Page 1
Validity of a self-administered food-frequency questionnaire in the estimation of
heterocyclic aromatic amines
Motoki Iwasaki, et al.
Supplementary Material
Laboratory analysis
Materials and reagents
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino
-1-(trideuteromethyl)-6-phenylimidazo[4,5-b]pyridine ([2H3C]PhIP) (99% isotopic
purity) were purchased from Toronto Research Chemicals (Toronto, ON, Canada).
Methanol-d4 was purchased from Sigma Aldrich Japan (Tokyo, Japan). 10%SDS solution
was purchased from Wako Pure Chemical (Osaka, Japan). InertSep® Slim J Aroma-Blue
was purchased from GL Sciences (Tokyo, Japan). Soluene 350 was purchased from
PerkinElmer (Waltham, MA, USA). LC/MS grade MeOH and acetic acid were purchased
from Wako Pure Chemical (Osaka, Japan). All other reagents were high purity from
Wako Pure Chemical (Osaka, Japan) and Sigma Aldrich Japan (Tokyo, Japan).
Standard solution and calibration curve
The stock standard solution of PhIP (1 mg/mL) was prepared in MeOH and then diluted
with MeOH to give a final concentration of 1 ng/μL and stored at -80 °C until use. The
stock standard solution of internal standard [2H3C]PhIP (1 mg/mL) was prepared in
methyl-d3 alcohol-d and stored at -80 °C until use. Internal standard was diluted in
MeOH to give a final concentration of 1 ng/μL each time the analyses were done.
Page 2
Quantification was conducted using internal standard calibration with [2H3C]PhIP. The
calibration curve was established as the area ratios between the analyte and internal
standard versus the added amount of analyte per sample. The standard solution was
prepared by adding the standards, diluted in mobile phase (40 μM ammonium acetate (pH
4.0): MeOH, 1:1, v/v), as follows: 0.027, 0.054, 0.1, 0.2, 0.4, 0.8 pg/μL for human hair
samples and 0.2, 0.4, 0.8, 1.67, 3.33, 16.7, 33.3, 40.0 pg/μL for rat fur samples. The
amount of internal standard was 4 pg/μL. The coefficient of determination values of the
slopes of the calibration curves exceeded 0.99. The limit of detection (LOD) and the limit
of quantification (LOQ) were set to 3 and 10 times the standard deviation (SD) of the
noise, respectively. The LOD and LOQ for the present analytical method were 13 and 45
pg/g hair, respectively.
Human hair and rat fur samples (preparation)
The sample was washed (about 1g for human hair and 5mg for rat fur) in 0.1% SDS
solution (100 mL) by ultrasonication for 5 min. The liquid was then decanted, and the
sample was washed in MeOH (100 mL) by ultrasonication for 5 min. The liquid was
again decanted, and the sample was washed 4 times with water (100 mL), once with
ethanol (50 mL), and dried at room temperature.
Extraction procedure
The dried sample was weighed (1 g for human hair and 2 mg for rat fur), and 1 mol/L
NaOH (100 mL) and internal standard ([2H3C]PhIP; 2 ng for human hair and 30 ng for rat
fur) were added. This solution was incubated at 80 °C for 1 h. After additional
centrifugation at 3100 rpm and 4 °C for 10 min, the supernatant was filtrated with
Page 3
ADVANTEC 5B filter paper. The filtrate was neutralized to pH 7-9 with 6 mol/L HCl,
and extracted using InertSep® SlimJ Aroma-Blue. A washing procedure with 60 mL H2O
was performed and the analytes were eluted with 20 mL of MeOH:28%NH3 (50:1, v/v)
(flow rate, 5 mL/min). The eluate was concentrated under vacuum. The residue was
dissolved in 1 mL of MeOH. After centrifugation of 3100 rpm and 4 °C for 10 min, the
supernatant was collected, and then concentrated under vacuum. The residue was
dissolved in 2 mL of 0.1 mol/L HCl. After washing with 2 mL of n-hexane, the aqueous
layer was adjusted to pH>10 with 28% NH3, and then extracted twice with 2 mL of
dichloromethane. The organic layer was concentrated under vacuum. For human hair
samples, the sample residue was dissolved in 500 μL of 40 μM ammonium acetate
(pH4.0): MeOH (1:1, v/v), filtrated with 45 μm filter (centrifugal filter units, Millipore,
Bedford, MA, USA), and an aliquot of 300 μL was injected onto the LC-ESI/MS/MS. For
rat fur samples, the sample residue was dissolved in 750 μL of 40μM ammonium acetate
(pH4.0): MeOH (1:1, v/v), filtrated with 45 μm filter, and the sample was diluted 10-fold
with solvent and injected at 200 μL/500 μL onto the LC-ESI/MS/MS.
LC-ESI/MS/MS analysis of PhIP
Chromatography was performed with a Shimadzu series VP model (Shimadzu, Kyoto,
Japan) equipped with an analytical column at a temperature of 40 °C. Chromatographic
separation was performed based on a previously described method (1). In brief, the
analytes were separated on a gradient with a column-switching technique. The mobile
phase consisted of A: 40 μM ammonium acetate in H2O (pH 4.0), B: MeOH, and C: 10
mM ammonium acetate in H2O. First, 300μL of the sample was injected, and loaded onto
the extraction column with C solvent at a flow rate of 2.0 mL/min for 8 min. The valve
Page 4
was then switched, and the analyte was introduced into the analytical column at a flow
rate of 0.2 mL/min. An Inertsil ODS-4 column (GL Sciences, Tokyo, Japan) was used to
improve peak shape for routine analysis (analytical column: 2.1 mm x 15 cm, 5 μm
particle size; extraction column: 3.0 mm x 10mm, 5 μm particle size). The gradient
program was as follows: 13% B (0-5 min) - 50% B (5.01-13 min).
The mass spectral data were obtained by electrospray ionization (ESI)-MS, which was
performed on an API 2000 (AB Sciex, Foster City, CA, USA) equipped with a modified
TurboIonSpray® source. Instrument settings for the mass spectrometer were set as
follows: declustering potential (DP) 36 V, entrance potential (EP) 10.5 V, collision
energy (CE) 39 V, nebulizer gas (GS1) 30 pounds per square inch (psi), turbo gas (GS2)
80 psi, curtain gas (CUR) 40 psi, collision gas (CAD) 8 psi, ion spray voltage (IS) 5000 V
and temperature 500 °C. Analysis of processed samples was performed in positive ion
mode using multiple reaction monitoring (MRM) with the following transitions: PhIP and
[2H3C]PhIP (precursor ion/product ion), 225.0/210.0 and 228.0/210.0. The instrument
operations and data manipulations were controlled with Analyst version 1.4.1 software.
Spectrophotometric characterization of melanin
Human hair and rat fur sample (1mg) and sepia melanin (1mg) were dissolved in 1 mL of
a mixture of Soluene 350:H2O (9:1,v/v), followed by heating at 95 °C for 45 min, as in a
previous study (1). Optical density was observed at 500 and 650 nm (A500 and A650) with
a Jasco V-550 (Japan Spectroscopic, Tokyo, Japan).
DNA adduct analysis
Rat colon DNA was extracted and purified using a Gentra® Puregene™ tissue kit
Page 5
(QIAGEN, Valencia, CA). The protocol was performed according to the manufacturer's
instructions. DNA samples in 40-μg aliquots were digested into their constituent
2'-deoxyribonucleoside-3'-monophosphate units by micrococcal nuclease, spleen
phosphodiesterase, nuclease P1 and alkaline phosphatase as described previously (2).
N2-(deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(dG-C8-PhIP) measurement was used under the same procedure as described previously
(3). Briefly, the DNA adduct-containing supernatant was purified by solid phase
extraction (SPE) using HYPERSEP C18 (50 mg) cartridges (Thermo Scientific,
Bellefonte PA, USA). The SPE cartridges were first washed with methanol containing
0.1% formic acid followed by conditioning with 10% methanol in 0.1% formic acid. The
DNA digest samples were loaded and unmodified 2′- deoxynucleosides were removed
with 10% methanol in 0.1% formic acid. The dG-C8-PhIP adduct was eluted with
methanol containing 0.1% formic acid. The eluent was evaporated to dryness by vacuum
centrifugation and resolved in 30% DMSO solution and subjected to liquid
chromatography tandem mass spectrometry (LC/MS/MS). LC/MS/MS analyses were
performed using a Waters 2795 LC system (Waters, Manchester, UK) interfaced with a
Quattro Ultima triple stage quadrupole MS (Waters). The LC column was eluted over a
gradient that began at a ratio of 20% acetonitrile in 0.1% acetic acid, changing to 95%
acetonitrile over a period of 30 min. Samples were separated on a Shim-pack FC-ODS
column (150 × 4.6 mm, 3 μm, Shimadzu, Kyoto, Japan) and eluted at a flow rate of 0.4
mL/min. Mass spectral analyses were carried out in positive ion mode with nitrogen as
the nebulizing gas. Ion source temperature was 120 °C, and desolvation gas temperature
was 350 °C. Nitrogen gas was also used as desolvation gas (700 L/h) and cone gas (85
L/h), and argon was used to provide a collision cell pressure of 1.5 × 10-3 mbar. Positive
Page 6
ions were acquired in multiple reaction monitoring mode. Multiple reaction monitoring
transitions were monitored, with respective cone voltage and collision energies of
490->374, 35 V, 20 eV, 490->357, 35 V, 50 eV, 490->329, 50 V, 50 eV, 490->250, 50 V,
50 eV.
REFERENCES
1.
Hashimoto H, Hanaoka T, Kobayashi M, Tsugane S. (2004) Analytical method
of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in human hair by
column-switching liquid chromatography-mass spectrometry. J Chromatogr B Analyt
Technol Biomed Life Sci. 803: 209-13.
2.
Kato T, Totsuka Y, Ishino K, et al. (2013) Genotoxicity of multi-walled carbon
nanotubes in both in vitro and in vivo assay systems. Nanotoxicology. 7: 452-61.
3.
Goodenough AK, Schut HA, Turesky RJ. (2007) Novel LC-ESI/MS/MS(n)
method for the characterization and quantification of 2'-deoxyguanosine adducts of the
dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D linear
quadrupole ion trap mass spectrometry. Chemical research in toxicology. 20: 263-76.