Accepted Manuscript

Title: Analysis of nicotine and cotinine in hair by on-line

in-tube solid-phase microextraction coupled with liquid
chromatography-tandem mass spectrometry as biomarkers of
exposure to tobacco smoke

Authors: Takehito Inukai, Sanae Kaji, Hiroyuki Kataoka

PII: S0731-7085(18)30642-3
DOI: https://doi.org/10.1016/j.jpba.2018.04.032
Reference: PBA 11931

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 17-3-2018

Revised date: 21-4-2018
Accepted date: 22-4-2018

Please cite this article as: Takehito Inukai, Sanae Kaji, Hiroyuki Kataoka, Analysis
of nicotine and cotinine in hair by on-line in-tube solid-phase microextraction
coupled with liquid chromatography-tandem mass spectrometry as biomarkers
of exposure to tobacco smoke, Journal of Pharmaceutical and Biomedical
Analysis https://doi.org/10.1016/j.jpba.2018.04.032

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Analysis of nicotine and cotinine in hair by
on-line in-tube solid-phase microextraction
coupled with liquid chromatography-tandem
mass spectrometry as biomarkers of exposure

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to tobacco smoke

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Takehito Inukai, Sanae Kaji, Hiroyuki Kataoka*

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School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan

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* Corresponding author. Hiroyuki Kataoka

Tel.: +81-86-271-8342; fax: +81-86-271-8342.

E-mail address: hkataoka@shujitsu.ac.jp

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Graphical abstract

Assessment of long-term exposure to nicotine and cotinine is possible from only 1 mg of hair.
Nicotine and cotinine concentrations in the hair of smokers were significantly higher than
those in non-smokers.

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Highlights
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 On-line in-tube solid-phase microextraction of nicotine and cotinine was optimized.

 Nicotine and cotinine were simultaneously determined by in-tube SPME coupled with
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liquid chromatography–tandem mass spectrometry.

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 This method is automated, simple, rapid, selective, and sensitive.

 This method was utilized successfully to analyze pg/mg levels of nicotine and cotinine
in only 1 mg of hairs of non-smokers and smokers.
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 The method is useful for the assessment of long-term exposure to tobacco smoke.
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Abstract
Smoking not only increases the risk of lung cancer but is strongly related to the onset of

cardiovascular disease. Particularly, passive smoking due to sidestream smoke is a critical

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public health problem. To assess active and passive exposure to tobacco smoke, we

developed a simple and sensitive method, consisting of on-line in-tube solid phase

microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry

(LC-MS/MS), to determine nicotine and its metabolite cotinine in hair samples. These

compounds were separated within 5 min using a Polar-RP80A column and detected in the

positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions

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were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Carboxen

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1006 capillary column as an extraction device. The extracted compounds in the stationary

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phase on the inner wall of the capillary could be dissolved easily into the mobile phase and

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transferred to an LC column. Using the in-tube SPME LC-MS/MS method, the calibration

curves were linear in the 5–1000 pg/mL ranges for nicotine and cotinine, and the detection

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limits (signal to noise ratio of 3) were 0.45 and 0.13 pg/mL, respectively. The intra-day and
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inter-day precisions were below 3.4% and 6.0% (n=5), respectively. This method was
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utilized successfully to analyze pg/mg levels of nicotine and cotinine in 1 mg of hairs
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without interference peaks, and good recoveries were obtained. The concentration of

cotinine in hair was two orders of magnitude lower than that of nicotine, but a good positive
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correlation was found between the concentrations of these compounds. This method can

automate the extraction, concentration and analysis of samples, and is useful for the
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assessment of long-term exposure to tobacco smoke.

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Keywords: Nicotine, Cotinine, Exposure biomarkers, Hair, In-tube solid-phase

microextraction, LC–MS/MS
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Abbreviations: WHO, World Health Organization; Nic, Nicotine; Cot, cotinine; GC–MS, gas

chromatography with mass spectrometry; HPLC, high performance liquid chromatography;

MS/MS, tandem mass spectrometry; ECD, electrochemical detection; SPME, solid phase

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microextraction; IS, internal standard; ESI, electro spray ionization; MRM, multiple reaction

monitoring; CUR, curtain gas; CAD, collision gas; DP, declustering potential; EP, entrance

potential; CE, collision energy; CXP, collision cell exit potential; LOD, limits of detection;

LOQ, limits of quantification; DCM, dichloromethane; HX, n-hexane; ON, overnight; RT,

room temperature; TMAH, tetramethylammonium hydroxide; CAN, acetonitrile; FA, formic

acid; LLE; liquid-liquid extraction; HS, headspace; MIP, molecular imprinted; API,

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atmospheric pressure ionization; UP, ultraperformance; UHP, ultra-high performance.

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1. Introduction

Tobacco smoking is regarded as a cause of various cancers and cardiovascular diseases,

making the use of tobacco products a critical public health problem [1,2]. In addition, passive

smoking, due to sidestream smoke, exhalation by smokers, and smoke adhering to clothes,

has been linked to various adverse health outcomes, particularly among non-smokers,

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including children and pregnant women [3–6]. Furthermore, the risk of onset of lung cancer

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has been reported to be 1.3-fold higher among persons who are than are not exposed to

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passive smoking [6], making measures to reduce passive smoking particularly important. To

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prevent or limit the deleterious effects of active and passive smoking, it is necessary to

determine the status of exposure to tobacco smoke. This requires the development of a

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selective and sensitive method of detecting biomarkers for exposure to tobacco smoke [1,7,8].
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Tobacco smoke contains more than 5000 chemical compounds, including at least 69
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carcinogens [1,2]. Nicotine (Nic) is the major alkaloid present in tobacco, constituting 98% of
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total alkaloids, with each cigarette containing a few milligrams of this compound. Nic is

absorbed rapidly in humans not only by inhalation in the lungs but also through the skin and
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mucosal lining of the mouth and nose. About 70%－80% of the Nic absorbed into the body is

metabolized to cotinine (Cot), and Nic and Cot can be detected in various biological fluids,
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including blood, saliva, and urine [1,8,9]. Therefore, these compounds have been widely used

as biomarkers to determine tobacco smoking status and estimate tobacco smoke exposure
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[1,7–9]. However, the half-lives of urinary Nic and Cot are 2–3 h and 7–40 h, respectively

[10], despite their concentrations increasing by active and passive smoking. Therefore,
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urinary concentrations of these compounds only indicate exposure to tobacco smoke during

the 3 or 4 days prior to sample collection. In contrast, hair has been used to assess and

monitor long-term human exposure to Nic and Cot [10–25], as compounds trapped within

hair shafts can accumulate for a long period of time, depending on the length of the hair [11].

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However, the levels of Nic and Cot are much lower in hair samples of passive than of active

smokers. Therefore, highly sensitive assays of Nic and Cot in hair are required to assess

exposure to tobacco smoke in active and passive smokers.

Nic and Cot in hair samples have been determined by gas chromatography with mass

spectrometry (GC–MS) [12–16], high performance liquid chromatography (HPLC) [17,18],

and LC with mass spectrometry (LC–MS) [19] or tandem mass spectrometry (LC–MS/MS)

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[10,20–22]. HPLC–UV and –electrochemical detection (ECD) are relatively insensitive,

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whereas GC–MS and LC–MS/MS are sensitive and selective methods of measuring Nic and

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its metabolites. However, most of these methods generally require time-consuming off-line

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sample preparation procedures, such as liquid-liquid extraction or solid-phase extraction, to

remove coexisting substances in biological samples prior to analysis.

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We have developed an in-tube solid phase microextraction (SPME) method using as an
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extraction device an open tubular fused-silica capillary column with an inner surface coating
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[20–22]. Sampling and pretreatment were found to be simple, rapid, miniaturized, and labor
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saving, and required very small volumes of organic solvents. Moreover, these methods can be

coupled easily on-line to HPLC and LC–MS. In-tube SPME allows convenient automation of
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the extraction process, and provides better precision and sensitivity than manual off-line

techniques. Using this in-tube SPME technique, we recently developed an on-line automated
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method for the analysis of various compounds in pharmaceuticals, biological samples, foods

and environmental samples [25–28]. Furthermore, we have previously developed an in-tube

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SPME LC−MS method for the analysis of Nic, Cot and related alkaloids in urine and saliva

samples of smokers and non-smokers [29]. This study describes our improvements in the
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performance and sensitivity of this method by using a stable isotope-labeled internal standard

(IS) and automated on-line in-tube SPME LC−MS/MS. Using this method, we have analyzed

Nic and Cot in hair samples to assess tobacco smoke exposure in active and passive smokers.

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2. Materials and methods

2.1. Materials

Nic and Cot were purchased from Sigma-Aldrich Japan (Tokyo, Japan). The stable

isotope-labeled internal standard (IS) compounds, Nic-d3 (isotopic purity 98.4%), and Cot-d3

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(isotopic purity 99.9%), were purchased from Toronto Research Chemicals Inc. (Toronto,

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Canada). Each compound was dissolved in methanol to make 1 mg/mL stock solutions,

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which were stored at 4°C. Immediately prior to use, aliquots of these stock solutions were

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diluted with pure water to the required concentrations. LC−MS grade methanol and water

used as mobile phases were purchased from Kanto Chemical (Tokyo, Japan). All other

chemicals were of analytical grade. U

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As extraction devices, non-coated fused silica, CP-Sil 5CB (100% polydimethylsiloxane,
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film thickness 5 μm), CP-Sil 19CB (14% cyanopropyl phenyl methylsilicone, film thickness
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1.2 μm), CP-Wax 52CB (polyethylene glycol, film thickness 1.2 μm) (Varian Inc., Lake

Forest, CA), Supel-Q PLOT (divinylbenzene polymer, film thickness 17 μm), and Carboxen
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1006 PLOT (Carboxen molecularsives, film thickness 17 μm) capillary columns were

purchased from Supelco (Bellefonte, PA, USA).

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2.2. Instrument and analytical conditions

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The LC system was a Model 1100 series (Agilent Technologies, Böblingen Germany),
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consisting of a binary pump, an online degasser, an autosampler, a column compartment, a

diode array detector, and an HP ChemStation. LC separation was performed on a Polar-RP

80A column (50 mm × 2.0 mm i.d., particle size of 4 μm) (Phenomenex; Torrance, CA, USA)

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at a column temperature of 30°C, with 2.5 mM ammonium formate/methanol (25/75, v/v) as

the mobile phase, at a flow rate of 0.2 mL/min..

Electro spray ionization (ESI)–MS/MS for Nic, Cot and their stable isotope-labeled

compounds was performed on an API 4000 triple quadruple mass spectrometer (Applied

Biosystems, Foster City, CA, USA), equipped with a turbo ion spray interface operated in

positive ion mode at 4000 V and 600°C by multiple reaction monitoring (MRM). A Kaken N2

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generator (System Instruments Co., Ltd., Tokyo, Japan) was used to generate nitrogen, which

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acted as a nebulizing and drying gas. The ion sources for gases 1 (GS1) and 2 (GS2) were set

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at 30 and 40 L/min, respectively. The curtain gas (CUR) and collision gas (CAD) flows were

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set at 30 and 4.0 L/min, respectively. Other setting parameters, including dwell time per

transition to detect ion pairs, declustering potential (DP), entrance potential (EP), collision

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energy (CE), and collision cell exit potential (CXP), are shown as Supplementary information
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(Table S1). Quantification and confirmation were performed by MRM of the protonated
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precursor molecular ions [M+H]+ and the related product ions for each compound.
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Quadrupoles Q1 and Q3 were set at unit resolution (Table S1, Supplementary Material).

LC−MS/MS data were processed using Analyst Software 1.3.1 (Applied Biosystems).
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2.3. In-tube solid-phase microextraction

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The in-tube SPME device consisted of a Carboxen 1006 PLOT capillary column (60 cm ×
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0.32 mm i.d.), placed between the injection loop and the injection needle of the autosampler

by connection with a 2.5-cm sleeve of 1/16-in. polyetheretherketone tubing, standard 1/16-in.

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stainless steel nuts, ferrules, and connectors. As described [25–29], the autosampler software

was programmed to control the in-tube SPME extraction, desorption, and injection (Table S2).

An outline of the in-tube SPME LC–MS/MS system is shown in Fig. S1 (Supplementary

Material).

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2.4. Collection and preparation of hair samples

Hair samples without dyeing were obtained from 66 healthy volunteers (19 males and 47

females, age 19–63), including 58 non-smokers and eight smokers. The experimental protocol

was approved by the ethics committee of Shujitsu University, and all volunteers provided

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written informed consent. For 50 non-smokers (nine males and 41 females), the frequency of

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passive smoking was self-reported by questionnaire. About 5 mg of each individual’s hair

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was obtained by normal hair cutting and stored in a sealed plastic bag at room temperature.

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The hair samples were subjected to three rounds of sonication in 1 mL dichloromethane for 3

min each and dried at room temperature. The dried hair was cut with scissors, and 1−2 mg of

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each sample was placed in a 7 mL screw-cap vial, to which was added 1.0 mL of distilled
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water and 4 µL of the IS solution, containing 200 pg of Nic-d3 and Cot-d3, followed by
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extraction at 80ºC for 30 min. After cooling to room temperature and centrifugation at 2500
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rpm for 1 min, an aliquot of supernatant was transferred to a 2.0 mL autosampler vial with a

septum, and its total volume was made up to 1 mL with distilled water. Then the vials were
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placed in the autosampler for in-tube SPME LC−MS/MS analysis, with the concentrations of

Nic and Cot calculated as pg/mg or ng/mg hair using a calibration curve created from stable
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isotope-labeled IS compounds. The results obtained from hair samples of non-smokers and

smokers were compared by Student’s t-tests.

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3. Results
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3.1. Optimization of in-tube solid-phase microextraction and desorption

We previously described an in-tube SPME LC−MS method for the determination of Nic,

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Cot and related alkaloids using a CP-Pora PLOT capillary as an extraction device [29].

Although this capillary showed higher extraction efficiency in comparison with other

capillary tested, the stationary phase coated onto the inner surface of the capillary tended to

come off. In testing six other commercially available capillary columns (60 cm length), we

found that the Carboxen 1006 PLOT column had superior extraction efficiency (Fig. S2-A).

Using this column, the optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL

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sample at a flow rate of 200 μL/min (Fig. S2B). The absolute amounts of these compounds

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extracted by the SPME capillary column were calculated by comparing peak area counts with

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the corresponding direct injection of the sample solution onto the LC column. At a sample

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concentration of 200 pg/mL, 7.54 ng (3.8%) of nicotine, and 25.6 ng (12.8%) of cotinine

were extracted onto the Carboxen 1006 PLOT column by in-tube SPME. Although the

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extraction yields of these compounds were relatively low, they showed good reproducibility
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(RSD < 5%) due to the autosampler. The extracted compounds were easily desorbed from the
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capillary by passage of the mobile phase, and no carryover was observed.
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3.2. LC−MS/MS analysis of nicotine, cotinine and their stable isotope-labeled compounds
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For MS/MS operation, we found that the ESI positive ion mode was the most effective for
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ionization of Nic, Cot and their stable isotope-labeled compounds. The optimum MS/MS

conditions for each compound are described in the Experimental section. Precursor and
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product ions consisted of protonated ions [M+H]+ (Q1 mass) and prominent fragment ions

(Q3 mass), respectively. These MRM transitions and optimal MS/MS parameters are shown
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in Table S1. Fragment ions were sufficiently separated by Q3 in unit resolution, and MRM

transitions were selected for confirmation and quantification.

Nic and Cot were separated within 5 min by LC on a Polar-RP 80A column using 2.5 mM

ammonium formate/methanol (25/75 v/v) as the mobile phase, at a flow rate of 0.2 mL/min.

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Nic, Cot and their stable isotope-labeled compounds were detected selectively in MRM mode.

Nic and Cot were automatically extracted and analyzed by the in-tube SPME LC−MS/MS

method within 25 min, making possible the automated analysis of about 50 samples per day

with overnight operation.

3.3. Linearity, sensitivity and precision

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Linearity was evaluated by triplicate analyses each at eight-point (5, 10, 20, 50, 100, 200,

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500 and 1000 pg/mL) of Nic and Cot, in the presence of 200 pg/mL of their stable

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isotope-labeled IS compounds. Calibration curves, constructed by comparing peak height

ratio with each IS, showed a linear relationship for each compound (eight-point calibration),

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with all correlation coefficients above 0.9992 (Table 1). Using these LC−MS/MS conditions,
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the limits of detection (LOD) (signal to noise ratio of 3) of Nic and Cot were 0.45 and 0.13
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pg/mL, respectively, with the in-tube SPME method showing a greater than 19-fold higher
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sensitivity than the direct injection method (10 µL injection) (Table 1). These sensitivities

were higher than those of previous LC−MS/MS methods [10, 20–22]. The intra-day and
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inter-day precisions (relative standard deviations) obtained from the analysis of 20 and 200

pg/mL standard mixtures containing Nic and Cot each were 1.62–3.37% and 2.35–5.97%,
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respectively (n=5) (Table 1).

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3.4. Application to the analysis of hair samples

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Because Nic and Cot may be deposited on the external surfaces of the cuticles following

exposure to tobacco smoke, the hair samples must be pre-rinsed to remove Nic and Cot on the

hair surface [1, 11]. Hair is usually sonicated in dichloromethane for analysis of Nic and Cot

[10, 12–19, 21]. In this study, hair samples were sonicated three times in dichloromethane to

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completely remove external Nic and Cot deposited on hair surfaces. To evaluate biological

exposure to tobacco smoke, internal Nic and Cot must be extracted from hair. Extraction in

distilled water at 80ºC for 30 min was efficient (Fig. S3). These extracts could be directly

analysed by the in-tube SPME/LC–MS/MS method without the need for any other

pre-treatment. The limits of quantification (LOQ) (signal to noise ratio of 10) of Nic and Cot

in hair were 7.5 and 4.4 pg/mg, respectively.

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Stable isotope-labeled compounds were added to hair samples prior to analysis to correct

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for the influence of the sample matrix. Fig. 1 shows chromatograms obtained from samples

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corresponding to 0.2 mg of hair. Nic and Cot in hair samples were detected successfully by

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in-tube SPME LC−MS/MS with MRM mode detection and without interference peaks. To

confirm the validity of this method, hair samples were spiked with known amounts of Nic and

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Cot, and the recoveries of these compounds were calculated. The overall recoveries of Nic
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and Cot were over 87% and relative standard deviations were below 6.3% (Table 2).
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3.5. Evaluation of tobacco smoke exposure in non-smokers and smokers

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To evaluate the utility of the developed method, we analyzed the concentrations of Nic

and Cot in hair samples of eight non-smokers and eight smokers. Each hair sample was
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assayed three times. Nic concentrations in non-smokers and smokers were 1.88±1.85 and

43.12±34.81 ng/mg, respectively. Cot concentrations in non-smokers and smokers were

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0.022±0.028 and 0.655±0.616 ng/mg, respectively. The concentrations of these compounds in

the hair of smokers were significantly higher than those in non-smokers. Another 50
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non-smokers reported the frequency of passive smoking by self-reported questionnaires, and

the concentrations of Nic and Cot in their hair samples were quantified by in-tube SPME

LC–MS/MS. Nic and Cot concentrations showed good correlations in these samples (Fig. 2).

Of the 50 non-smokers, 20 reported no exposure to passive smoking, 22 to sometimes

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exposure, and eight to frequent exposure; the concentrations of hair Nic and Cot in each

subgroup are shown in Table 3. There were no significant differences between those who

self-reported no and sometimes exposure, although many subjects who reported no exposure

were positive for Nic and Cot. In contrast, hair Nic and Cot concentrations were significantly

higher in subjects who reported frequent exposure than in the other groups (p<0.01).

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4. Discussion

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Hair may be promising for assessing long-term exposure to Nic and Cot, because hair

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retains Nic and Cot for longer periods of time than other biological specimens, including

urine and blood, and because Nic and Cot concentrations in hair are thought to reflect

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exposure levels [11]. Moreover, hair can be collected non-invasively and stored without
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deterioration at room temperature for at least 5 years [18]. Chromatographic methods for the
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analysis of Nic and Cot in hair are summarized in Table 4. Previously reported methods
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require relatively large sample sizes, as well as time-consuming and laborious sample

preparation methods to isolate and preconcentrate Nic and Cot. The on-line in-tube
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SPME/LC−MS/MS method presented in the present study resulted in the continuous

extraction and concentration of Nic and Cot in hair samples, allowing their selective and
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sensitive analyses by LC−MS/MS. The method described in this study can assess exposure by

analyzing a few hair strands, weighing as little as 1 mg, whereas other methods require a few
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to 100 mg hair [10, 12–22]. Furthermore, Nic and Cot in hair are usually extracted by

digestion with an alkaline or organic solvent at temperatures ranging from room temperature
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to 80ºC for 30 min to 18 h [10, 12–22], but these compounds may be unstable and partially

decompose under these conditions. This study showed that Nic and Cot could be successfully

extracted from hair sample by incubation in distilled water at 80ºC for 30 min. In addition,

this method accurately analyzed pg/mg levels of Nic and Cot in single hair samples and

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showed higher sensitivity than other methods.

4. Conclusion

The on-line in-tube SPME/LC–MS method developed in this study can continuously

extract and concentrate Nic and Cot from hair samples, followed by their analysis by LC-MS.

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This method is automated, simple, rapid, selective, and sensitive, and can be applied easily to

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the analysis of small amounts of hair samples without pre-treatment. This method is a very

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useful tool for monitoring tobacco smoking and for the assessment of long-term exposure to

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tobacco smoke over days or months in active and passive smokers.

Acknowledgements U
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This study was supported by Smoking Research Foundation.
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Declaration of interest
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The authors declare no conflict of interest.

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and its application – a review, Anal. Bioanal. Chem. 373 (2002) 31−45.

T
[24] H. Kataoka, New trends in sample preparation for clinical and pharmaceutical analysis,

IP
(Review) Trends in Anal. Chem. 22 (2003) 232−244.

R
[25] H. Kataoka, A. Ishizaki, Y. Nonaka, K. Saito, Developments and applications of

SC
capillary microextraction techniques: A review, Anal. Chim. Acta 655 (2009) 8−29.

[26] H. Kataoka, Recent developments and applications of microextraction techniques in drug

analysis, Anal. Bioanal. Chem. 396 (2010) 339−364. U

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[27] H. Kataoka, K. Saito, Recent advances in SPME techniques in biomedical analysis, J.
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Pharm. Biomed. Anal. 54 (2011) 926−950.
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[28] H. Kataoka, A. Ishizaki, K. Saito, Solid-phase microextraction for pharmaceutical and

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ED

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T
EP

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(2009) 108−114.
CC
A

17
Figure captions

Fig. 1. MRM chromatograms obtained from hair samples of (A) a non-smoker and (B) a

smoker. In-tube SPME LC−MS/MS conditions are described in the Experimental section.

Fig. 2. Correlation of concentrations of nicotine and cotinine in hair samples of 50

non-smokers.

T
R IP
Table captions

SC
Table 1 Linearity, sensitivity and precisions of nicotine and cotinine by in-tube SPME

LC−MS/MS. U
N
Table 2 Recoveries of nicotine and cotinine spiked into hair samples.
A
Table 3 Relationship between nicotine and cotinine levels in hair samples and self-reported
M

frequency of passive smoking.

T ED
EP
CC
A

18
 T
R IP
SC
Fig. 1. MRM chromatograms obtained from hair samples of (A) a non-smoker and (B) a

U
smoker. In-tube SPME LC−MS/MS conditions are described in the Experimental section.
N
A
M
T ED
EP
CC
A

19
Fig. 2. Correlation of concentrations of nicotine and cotinine in hair samples of 50
non-smokers.

T
R IP
SC
U
N
A
M
T ED
EP
CC
A

20
Table 1
Linearity, sensitivity and precisions of nicotine and cotinine by in-tube SPME
LC−MS/MS.

T
Precision (RSD
Linearity LOD b) (pg/mL) c)
%), (n = 5)

IP
Compound Concentration
Correlation
Range Direct In-tube (pg/mL)

R
coefficient Intra-day Inter-day
(pg/mL) a)
injection SPME

SC
5 ─ 20 1.62 2.42
Nicotine 0.9997 8.56 0.45 200 1.64 5.97
1000
5 ─ 20 3.37 2.35
Cotinine
1000
0.9992 2.68 0.13
U
200 3.12 2.49
N
a)
n = 24.
b)
A
Limits of detection (signal to noise ratio of 3).
c)
RSD, relative standard deviation.
M
T ED
EP
CC
A

21
Table 2
Recoveries of nicotine and cotinine spiked into hair samples.
Concentration (ng/mg hair) Recovery

T
Compound
Spiked Mean ± SD (n=3) (%)

IP
0 0.123 ± 0.008

R
Nicotine 0.10 0.218 ± 0.006 95.0

SC
1.00 1.084 ± 0.001 96.1
0 0.070 ± 0.002
Cotinine 0.10
1.00
0.157 ± 0.002
U
1.036 ± 0.009
87.0
96.6
N
A
M
T ED
EP
CC
A

22
Table 3
Relationship between nicotine and cotinine levels in hair samples and

T
self-reported frequency of passive smoking.

IP
Concentration in hair (ng/mg), Mean±SD / (Range)

R
Not at all (n=20, age Sometimes (n=22, age Frequently (n=8, age
20–62) 19–63) 20–34)

SC
1.86±1.03 /
Nicotine 1.86±1.12 / (0.34–4.55) 9.20±13.85 / (0.72–40.0)
(0.87–4.54)

Cotinine
0.008±0.003 / 0.009±0.003
U / 0.050±0.074 /
N
(0.004–0.015) (0.005–0.015) (0.007–0.213)
A
M
T ED
EP
CC
A

23