Subpicogram per milliliter determination of the tobacco-specific carcinogen metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine using liquid chromatography-tandem mass spectrometry

Article abstract of DOI:10.1021/ac8009005

Exposure to secondhand tobacco smoke (SHS) has been linked to increased risk for a number of diseases, including lung cancer. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is of particular interest due to its potency and its specificity in producing lung tumors in animals. The NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanol (NNAL) in urine is frequently used as a biomarker for exposure. Due to its long half-life (40-45 days), NNAL may provide a long-term, time-averaged measure of exposure. We developed a highly sensitive liquid chromatography- tandem mass spectrometry method for determination of NNAL in human urine. The method involves liquid-liquid extraction followed by conversion to the hexanoate ester derivative. This derivative facilitates separation from interfering urinary constituents by extraction and chromatography and enhances detection with electrospray ionization mass spectrometry. The lower limit of quantitation is 0.25 pg/mL for 5-mL urine specimens. Applications to studies of people with a range of different SHS exposure levels is described.

Full text of DOI:10.1021/ac8009005

Anal. Chem. 2008, 80, 8115–8121
Subpicogram per Milliliter Determination of the
Tobacco-Specific Carcinogen Metabolite
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in
Human Urine Using Liquid
Chromatography-Tandem Mass Spectrometry
Peyton Jacob III,*,† Christopher Havel,† Do-Hoon Lee,‡ Lisa Yu,† Mark D. Eisner,§ and
Neal L. Benowitz†
Division of Clinical Pharmacology and Experimental Therapeutics, the Departments of Medicine, Psychiatry, and
Biopharmaceutical Sciences, San Francisco General Hospital Medical Center, 1001 Potrero Avenue, University of
California, San Francisco, UCSF Box 1220, San Francisco, California 94143-1220, Diagnostic Laboratory, National
Cancer Center, Goyang-si, Gyeonggi-do, South Korea 410-769, and Division of Occupational and Environmental
Medicine, Department of Medicine, University of California, San Francisco, UCSF Box 0924,
San Francisco, California 94113-0924
Exposure to secondhand tobacco smoke (SHS) has been
linked to increased risk for a number of diseases, includ-
ing lung cancer. The tobacco-specific nitrosamine 4-(me-
thylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is of
particular interest due to its potency and its specificity in
producing lung tumors in animals. The NNK metabolite
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in
urine is frequently used as a biomarker for exposure. Due
to its long half-life (40-45 days), NNAL may provide a
long-term, time-averaged measure of exposure. We de-
veloped a highly sensitive liquid chromatography-tandem
mass spectrometry method for determination of NNAL in
human urine. The method involves liquid-liquid extrac-
tion followed by conversion to the hexanoate ester deriva-
tive. This derivative facilitates separation from interfering
urinary constituents by extraction and chromatography
and enhances detection with electrospray ionization mass
spectrometry. The lower limit of quantitation is 0.25 pg/
mL for 5-mL urine specimens. Applications to studies of
people with a range of different SHS exposure levels is
described.
causes of morbidity and mortality.4 The association between
cigarette smoking and lung cancer has been known for many
years, well documented and publicized initially in the 1964
Surgeon General’s Report.5 A number of epidemiological studies
have demonstrated an increased risk of lung cancer in nonsmok-
ers having long-term exposure to SHS.4,6
Based on toxicology studies in animals, it has been proposed
that polycyclic aromatic hydrocarbons and tobacco-specific nit-
rosamines (TSNAs are the major lung carcinogens in tobacco
smoke.7 The TSNA 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK) has received particular interest due to its potency as a
carcinogen and its specificity in producing lung tumors in
laboratory animals.7,8 NNK is specific to tobacco and tobacco
smoke, formed from nicotine by oxidation and nitrosation during
the curing process, as well as being formed pyrosynthetically from
nicotine during smoking (Figure 1).8-10
In humans and in laboratory animals, a major route of NNK
metabolism is reduction of the keto group to give the secondary
alcohol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL (Fig-
ure 1). NNAL is further metabolized to N- and O-glucuronide
conjugates, which are excreted in urine along with free NNAL.7,8
A number of studies have documented the presence of NNAL
and its glucuronides in urine of smokers and in urine of
nonsmokers exposed to SHS. Since these are quantitatively
Tobacco and tobacco smoke contain numerous toxic sub-
stances, including ∼70 carcinogens.1 More than 400 000 people
in the United States and about 5 000 000 people worldwide die
from tobacco-related diseases each year.2,3 Most of these deaths
occur in cigarette smokers, but other forms of tobacco use and
exposure to secondhand tobacco smoke (SHS) are also significant
(4) Department of Health and Human Services, P. H. S. The Health Conse-
quences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon
General. DHHS (CDC) Publication No. 87-8398; US Government Printing
Office: Washington DC, 2006.
(5) Smoking and Health: A Report of the Advisory Committee to the Surgeon
General, Public Health Service Publication No. 1103 ed.; U.S. Department
of Health and Human Services: Bethesda, MD, 1964.
* To whom correspondence should be addressed. E-mail: peyton.jacob@
ucsf.edu. Phone: (415) 282-9495. Fax: (415) 206-5080.
† Division of Clinical Pharmacology, University of California, San Francisco.
‡ National Cancer Center.
(6) International Council for Research on Cancer IARC Sci. Publ. 2004, 83.
(7) Hech, S. S. Chem. Res. Toxicol. 1998, 11, 559–603
.
§ Division of Occupational and Environmental Medicine, University of
California, San Francisco.
(8) Hecht, S. S.; Tricker, A. R. In Analytical Determination of Nicotine and
Related Compounds and Their Metabolites; Gorrod, J. W., Jacob, P., III, Eds.;
Elsevier: Amsterdam, 1999; pp 421-488.
(1) Hoffmann, D.; Hoffmann, I.; El-Bayoumy, K. Chem. Res. Toxicol. 2001,
14, 767–790
.
(9) Hecht, S. S.; Chen, C. B.; Ornaf, R. M.; Jacobs, E.; Adams, J. D.; Hoffmann,
(2) MMWR Morb. Mortal. Wkly. Rep. 2005, 54, 625-628.
(3) Benowitz, N. L. Clin. Pharmacol. Ther. 2008, 83, 531–541
D. J. Org. Chem. 1978, 43, 72–76
.
.
(10) Adams, J. D.; Lee, S. J.; Hoffmann, D. Carcinogenesis 1984, 5, 221–223.
10.1021/ac8009005 CCC: $40.75 2008 American Chemical Society
Published on Web 10/08/2008
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8115

steps with a solid-phase extraction.20 LC-MS/MS has been used
to quantify NNAL in urine of smokers.21,22 Recently, an LC-MS/
MS method having sufficient sensitivity (detection limit of 3 pg/
mL) to determine NNAL in urine of SHS-exposed nonsmokers
was reported. This method utilizes molecularly imprinted polymer
(MIP) columns for selective extraction of NNAL and removal of
interfering urinary constituents.23
As part of the studies on the biological consequences of SHS
exposure, we required a method for determination of NNAL in
urine of people with relatively low exposure levels. In addition, in
some studies large volumes of urine were not available, which
required a very sensitive method. In this report, we describe an
LC-MS/MS method for quantitation of NNAL at subpicogram
per milliliter concentrations in 5-mL urine specimens. The method
involves conversion of the hydroxy group of NNAL to the
hexanoate ester derivative, which facilitates the removal of
interfering urinary constituents and enhances detection using
electrospray ionization mass spectrometry.
Figure 1. Formation and metabolism of 4-(methylnitrosamino)-1-
(3-pyridyl)-1-butanone (NNK).
important metabolites of NNK, their determination in urine is of
considerable utility in studies of human exposure to this tobacco-
specific carcinogen.11,12 In addition, the very long biologic half-
live of NNK, 40-45 days,13 makes its metabolites useful as
biomarkers for studies of long-term exposure to this carcinogen
and to tobacco smoke in general. This is especially useful in
studies of SHS exposure, in which exposure patterns are often
irregular and the impact of exposure on disease outcomes occurs
over a relatively long time period.
EXPERIMENTAL SECTION
Instrumentation. LC-MS/MS analyses were carried out with
a Surveyor HPLC interfaced to a TSQ Quantum Ultra triple-stage
quadrupole mass spectrometer (Thermo-Finnigan, San Jose, CA),
with a heated electrospray ion source (HESI). An HSF5 column
(2.1 × 150 mm, 5 µm, Supelco, Sigma-Aldrich, Saint Louis, MO)
was used for the chromatography. Solvent evaporation was carried
out using a Savant SpeedVac model SC210A (Thermo-Savant,
Marietta, OH).
Depending upon the brand and the smoking conditions, a
cigarette delivers 50-220 ng of NNK in the mainstream smoke,
which is inhaled by the smoker8,14-16 and 50-1440 ng in the
sidestream smoke.6,8,12 This results in urine concentrations (total
NNAL, free + glucuronide) ranging from ∼50 to ∼3000 pg/mL,
with means of ∼400-600 pg/mL in habitual smokers.17 In
nonsmokers, concentrations are much lower, from undetectable
to ∼100 pg/mL, with means ranging from 2 to 20 pg/mL
depending on the exposure levels.18 Measuring low parts per
trillion levels of NNAL in a complex biological matrix such as
urine presents a considerable analytical challenge. In the pioneer-
ing work by Hecht and colleagues, GC with a nitrosamine-selective
detector (thermal energy analyzer, TEA) was used. This method
required extraction of large volumes of urine (50-100 mL),
purification of the extract using two preparative HPLC steps, and
conversion of the secondary hydroxy group to a trimethylsilyl
ether derivative prior to GC analysis using a TEA detector.19
Recently, the method has been streamlined by simplifying the
extraction procedure and replacing one of the HPLC purification
Chemicals. NNAL-d3 was a generous gift from Dr. Stephen
Hecht, University of Minnesota. NNAL-d0 was obtained from
Toronto Research Chemicals, North York, ON, Canada. HPLC
grade methanol and water from Burdick and Jackson (Muskegon,
MI) were used to prepare the LC mobile phase. HPLC grade
toluene, 1-butanol, pentane, and ethyl acetate from Fisher were
used for extractions. ꢀ-Glucuronidase type IXA from Escherichia
coli (3660 units/mg), hexanoic anhydride, and 4-(dimethylami-
no)pyridine (DMAP)were purchased from Sigma-Aldrich.
Preparation of Standards and Controls. A stock standard
solution of NNAl-d0 was prepared in 12 mM HCl and stored frozen
at -20 °C. A 500 pg/mL solution of NNAl-d3 was prepared in 10%
methanol containing 12 mM HCl. Pooled urine, collected over
several days, (2-L batches) from a nonsmoker with no known SHS
exposure was used to prepare the standards and QCs. Standards
and controls were stored frozen at -20 °C until use.
Sample Preparation. The 50 µL of 500 pg/mL NNAl-d3
internal standard solution was added to 5-mL urine samples,
standards, and QCs in 50-mL polypropylene centrifuge tubes
(Fisherbrand) followed by brief vortex mixing. Sodium potassium
phosphate buffer (0.5 mL of 2 M, pH 7) was added followed by
the addition of 50 µL of 50 mg/mL glucuronidase dissolved in
0.1 M phosphate buffer. The samples were then incubated for
20-24 h at 37 °C to convert the NNAL glucuronides to free NNAL.
(11) Hecht, S. S. Carcinogenesis 2002, 23, 907–922
(12) Hecht, S. S. Tob. Control 2004, 13, i48–56
(13) Hecht, S. S.; Carmella, S. G.; Chen, M.; Dor Koch, J. F.; Miller, A. T.;
.
.
Murphy, S. E.; Jensen, J. A.; Zimmerman, C. L.; Hatsukami, D. K. Cancer
Res. 1999, 59, 590–596
(14) Wu, W.; Ashley, D. L.; Watson, C. H. Anal. Chem. 2003, 75, 4827–4832
(15) Harris, J. E. Public Health Rep. 2001, 116, 336–343
(16) Wagner, K. A.; Finkel, N. H.; Fossett, J. E.; Gillman, I. G. Anal. Chem. 2005,
77, 1001–1006
.
.
.
.
(20) Carmella, S. G.; Han, S.; Fristad, A.; Yang, Y.; Hecht, S. S. Cancer Epidemiol.
(17) Hecht, S. S.; Carmella, S. G.; Murphy, S. E.; Riley, W. T.; Le, C.; Luo, X.;
Biomarkers Prev. 2003, 12, 1257–1261
(21) Byrd, G. D.; Ogden, M. W. J. Mass Spectrom. 2003, 38, 98–107
(22) Pan, J.; Song, Q.; Shi, H.; King, M.; Junga, H.; Zhou, S.; Naidong, W. Rapid
Commun. Mass Spectrom. 2004, 18, 2549–2557
(23) Xia, Y.; McGuffey, J. E.; Bhattacharyya, S.; Sellergren, B.; Yilmaz, E.; Wang,
L.; Bernert, J. T. Anal. Chem. 2005, 77, 7639–7645
.
Mooney, M.; Hatsukami, D. K. Cancer Epidemiol. Biomarkers Prev. 2007,
.
16, 1567–1572
(18) Hecht, S. S. Prev. Med. 2006, 43, 256–260
(19) Hecht, S. S.; Carmella, S. G.; Murphy, S. E.; Akerkar, S.; Brunnemann,
K. D.; Hoffmann, D. N. Engl. J. Med. 1993, 329, 1543–1546
.
.
.
.
.
8116 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

(Using twice the enzyme concentration did not increase the
measured amount of NNAL.) Potassium carbonate (0.5 mL of 50%
w/v) was added, and the samples were extracted by vortex mixing
for 5 min with 8 mL of toluene/1-butanol (70:30). The two phases
were separated by centrifugation at 4000g for 5 min. After freezing
the tubes in a dry ice-acetone bath, the organic upper layers were
poured into 16 × 125 mm glass culture tubes containing 0.8 mL
of 1 M H2SO4. The tubes were then vortexed 5 min and
centrifuged as described above. The aqueous layers were frozen
with a dry ice-acetone bath, and the organic layers were poured
off and discarded. The acid layer was then washed with 5 mL of
ethyl acetate/toluene (2:1) by vortex mixing 5 min. The tubes
were centrifuged, the aqueous acid layers were frozen in a dry
ice-acetone bath, and the upper wash layer was discarded. The
acid phases were made basic with 0.8 mL of 50% (w/v) K2CO3
and extracted by vortex mixing 5 min with 4 mL of ethyl acetate/
toluene (1:2). After centrifugation and freezing the aqueous
layers, the organic layers were transferred to 13 × 100 mm glass
culture tubes. The organic extracts were then evaporated to
dryness using the SpeedVac at high heat setting. Hexanoic
anhydride (50 µL) and 10 µL of 50 mg/mL DMAP in toluene were
then added to the dry extracts; the tubes were then tightly capped
and heated at 70 °C for 15 min. Saturated aqueous sodium
bicarbonate (0.5 mL) and 4 mL of 10% ethyl acetate in pentane
were added, and the derivatives were extracted by vortex mixing,
centrifugation, and freezing the aqueous layers. The organic layers
were transferred to 13 × 100 mm tubes containing 0.5 mL of 1 M
H2SO4, and the organic layers were discarded after vortexing,
centrifuging, and freezing. The acid layers were made basic with
0.5 mL of 50% (w/v) K2CO3 and then extracted with 4 mL of 10%
ethyl acetate in pentane by vortex mixing, centrifugation, and
freezing the aqueous layers. The organic layers were transferred
to a new set of 13 × 100 mm tubes and evaporated using the
SpeedVac at medium heat. The samples were reconstituted in 125
µL of 10% methanol containing 12 mM HCl and transferred to
300-µL polypropylene insert vials for analysis. Extraction efficiency
was determined by spiking NNAL-d0 and NNAL-d3 into blank
extract prior to evaporation and derivatization. A typical run
consisted of 46 study samples plus 26 standards and QCs for a
total of 72 samples. One analyst can carry out the enzyme
incubation, extraction, and derivatization steps in 3 days, and the
entire run, including LC-MS/MS analysis can be completed in 1
week.
of 20 eV were used for the hexanoate derivatives of NNAL and
the internal standard, NNAL-d3, respectively. The mass resolution
(fwhm) was set at 0.5 amu for both Q1 and Q3.
Data Analysis. The Finnigan XCalibur/LC Quan software was
used to generate calibration curves (linear regression, 1/X
weighting) and calculate concentrations using peak area ratios of
analyte/internal standard. Two sets of six standards, spanning the
range of 0.25-50 pg/mL were included in each run of 40-45
clinical samples. For studies of low-level SHS exposure, three sets
of quality control specimens at 0, 0.5, 1.0, and 2.0 pg/mL were
included. Analysis of spiked urine samples indicated that the
method is linear to at least 500 pg/mL.
Clinical Samples. Urine specimens were obtained from
nonsmokers participating in research studies, described else-
where,24,25 and were stored frozen at -20 °C prior to analysis.
RESULTS AND DISCUSSION
Method Development. Using LC-ESI-MS/MS methods simi-
lar to published methods,17,21-23 we had previously been able to
achieve a detection limit of 5 pg/mL for 3-mL urine specimens.26
However, some of our studies involved subjects with low-level
exposure to SHS,24 and a method with higher sensitivity was
required. The limiting factor was not instrument sensitivity, but
rather trace amounts of coeluting substances in the urine extracts.
Attempts to separate these interfering substances chromatographi-
cally and with further MIP column purification23 of the liquid/
liquid solvent extracts were unsuccessful.
NNAL is a moderately polar organic compound, and it occurred
to us that, by esterification of the hydroxy group, it could be
converted to a relatively nonpolar substance. Doing so might be
expected to improve sensitivity in LC-MS/MS analysis for
the following reasons: (1) A relatively nonpolar substance could
be partitioned between an aqueous phase and a nonpolar organic
solvent to remove many of the multitude of potentially interfering
polar organic compounds present in urine. (2) The relatively
nonpolar ester derivative would be better retained on reversed-
phase HPLC columns than the parent NNAL, and this could be a
mechanism for separation of more polar, potentially interfering
substances in the sample. (3) More efficient separation from other
ionizable substances in urine, by extraction and chromatography,
would be expected to reduce the extent of matrix suppression of
ionization often occurring in electrospray ionization (ESI) of
biological extracts.27-29 (4) Ionization efficiency of the ester
derivative in an ESI source might be enhanced compared to
NNAL, due to more efficient desolvation. The hydroxy group of
NNAL would be expected to hydrogen bond strongly with the
usual reversed-phase HPLC solvents water, methanol, and aceto-
nitrile, but an ester would be expected to do so less avidly. (5)
Increasing the mass by derivatization might be expected to
Liquid Chromatography. The extracts (50 µL) were chro-
matographed with a methanol and water solvent system containing
10 mM ammonium formate at 0.333 mL/min using a linear
gradient from 33 to 100% methanolic buffer over 11 min. The
column was then held at 100% methanol for 1 min and then
ramped back to the initial conditions over 1 min. Re-equilibration
time was 7 min for a total run time of 20 min per sample.
Mass Spectrometry. The mass spectrometer was operated
using electrospray ionization, in the positive ion mode. The ion
source parameters were optimized by infusing a solution of the
hexanoate ester derivative of NNAL into the ion source via a
syringe pump. The vaporizer temperature was 372 °C, the heated
capillary temperature was 265 °C, and the spray voltage was 3500
mV. Data was acquired in the selected reaction monitoring (SRM)
mode. The transitions 308 to 84 and 311 to 87 at a collision energy
(24) Eisner, M. D.; Balmes, J.; Yelin, E. H.; Katz, P. P.; Hammond, S. K.;
Benowitz, N.; Blanc, P. D. BMC Pulm. Med. 2006, 6, 12
(25) Repace, J.; Hughes, E.; Benowitz, N. Nicotine Tob. Res. 2006, 8, 701–711
(26) Benowitz, N. L.; Hall, S. M.; Stewart, S.; Wilson, M.; Dempsey, D.; Jacob,
P., 3rd Cancer Epidemiol. Biomarkers Prev. 2007, 16, 2479–2485
(27) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem.
1998, 70, 882–889
(28) Jacob, P., 3rd; Wilson, M.; Yu, L.; Mendelson, J.; Jones, R. T. Anal. Chem.
2002, 74, 5290–5296
(29) Liang, H. R.; Foltz, R. L.; Meng, M.; Bennett, P. Rapid Commun. Mass
Spectrom. 2003, 17, 2815–2821
.
.
.
.
.
.
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8117

Table 1. Comparison of ESI Sensitivity for NNAL, NNAL
Acetate, and NNAL Hexanoate with Different HPLC
Mobile-Phase Compositions
% of NNALa
analyte
isocraticb
fast gradientc
slow gradientd
NNAL
100
113
154
100
154
212
100
112
231
NNAL acetate
NNAL hexanoate
a Peak areas as percent of the peak area for NNAL. b 10 mM
ammonium formate in 90:10 methanol/water. c Linear gradient of 10
mM ammonium formate, 100% aqueous changing to 100% methanolic
over 2 min followed by 6 min methanolic. d Gradient described in
Experimental Section used for urine sample analyses.
confirmation ion would be an advantage, none of the alternative
product ions we investigated had enough specificity for detection
in urine below ∼3 pg/mL, and unfortunately, most of our study
samples had concentrations below this.
A liquid-liquid extraction/derivatization procedure was de-
vised to (1) utilize acid-base partitioning of the weakly basic
NNAL, which has pyridine ring, to remove neutral and acidic
substances; (2) esterify the hydroxy group with hexanoic anhy-
dride to provide the hexanoate derivative; (3) remove excess
hexanoic anhydride and other remaining neutral and acidic
substances by acid-base partitioning; and (4) a final extraction
with a solvent of low polarity (90-10 pentane-ethyl acetate)
followed by evaporation and reconstitution in HPLC mobile phase.
The efficiency of extraction was determined by spiking urine with
both NNAL-d0 and NNAL-d3 and comparing the peak areas with
urine extracts spiked prior to the derivatization step. The extrac-
tion recovery was ∼60%.
Figure 2. Product ion spectra of NNAL, NNAL acetate, and NNAL
hexanoate.
increase selectivity in mass spectrometric detection, since poten-
tially interfering substances in urine would be expected to have
relatively low molecular weight. Consequently, we prepared the
acetate and hexanoate derivatives and evaluated them for sensitiv-
ity and specificity of detection in urine extracts.
Method Validation. Standard curves were linear from 0.25
to 50 pg/mL with typical r2 values >0.99 using linear regression
with 1/x weighting. A standard curve constructed from means of
six replicates is presented in Figure 4. Precision, accuracy, and
lower limit of quantitation (LLOQ) were determined by analyzing
urine from a nonsmoker, who had no known exposure to SHS,
spiked with NNAL. Over the range of 0.25-50 pg/mL, precision
ranged from 3.2 to 15.0% (percent coefficient of variation, CV) and
accuracy ranged from 88.0 to 107.1% (percent of expected) (Tables
2 and 3). Using the criteria of Shah et al., the LLOQ was
determined to be 0.25 pg/mL.30 Determining the limit of detection
(LOD) in urine is complicated by the ubiquitous presence of
tobacco smoke residues in the environment, and consequently,
nearly everyone will have at least a minute exposure to the
precursor substance NNK. Therefore, truly NNAL-free urine may
be hard to verify, and it may be not be possible to distinguish
NNAL from a coeluting substance with the same product ion.
Consequently, we investigated the LOD by extracting, derivatizing,
and analyzing standards prepared in 0.01 N HCl. The blank value
was positive, as the intercept of the standard curve indicated. A
5-mL 0.0625 pg/mL standard prepared in 0.01 N HCl, following
extraction, derivatization, and injection of an aliquot, gave rise to
a peak with about twice the peak area of a corresponding peak in
the blank. We also estimated the instrument sensitivity by injecting
ESI was used, and the most abundant ion transitions of each
derivative (Figure 2) were evaluated in the SRM mode. HPLC
separation was carried out using a gradient of water-methanol
with ammonium formate (pH ∼6) buffer. Both derivatives pro-
vided better sensitivity than underivatized NNAL, but the hex-
anoate was by far the best, presumably owing to its much lower
polarity, facilitating chromatographic separation from coextracted
substances in urine, as well as more efficient desolvation in the
electrospray ion source. Depending on the mobile-phase composi-
tion, the ESI response was 1.5-2.3 times greater for the hexanoate
derivative than for underivatized NNAL, with the response for the
acetate derivative between the two. Not surprisingly, the differ-
ences were greater under conditions of gradient elution, in which
the hexanoate derivative eluted with a lower percentage of water
in the mobile phase than the acetate or underivatized NNAL
(Table 1). These data support the notion that these less polar
derivatives bind less avidly to water and methanol, facilitating
desolvation and increasing transmission of ions into the
mass spectrometer. However, the most important factor affecting
sensitivity was separation from interfering urinary constituents,
since the intrinsic ESI response was not the factor limiting the
sensitivity of the method (Figure 3). By comparing spiked urine
to aqueous standards, suppression of ionization was estimated to
be ∼25%. Of the SRM transitions evaluated, m/z 308 to m/z 84 of
the hexanoate derivative provided the best selectivity in terms of
minimizing interference by urinary constituents. Although a
(30) Shah, V. P.; Midha, K. K.; Findlay, J. W.; Hill, H. M.; Hulse, J. D.;
McGilveray, I. J.; McKay, G.; Miller, K. J.; Patnaik, R. N.; Powell, M. L.;
Tonelli, A.; Viswanathan, C. T.; Yacobi, A. Pharm. Res. 2000, 17, 1551–
1557
.
8118 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

Figure 3. Chromatograms of urine extracts containing NNAL, NNAL acetate, and NNAL hexanoate. Mobile phase (10 mM ammonium formate
in methanol-water), column, and flow rate as described in experimental section. A was a linear gradient from 10% methanol to 70% methanol
over 15 min; B was a linear gradient from 33% methanol to 100% methanol over 11 min.
Table 2. Within-Run Precision and Accuracy for
Determination of NNAL in Urinea
added
amount (pg/mL)
measured
mean (pg/mL)
accuracy
(% of expected)
precision
CV (%)
0
<LLOQ
0.24
12.3b
12.6
12.5
10.2
8.3
0.25
0.50
1.00
3.00
10.00
50.00
95.2
94.1
0.47
1.08
2.87
10.10
49.99
107.9
95.8
101.0
100.0
4.2
3.2
a Six replicate analyses of urine from a nonsmoker with no known
SHS exposure spiked with NNAL. b For blank urine, precision is based
on the area ratios of peak corresponding to analyte/peak of internal
standard.
Table 3. Between-Run Precision and Accuracy for
Determination of NNAL in Urinea
added amount
(pg/mL)
measured
mean (pg/mL)
accuracy
(% of expected)
precision
CV (%)
0.50
1.00
2.00
0.487
0.950
1.76
97.4
95.0
88.0
15.0
8.8
4.4
Figure 4. Calibration curve for NNAL in urine. Six replicates at each
concentration, linear regression, 1/x weighting. Lower panel is
expanded scale for range of 0-1 pg/mL.
a Seven replicate analyses (three different runs) of urine from a
nonsmoker with no known SHS exposure spiked with NNAL.
known amounts of NNAL hexanoate. Injecting an aliquot of a
methanol solution containing 62.5 amol of NNAL hexanoate
(which would correspond to ∼13 fg of NNAL) resulted in a peak
of about twice the area of a corresponding peak in the methanol
blank.
Specificity was determined by analyzing urine samples from
eight nonsmokers with no known SHS exposure. All had NNAL
concentrations below the limit of quantitation of 0.25 pg/mL. A
urine specimen from a person reporting a brief exposure to SHS
17 h prior to the sample collection had a concentration of 0.99
pg/mL. Chromatograms of urine extracts from three people with
varying levels of SHS exposure are presented in Figure 5.
Application of the Method. Urine specimens from 73
nonsmoking research subjects who suffered from chronic obstruc-
tive pulmonary disease24 were analyzed for NNAL. Concentrations
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8119

Figure 5. Chromatograms of nonsmokers’ urine extracts. (A) Person with little or no SHS exposure; (B) subject in study of SHS exposure in
people with chronic obstructive pulmonary disease;24 (C) college student after SHS exposure in a bar.25
Figure 6. Distribution of NNAL concentrations in urine of 73 research
subjects with chronic obstructive pulmonary disease.24 BLOQ ) below
limit of quantitation.
ranged from below the LOQ (26 subjects) to 153 pg/mL, with a
mean of 5.5 pg/mL, and median of 0.87 pg/mL. (Figure 6). The
Figure 7. Concentrations of NNAL in urine of eight college students
mean is similar to that reported by other investigators for
nonsmokers.18 We also analyzed urine samples from eight
nonsmoking college students before and after they spent 6 h in
smoking-allowed bars. In all eight subjects, concentrations of
NNAL in urine collected ∼16 h after entering the bar were higher
than those from samples collected prior to entering the bar (Figure
7).
prior to and after spending 6 h in smoking-allowed bars.25 In the lower
panel, concentrations are normalized to creatinine to adjust for
differences in urinary flow.
CONCLUSION
A method for determination of the tobacco-specific carcinogen
biomarker NNAL in human urine at subpicogram per milliliter
8120 Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

concentrations has been developed. Key to the success of this
method was conversion of the analyte to a relatively nonpolar
derivative, which facilitates removal of interfering substances in
the sample matrix by chromatography and by the extraction
procedure, and enhances detection by electrospray ionization mass
spectrometry. This method appears to be the most sensitive
method yet reported for determination of NNAL in urine. Ap-
plicability of the method to determination of NNAL in urine of
humans exposed to low levels of secondhand tobacco smoke has
been demonstrated. Although derivatization adds extra steps to
the sample preparation procedure, conversion of hydrophilic
analytes to more hydrophobic derivatives has the potential to
greatly improve the sensitivity and specificity of ESI mass
spectrometric analyses.
ACKNOWLEDGMENT
The authors thank Margaret Wilson, who carried out much
of the development work for the extraction procedure, Yan Chen
and Kefei Wang of Thermo-Fisher Corporation for providing the
heated ESI source used in these studies, and Marc Olmsted for
editorial assistance. Financial support from the Flight Attendant
Medical Research Institute, The National Institutes of Health
(DA12393, CA78603, DA02277), and the California Tobacco
Related Disease Research Program (10RT-0215) is gratefully
acknowledged.
Received for review May 1, 2008. Accepted August 22,
2008.
AC8009005
Analytical Chemistry, Vol. 80, No. 21, November 1, 2008 8121