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DETERMINATION OF PAHS IN LUNG TISSUE SAMPLES

USING SPECIFIC CHROMATOGRAPHIC TECHNIQUES:
METHOD DEVELOPMENT AND VALIDATION
a b c d
Bogdan. I. Cioroiu , Mona. E. Cioroiu , Doina Tarcau , Alina M. Tomoiaga & Mihai I.
a
Lazar
a
Department of Drug Analysis , University of Medicine and Pharmacy “Gr.T. Popa” Iasi , Iasi ,
Romania
b
Department of Clinical Biochemistry, Clinical Hospital of Pulmonary Disease , Iaşi ,
Romania
c
Faculty of Agriculture , “Ion Ionescu De La Brad” University of Agricultural Science and
Veterinary Medicine of Iaşi , Iaşi , Romania
d
Department of Chemistry , “Al. I. Cuza” University of Iaşi , Iaşi , Romania
Accepted author version posted online: 16 May 2013.

To cite this article: Journal of Liquid Chromatography & Related Technologies (2013): DETERMINATION OF PAHS IN LUNG
TISSUE SAMPLES USING SPECIFIC CHROMATOGRAPHIC TECHNIQUES: METHOD DEVELOPMENT AND VALIDATION, Journal of Liquid
Chromatography & Related Technologies

To link to this article: http://dx.doi.org/10.1080/10826076.2012.758139

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DETERMINATION OF PAHs IN LUNG TISSUE SAMPLES USING SPECIFIC
CHROMATOGRAPHIC TECHNIQUES: METHOD DEVELOPMENT AND
VALIDATION

Bogdan. I. Cioroiu1,, Mona. E. Cioroiu2 ,Doina Tarcau3,Alina M. Tomoiaga4,Mihai I.

Lazar1
1
Department of Drug Analysis, University of Medicine and Pharmacy “Gr.T. Popa" Iasi,
Iasi, Romania 2Department of Clinical Biochemistry, Clinical Hospital of Pulmonary
Disease, Iaşi, Romania 3Faculty of Agriculture, "Ion Ionescu De La Brad" University of
Agricultural Science and Veterinary Medicine of Iaşi, Iaşi, Romania 4Department of
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Chemistry, "Al. I. Cuza" University of Iaşi, Iaşi, Romania

Author of correspondence: Bogdan. I. Cioroiu, Department of Drug Analysis, University

of Medicine and Pharmacy “Gr.T. Popa" Iasi, Universitatii street, No. 16, Iasi, Romania
E-mail: ionel.cioroiu@d.umfiasi.ro

Abstract
Quantification of polycyclic aromatic hydrocarbons (PAHs) in lung tissue samples for the
estimation of clinical impact was carried out. Methods and materials. A high pressure
liquid chromatography system (HPLC) with ultraviolet-visible diode array detector
(DAD) and fluorescence (FLD) detection was used. An automated solvent extraction
(ASE) procedure with sample clean-up was employed for the extraction of PAHs from
the lung tissue samples. The chromatographic method allowed identification of a total
number of 16 PAHs: naphtalene, acenaphthene, fluorene, phenanthrene, pyrene,
benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene,
benzo(ghi)perylene, indeno(1,2,3-cd)pyrene using fluorescence detection, while
acenaphtylene was identified using a diode array detector (DAD).Results. The linearity
domain for the method was between 5pg/µl and 400pg/µl for each compound. The
quantification limits were between 0.5 pg/µl and 20 pg/µl. Automated solvent extraction
was used for obtaining recoveries between 82-120%.

KEYWORDS: Polycyclic aromatic hydrocarbons, accelerated solvent extraction, HPLC,

lung tissue

INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants containing

from two up to eight fused benzene rings. Exposure to these compounds is a public health

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concern, since they are considered as priority pollutants due to their high toxicity,

mutagenicity and carcinogenicityand their effects have been repeatedly evaluated by

different authors.[1–4] These compounds are produced in all incomplete combustion

processes of organic matter and they have been found in cooked food, in dietary

products[5] or tobacco smoke.[6] PAHs have been determined also in the atmosphere, in

terrestrial and aquatic ecosystems.[7]

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The great importance on the influence of human health is determined by their interactions

revealed at the level of liver or lung.[8,9] It was demonstrated that they are toxic,

dibenzo[a,l]pyrene has a 10 fold higher toxicity than benzo[a]pyrene, which is considered

as a standard on the definition of potency equivalency factors (PEFs).[10]

PAHs are highly lipophilic compounds, so it is necessary to use non-polar organic

solvents. Accelerated solvent extraction represents a alternative as extraction method, the

major advantages are reduced solvent consumption, high recovery factors due to the

temperature/pressure control capabilities and high precision in conditions of standardized

extraction parameters.[11] Several separation techniques have been used and also a high

number of chromatographic methods have been developed for PAHs monitoring in

human matrices, including HPLC,[12] GC-MS[13] and LC–MS.[14] Most of these methods

refer to a limited number of compounds from trace environmental species like aliphatic

hydrocarbons, single-ring aromatic compounds, and polycyclic aromatic

hydrocarbons.[15,16] In a recent study on the assessment of the effect of exposure to 22

speciated PAHs to human subjects, the authors developed a specific method for

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determination of 2–6-ring PAHs using liquid–liquid extraction from blood and plasma

samples followed by gas chromatography–mass spectrometry analysis (GC–MS).[17] Only

limited data are available on the PAHs assessment in lung samples, although PAHs

potency to induce lung cancer is well documented.[18]

In this study, the main objective was to establish a sensitive, reliable and selective
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analytical method for the determination of 16 PAHs in lung tissue samples, specific to

respiratory carcinogenic diseases. Thus we developed an HPLC method with DAD and

fluorescence detection following accelerated solvent extraction of PAHs. The

proposedmethod was validated and successfully applied to the elucidation of the

concentration of existing PAHs in human lung tissue samples.

MATERIALS AND METHODS

Samples

A total of 31 subjects from Clinical Hospital of Pneumology, Iasi, Romania were

included in this study. Lung tissue samples were collected after biopsy analysis and were

procured from the anatomical pathology laboratory. Additionally institutional ethics

committee clearance was also obtained for collecting the samples. All subjects were

diagnosed with pulmonary diseases confirmed by histological examination.

Chemical And Reagents

The mixture of sixteen PAHs: naphthalene (N), acenaphtene (Ace), acenaphthylene

(Acy), fluorene (Fl), phenanthrene (P), anthracene (A), fluoranthene (Flu), pyrene (Py),

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benzo(a)anthracene (BA), benzo(b)fluoranthrene (BbFlu), benzo(k)fluoranthrene

(BkFlu), benzo(a)pyrene (BaPy), dibenzo(a,h)anthracene (DahA), benzo(g,h,i)perylene

(BghiP), chrysene (Ch), indeno1,2,3-cd-pyren (Ipy) was purchased from Sigma-Aldrich

Chemical Co. (St. Louis, USA) The quantity of each component in the mixture was 10

µg, and their purity was 99,99 %. Methanol (MeOH), acetonitrile (ACN), acetone (ACT),

dichloromethane (DCM), hexane (HXN) were HPLC purity and were provided by Merck
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– Germany. Ultrapure water was obtained from a MilliQ apparatus from Millipore

(Milford USA). Hydromatrix, a type of diacetameous earth, was purchased from Dionex

(USA), aluminum oxide (Al2O3) (80-325 mesh), and silica-gel (SiO2) (100-200 mesh)

were supplied by Sigma Aldrich Chemical Co. (St. Louis, USA).

Apparatus And Running Conditions

Accelerated extraction process was carried out on a DIONEX 300 Automated solvent

extraction system. Chromatographic determinations were performed on a high

performance liquid chromatography Surveyor Plus system provided by Thermo Fisher

Scientific – USA, equipped with Surveyor LC – Pump, a Surveyor Autosampler with

solvent degasser and two different detectors: a Surveyor Fluorescence (FLD) detector and

a ultraviolet-visible, with 650 photo-sensible diode array detector (DAD). Data

acquisition and spectra recording were performed using ChromQuest software.

The mobile phase consisted of water (solvent A) and acetonitrile (solvent B). The elution

conditions were: starting composition: 50% solvent A: 50% solvent B; 0-30 minutes –

linear gradient 50 to 100% solvent B; 30 – 40 minutes 100% solvent B isocratic. Column

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reconditioning was monitored for 15 minutes, using the baseline check option of the

ChromQuest software. The flow rate was maintained at 1 ml/min, the injection volume

was 5 µl and column was thermostated at 25ºC.

Total chromatographic time was 40 minutes, enough to ensure detection and separation

of all interested compounds and all matrix components, avoiding column overload, cross
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contamination and carryover during samples analysis (figure (1a)).

Detection was performed on DAD detector at 228 nm for acetylnaphtylene, while all

other 15 PAH compounds were monitored on FLD detector using the excitation/emission

program presented in table 1.

Preparation Of Standard Stock Solutions

Starting stock solution of 10 ng/µl for each componentwas prepared by dissolving the

PAHs mixture in 1 ml acetonitrile. Working standards for the calibration curve were

prepared by adequate dilution in acetonitrile. Calibration curves were designed on 7

points in the concentration range 5 pg/µl to 400 pg/µl of each PAH compound. The

solutions were stored at -18 0C, for maximum 5 days.

RESULTS

Optimization Of The Chromatographic Conditions

Method development and optimization was carried by detection using DAD detector due

to the possibility to identify all compounds without concerning their absorption

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wavelength.

Three HPLC columns types were tested in the same experimental conditions in order to

find other alternatives for separation of the 16 compounds mixture: Agilent Zorbax XDB

column (250 length, 4.6 mm internal diameter, 5µm particle size); Phenomenex® Luna2

column (250 length, 4.6 mm internal diameter, 10µm particle size) and one Thermo
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Fisher Scientific C18Hypersil Green PAH column with a length of 150 mm, 4.6 mm

internal diameter and 3 µm particle diameter. The best selectivity was achieved on the

Hypersil Green PAH, although good results were obtained on the Phenomenex Luna

Column.

Sensitivity of the method was developed by comparison between DAD and FLD

detection methods. Identification of all analyzed compounds was possible by

DADdetection on the 228 nm acquisition channel.

Detection of acetylnaphtylene using a 289 nm excitation wavelength and a 321 nm

emission wavelength it was considered,[19] but it was analyzed on the DAD detector, due

to absence of fluorescence signal. On DAD detection channel, identification was

performed at 228 nm, and the molecular spectrum was monitored during the analysis on

the UV interval 190 – 360 nm. Confirmation of the ACY signal in the lung tissue samples

was evaluated by comparing spectra at apex of each peak in the sample to that of the

library maintained for reference. Similarity factors determined were included between

0.63 and 0.95, correlated with concentration of ACY in samples.

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The chromatographic method specificity was improved by proper selection of the

excitation/emission wavelengths based on the fluorescence parameters from Standard SR

EN ISO 17993.[20] The elution order of each compound was established as a function of

their distribution in the analyzed mixture.

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The correlation between excitation/emission wavelengths and PMT voltage

(photomultiplier voltage) was established. For each compound, necessary modifications

were performed on the ratio between excitation/emission wavelengths used for detection.

All 15 aromatic polycyclic hydrocarbons showed significant fluorescence, with an

excitation wave-length range between 275 and 290 nm and an emission wave-length

between 350 and 470 nm. Table 1 shows the optimized excitation, emission wave-length

and PMT Voltage applied on xenon fluorescence detector lamp. These values were used

further throughout method validation and application to biological samples. Comparative

chromatograms plots of DAD and Fluorescence channels are presented in the figures 1(a)

and 1(b).

Sample Pre-Treatment-Extraction And Clean-Up Procedure

Lung Tissue Samples

The optimization and validation of the human lung tissue extraction procedure was

performed on spiked samples. The extraction of polycyclic aromatic hydrocarbons from

lung tissue samples was carried out by accelerated solvent extraction (ASE), according to

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the procedure proposed by Donell with some modifications.[21] The lung tissue samples

were defrosted and dehydrated with sodium sulphate. Aamount of 2gwas mixed with 5

grams of Hydromatrix, followed by transfer to the extraction cell (33 ml). Supplementary

quantity of Hydromatrix support material was added allowing packing into the extraction

cell with minimum loss.

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Accelerated extraction process was carried in two extraction cyclesusing a (1:1)

DCM/ACT mixture at a 100°C temperature and a pressure of 1500 psi. Extraction solvent

was evaporated under controlled temperature of 40 °C using a rotaevaporator up to a

volume of 3 ml.

The final volume was processed in clean-up procedure in order to eliminate the

interfering components from the extracted matrix. The column bed was of 6 g of mixture

of (1:1) (Al2O3:SiO2)and 2 g of anhydrous sodium sulphate placed on top. This was

determined by the higher retaining capacity of aluminum oxide for lipids and proteins

extracted in accelerated solvent extraction step and the specificity of the PAHs which is

proven to be highly non-polar.[22]

The cartridge was conditioned with 10 ml DCM and then with 10 ml high purity HXN.

The sample was loaded and the column was washed with 20 ml of mixture of (1:1 v/v)

DCM:HXN with a flow rate of 1 ml/min. The eluent was collected and evaporated to 3

ml using a rotor evaporator at 40 °C. The final eluate was concentrated under gentle

nitrogen stream at room temperature until near dryness and resolubilized in 100 µl

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ACN.A analysis workflow with sample preparation and chromatographic detection

conditions is presented in the figure 2:

HPLC Method Validation

System Suitability

System suitability was considered according to the methodology of the validation of the
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analytical methods proposed by official monographs of ICH guideline Q2B.[23] Relative

standard deviation (RSD) of retention times was less than 2% on three replicates from the

same sample, the average theoretical plates per column was higher than 700 and the

asymmetry factor was less than 1.5. Capacity factor for first compound, naphthalene, is

3.86,resolution was higher than 2 for all analysts, except the resolution between

compound 15(BghiP) and 16(Ipy) which was 1.52. Results are presented in the table 2.

Method Selectivity

The selectivity of the method was assessed by monitorization of the interference from the

endogenous materials in lung tissue. The selectivity was determined by injecting samples

containing mixture of the 16 analytes, blank sample, spiked sample and were

chromatographed according to the experimental method. The results for spiked samples

along with a blank sample for lung tissue are shown in figure 3and confirm the

separation.

Linearity Range

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A 7 points calibration curve was constructed from 5to 400 pg/µl for each compound.

These standard solutions were analyzed in three replicates. Relative standard deviation

was calculated. For all 16 PAH compounds, the correlation coefficient were minimum

0.999, leading to the conclusion that standard calibration plots reflected good linearity of

the method. Statistical models were applied in order to verify if the correlation is good to

produce the best approximation of the data and correlation coefficient, F test value, t test
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value and p value were calculated by Fischer statistics.[24] The results show that p-valueof

the slopeis less than 0.05, so the regression slope approximates correctly all studied data.

Limit Of Detection (LOD) And Limit Of Qualification (LOQ)

Limit of detection (LOD) and limit of quantification (LOQ) were calculated in order to

assess the sensitivity for the chromatographic method. For evaluation, three replicates of

the blank solutions were recorded in order to verify the absence of possible interference

signals. Methods of calculation were evaluated according to J. Ermer et al.[24]

Mathematical calculations were performed using a threshold of a blank for a hmax of 1000

mFlu units (for FLD detection) and 1000 mAu (for DAD detection module).

The LOD and LOQ obtained for each PAH compound under study are summarized in

table 3. The values obtained were comparable to those who were reported in literature in

other similar studies.[25]

Precision And Accuracy

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The accuracy of the method was established by calculating the recovery factors and was

assessed by spiking blank samples with known quantities of the standard mixture. Spiked

samples with 400, 200 and 100pg/µl of every compound were prepared. In table 4 are

presented recovered concentrations along with recovery factors on every compound.

The significance of matrix effect was evaluated with T test applied to intercept which
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showed there are no differences in respect with 0. Standard deviation variances applied to

intercept along with calculated value for t-testare less then theoretical statistical values

with a confidence level of 95% (p<0.05). The intercept is not significant different of 0.

Experimental recovered values are not different of theoretical values, with confidence

level of 95%, so the extraction method has the necessary accuracy.[24] As it was

previously mentioned, the use of ASE method had the advantage of higher precision, this

was confirmed by the better recoveries whichwere situated between of 82 -120%,

comparing with other publications which reported recovery factors between 42 and 101%
[26]
or62 and 107%, using same analysis techniques.[27]

Intermediate Precision

Intermediate precision was evaluated by analyzing extracted samples of 100 pg/µl in two

different days in the same conditions on the same analytical instrument and by two

different analysts. Within days, the relative standard deviation was less than 2% for every

sample type. Sample stability at the room temperature was also tested. A total number of

6 determinations were performed. Intermediate precision regarding the stability of the

signal area showed an increase for relative standard deviation more than 2% for

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compounds Ipy, BghiP, DahA and A. Relative standard deviation for anthracene showed

a cumulative value of 5.46%. Temperature stability of the sample in room conditions

showed a decrease of the values determined for some compounds. A significant

decreasing was registered for N-3.31%, Fl-4.85%, A-8.52%, P-3.88%, Ch-3.30% and

Ipy-2.34%.
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DISCUSSIONS

Level Of Pahs Found In Lung Tissue Samples.

These methods were tested on 31 individual human samples from patients diagnosed with

lung cancer as primary diagnostic and subjected to PAH determination study.

The results with clinical meaning of the study were published elsewhere.[2] In this

situation, from the analytical perspective, due to high affinity to this type of matrix, we

found high concentrations of all carcinogenic and non-carcinogenic types of PAHs. Also,

there was proven that these two kinds of PAHs to be present in all cases included in this

study. Limits of content of each target compound were different and also, were in direct

correlation with other parameters which proved to be of great importance in pulmonary

cancer assessment.

ACKNOWLEDGMENTS

The authors thank Patricia Fiterman, executive manager at FitermanPharma Iasi,

Romania for the opportunity to extend our study in the company’s Quality Control

Department where we found all necessary techniques for fully developing of the project.

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CONCLUSIONS

A rapid and sensitive method was developed and validated for simultaneous

determination of 16 PAH compounds in biological samples. Specifically, HPLC with

FLD detection was used for detection and quantification of 15 PAH compounds, while

DAD was applied for quantification of Acy. The method was successfully applied to
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assess the PAH levels in the lung tissue samples.

Best recovery and selectivity on determination of the sixteen compounds were

determined using the 1:1 mixture aluminum oxide:silicagel due to the higher retaining

capacity of aluminum oxide:silica gel for lipophilic compounds.

Accelerated solvent extraction (ASE) was employed for human lung tissue samples, The

developed method provided good sensitivity and specificity to allow the quantitative

profiling of PAH levels in human lung tissue samples. In addition, this method uses a

suitable run time and was fully validated to ensure the reliability of the results with a high

degree of accuracy.

The assessed amounts of PAHs in the biological samples under study and investigation

were correlated with the effect of the residential area, smoking habits, age, ABO

phenotypes and will be presented in a further paperwork.

Also, a further study for better elucidation of structure and bio-transformation products of

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these compounds will be provided using high resolution mass–spectrometry liquid

chromatography hyphenated techniques.

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[24] Ermer, J.; Miller, H. Mc. B. 2005. Method Validation in Pharmaceutical Analysis.

Boschstrasse- Weinheim: Wiley-VchVerlag GmbH & Co. KGaA.

[25] Singh, V. K.; Singh, J.; Anand, M.; Kumar, P.; Patel, D. K.; Krishna-Reddy, M. M.;

Javed-Siddiqui, M. K. Comparison of polycyclic aromatic hydrocarbon levels in

placental tissues of Indian women with full- and preterm deliveries. International Journal

of Hygiene and Environmental Health 2008, 211 (5-6), 639-47.

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[26] Del Bubba, M.; Zanieri, L.; Galvan, P.; Donzelli, G. P.; Checchini, L.; Lepri, L.

Determination of polycyclic aromatic hydrocarbons (PAHs) and total fats in human

milk. Annali Di Chimica 2005, 95 (9-10), 629–641.

[27] Jonssona, G.; Tabana, I. C.; Jørgensenb, K. B.; Sundta, R. C. Quantitative

determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence

and GC–MS. Chemosphere 2004, 54 (8), 1085–1097.

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Subject category: chromatographic techniques

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Table 1. Florescence parameters used in the FLD-HPLC for the detection of PAHs in

biological samples

Compound Excitation Emission PMT

(nm) (nm) Voltage

Naphtalene, Fluorene, Antracene, 275 350 Medium

FluorantheneAcenaphtene,Phenantrene 270 440 Medium

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Pyrene, Benzo(a)antracene, Chrysene, 260 420 Medium

Benzo(b)fluoranthene, 260 420 Low

Benzo(a)pyrene, 290 470 Medium

Dibenzo(a,h)anthracene,Benzo(k)fluoranthene,

Benzo(g,h,i)perylene,

Indeno(1,2,3,-c,d)pyrene,

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Table 2 System suitability parameters for the 16 analysed polycyclic aromatic

hydrocarbons.

Retention Capacity Resolutio Asymmetr Theoretical

time factor n y plates

Unret. 1.83

comp.
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N 8.90 3.86 28.28 1.42 7033

Acy 10.34 4.65 5.76 1.28 9494

Ac 12.57 5.86 8.92 1.26 14030

Fl 13.08 6.14 2.04 1.28 15192

A 14.78 7.07 6.80 1.36 19398

P 16.31 7.91 6.12 1.38 23622

Flu 18.18 8.93 7.48 1.29 29349

Py 19.66 9.74 5.92 1.46 34322

Ba 23.70 11.95 16.16 1.42 49878

Ch 24.53 12.40 3.32 1.36 53432

BbFlu 28.27 14.44 14.96 1.55 70968

BkFlu 29.58 15.16 5.24 1.50 77697

BaPy 31.11 16.00 6.12 1.26 85943

DahA 32.90 16.97 7.16 1.39 96118

BghiP 35.55 18.39 10.40 1.42 111910

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IPy
35.88
18.60
1.52

21
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114318

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Table 3. Limits of detection (LOD) and limit of quantification (LOQ)

Compoun LOQ LOD

d (pg/µl) (pg/µl)

N 1.89 0.56

Acy 77.83 23.23

Ac 0.96 0.29
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Fl 2.27 0.68

A 1.75 0.52

P 1.36 0.40

Flu 2.07 0.62

Py 1.92 0.57

Ba 0.83 0.25

Ch 1.93 0.58

BbFlu 1.03 0.31

BkFlu 1.37 0.41

BaPy 1.82 0.54

DahA 2.53 0.76

BghiP 6.52 1.95

IPy 7.41 2.22

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Table 4. Quality parameters for spike samples at 400, 200 and 100 pg/µl for target

compounds

Analyte Spiked level Recovery ± SD Analyte Spiked level Recovery ± SD

N 400 97.98 3.56 Ba 400 92.12 1.92

200 99.95 0.76 200 97.25 0.02

100 88.46 0.77 100 91.43 0.26

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Acy 400 86.35 7.88 Ch 400 120.85 2.32

200 90.26 1.06 200 115.32 1.26

100 89.47 0.95 100 90.55 0.17

Ac 400 83.19 2.56 BbFlu 400 90 1.16

200 85.06 0.38 200 101.2 2.08

100 90.58 0.71 100 92.32 1.45

Fl 400 95.51 6.32 BkFlu 400 82.62 4.28

200 91.5 0.5 200 92.45 0.86

100 89.45 1.31 100 87.39 0.39

A 400 84.89 3.02 Ba 400 82.71 3.84

200 97.74 3.03 200 83.55 1.70

100 89.96 1.36 100 86.45 1.23

P 400 103.7 5.48 DahA 400 82.45 1.09

200 104.4 2.55 200 89.48 3.02

100 83.51 0.79 100 85.82 1.09

BaPy 400 102.66 4.07 BghiP 400 82.61 1.06

200 97.77 3.07 200 83.7 0.57

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100 90.8 0.44 100 83.65 1.93

Py 400 89.87 3.16 IPy 400 87.98 2.73

200 90.13 1.28 200 93.54 3.92

100 88.68 0.16 100 89.72 1.01

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Figure 1 PAHs distribution in (a) fluorescence detection and (b) in UV-VIS detection,

PDA-228 nm channel for the stock solution with containing all 16 PAHs with a nominal

concentration of 10 µg/pl for each compound.

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Figurre 2 Complette workflow
w for the deteermination of PAHs withh sample preeparation andd

HPLC
C analysis.
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Figure 3 (a) Spike for PAHs (100 pg/µl) in lung tissue and (b) blank for sample

determinations.
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