Journal of Chromatography B, 877 (2009) 1185–1192

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of four tobacco-specific N-nitrosamines

(TSNA) in human urine
Dominique Kavvadias a , Gerhard Scherer a,∗ , Michael Urban a , Francis Cheung b ,
Graham Errington b , Jim Shepperd b , Mike McEwan b
a
Analytisch-biologisches Forschungslabor GmbH, Goethestrasse 20, 80336 Munich, Germany
b
British American Tobacco, Group Research and Development, Regents Park Road, Southampton SO15 8TL, UK

a r t i c l e i n f o a b s t r a c t

Article history: Tobacco-specific N-nitrosamines (TSNA) include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Received 20 December 2008 (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT). TSNA are
Received in revised form 2 March 2009 suggested to play an important role in tobacco smoke carcinogenesis. We have developed and validated
Accepted 8 March 2009
an LC–MS/MS method for the determination of total (free and conjugated) TSNA in human urine. The lim-
Available online 16 March 2009
its of detection (LOD) were 2.0, 0.8, 1.1 and 0.7 pg/ml for NNAL, NNN, NAB and NAT, respectively. Smokers
were found to have significantly higher levels of TSNA in their urine than nonsmokers. In conclusion, the
Keywords:
newly developed method is suitable for assessing the tobacco use-related exposure to NNK, NNN, NAB
Tobacco-specific N-nitrosamines (TSNA)
4-(Methylnitrosamino)-1-(3-pyridyl)-1-
and NAT.
butanone
© 2009 Elsevier B.V. All rights reserved.
(NNK)
4-(Methylnitrosamino)-1-(3-pyridyl)-1-
butanol
(NNAL)
N-nitrosonornicotine (NNN)
N-nitrosoanabasine (NAB)
N-nitrosoanatabine (NAT)
Biological monitoring
Tobacco smoking
Human urine

1. Introduction to be involved in the development of lung, pancreatic and possibly

Tobacco-specific N-nitrosamines (TSNA) include 4-(methylni-
trosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine
(NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT).
They are formed from tobacco alkaloids during the curing pro-
cess and occur in tobacco and tobacco smoke [1–3]. TSNA levels
in tobacco vary widely and are significantly correlated with
the amount of nitrate in tobacco [4]. Median and ranges of
mainstream smoke levels determined in the 1999 Massachusetts
Benchmark study on 18 leading cigarette brands in the USA
were found to be 147.3 (53.5–220.7), 199.1 (99.9–317.3) and 26.2
(14.2–45.3) ng/cigarette for NNK, NNN and NAB, respectively [5].
The predicted mean mainstream smoke yield for NAT was 231 (95%
confidence interval: 154–308) ng/cigarette [5].
NNK and NNN are IARC Class 1 carcinogens (‘carcinogenic to
humans’) [6]. Based on previous animal studies, NNK is believed
∗ Corresponding author. Tel.: +49 89 535395; fax: +49 89 5328039.
E-mail address: gerhard.scherer@abf-lab.com (G. Scherer).
other cancers, whereas NNN may play a role in causing oesophageal
cancer in smokers [7]. They have also been implicated in the devel-
opment of oral cancer in smokeless tobacco users [6]. The IARC Class
3 carcinogen (‘not classifiable for human carcinogenicity’) NAB is a
weak oesophageal carcinogen in rats, whereas NAT (also a Class 3
carcinogen) is inactive in rats [8].
The metabolism of NNK has been investigated extensively (for
review, see [8]). NNK is rapidly reduced to 4-(methylnitrosamino)-
1-(3-pyridyl)-1-butanol (NNAL), which is also a carcinogen. NNAL
can be detoxified by O- or N-pyridine-glucuronidation. Experiments
using monkeys suggest that smokers might excrete about 20–30%
of the absorbed NNK dose as NNAL and NNAL-glucuronide (total
NNAL) in their urine [9,10]. Total urinary NNAL has been shown
to be a specific biomarker for exposure to NNK in smokers, users
of smokeless tobacco, and nonsmokers exposed to environmental
tobacco smoke (for review, see [11]).
NNN metabolism in vivo has been extensively investigated in
laboratory animals (for review, see [8]). A pharmacokinetic study
in patas monkeys revealed NNN-N-oxide and norcotinine, nor-
cotinine-1-N-oxide, 3 -hydroxynorcotinine and its O-glucuronide

1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2009.03.009
1186 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192

Fig. 1. Chemical structure of the free bases of tobacco-specific nitrosamines determined in human urine.

as well as keto alcohol, keto acid, lactol, lactone and hydroxy acid as
metabolites in urine [12]. Only recently, NNN-N-glucuronide, along
with NNN, was identified in urine of tobacco users [13].
The major urinary metabolite of NAB was found to be NAB-N-
oxide (about 25–30%) [8]. No metabolic studies on NAT have been
reported. Recently, the pyridine-N-glucuronides of NAB and NAT
have been identified in urine of tobacco users along with the cor-
responding free bases [13].
The aim of our investigation was to develop and validate an ana-
lytical method for the simultaneous determination of total (sum of
free base and N-glucuronide) NNAL, NNN, NAB and NAT in human
urine. The chemical structures of the free bases are shown in
Fig. 1.
2. Experimental
2.1. Chemicals
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), meth-
yl-deuterated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
(NNAL-d3 ), N -nitrosonornicotine (NNN), pyridine-ring-
deuterated N -nitrosonornicotine (NNN-d4 ), N -nitrosoanabasine
(NAB), pyridine-ring-deuterated N -nitrosoanabasine (NAB-d4 ),
N -nitrosoanatabine (NAT), and pyridine-ring-deuterated N -
nitrosoanatabine (NAT-d4 ) were purchased from Toronto Research
Chemicals, North York, Ontario, Canada.
De-ionized water was irradiated with ultra-violet light
(254 nm) for at least 12 h directly before use, in order to
destroy any N-nitrosamines. Acetonitrile/0.1% formic acid (for
LC–MS), hydrochloric acid (>37%), de-ionized water, de-ionized
water/0.1% ammonium acetate (both irradiated at 254 nm for
>12 h) were supplied by Riedel-de Haën/Sigma–Aldrich, Steinheim,
Germany. Ammonium hydroxide (25% in water) was obtained
form Fluka/Sigma–Aldrich, Steinheim, Germany. Dichloromethane,
hexane, methanol (all nitrosamine free), were purchased from Pro-
mochem GmbH, Wesel, Germany. Disodiumhydrogen phosphate
(≥99.5%), heptane (99%), toluene (≥99.9%) and ␤-glucuronidase
(type IX-A, E. coli) were obtained from Sigma–Aldrich, Steinheim,
Germany. Potassium dihydrogen phosphate was purchased from
Merck, Darmstadt, Germany.
2.3. Sample preparation
Two millilitres of 0.67 M phosphate buffer (pH 7.2) and 20 ␮l
methanol containing 25 ng/ml of each NNAL-d3 , NNN-d4 and NAB-
d4 as well as 250 ng/ml NAT-d4 and 40 ␮l ␤-glucuronidase in
phosphate buffer (pH 7.2, 250 units/␮l) were added to 6 ml of urine.
The mixture was incubated overnight (14 h) at 37 ◦ C in the dark.
The hydrolysate was applied to a TSNA-molecular-imprinted-phase
(MIP) cartridge (50 mg, 10 ml, Supelco, Taufkirchen, Germany),
which was preconditioned with 10 ml methanol/dichloromethane
(1:9), 1 ml methanol and 1 ml water. The cartridge was washed
with 4 ml ammonium acetate (10 mM), 2 ml heptane, 1 ml hexane
and eluted with 3 ml dichloromethane/toluene (1:1). The extract
was reduced to dryness in a SpeedVac evaporator (Thermo-Fisher,
Dreieich, Germany) and re-dissolved in 1 ml 0.67 M phosphate
buffer, pH 7.2. The extract was applied to a cation-exchange
cartridge (Oasis MCX, 60 mg, 3 ml, Waters GmbH, Darmstadt, Ger-
many) pre-conditioned with 2 ml methanol, 1 ml methanol/25%
ammonium hydroxide (9:1) and 2 ml water. The cartridge was
washed with 2 ml water, 2 ml 0.1 N HCl, 2 ml methanol and
eluted with 2 ml methanol/25% ammonium hydroxide (9:1). The
eluate was evaporated to dryness and re-dissolved in 100 ␮l
0.1% ammonium acetate in water/0.1% formic acid in acetonitrile
(9:1).
2.4. LC–MS/MS analysis
Ten microlitres of the extract were injected into the LC–MS/MS
system. Chromatography was performed on a Luna C18 column
(250 mm × 3.0 mm, 3 ␮m particle size, Phenomenex, Aschaffen-
burg, Germany) at 50 ◦ C with a flow rate of 0.45 ml/min, applying a
gradient consisting of 0.1% ammonium acetate in water (A) and 0.1%
formic acid in acetonitrile (B). Gradient conditions were as follows:
0–1 min: 90% A, 1–5 min: 90–40% A, 5–6 min: 40–30% A, 6–10 min:
30% A, 10–11 min: 30–90% A, 11–17 min: 90% A. Positive electro-
spray ionization (ESI+) was applied with an ion source temperature
of 650 ◦ C. The MS/MS system was run in the multiple-reaction
monitoring (MRM) mode. Retention times and mass transitions for
quantification and qualification of analytes and internal standards
are shown in Table 1.

2.2. Instrumentation
Table 1
The LC–MS/MS system consisted of a Model 1100 HPLC device Retention times and mass transitions of quantifiers and qualifiers of the analytes
(Agilent Technologies, Waldbronn, Germany) and an atmospheric and the deuterated internal standards.
pressure ionization triple quadrupole mass spectrometer (Sciex API Mass transition (m/z)
4000, Applied Biosystems, Darmstadt, Germany). The HPLC sys-
RT (min) Quantifier Qualifier
tem comprised a binary pump (Model G1312 A), a column oven
(G1316 A), a degasser (G1379 A) and a temperature-controlled NNAL 7.9 210 → 180 210 → 93
autosampler with a Peltier cooled trayholder (HTC PAL, Axel Sem- NNAL-d3 7.9 213 → 183 –
NNN 8.5 178 → 148 178 → 120
rau, Sprockhoevel, Germany). The MS/MS system was equipped NNN-d4 8.5 182 → 152 –
with an electrospray ionization (ESI) source. Nitrogen was sup- NAT 9.1 190 → 160 190 → 79
plied by a nitrogen generator (Model NGM 22, CMC Instruments, NAT-d4 9.1 194 → 164 –
Eschborn, Germany) equipped with a compressor (Model SF 4-10 NAB 9.3 192 → 162 192 → 106
NAB-d4 9.3 196 → 166 –
FF, Atlas Copco, Essen, Germany).
 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192
2.5. Calibration
The method was calibrated by spiking a nonsmoker pooled
urine with 5, 25, 50, 250, 750, 1500 pg/ml NNAL, 2, 10, 25, 50,
100, 250 pg/ml NNN, 5, 10, 25, 50, 100, 250 pg/ml NAB and 2, 10,
100, 250, 750, 1500 pg/ml NAT. Calibration of NAT led to a non-
linear calibration function when 10 ␮l of the extract was injected.
After injection of 2 ␮l of the extract, a linear calibration func-
tion was achieved for NAT. Method validation was performed with
an injection volume of 10 ␮l for NAB, NNAL and NNN and 2 ␮l
for NAT. The background peak area ratio in unspiked urine was
zero for NNAL. The background peak area ratios for NAB, NAT
and NNN in unspiked urine were subtracted from all spiked cal-
ibrators. Each calibrator was analyzed twice. The means of the
analyte/internal standard ratios were used for calculation of the
regression function. The regression curves were forced through the
origin.
2.6. Method validation
The method was validated according to the U.S. Food and
Drug Administration (FDA) guidelines for bioanalytical methods
[14].
2.6.1. Specificity
Six different analyte-free urine matrices were checked for pos-
sible interferences of the analytes and the internal standards.
For NNAL, in 4 urine matrices which were not analyte-free, the
peak identity was confirmed by comparing the peak area ratios
(quantifier/qualifier) with those of the authentic standards. As an
acceptance criterion, ratios in matrix should not deviate by more
than ± 25% (±35% at LOQ) from the mean ratio in the standard solu-
tion. In addition, the accuracy at a medium level was checked in 6
different urine matrices for NNAL, NNN, NAB and NAT.
Fig. 2. Product ion mass spectra with suggested chemical structures of fragments derived from NNAL, NNN, NAB and NAT.
1187
2.6.2. Precision
Precision was determined by measuring NNAL, NNN, NAB and
NAT at three levels. All samples used for the determination of the
precision were authentic human urine matrices, which were partly
spiked with the analytes to achieve the required levels. For intra-day
precision, each sample was analyzed 5 times. For inter-day pre-
cision, samples were analyzed once on 6 different days within 2
weeks.
2.6.3. Accuracy
Accuracies at low, medium and high levels of NNAL, NNN, NAB
and NAT were determined with spiked human urine samples. Each
level was analyzed 5 times.
2.6.4. Recovery
Recovery rates were determined by comparing the peak areas
obtained when spiking (at three levels) the analytes before the
LC–MS/MS measurements (reference = 100%) with the peak areas
obtained when spiking at the start of the sample clean-up proce-
dure. A nonsmoker urine pool was used for the low, medium and
high levels.
2.6.5. Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ were estimated with the signal/noise method
(standard deviation of noise method, integrated in the Analyst soft-
ware Version 1.4.2 of the LC–MS/MS system). Signal/noise ratios of
3 and 9 were applied for estimating the LOD and LOQ, respectively.
A second criterion for the LOQ level was that the deviation from
the nominal concentration should not exceed 20%. A third criterion
was the precision of <20% at the LOQ level.
2.6.6. Stability of analytes
Short-term (24 h) stability at 21 ◦ C of NNAL, NNN, NAB and
NAT in urine at two levels was tested. Long-term stability (up
to 8 months) was tested at −20 ◦ C in urine, at two levels. Three
1188 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192

freeze/thaw cycles at low and high concentrations of TSNA were
checked for any losses. A post-preparative stability check, i.e sta-
bility of analytes and internal standards in extracts ready for
measurement under autosampler conditions (6 d, 10 ◦ C) was also
performed.
2.6.8. Matrix effect
The matrix effects were determined by comparing the peak
areas of the analytes (at low and high spiking levels) and of the inter-
nal standards (at the usual spiking levels) obtained after spiking
an analyte-free matrix extract with the corresponding peak areas
obtained in solvent.

2.6.7. Carry-over effects
Carry-over effects in the chromatographic system were tested
by injecting extracts with high analyte concentrations 5 times, fol-
lowed by a low concentration sample at about LOQ level. This cycle
was repeated 3 times.
2.6.9. Artifactual formation of TSNA during the analytical
procedure
The artifactual formation of TSNA during the sample work-up
was checked by spiking a nonsmoker pool urine with the precursor
compounds anabasine, anatabine and nornicotine at concentra-

Fig. 3. Chromatograms of the ion transitions for NNAL (119.0 pg/ml), NNN (17.2 pg/ml), NAB (69.9 pg/ml) and NAT (327.4 pg/ml) (left panel) and their respective deuterated
internal standards (right panel) in a urine sample of a smoker. The mass transitions used are the quantifier ions shown in Table 1.
 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192 1189

Fig. 3. (Continued ).

tions of 215 ng/ml, 217 ng/ml and 253 ng/ml, respectively, which
is about 10% of the smoking-related urinary nicotine level. The
samples were treated as described for the analytical procedure,
and analyzed in duplicate.
2.7. Human urine samples
Fourteen urine samples (7 from healthy nonsmokers and 7 from
healthy smokers) were analyzed for TSNA. Smokers collected a
24-h urine and were asked to report their daily cigarette consump-
tion.
3. Results
3.1. Product ion mass spectra and chromatograms
The product ion mass spectra and the structures for the sug-
gested ion fragments of the four TSNA are shown in Fig. 2. Loss of
NO (m/z 30) is the predominant mass transition, which is used as
quantifier for all analytes and internal standards.
Typical chromatograms of the mass transitions for NNAL, NNN,
NAB, NAT and the corresponding deuterated internal standards for
a urine sample of a smoker are shown in Fig. 3.
1190 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192

103.9%
3.2. Method performance

101.8%
93.5%

46.3%
60.4%

89.8%
95.4%
86.4%
13.2%

77.4%
2.2%

8.8%
6.7%
2.5%
1.4%

2 pg/ml (CV = 7.2%)

Method performance data are summarized in Table 2.

∼0.7 pg/ml
The specificity test revealed that in all matrices tested, the NAB,
NAT and NNN interferences were below 20% of the LOQ concentra-

IS (833 pg/ml):
tion and the internal standard interferences were always below 5%

1422.9 pg/ml:

1422.9 pg/ml:

1350.0 pg/ml:

1350.0 pg/ml:
750.0 pg/ml:

750.0 pg/ml:
441.6 pg/ml:

441.6 pg/ml:

52.0 pg/ml:
600 pg/ml:
of the peak area. For NNAL, three of the investigated matrix sam-

4.3 pg/ml:

4.3 pg/ml:

4.9 pg/ml:

4.9 pg/ml:
ples were almost free of analyte with interferences <20% of the LOQ

NAT
concentration. In 4 urine matrices with interferences >20% of the
LOQ, the peak identity was confirmed by comparing the peak ratios
(quantifier/qualifier) with those of the authentic standards. Accura-
cies determined in 6 different urine matrices were between 85 and
115% and thus regarded as acceptable. Taken together, specificity

88.2%
90.8%

42.0%

78.6%
78.8%
73.5%
76.3%
97.2%

61.6%
11.7%

3.8%
6.5%
3.5%
1.1%
1.1%
was found to be acceptable according to the FDA criteria [14] for all

5 pg/ml (CV = 10.0%)

four analytes.

∼1.1 pg/ml
Precision (both intra- and inter-day) of the method was found
to be acceptable (CV < 15%). Accuracies for all analytes and lev-

IS (83.3 pg/ml):
191.6.8 pg/ml:
els were within the acceptable range of 85–115%. Recovery rates

100.0 pg/ml:
123.3 pg/ml:

123.3 pg/ml:

180.0 pg/ml:

180.0 pg/ml:
191.6 pg/ml:

100.0 pg/ml:

80.0 pg/ml:
11.0 pg/ml:

11.0 pg/ml:
of the whole analytical procedure at low, medium and high lev-

8.9 pg/ml:

8.9 pg/ml:

5.0 pg/ml:
els were 32.1–45.5, 37.9–56.8, 42.0–76.3 and 46.3–77.4% for NNAL,

NAB
NNN, NAB and NAT, respectively. The lowest recovery rates were
usually observed at the lowest concentration. LODs (LOQs) were
2.0 (5.0), 0.8 (2.0), 1.1 (5.0) and 0.7 (2.0) pg/ml for NNAL, NNN, NAB
and NAT, respectively.
The method was linear over the calibration ranges of 5–1500,

66.3%
56.8%

63.4%
90.8%
90.3%

40.2%
37.9%

67.1%
11.4%

91.1%
4.4%

3.1%

5.1%
1.9%

7.1%
2–250, 5–250 and 2–1500 pg/ml for NNAL, NNN, NAB and NAT,

2 pg/ml (CV = 13.6%)

LOQ was determined from the lowest calibrator fulfilling two conditions: (1) deviation from nominal value <20%, (2) CV < 20% (N = 2).
respectively. Injection volume for NAT analysis was reduced from
10 ␮l to 2 ␮l in order to achieve linearity.

0.8 pg/ml
All analytes were found to be stable in urine under short-term

IS (83.3 pg/ml
(24 h, 21 ◦ C) and long-term storage conditions (8 months, −20 ◦ C).
224.6 pg/ml:

224.6 pg/ml:

100.0 pg/ml:

100.0 pg/ml:
180.0 pg/ml:

180.0 pg/ml:
90.0 pg/ml:

90.0 pg/ml:
4.3 pg/ml:

4.3 pg/ml:

4.1 pg/ml:

4.1 pg/ml:

80.0 pg/m
Three freeze/thaw cycles did not lead to measurable losses of the

2.0 pg/m
analytes. Sample extracts were stable under autosampler condi-
NNN

tions (6 d, 10 ◦ C).
No carry-over effects during LC–MS/MS analysis were observed
for any of the four analytes.
Slightly negative matrix effects, i.e. suppression of the responses
100.4%
109.7%
110.7%

45.5%

75.9%
72.2%
39.1%
32.1%

70.1%
11.9%

11.5%

by 30, 35, 23 and 15%, were observed for NNAL, NNN, NAB and NAT,
8.3%

3.9%

9.9%
6.0%

respectively, and their deuterated internal standards.

5 pg/ml (CV = 0.3%)

No artifactual formation of NNN, NAB and NAT was detected

∼2.0 pg/ml
during the analytical procedure in the presence of the precursor
alkaloids nornicotine, anabasine and anatabine.

IS (83.3 pg/ml):
1252.0 pg/ml:

1252.0 pg/ml:

1350.0 pg/ml:

1350.0 pg/ml:
590.6 pg/ml:

590.6 pg/ml:

750.0 pg/ml:

750.0 pg/ml:

600 pg/ml:
8.5 pg/ml:

8.5 pg/ml:

5.0 pg/ml:
7.8 pg/ml:

7.8 pg/ml:
Performance data of the analytical method for NAB, NAT, NNAL and NNN in urine.

3.3. TSNA in human urine

NNAL

The method was applied to 14 urine samples derived from non-

smokers and smokers. The individual concentration of the urinary
TSNA as well as the means and standard deviations are shown in
Table 3. NNN was < LOD in one of the smoker’s urine samples.
Matrix effect (3 different urines, low and high spiking levels)

In another smoker urine sample, NNAL, NNN, NAB and NAT were
between LOQ and LOD.
For smokers, there was a significant correlation between the
TSNA (expressed as amount excreted in 24 h) and the reported daily
cigarette consumption (NNAL: r = 0.92, p = 0.003; NNN: r = 0.93,
p = 0.002; NAB: r = 0.71, p = 0.05; NAT: r = 0.85, p = 0.015).
Recovery (whole analytical procedure)

4. Discussion

To our knowledge, this is the first method which allows the

simultaneous determination of NNAL, NNN, NAB and NAT in human
urine. The newly developed LC–MS/MS method includes the fol-
Inter-day (6 days)
Intra-day (N = 5)

Intra-day (N = 5)

lowing steps: (i) Addition of the four corresponding deuterated

internal standards to 6 ml urine, (ii) release of the free-base TSNA
from the glucuronides by treatment with ␤-glucuronidase, (iii)
Precision

Accuracy
Table 2

solid phase extraction on a TSNA-specific MIP, (iv) solid phase

LOQa
LOD

extraction on a cation-exchange resin, (v) LC–MS/MS analysis with

 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192 1191

Table 3
TSNA concentrations (pg/ml) in urine samples of nonsmokers and smokers.

Cigarettes/d NNAL NNN NAB NAT

Nonsmokers
1 – <LOD <LOD <LOD <LOD
2 – 2.3 (<LOQ) <LOD <LOD <LOD
3 – <LOD <LOD <LOD <LOD
4 – 8.34 <LOD <LOD <LOD
5 – <LOD <LOD <LOD <LOD
6 – <LOD <LOD <LOD <LOD
7 – 5.47 <LOD <LOD <LOD
Mean ± SDa – 2.68 ± 3.09 <LOD <LOD <LOD

Smokers (arranged in increasing order of cigarette consumption)

12 6 10.6 <LOD 3.9 (<LOQ) 15.0
8 12 3.8 (<LOQ) 0.52 (<LOQ) 1.1 (<LOQ) 1.0 (<LOQ)
11 14 94.5 4.74 26.5 96.7
14 18 374.9 17.64 126.8 422.9
13 20 247.3 11.48 97.6 288.0
10 25 160.6 6.27 36.0 132.9
9 30 176.1 9.33 37.0 171.5
Mean ± SDa 17.9 ± 8.1 152.5 ± 132.1 7.20 ± 6.19 47.0 ± 47.5 161.1 ± 150.9
a
For calculation of the mean and standard deviation (SD) for samples with <LOD concentrations, 1/2 LOD was used.

positive electrospray ionization and quantification in the MRM
mode.
The observed product ion fragments for NNN, NAB and NAT
(Fig. 2) were similar to those reported earlier [15]. The molecular
ions (MH+ ) with m/z 210, 178, 192 and 190 of NNAL, NNN, NAB and
NAT, respectively, are visible in the four individual spectra (Fig. 2).
The major fragment ions [MH–30]+ , derived from loss of NO, appear
in all four spectra. The same is true for the pyridyl radical cation (m/z
79) and the methylpyridine radical cation (m/z 93). The deuterated
internal standards show mass fragmentations analogous to those
of the undeuterated compounds. The NO loss fragmentation was
used for quantification of both analytes and internal standards.
The method performance (Table 2) meets the criteria stated in
the guidelines of the U.S. Food and Drug Administration for bio-
analytical methods [14]. The recovery over the whole analytical
procedure was approximately 35–80%. The recovery was somewhat
lower for NNAL and NNN compared to NAB and NAT, but still within
an acceptable range. This is particularly true because any variations
in the recovery rate from sample to sample are controlled by the
use of the deuterated internal standards. The same is true for the
moderate negative matrix effect yielding a 5–30% suppression of
the response. Suppression by urinary matrix increased in the order
NAT < NAB < NNAL < NNN. LOQs for the four TSNA in urine are in the
low (2–5) pg/ml range, which allows the quantification of NNAL,
NNN, NAB and NAT in urine samples of tobacco users (i.e. com-
bustible and smokeless) without difficulty. Quantification of total
NNAL in urine samples of nonsmokers exposed to environmental
tobacco smoke is also possible with our method, since reported
concentrations for this group are in the range of 6–12 pg/ml [5].
We observed no detectable artifactual formation of NNN, NAB and
NAT when the corresponding precursors were added to urine in
concentrations of 200–250 ng/ml prior to the analysis. It is interest-
ing to note that significant artifactual formation of NAT and NNN
was observed when the order of the two SPE steps was reversed
(first SPE on cation-exchange cartridges, then MIP-SPE). We pre-
sume that the acidic conditions in the presence of nitrosating agents
in urine (probably nitrite), combined with the alkaloids present dur-
ing washing of the cation-exchange cartridge, were responsible for
the artifactual formation.
Stepanov and Hecht [13] were the first to detect NNN, NAB and
NAT, as well as their corresponding pyridine-N-glucuronides, in
urine of smokers and smokeless tobacco users. Their method for
determining total NNN, NAB and NAT comprised enzymatic hydrol-
ysis of the N-glucuronides, two liquid/liquid distributions, two solid
phase extractions and analysis with GC-TEA (gas chromatography
with a nitrosamine-specific thermal energy analyzer). 5-Methyl-
N -nitrosonornicotine was added as an internal standard after the
enzymatic hydrolysis. GC-TEA peak identity was confirmed in some
urine samples by GC-MS/MS using two mass transitions for each
analyte. The reported method performance was to a large extent
similar to our method. Recovery of NNN in urine was 79%, which
is higher than that in our study (38–57%). The reported LODs for
NNN, NAB and NAT were 5.7, 3.4 and 2.6 pg/ml, respectively, which
is also somewhat higher than in our method. Stepanov and Hecht
[13] reported no artifactual formation of NNN, NAB and NAT during
the analytical procedure.
Comparing the urinary TSNA levels of smokers reported by
Stepanov and Hecht [13] to smokers in our study, the average con-
centrations of total NNN (47.2 versus 7.2 pg/ml) and total NNAL
(473.8 pg/ml versus 152.5) were lower in our study, whereas the
concentrations of total NAT (53.2 versus 161.1 pg/ml) and total
NAB (11.3 versus 47.0 pg/ml) were lower in the Stepanov study.
Presently, we cannot provide an explanation for these discrepan-
cies, although differences in the tobacco product yields may be a
possibility.
We observed significant correlations between the daily cigarette
consumption and the daily excretion of total NNAL (r = 0.92,
p = 0.003), NNN (r = 0.93, p = 0.002), NAB (r = 0.71, p = 0.05) and NAT
(r = 0.85, p = 0.015). This is in agreement with correlations reported
by Stepanov and Hecht [13]. Similarly, we confirmed the very
strong bivariate correlations between the urinary TSNA found in
the Stepanov study (r > 0.95).
Total NNAL was quantifiable in 2 of 7 nonsmoker urine samples,
with 1 sample being between LOD and LOQ of the method (Table 3).
The observed concentrations of total NNAL in urine of nonsmokers
is in agreement with the range of 6–12 pg/ml reported in previous
studies [5]. NNN, NAB and NAT were not detectable in any of the
nonsmoker urine samples. We are not aware of any published data
for NNN, NAB or NAT in the body fluids of nonsmokers.
Urinary TSNA levels of smoker no 8 were below the LOQ, despite
the fact that this individual smoked 12 cigarettes/d (Table 3). A
check of the additional information on the subject revealed that
this subject had also a very low salivary cotinine level (12.5 ng/ml),
suggesting a non-inhaling smoking behaviour.
In conclusion, an analytical method for the simultaneous deter-
mination of total NNAL, NNN, NAB and NAT in human urine was
developed. The method performance data meet the commonly
accepted criteria for bioanalytical methods. The detection sensi-
1192 D. Kavvadias et al. / J. Chromatogr. B 877 (2009) 1185–1192
tivity for total NNAL is similar to previously reported methods
and, therefore, sufficient to detect the exposure of nonsmokers
to environmental tobacco smoke. All four TSNA were found to be
significantly higher in the urine of smokers compared to nonsmok-
ers. We conclude that the newly developed method is suitable to
quantify the tobacco use-related exposure to NNK, NNN, NAB and
NAT.
References
[1] K.D. Brunnemann, D. Hoffmann, Toxicology 21 (1991) 235.
[2] S.S. Hecht, C.H.B. Chen, R. Young, D. Hoffmann, Beitr. Tabakforsch. Int. 11 (1981)
57.
[3] A. Wiernik, A. Christakopoulos, L. Johansson, I. Wahlberg, Recent Adv. Tobacco
Sci. 21 (1995) 39.
[4] S. Fischer, B. Spiegelhalder, R. Preußmann, Carcinogenesis 10 (1989) 1511.
[5] International Agency for Research on Cancer, IARC Monographs on the Evalua-
tion of Carcinogenic Risks to Humans, 83 (2004) 1.
[6] International Agency for Research on Cancer, IARC Monographs on the Eval-
uation of the Carcinogenic Risk of Chemicals to Humans—Tobacco Habits
Other than Smoking; Betel-Quid and Areca-Nut Chewing; and Some Related
Nitrosamines, IARC Press, Lyon, 1985.
[7] S.S. Hecht, D. Hoffmann, Cancer Surv. 8 (1989) 273.
[8] S.S. Hecht, Chem. Res. Toxicol. 11 (1998) 559.
[9] S.S. Hecht, N. Trushin, A.R. Reid-Quinn, E.S. Burak, A.B. Jones, J.L. Southers,
C.T. Gombar, S.G. Carmella, L.M. Anderson, J.M. Rice, Carcinogenesis 14 (1993)
229.
[10] M. Meger, E. Richter, W. Zwickenpflug, C. Oehlmann, M.B. Hargaden, Y.I.A.
Rahim, E.S. Vesell, Drug Metab. Dispos. 27 (1999) 471.
[11] S.S. Hecht, Carcinogenesis 23 (2002) 907.
[12] P. Upadhyaya, C.L. Zimmerman, S.S. Hecht, Drug Metab. Dispos. 30 (2002) 1115.
[13] I. Stepanov, S.S. Hecht, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 885.
[14] Food and Drug Administration (FDA), U.S. Department of Health and Human
Services, 2001, p. 1.
[15] C. Jansson, A. Paccou, B.G. Osterdahl, J. Chromatogr. A 1008 (2003) 135.