Quantification of Urinary Mono-hydroxylated Metabolites of
Polycyclic Aromatic Hydrocarbons by on-line Solid Phase
Extraction-High Performance Liquid Chromatography-Tandem
Mass Spectrometry
Yuesong Wang
*
, Lei Meng, Erin N. Pittman, Alisha Etheredge, Kendra Hubbard, Debra A.
Trinidad, Kayoko Kato, Xiaoyun Ye, and Antonia M. Calafat
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease
Control and Prevention, Atlanta 30341, Georgia, USA
Abstract
Human exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring
of urinary mono-hydroxylated PAHs (OH-PAHs). Gas chromatography (GC) has been widely used
to separate OH-PAHs before quantification by mass spectrometry in biomonitoring studies.
However, because GC requires derivatization, it can be time consuming. We developed an on-line
solid phase extraction coupled to isotope dilution-high performance liquid chromatography-
tandem mass spectrometry (on-line-SPE-HPLC-MS/MS) method for the quantification in urine of
1-OH-naphthalene, 2-OH-naphthalene, 2-OH-fluorene, 3-OH-fluorene, 1-OH-phenanthrene, the
sum of 2-OH and 3-OH-phenanthrene, 4-OH-phenanthrene, and 1-OH-pyrene. The method, which
employed a 96-well plate platform and on-line SPE, showed good sensitivity (i.e., limits of
detection ranged from 0.007 to 0.09 ng/mL) and used only 100 μL of urine. Accuracy, calculated
from the recovery percentage at three spiking levels, varied from 94% to 113%, depending on the
analyte. The inter- and intra-day precision, calculated from 20 repeated measurements of two
quality control materials, varied from 5.2% to 16.7%. Adequate method performance was also
confirmed by acceptable recovery (83–102%) of two NIST standard reference materials (3672 and
3673). This high-throughput online-SPE-HPLC-MS/MS method can be applied in large scale
epidemiological studies.
Keywords
polycyclic aromatic hydrocarbons (PAHs); OH-PAHs; exposure; HPLC-MS/MS; urine
*
Corresponding author: Yuesong Wang, Centers for Disease Control and Prevention, 4770 Buford Hwy, Mailstop F53, Atlanta,
Georgia 30341, USA, Telephone: 770-488-0336, Fax: 770-488-0333, ywang6@cdc.gov.
Compliance with Ethical Standards
The authors declare that they have no conflict of interest. This research involved human participants. The Centers for Disease Control
and Prevention (CDC) Institutional Review Board approved the anonymous collection of urine, and waived informed consent under 45
CFR46.116 (d).
Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the
Centers for Disease Control and Prevention (CDC). Use of trade names is for identification only and does not imply endorsement by
the CDC, the Public Health Service, or the US Department of Health and Human
Ser
vices. The authors declare they have no actual or
potential competing financial interests. The authors complied with all needed research requirements regarding human subjects.
HHS Public Access
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Anal Bioanal Chem
. Author manuscript; available in PMC 2017 August 23.
Published in final edited form as:
Anal Bioanal Chem
. 2017 February ; 409(4): 931–937. doi:10.1007/s00216-016-9933-x.
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Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants linked to
a variety of adverse health effects (1–3). Humans may be exposed to PAHs through
occupation, such as in work involving diesel fuels and coal tars (4, 5), as well as through diet
and other lifestyle activities (e.g., smoking) (6–8). The urinary concentrations of PAH
metabolites, specifically mono-hydroxylated PAHs (OH-PAHs), have been used as
biomarkers of human exposure to PAHs (7, 9, 10).
The pioneering quantification of OH-PAHs in urine was conducted by high performance
liquid chromatography (HPLC) coupled with fluorescence detection (4). Thanks in part to
technology advances of the last few decades, isotope dilution gas chromatography-mass
spectrometry (GC-MS) became widely used for the determination of urinary OH-PAHs (11–
16). GC-MS showed improved accuracy, sensitivity, and precision, mainly because of the
high specificity of mass spectrometry detection. However, sample preparation involved
derivatization and solvent evaporation steps, and was, therefore, labor intensive and time
consuming. More recently, HPLC-tandem MS (HPLC-MS/MS), introduced to measure OH-
PAHs (17–26), eliminated the derivatization step and yet maintained the high specificity of
mass spectrometry. Still, a relatively large volume of urine, e.g., 2–5 mL, was required in
HPLC-MS/MS methods, and automated sample preparation was not fully applied (20, 21).
These conditions have limited HPLC-MS from application in large scale biomonitoring
studies when matrix volume is often limited.
For the present study, we developed a fully automatic on-line solid phase extraction coupled
with isotope dilution-high performance liquid chromatography-tandem mass spectrometry
(on-line SPE HPLC-MS/MS) method for the accurate and reliable measurement of nine OH-
PAHs in human urine.
EXPERIMENTAL SECTION
Materials and Methods
All solvents were HPLC grade, and chemicals were reagent grade. We purchased
acetonitrile, ethanol, 0.1% formic acid in water, methanol, water, and ammonium fluoride
from Thermo Fisher Scientific (Waltham, MA, USA); ascorbic acid, sodium acetate, and
Helix pomatia β-glucuronidase type H-1 (β-glucuronidase ≥300,000 units/g, sulfatase
≥10,000 units/g) from Sigma-Aldrich (St. Louis, MO, USA). We obtained 1-
hydroxynaphthalene (1-OH-NAP), 2-hydroxynaphthalene (2-OH-NAP), 2-hydroxyfluorene
(2-OH-FLU), 3-hydroxyfluorene (3-OH-FLU), 1-hydroxyphenanthrene (1-OH-PHE), 2-
hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene (4-OH-PHE), 1-
hydroxypyrene (1-OH-PYR), and their corresponding
13
C-labeled internal standards (IS,
listed in Table 1) from Cambridge Isotope Laboratories (Andover, MA, USA).
We purchased smokers’ urine samples from BioreclamationIVT (Westbury, NY, USA). We
also collected urine anonymously in 2015 from non-smoker adult volunteers with no
documented occupational exposure to PAHs in Atlanta, GA. We obtained two Standard
Reference Materials® (SRMs), SRM 3672 (smoker urine) and SRM 3673 (non-smoker
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urine), from the US National Institute of Standards and Technology (NIST) (Gaithersburg,
MD, USA). All urine specimens were stored upon collection or arrival at −70 °C until use.
Appropriate safety control measures (including engineering, administrative, and personal
protective equipment) were used for all procedures based on a site-specific risk assessment
that identified physical, health, and procedural hazards.
Preparation of standard stock solutions and quality control materials—We
prepared the stock solutions of individual analytical standard in ethanol. Standards with all
nine OH-PAHs were generated by serial dilution of the individual stock in 40% ethanol/60%
water. The final concentrations of the mixed stock standards ranged from 0.08 – 200 ng/mL
(1-OH-NAP and 2-OH-NAP) and 0.005 – 50 ng/mL (all other analytes). The standard
solutions were aliquoted into 2 mL silanized amber glass vials and stored at 4 °C until use.
The internal standard solution with
13
C-labeled analytes was prepared in water with 0.2%
acetonitrile so that a 50 μL spike would result in approximate concentrations of 32 ng/mL
(
13
C-1-OH-NAP and
13
C-2-OH-NAP) or 8 ng/mL (other
13
C-labeled analytes). Internal
standards were aliquoted into 15 mL amber glass vials and stored at −70 °C.
Two levels of quality control (QC) materials, QC low (QCL) and QC high (QCH), were
prepared by pooling urines from smokers and non-smokers. The QC concentrations were
fortified, as needed, with native target compounds to encompass the ranges described for the
U.S. general population (
27). All QC materials were stored in 4 mL amber glass vials at
−70 °C until used. The stability of spiked material stored at −70 °C has been previously
evaluated for up to one year (data not shown), and no obvious degradation of OH-PAHs was
observed. The QC materials stored at −70 °C for more than one year will be re-evaluated for
their stability.
Sample Preparation—Urine samples were thawed and mixed at room temperature. QC
samples, reagent blanks, and standards were processed the same way as urine samples,
going through all of the sample preparation steps. Sample preparation was automatically
conducted on a Perkin-Elmer Staccato® System (controlled by the Perkin Elmer iLink and
Maestro software) (Waltham, MA, USA). The robotic system included six main
components: Sciclone G3/G3T, Fluidx CESD-24PRO decapper, Hettich Rotanta 460
centrifuge, ThermoScienfic ALPS 3000 sealer, IVD Inheco Incubator shaker DWP, and
Mitsubishi robotic arm. We programmed this system to aliquot urine samples, standards,
QCs and reagent blanks (100 μL) into a 96-well plate (Corning, NY, USA), and
subsequently add ascorbic acid solution (20 μL, ~12.5 mg/mL), internal standards solution
(50 μL), and sodium acetate buffer (50 μL, ~1 mol/L, pH 5.5) containing ~10 mg/mL β-
glucuronidase/arylsulfatase. The accuracy and precision of automatic aliquating by Sciclone
robotic system was previously evaluated, and good aliquoting accuracy (recovery rate from
95% to 105%) and precision (CV<3 %) were achieved from current procedures. Details
regarding enzymatic de-conjugation were previously described (16). The robotic system then
sealed and transferred the plate for overnight incubation at 37±2 °C. After enzymatic
hydrolysis, the robotic system automatically added methanol (175 μL) to all sample wells,
mixed the solution, resealed the plate, and centrifuged for ~15 minutes at 5000 rpm (5900
rcf). Finally, the robotic system transferred 200 μL of the supernatant in each well to a new
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96-well plate, and added 350 μL of water to each well before on-line SPE-HPLC-MS/MS
analysis. Non-spiked and spiked synthetic urine (28) used for determining the limit of
detection (LOD) was prepared as study samples.
Online SPE-HPLC-MS/MS—The on-line SPE-HPLC-MS/MS system consisted of a
Sciex 5500 or 6500 triple quadrupole mass spectrometer (Foster City, CA, USA) equipped
with an electrospray ion source and controlled by AB Sciex Analyst
TM
software, and one
Agilent 1260 pump and one degasser (Santa Clara, CA, USA), and an on-line SPE Spark
Holland system (Glassboro, NJ, USA) controlled by the Sparklink
®
software (iChrom
Symbiosis system).
After injection (300 μL), the sample was loaded onto an Oasis WAX on-line SPE cartridge
with 0.1% formic acid in water (1.5 mL), and the cartridge was washed with acetonitrile/
methanol/water (~0.4 mL, 1/1/2, v/v/v). The target analytes were eluted with methanol (350
μL), and focused on a Chromolith HighResolution RP-18 endcapped guard column (5×4.6
mm, Merck KGaA, Darmstadt, Germany) with the initial HPLC gradient.
We separated the target analytes on a pair of Chromolith HighResolution RP-18 endcapped
HPLC columns (100×4.6 mm, Merck KGaA) by a programmed HPLC gradient (Table 2).
The mobile phases were water with 0.1 mM ammonium fluoride (A) and methanol with 0.1
mM ammonium fluoride (B).
The ionspray voltage and source temperature were −3.0 kV and 500 °C, respectively.
Curtain gas, ion source gas 1, ion source gas 2, and collision gas were 35 psi, 50 psi, 70 psi,
and 9 psi, respectively. The representative decluster, entrance and exit potentials were −120
V, −3 V and −12 V, respectively. We quantified OH-PAHs by selected reaction monitoring in
the negative ion mode by the ion transitions listed in Table 1, and optimized the collision
energies for all ion transitions (Table 1).
Data Analysis—We used Analyst (version 1.6.2, Sciex, Foster City, CA, USA) and
MultiQuant (version 3.0, Sciex, Foster City, CA, USA) for data processing. We defined
quality control limits, and evaluated analytical runs using SAS (version 9.3, SAS Institute
Inc.; Cary, NC, USA) with a multi-rule quality control approach (29).
Results and discussion
OH-PAHs were enriched and extracted from the urine matrix by on-line SPE. We separated
the target analytes on a pair of monolithic RP-18 column by using a gradient of water and
methanol, including ammonium fluoride (0.1 mM) in both mobile phases. We used
ammonium fluoride to improve method sensitivity in the negative ion mode (30). An
example of LC-MS/MS selected ion chromatogram is shown in Figure 1. Within 27 mins,
we were able to separate several pair of isomers: 2-OH-NAP, 1-OH-NAP, 3-OH-FLU, 2-OH-
FLU, 1-OH-PHE, 4-OH-PHE, and 1-OH-PYR, but 2-OH-PHE and 3-OH-PHE were eluted
together so we had to measure these two analytes as a sum (Σ2,3-OH-PHE). 9-
hydroxyphenanthrene and 3-hydroxyfluoranthene could also be separated from the other
target metabolites (data not shown).
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Method accuracy was assessed by repeated analyses (n=7) of synthetic urine spiked with the
target analytes at three spiking concentrations. Accuracy, expressed as a percentage of
recovery, was 105–113% (level 1), 94–100% (level 2), and 98–102% (level 3), depending on
the analyte (Table 3). Furthermore, accuracy was evaluated by analyzing two NIST SRMs,
SRM 3672 and SRM 3673. The calculated OH-PAHs concentrations were in good
agreement with the certified concentrations (31), and accuracy ranged from 83 to 102%,
depending on the analyte (Table 3).
We determined the method precision from repeated measurements of low and high QC pools
by following the CLSI protocol EP5-A2 (32) on 51 different days (two results from each of
two daily runs) over a period of 8 months that involved multiple analysts. The relative
standard deviations (RSDs), which reflect the within- and between-run variability, ranged
from 3.2% to 12.1% (within-run) and 4.8% to 13.0% (within and between runs) for all
analytes (Table 4).
The LOD was determined according to procedures previously described (33) from 60
repeated measurements of non-spiked and spiked synthetic urine analyzed by multiple
operators and using four different mass spectrometers (Sciex 5500 or 6500). The LODs
ranged from 0.007 ng/mL to 0.09 ng/mL for all analytes (Table 4), indicating the good
sensitivity of the method, especially considering the relatively low volume of urine used
(100 μL). The method also provided wide dynamic ranges, with upper linearity of the
method at 200 ng/mL (2-OH-NAP, 1-OH-NAP); 25 ng/mL (2-OH-FLU); 20 ng/mL (Σ2,3-
OH-PHE); and 10 ng/mL (3-OH-FLU, 1-OH-PHE, 4-OH-PHE, 1-OH-PYR).
SPE recovery was calculated as previously described (34). The mean recoveries of three
repeated measurements were 67±4% (2-OH-NAP), 67±5% (1-OH-NAP), 81±4% (3-OH-
FLU), 64±2% (2-OH-FLU), 63±2% (Σ2,3-OH-PHE), 72±5% (1-OH-PHE), 55±2% (4-OH-
PHE), and 49±6% (1-OH-PYR). These recoveries, which are comparable with those
reported before using a GC-MS method (16), are adequate for quantitative measurement of
OH-PAHs in urine from both smokers and non-smokers. We also evaluated matrix effects
using a matrix factor, defined as the ratio of IS peak area in the presence of urine matrix to
the IS peak area in the absence of urine matrix (34). The matrix factor, calculated from 2
different QC concentrations of three repeated measurements varied from 63% to 101%,
depending on the analyte.
To validate the method, we measured OH-PAHs in 36 non-smokers and 36 self-identified
smokers’ urine samples (Table 5). Among non-smokers, detection frequencies were 56% for
1-OH-PYR, 14% for 4-OH-PHE, 94% for 3-OH-FLU, and 100% for the other analytes. For
smokers, detection frequencies were 97% for 1-OH-PYR, 53% for 4-OH-PHE, and 100%
for the rest of analytes. The geometric mean concentrations of OH-PAHs were 2.5–12.4
(average 6.6) times higher in smokers than non-smokers (Table 5). 2- and 3-OH-FLU and 1-
and 2-OH-NAP showed the largest concentration differences between smokers and non-
smokers.
Compared with our previous GC-MS method (13, 16), the current approach eliminated the
derivatization and solvent evaporation steps, simplified the sample processing procedure,
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