ mL for studies of SHS exposure, from 1 to 500 ng/ mL analyses of smokers’ plasma or saliva, and from 10 to 10,000 ng/ mL for analyses of smokers’ urine. Aqueous working standards were used because nicotine metabolites are present in biofluids from virtually all individuals due to environmental exposure to nicotine. Controls were prepared from plasma, saliva, and urine of non -smokers spiked with stock solutions of the analytes, or in the case of low-level urine controls, an “artificial urine” was used as the matrix,[50] prepared from major components reported in human urine.[51]

2.5. Method Variation 1: Biofluids from Non-Smokers

2.5.1 Extraction Procedure 1: Plasma, Saliva, and Urine from Non-Smokers

To 1mL of biofluid sample, standard, or control contained in 13 × 100 mm glass culture tube was added 100 μL of a solution of the internal standards in 0.01 M HCl. The internal standard solution contained 20 ng/ mL of cotinine-d9, and 20 ng/ mL trans-3′-hydroxycotinine-d9. The tube was briefly vortex-mixed, and 100 μL of 30% perchloric acid was added to precipitate protein. (Although urine should not contain protein, for some samples addition of perchloric acid resulted in less emulsion and cleaner separation of the organic layer in the subsequent extraction step, and therefore it was routinely added to urine samples as well as plasma and saliva.) After vortex-mixing and centrifugation, the supernate was decanted to a 16 × 100 mm culture tube. Tripotassium phosphate, 2 mL of 50% (w/ v, pH ~ 14), and 6 mL of methylene chloride was added. The tube was vortex-mixed for 5 min, centrifuged, and placed in a dry ice-acetone mixture to freeze the aqueous layer. The organic (upper) layer was poured to a 13 × 100 mm culture tube, 100 μL of 10% HCl in methanol was added, and the extract was evaporated to dryness in a centrifugal vacuum evaporator. The dried extract was reconstituted in 150 μL of 100 mM aqueous ammonium formate, and transferred to an autosampler vial for LC-MS/ MS analysis. Twenty microliters were injected.

Extraction recovery was estimated by adding analytes to extracts of blank plasma, and comparing peak areas to those from spiked plasma carried through the extraction procedure.

2.5.2 Liquid Chromatography System 1

The chromatography was carried out using a 4.6mm × 150 mm Phenomenex Synergi Polar RP column (4 micron) fitted with a Phenomenex Polar -RP guard column, 4mm L × 3.0 ID. A binary, linear gradient elution with 10 mM ammonium acetate/ 0.1 % acetic acid in water (solvent A) and 10 mM ammonium acetate/ 0.1 % acetic acid in methanol (solvent B) was used at a flow rate was 0.7 mL/ min with the following program: The initial composition was 80% A, changing to 100% B over 6.5 min. 100% B was maintained from 6.5-8 min, then changed to 80% A at 8.1 min and maintained at this composition until the end of run at 13 min (Figures (Figures3,3, ,4,4, and and55).

Figure 3
SRM chromatograms of non-smokers’ plasma
Figure 4
SRM chromatograms of non-smokers’ saliva
Figure 5
SRM chromatograms of non-smokers’ urine

2.5.3 Mass Spectrometry Parameters 1

The mass spectrometer was operated in the positive ion mode using atmospheric pressure chemical ionization (APCI). The ion source parameters were optimized by infusing an aqueous solution of cotinine via syringe pump. The vaporizer temperature was 450° C, the heated capillary temperature was 250° C, and the corona discharge current was set at 5 μamps. Cotinine and 3HC solutions were infused to determine appropriate ion transitions and the optimum collision energies for CID. The collision gas (argon) pressure was 1.5 mTorr. The collision energy was set at 30 eV for cotinine and cotinine-d9, and at 35 eV for 3HC and 3HC-d9. The SRM transitions monitored were as follows: m/ z 177 to m/ z 80 for cotinine, m/ z 193 to m/ z 80 for trans-3′-hydroxycotinine and the transitions m/ z 186 to m/ z 84 and m/ z 202 to m/ z 84 for the respective internal standards. The resolution of the first quadrupole, FWHM, was set at 0.2 amu, the resolution of the third quadrupole was set at 0.7 amu FWHM.

2.6. Method Variation 2: Biofluids from Smokers

2.6.1 Extraction Procedure 2: Plasma, Saliva, and Urine from Smokers

The procedure was identical to Extraction Procedure A, except that 100 μL of biofluid was used, and it was diluted with 900 μL of HPLC grade water. For plasma and saliva samples, the evaporated extract was reconstituted in 150 μL of 100 mM aqueous ammonium formate; for urine the volume was 1 mL.

2.6.2 Liquid Chromatography System 2

The chromatography was carried out using a 4.0 mm × 150 mm Supelco Discovery HSF5 column (5 micron) fitted with an HSF5 guard column, 4.0 mm × 20 mm. The mobile phase flow rate was 0.7 mL/ min, and injection volume was 50 μL. A binary, linear gradient elution with 10 mM ammonium formate in water (solvent A) and 10 mM ammonium formate in methanol (solvent B), flow rate of 0.7 mL/ min with the following program: The initial composition was 80% A, changing to 100% B over 6.5 min. 100% B was maintained from 6.5-8 min, then changed to 80% A at 8.1 min and maintained at this composition until the end of run at 10 min. 3HC and cotinine eluted at approximately 5.3 and 6.2 min, respectively.

2.6.3 Mass Spectrometry Parameters 2

All Parameters were the same as described for Method Variation 1, except that the resolution of both quadrupoles was set at 0.7 amu FWHM.

2.7. Data Analysis

The Finnigan XCalibur/ LC Quan software was used to generate calibration curves and calculate concentrations using peak area ratios of analyte/ internal standard. Linear regression with 1/ X weighting, “ignore origin” was used. Blanks were included in the standard curves and “ignore origin” was used to correct for the small amounts of cotinine present in solvents and reagents. (See Section 3.5). Standard curves were linear from 0.01 to 20 ng/ mL for SHS exposure studies, from 1 to 500 ng/ mL analyses of smokers’ plasma or saliva, and from 10 to 10,000 ng/ mL for analyses of smokers’ urine. Eight concentrations spanning each range were used, and standards were run in duplicate. Typically, one set of standards was injected at the beginning of the run, and one set following injection of the clinical study samples. Equations and correlation coefficients for representative standard curves are below.

Cotinine, Non-SmokersPlasma or Saliva(0.022ng/mL):Y=0.00570+0.06318*Xr2=0.9997
3-HC,Non-SmokersPlasma or Saliva(0.022ng/mL):Y=0.000351+0.0445*Xr2=0.9998
Cotinine, Non-SmokersUrine(0.02525ng/mL):Y=0.00693+0.141*Xr2=0.9998
Cotinine, SmokersPlasma or Saliva(1500ng/mL):Y=0.00564X+0.00724r2=0.9991
Cotinine, SmokersUrine(1010,000ng/mL):Y=0.0119+0.00624*Xr2=0.9995

2.8. Validation

Precision, accuracy, and limits of quantitation were determined by replicate analysis of spiked plasma, saliva, and urine samples, at concentrations spanning the expected concentration ranges (Tables (Tables11--5)5) as described by Shah et al.[52] and Viswanathan et al.[53] In addition, 54 plasma samples obtained from smokers were analyzed for cotinine by GC using nitrogen-phosphorus detection[27] modified for use with a capillary column.[29] These results were compared with results obtained using the method described here (Figure 6).

Figure 6
Correlation of cotinine concentrations in smokers determined by GC (27, 29) with concentrations determined by the method described in this paper. The analytical data were used for a published study (44)
Table 1
Within-run precision and accuracy for determination of cotinine and trans-3′-hydroxycotinine in plasma
Table 5
Between-run precision and accuracy for determination of cotinine and trans-3′-hydroxycotinine in urine

3. Results and Discussion

The goal of this study was to develop a versatile method with very high sensitivity (LLOQs less than 0.1 ng/ mL) that would be practical for various large-scale clinical studies. Consequently, considerable effort was put into optimizing the chromatography, mass spectrometry, and extraction procedure.

3.1. Extraction

Cotinine and 3HC are hydrophilic substances that are relatively difficult to extract from aqueous matrices. Both solid -phase and liquid/ liquid extraction procedures have been employed for cotinine and 3HC.[11; 31; 39; 42; 54] We found that the combination of high concentration (50% w/ v) tripotassium phospha te as a base and methylene chloride as solvent was convenient and efficient for extraction of the two analytes from biofluids. The high base concentration makes the density of the aqueous phase greater than that of methylene chloride. Therefore, the meth ylene chloride extract constitutes the upper layer, facilitating phase separation by the freeze and pour technique. Furthermore, the high base concentration improves recovery of the highly polar 3HC[39] by a “salting out” effect. Prior to evaporation of the methylene chloride, hydrochloric acid is added to convert the bases to non-volatile salts, since some losses occurred during vacuum evaporation if no acid was added. Extraction recovery from plasma was about 53% for 3HC and 65% for cotinine.

3.2. Chromatography

Several column and mobile phase combinations were evaluated. Extracts of plasma and urine from persons with little or no SHS exposure (do not live or work with a smoker) were used to test for interference derived from the matrix (Table 6). The column that provided the best separation of the analytes from traces of matrix-derived substances, particularly in urine, was a 4.6 × 150 mm Phenomenex Synergi Polar RP (embedded phenoxypropyl group) column using a water - methanol gradient with 10 mM ammonium acetate/ 0.1% acetic acid buffer. A 4.0 × 150 mm Supelco HSF5 (pentafluorophenylpropylsilane) column using a water - methanol gradient with 10 mM ammonium formate was satisfactory for plasma samples, and for urine concentrations above about 0.1 ng/ mL.

Table 6
Cotinine and 3HC concentrations in urine and saliva of non-smokers with little or no exposure to SHSa

3.3. Mass Spectrometry

Both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have been utilized in mass spectrometric methods for determination of nicotine metabolites. Although ESI is generally more sensitive than APCI for detection of basic substances, it is also more prone to matrix suppression of ionization.[55; 56; 57] We found that sensitivity for an aqueous cotinine standard was about 10X higher using ESI than it was using APCI. However, when applied to urine extracts, suppression of ionization of 90% or more sometimes occurred with ESI. Therefore, APCI was evaluated, and it was found that significant matrix suppression of ionization rarely occurred. When it did occur, with some very concentrated urine samples, the stable isotope-labeled internal standard corrected for ionization suppression. This was demonstrated by re-analysis of the sample, diluted 10-fold with HPLC grade water, in which case the internal standard peak area was similar to that of the standards, and the analytical result agreed within ± 10% of the original analysis. Sensitivity using APCI was adequate for measuring low picogram per milliliter concentrations.

As reported for previous studies, we found that the most suitable SRM transitions for quantitation were m/z 177 to m/z 80 for cotinine and m/z 193 to m/z 80 for 3HC [22; 31; 32; 33; 34; 35], as these produced the most abundant product ions. The corresponding transitions for the internal standards were used, m/z 186 to 84 for cotinine-d9 and m/z 202 to 84 for 3-HC-d9. Neutral loss of the pyridine ring was also a significant pathway for cotinine, resulting m/z 98. This ion has been used for confirmation in previous studies [22; 31] but its abundance was too low to use for confirmation in our methods for low-level SHS exposure, which required maximum sensitivity. Likewise, the abundances of other product ions of m/z 193 for 3HC (e.g., m/z 86, 106, 114, 134) were too low to be used for confirmation at low concentrations in samples from many non-smokers. At higher concentrations, these transitions would probably be suitable for confirmation. Chromatograms of plasma, saliva, and urine samples are shown in Figures Figures33--55.

3.4. Calibration

Instrument response was found to be linear to at least 25 ng/ mL for cotinine and to 50 ng/ mL for 3HC for 1 mL sample extracts (Method Variation 1). For studies in active smokers, in which concentrations were higher than 50 ng/ mL, a smaller sample size, 100 μL, was used, and linearity was demonstrated to 20,000 ng/ mL (Method Variation 2). Calibration curves were generated using linear regression with 1/ X weighting, using the peak area ratio of analyte/ internal standard as response. Equations for typical standard curves are in the Methods Section.

3.5. Validation

We used the criteria of Shah et al[52] and Viswanathan et al.[53] to validate the methods for precision, accuracy, and to determine the lower limits of quantitation (LLOQs). These are precision (CV) of ± 15% and accuracy within ± 15% of the expected amount, except at the lower limit of quantitation, for which ± 20% is considered acceptable. LLOQs were determined using within-run precision and accuracy data. Between-run data also met the above criteria, with the exception of precision at the LLOQs for plasma, which were based on thirteen analytical runs carried out over a 5 month period (Table 2). Within-run precision and accuracy was evaluated by analyzing six replicate samples at three or more concentrations spanning the expected range (Tables (Tables1,1, ,3,3, and and4).4). Between-run precision and accuracy was determined from QC specimens analyzed along with clinical study samples (Tables (Tables22 and and5).5). For Method Variation 1, the LLOQs for cotinine are 0.02, 0.02, and 0.05 for plasma, saliva, and urine, respectively; the LLOQs for 3HC are 0.02, 0.02, and 0.10 for plasma, saliva, and urine, respectively. For Method Variation 2, the LLOQs for cotinine are 1 and 10 ng/ mL for plasma and urine, respectively; the LLOQs for 3HC are 1 and 10 ng/ mL for plasma and urine, respectively.

Table 2
Between-run precision and accuracy for determination of cotinine and trans-3′-hydroxycotinine in plasma
Table 3
Within-run precision and accuracy for determination of cotinine and trans-3′-hydroxycotinine in saliva
Table 4
Within-run precision and accuracy for determination of cotinine and trans-3′-hydroxycotinine in urine

Determining specificity is complicated by nicotine metabolites being present in virtually all biofluid specimens, due to the presence of nicotine in the environment [27; 58] and its ingestion, resulting in its metabolism and excretion of the metabolites. For example, in six people who reported little or no exposure to SHS, urine cotinine concentrations were above the LLOQ in four of the six, and 3HC concentrations were above the LLOQ in all six (Table 6). In six saliva samples from people who reported little or no exposure, only one had cotinine concentrations above the LLOQ, and two had 3HC concentrations above the LLOQ. We find peaks corresponding to cotinine, but not 3HC in our “blanks” (Figures (Figures44 and and5).5). It has been reported that nicotine can be converted to cotinine under typical environmental conditions, [59] which may explain its presence in solvents and reagents. Specificity was also evaluated by analyzing four serum samples, spiked with different concentrations of cotinine, that were being developed as serum reference materials by the US Centers for Disease Control (CDC). The results were in excellent agreement with the expected concentrations and with those determined [31] in the laboratories of the CDC (Table 7). For plasma samples with cotinine concentrations high enough to be measured by GC we compared analytical results determined by the described LC-MS/ MS method with the results determined using GC. For 54 plasma samples with concentrations ranging from 2.4 to 514 ng/ mL, there was excellent agreement, r2 = 0.991 (Figure 6).

Table 7
Cotinine concentrations in a serum reference material determined by the method described in this paper and by the method of Bernert et al. (30)

4. Conclusions

Our method builds on the pioneering LC-MS/ MS method developed by Bernert et al. at the CDC.[31] In our opinion, this was the first reported method with adequate sensitivity and specificity for determination of cotinine to evaluate low-level exposure to SHS. Application of this method has provided a large data base of serum cotinine levels in Americans who participated in the Third National Health and Nutrition Examination Survey (NHANES III)[60] and other studies. The two major applications of our methods are 1.] assessing exposure to secondhand smoke, and 2.] using the 3HC/ cotinine ratio, phenotyping individuals for CYP2A6 activity to optimize pharmacotherapies for tobacco dependence. [25; 26; 45; 61; 62] Metabolic activation of some carcinogenic nitrosamines, including some present in tobacco, is mediated by CYP2A6[63; 64], and that is another reason for interest in this phenotypic marker.

Exposure to SHS in the United States and many developed countries is declining, especially in persons living in areas where smoking bans are widespread and in people who are concerned about the health risks of SHS exposure. We found that many human subjects participating in studies of SHS exposure had cotinine concentrations below 0.1 ng/ mL in plasma and urine, despite reporting some SHS exposure. Even at low exposure levels, there was a positive correlation between symptoms of chronic obstructive pulmonary disease (COPD) and urine cotinine concentrations, which were below 0.2 ng/ mL for 73% of the samples.[43] In contrast, cotinine concentrations in urine of non-smokers in Mexico City, prior to SHS exposure in discotheques,[44] were below 0.2 ng/ mL for only 20% of the subjects (Table 8). Data from these studies also illustrates the generally higher concentrations of 3HC in urine, sug gesting that it may be a more sensitive biomarker for low-level exposure (Table 8).

Table 8
Concentrations of cotinine and 3HC in urine of non-smokers in two geographical areas.

In summary, very sensitive and versatile methods for determination of the nicotine metabolites cotinine and 3HC in plasma, saliva, and urine has been developed and valid ated. Precision and accuracy are excellent, and the methods are suitable for large-scale studies. The methods have been used to analyze several thousand biofluid samples for studies of SHS exposure, and appear to be the most sensitive methods yet reported for these two important analytes.


This study was supported by a grant from the Flight Attendant Medical Research Institute, grants DA02277 and DA12393 from the National Institute on Drug Abuse, National Institutes of Health, and the California Tobacco-Related Disease Research Program (10RT-0215). Some of the studies were carried out at the General Clinical Research Center at San Francisco General Hospital Medical Center with support from NIH/ NCRR UCSF-CTSI Grant Number UL1 RR024131. The authors thank Dr John T. Bernert of the US Centers for Disease Control and Prevention, Atlanta for allowing us to publish his data on cotinine concentrations in the serum reference materials, and Marc Olmsted for editorial assistance.

Funding for this research was provided by the Flight Attendant Medical Research Institute (to the UCSF Bland Lane Center of Excellence in Secondhand Smoke), the National Institutes of Health (DA02277 and DA12393), and from the California Tobacco-Related Disease Research Program (10RT-0215). Some of the studies were carried out at the General Clinical Research Center at San Francisco General Hospital Medical Center with support from NIH/ NCRR UCSF-CTSI Grant Number UL1 RR024131. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Non-standard abbreviations

secondhand smoke
chronic obstructive pulmonary disease


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


[1] Benowitz NL. Clinical pharmacology of nicotine: implications for understanding, preventing, and treating tobacco addiction. Clin Pharmacol Ther. 2008;83:531–541. [PubMed]
[2] Hecht SS, Carmella SG, Murphy SE, Riley WT, Le C, Luo X, Mooney M, Hatsukami DK. Similar exposure to a tobacco-specific carcinogen in smokeless tobacco users and cigarette smokers. Cancer Epidemiol Biomarkers Prev. 2007;16:1567–1572. [PubMed]
[3] P.H.S. Department of Health and Human Services The Health Consequences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon General. Washington DC: US Government Printing Office; 2006. DHHS (CDC) Publication No. 87-8398.
[4] IARC Monograph on the Evaluation of Carcinogenic Risks to Humans. Lyon, France: WHO; 2004. [PubMed]
[5] Benowitz NL. Pharmacologic aspects of cigarette smoking and nicotine addiction. New England Journal of Medicine. 1988;319:1318–1330. [PubMed]
[6] Benowitz NL, Jacob P, III, Ahijevych K, Jarvis MJ, Hall S, LeHouezec J, Hansson A, Lichtenstein E, Henningfield J, Tsoh J, Hurt RD, Velicer W. Biochemical verification of tobacco use and cessation. Nicotine and Tobacco Research. 2002;4:149–159. [PubMed]
[7] Jacob P, III, Hatsukami DK, Severson H, Hall S, Yu L, Benowitz NL. Anabasine and anatabine as biomarkers for tobacco use during nicotine replacement therapy. Cancer Epidemiol Biomarkers Prev. 2002;11:1668–1673. [PubMed]
[8] Hecht SS. Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer. Carcinogenesis. 2002;23:907–922. [PubMed]
[9] Benowitz NL. Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol. Rev. 1996;18:188–204. [PubMed]
[10] Bernert JT, Jacob P, 3rd, Holiday DB, Benowitz NL, Sosnoff CS, Doig MV, Feyerabend C, Aldous KM, Sharifi M, Kellogg MD, Langman LJ. Interlaboratory comparability of serum cotinine measurements at smoker and nonsmoker concentration levels: a round -robin study. Nicotine Tob Res. 2009;11:1458–1466. [PMC free article] [PubMed]
[11] Jacob P, III, Byrd GD. Use of gas chromatographic and mass spectrometric techniques for the determination of nicotine and its metabolites. In: Gorrod JW, Jacob P III, editors. Analytical Determination of Nicotine and Related Compounds and Their Metabolites. Elsevier; Amsterdam: 1999. pp. 191–224.
[12] Al-Delaimy WK. Hair as a biomarker for exposure to tobacco smoke. Tob Control. 2002;11:176–182. [PMC free article] [PubMed]
[13] Al-Delaimy WK, Mahoney GN, Speizer FE, Willett WC. Toenail nicotine levels as a biomarker of tobacco smoke exposure. Cancer Epidemiol Biomarkers Prev. 2002;11:1400–1404. [PubMed]
[14] Ryu HJ, Seong MW, Nam MH, Kong SY, Lee DH. Simultaneous and sensitive measurement of nicotine and cotinine in small amounts of human hair using liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:2781–2782. [PubMed]
[15] Stepanov I, Hecht SS, Lindgren B, Jacob P, 3rd, Wilson M, Benowitz NL. Relationship of human toenail nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol to levels of these biomarkers in plasma and urine. Cancer Epidemiol Biomarkers Prev. 2007;16:1382–1386. [PubMed]
[16] Jarvis MJ, Primatesta P, Erens B, Feyerabend C, Bryant A. Measuring nicotine intake in population surveys: comparability of saliva cotinine and plasma cotinine estimates. Nicotine Tob Res. 2003;5:349–355. [PubMed]
[17] Bernert JT, Jr., McGuffey JE, Morrison MA, Pirkle JL. Comparison of serum and salivary cotinine measurements by a sensitive high -performance liquid chromatography-tandem mass spectrometry method as an indicator of exposure to tobacco smoke among smokers and nonsmokers. J Anal Toxicol. 2000;24:333–339. [PubMed]
[18] Jarvis MJ, Tunstall-Pedoe H, Feyerabend C, Vesey C, Saloojee Y. Comparison of tests used to distinguish smokers from nonsmokers. Am. J. Public Health. 1987;77:1435–1438. [PubMed]
[19] Benowitz NL, Jacob P, III, Fong I, Gupta S. Nicotine metabolic profile in man: Comparison of cigarette smoking and transdermal nicotine. J. Pharmacol. Exp. Ther. 1994;268:296–303. [PubMed]
[20] Hukkanen J, Jacob P, 3rd, Benowitz NL. Metabolism and disposition kinetics of nicotine. Pharmacol Rev. 2005;57:79–115. [PubMed]
[21] Benowitz NL, Jacob P., III Trans-3′-hydroxycotinine: Disposition kinetics, effects and plasma levels during cigarette smoking. Br J Clin Pharmacol. 2001;51:53–59. [PMC free article] [PubMed]
[22] Bentley MC, Abrar M, Kelk M, Cook J, Phillips K. Validation of an assay for the determination of cotinine and 3-hydroxycotinine in human saliva using automated solid -phase extraction and liquid chromatography with tandem mass spectrometric detection. J Chromatogr B Biomed Sci Appl. 1999;723:185–194. [PubMed]
[23] Nakajima M, Yamamoto T, Nunoya K, Yokoi T, Nagashima K, Inoue K, Funae Y, Shimada N, Kamataki T, Kuroiwa Y. Characterization of CYP2A6 involved in 3′-hydroxylation of cotinine in human liver microsomes. J. Pharmacol. Exp. Ther. 1996;277:1010–1015. [PubMed]
[24] Messina ES, Tyndale RF, Sellers EM. A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J. Pharmacol. Exp. Ther. 1997;282:1608–1614. [PubMed]
[25] Dempsey D, Tutka P, Jacob P, 3rd, Allen F, Schoedel K, Tyndale RF, Benowitz NL. Nicotine metabolite ratio as an index of cytochrome P450 2A6 metabolic activity. Clin Pharmacol Ther. 2004;76:64–72. [PubMed]
[26] Johnstone E, Benowitz N, Cargill A, Jacob R, Hinks L, Day I, Murphy M, Walton R. Determinants of the rate of nicotine metabolism and effects on smoking behavior. Clin Pharmacol Ther. 2006;80:319–330. [PubMed]
[27] Jacob P, 3rd, Wilson M, Benowitz NL. Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids. J Chromatogr. 1981;222:61–70. [PubMed]
[28] Hariharan M, VanNoord T, Greden JF. A high-performance liquid -chromatographic method for routine simultaneous determination of nicotine and cotinine in plasma. Clin Chem. 1988;34:724–729. [PubMed]
[29] Jacob P, III, Yu L, Wilson M, Benowitz NL. Selected ion monitoring method for determination of nicotine, cotinine, and deuterium -labeled analogs. Absence of an isotope effect in the clearance of (S)-nicotine-3′-3′-d2 in humans. Biological Mass Spectrometry. 1991;20:247–252. [PubMed]
[30] Ji AJ, Lawson GM, Anderson R, Dale LC, Croghan IT, Hurt RD. A new gas chromatography-mass spectrometry method for simultaneous determination of total and free trans-3′-hydroxycotinine and cotinine in the urine of subjects receiving transdermal nicotine. Clin Chem. 1999;45:85–91. [PubMed]
[31] Bernert JT, Jr., Turner WE, Pirkle JL, Sosnoff CS, Akins JR, Waldrep MK, Ann Q, Covey TR, Whitfield WE, Gunter EW, Miller BB, Patterson DG, Jr., Needham LL, Hannon WH, Sampson EJ. Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/ atmospheric pressure ionization tandem mass spectrometry. Clin. Chem. 1997;43:2281–2291. [PubMed]
[32] Meger M, Meger-Kossien I, Schuler-Metz A, Janket D, Scherer G. Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biom ed Life Sci. 2002;778:251–261. [PubMed]
[33] Xu X, Iba MM, Weisel CP. Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry. Clin Chem. 2004;50:2323–2330. [PubMed]
[34] Shakleya DM, Huestis MA. Simultaneous and sensitive measurement of nicotine, cotinine, trans-3′-hydroxycotinine and norcotinine in human plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:3537–3542. [PMC free article] [PubMed]
[35] Miller EI, Norris HR, Rollins DE, Tiffany ST, Wilkins DG. A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid -phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878:725–737. [PMC free article] [PubMed]
[36] Niedbala RS, Haley N, Kardos S, Kardos K. Automated homogeneous immunoassay analysis of cotinine in urine. J Anal Toxicol. 2002;26:166–170. [PubMed]
[37] Zuccaro P, Pichini S, Altieri I, Rosa M, Pellegrini M, Pacifici R. Interference of nicotine metabolites in cotinine determination by RIA. Clin Chem. 1997;43:180–181. [PubMed]
[38] Schepers G, Walk R. Cotinine determination by immunoassays may be influenced by other nicotine metabolites. Arch. Toxicol. 1988;62:395–397. [PubMed]
[39] Jacob P, III, Shulgin A, Yu L, Benowitz NL. Determination of the nicotine metabolite trans–3′–hydroxycotinine in smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring. Journal of Chromatography. 1992;583:145–154. [PubMed]
[40] Tiller PR, Romanyshyn LA, Neue UD. Fast LC/ MS in the analysis of small molecules. Anal Bioanal Chem. 2003;377:788–802. [PubMed]
[41] Maurer HH. Multi-analyte procedures for screening for and quantification of drugs in blood, plasma, or serum by liquid chromatography-single stage or tandem mass spectrometry (LC-MS or LC-MS/ MS) relevant to clinical and forensic toxicology. Clin Biochem. 2005;38:310–318. [PubMed]
[42] Heavner DL, Richardson JD, Morgan WT, Ogden MW. Validation and application of a method for the determination of nicotine and five major metabolites in smokers’ urine by solid -phase extraction and liquid chromatography-tandem mass spectrometry. Biomed Chromatogr. 2005;19:312–328. [PubMed]
[43] Eisner MD, Balmes J, Yelin EH, Katz PP, Hammond SK, Benowitz N, Blanc PD. Directly measured secondhand smoke exposure and COPD health outcomes. BMC Pulm Med. 2006;6:12. [PMC free article] [PubMed]
[44] Lazcano-Ponce E, Benowitz N, Sanchez-Zamorano LM, Barbosa-Sanchez L, Valdes-Salgado R, Jacob P, 3rd, Diaz R, Hernandez-Avila M. Secondhand smoke exposure in Mexican discotheques. Nicotine Tob Res. 2007;9:1021–1026. [PubMed]
[45] Lerman C, Tyndale R, Patterson F, Wileyto EP, Shields PG, Pinto A, Benowitz N. Nicotine metabolite ratio predicts efficacy of transdermal nicotine for smoking cessation. Clin Pharmacol Ther. 2006;79:600–608. [PubMed]
[46] Schneider NG, Jacob P, 3rd, Nilsson F, Leischow SJ, Benowitz NL, Olmstead RE. Saliva cotinine levels as a function of collection method. Addiction. 1997;92:347–351. [PubMed]
[47] Jacob P, 3rd, Benowitz NL, Shulgin AT. Synthesis of optically pure deuterium -labeled nicotine, nornicotine and cotinine. Journal of Labeled Compounds and Radiopharmaceuticals. 1988;25:1117–1128.
[48] Jacob P, III, Shulgin AT, Benowitz NL. Synthesis of (3′R,5′S)–trans–3′–hydroxycotinine, a major metabolite of nicotine. Metabolic formation of 3′-hydroxycotinine in humans is highly stereoselective. Journal of Medicinal Chemistry. 1990;33:1888–1891. [PubMed]
[49] Jacob P, III, Benowitz NL. Pharmacokinetics of (S)-nicotine and metabolites in humans. In: Gorrod JW, Wahren J, editors. Nicotine and Related Alkaloids: Absorption, Distribution, Metabolism and Excretion. Chapman and Hall; London: 1993. pp. 197–218.
[50] Jacob P, 3rd, Wilson M, Benowitz NL. Determination of phenolic metabolites of polycyclic aromatic hydrocarbons in human urine as their pentafluorobenzyl ether derivatives using liquid chromatography-tandem mass spectrometry. Anal Chem. 2007;79:587–598. [PubMed]
[51] Putnam DF. McDonnell Douglas Astronautics Company, Report # NASA CR-1082. National Information Service; Springfield, Virginia: 1971. Composition and Concentrative Properties of Human Urine.
[52] Shah VP, Midha KK, Findlay JW, Hill HM, Hulse JD, McGilveray IJ, McKay G, Miller KJ, Patnaik RN, Powell ML, Tonelli A, Viswanathan CT, Yacobi A. Bioanalytical method validation --a revisit with a decade of progress. Pharm Res. 2000;17:1551–1557. [PubMed]
[53] Viswanathan CT, Bansal S, Booth B, DeStefano AJ, Rose MJ, Sailstad J, Shah VP, Skelly JP, Swann PG, Weiner R. Quantitative bioanalytical methods validation and implementation: best practices for chromatograp hic and ligand binding assays. Pharm Res. 2007;24:1962–1973. [PubMed]
[54] Tyrpien K, Wielkoszynski T, Janoszka B, Dobosz C, Bodzek D, Steplewski Z. Application of liquid separation techniques to the determination of the main urinary nicotine metabolites. J Chromatogr. 2000;A 870:29–38. [PubMed]
[55] Matuszewski BK, Constanzer ML, Chavez-Eng CM. Matrix effect in quantitative LC/ MS/ MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per milliliter concentrations. Anal Chem. 1998;70:882–889. [PubMed]
[56] Liang HR, Foltz RL, Meng M, Bennett P. Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/ tandem mass spectrometry. Rapid Commun Mass Spectrom. 2003;17:2815–2821. [PubMed]
[57] Mallet CR, Lu Z, Mazzeo JR. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid -phase extracts. Rapid Commun Mass Spectrom. 2004;18:49–58. [PubMed]
[58] Gruenke LD, Beelen TC, Craig JC, Petrakis NL. The determination of nicotine in biological fluids. Analyt. Biochem. 1979;94:411–416. [PubMed]
[59] Sleiman M, Gundel LA, Pankow JF, Jacob P, 3rd, Singer BC, Destaillats H. Formation of carcinogens indoors by surface-mediated reactions of nicotine with nitrous acid, leading to potential thirdhand smoke hazards. Proc Natl Acad Sci U S A. 2010;107:6576–6581. [PubMed]
[60] Pirkle JL, Flegal KM, Bernert JT, Brody DJ, Etzel RA, Maurer KR. Exposure of the US Population to Environmental Tobacco Smoke. The Third National Health and Nutrition Examination Survey, 1988 to 1991. J. Am. Med. Assn. 1996;275:1233–1240. [PubMed]
[61] Gu DF, Hinks LJ, Morton NE, Day IN. The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit. Ann Hum Genet. 2000;64:383–390. [PubMed]
[62] Fujieda M, Yamazaki H, Saito T, Kiyotani K, Gyamfi MA, Sakurai M, Dosaka-Akita H, Sawamura Y, Yokota J, Kunitoh H, Kamataki T. Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. Carcinogenesis. 2004;25:2451–2458. [PubMed]
[63] Loriot MA, Rebuissou S, Oscarson M, Cenee S, Miyamoto M, Ariyoshi N, Kamataki T, Hemon D, Beaune P, Stucker I. Genetic polymorphisms of cytochrome P450 2A6 in a case-control study on lung cancer in a French population. Pharmacogenetics. 2001;11:39–44. [PubMed]
[64] Xu C, Goodz S, Sellers EM, Tyndale RF. CYP2A6 genetic variation and potential consequences. Adv Drug Deliv Rev. 2002;54:1245–1256. [PubMed]