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GFAAS Determination of Arsenic Levels in Biological Samples of Workers

Occupationally Exposed to Metals: An Application in Analytical Toxicology

Article in Atomic Spectroscopy · July 2015

DOI: 10.46770/AS.2015.04.004

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 GFAAS Determination of Arsenic Levels in Biological
Samples of Workers Occupationally Exposed to Metals:
An Application in Analytical Toxicology
*Bayram Yüksela,b, Zeliha Kayaaltia, Tülin Söylemezoglua, Vugar Ali Türksoya, and Engin Tutkunc
a
Ankara University Institute of Forensic Sciences, Dikimevi, 06590 Ankara, Turkey
b Ankara Police Forensic Laboratory, Gölba ı, 06830 Ankara, Turkey
c
Ankara Occupational Diseases Hospital, Keçiören, 06280 Ankara, Turkey

diovascular and peripheral vascular

ABSTRACT sis, the samples were pre-treated diseases, neurological disorders,
with an acid digestion procedure. diabetes mellitus, and various forms
Arsenic exposure in humans The method showed linearity in of cancer (2–4). Arsenic is found in
has been associated with adverse the range of 0–100 µg/L, with a
health effects such as neurologi-
inorganic and organic forms with
detection and quantification limit
cal and cardiovascular effects, of 0.37 µg/L and 1.1 µg/L, respec- different valence or oxidation states
diabetes mellitus, skin lesions, tively. The calibration curve was in the environment. Unlike inorganic
skin, lung, kidney and liver can- characterized by a high correla- arsenic, organic arsenic compounds
cers. Occupational exposure to tion coefficient (r=0.9991). Vali- in the pentavalent oxidation state
arsenic usually occurs with dation of the method was per- are much less toxic because con-
inhalation of arsenic-containing formed in terms of precision and sumption of these organic arseni-
particles in the mining industry. accuracy with the use of refer- cals are not immediately accepted
A simple and sensitive method ence materials. The method was into the cells, and meet with lim-
was developed and validated for applied to the analysis of certified ited metabolism (5, 6). Some impor-
the determination of arsenic lev- reference material samples with
els in biological samples by
tant arsenic species are listed in
satisfactory results (96.77–97.50%).
graphite furnace atomic absorp- The arsenic levels of the biologi- Figure 1.
tion spectrometry (GFAAS), cal samples of the metal workers Inorganic arsenic occurs natu-
equipped with a Zeeman back- ranged between 3.83–52.44 µg/L rally in soil and many kinds of rock,
ground correction system. Blood, in blood; 1.26–27.54 µg/L in
urine, and hair samples are
especially in minerals and ores that
urine; and 0,06–7.90 mg/kg As in
known to be the hair. The mean arsenic levels in contain copper, lead, cobalt, silver,
best biomarkers to assess arsenic the blood, urine, and hair samples and gold. Arsenic trioxide is
exposure in humans. Samples of the silver metal workers were volatilized during smelting and
were collected from 95 metal found at 21.25±12.47 µg/L, accumulates in flue dust, which
workers who were admitted at 6.43±4.99 µg/L, and 1.81±1.79 may contain up to 30% arsenic tri-
the Ankara Occupational Diseases mg/kg As, respectively. oxide (8). Thus, inhalation of indus-
Hospital in Turkey. Prior to analy trial soil and dust causes arsenic
exposure in metal workers. Occu-
pational exposure to chemicals
occurs most commonly via inhala-
INTRODUCTION
Arsenic (As) is an extremely poi-
sonous element and has been classi-
fied as a human carcinogenic sub-
stance, group 1, by the International
Agency for Research on Cancer (1).
Arsenic exposure in humans is gen-
erally associated with the consump-
tion of drinking water contaminated
from natural, geological sources of
inorganic arsenic. Chronic expo-
sure to arsenic in humans has been
related to the development of
adverse health effects such as car-

Fig 1. (Yüksel et al.) Some important arsenic species. Common inorganic arsenicals
*Corresponding author. and their metabolites are listed in top row, while organic arsenicals found in
E-mail: [email protected]
seafood are listed in bottom row.

Atomic Spectroscopy 171

Vol. 36(4), July/August 2015
tion. It can be possible to gather a ICP-MS (15-17) is widely used samples obtained voluntarily from
more accurate prediction of total because of its multi-element capa- metal workers. The samples were
dose by using biomarkers (9) such bilities, but it is also one of the taken from them at the Ankara
as blood, urine, and hair. Since the most expensive instruments (18). Occupational Diseases
main route of arsenic excretion Graphite furnace atomic absorption Hospital, Turkey (21).
takes place in the kidneys, the level spectrometry is more economical
of arsenic in urine can be used to and is a good choice due to its EXPERIMENTAL
predict exposure (10). Blood- selectivity and sensitivity in the
arsenic is peculiarly employed only detection of a wide range of metals Instrumentation
as a sign of very current or compar- and non-metals, including arsenic The measurements for arsenic
atively high-level exposure because (19). determination were performed
inorganic arsenic is quickly elimi- using a Varian AA240Z atomic
The Zeeman effect (20) is based
nated from the blood (11). Hair has absorption spectrometer (Varian,
on the shift of energy of atoms and
a unique potential to reveal retro- Victoria, Australia), equipped with
molecules in a magnetic field. If a
spective information about the a Zeeman background correction
magnetic field is generated at the
exposure of subjects (12). Addition- system. A boosted-discharge hollow
atomizer (graphite furnace), the
ally, once incorporated into keratin, cathode lamp (Agilent, Australia)
absorption lines of the analyte
arsenic has limited mobility, so it was used as the excitation source
atoms are split into three compo-
is known to be deposited in nails for arsenic. The digestion proce-
nents. Two of these components
and especially in hair (13). Arsenic dure for the blood and hair samples
(σ–components) are shifted to
levels in blood, urine, nail, and hair was carried out using a Mars
slightly lower and higher wave-
of unexposed human adults are Xpress microwave system (CEM,
lenghts, repectively, whereas the
usually below 1 µg/L, 100 µg/L, Matthews, NC, USA) with PTFE
third component (π–component)
1 mg/kg, and 1 mg/kg, respectively microwave digestion vessels. The
remains largely unchanged. The
(8). operating parameters for the
π–component can be removed
The determination of arsenic from the spectrum using a polarizer GFAAS system are listed in Table I.
levels in biological samples can be (Figure 2).
Standard Solutions and
performed by methods such as neu- Reagents
The main goal of this study was
tron activation, X-ray fluorescence,
to develop and validate a sensitive A 1000-µg/mL arsenic stock solu-
atomic absorption and fluorescence
method with graphite furnace tion was obtained from SCP
spectrometry, and inductively cou-
atomic absorption spectrometry, Science (Courtaboeuf, France).
pled plasma atomic emission and
equipped with a Zeeman-effect Triton® X-100, polyethylene glycol
mass spectrometry (ICP-AES and
background correction system, to mono (p-1,1,3,3-tetramethylbutyl-
ICP-MS) (14). In recent years,
determine arsenic concentrations phenyl) ether, was obtained from
graphite furnace atomic absorption
in biological samples for routine Scharlau (Barcelona, Spain). Nitric
spectrometry (GFAAS), hydride
toxicological analytical application. acid (HNO3, 65%) was purchased
generation atomic absorption spec-
The method developed for the from Merck (Darmstadt, Germany).
trometry (HGAAS), and ICP-MS
determination of arsenic was All chemicals used were of analyti-
have become the leading techniques.
applied to blood, urine, and hair cal reagent grade unless otherwise

resistivity of 18 MΩ.cm, was used

specified. Ultrapure water (Human
UP 900 Scholar-UV, Korea), with a

to prepare the solutions for the

experimental process. Argon gas
with a purity of 99.999% was pur-
chased from a local supplier (Vasak
Gaz, Ankara, Turkey). The reference
materials used were BCR-CRM 397
Human Hair Powder (Community
Bureau of Reference BCR, Institute
for Reference Materials and Measure-
ment, Belgium) and Seronorm™
Trace Elements Whole Blood L-2
(Sero AS, Billingstad, Norway).
Fig. 2. (Yüksel et al.) Schematic diagram of Zeeman effect.

172
 Vol. 36(4), July/August. 2015

Sample Collection Procedure Optimization and Sample

Blood, urine, and hair samples In order to prepare calibration Treatment
were collected from 95 metal work- standards at the concentrations of Important parameters were
ers (volunteers) at the Ankara 3, 6, 9, 12, and 15 µg/L, a 1000- adjusted to obtain the best perfor-
Occupational Diseases Hospital, µg/mL arsenic stock solution was mance from this spectrometric
Turkey. The patients ranged in age diluted in 5% (v:v) HNO3. All glass- analysis. Selection of the digestion
from 18–61 years. This study was ware was kept in 10% (v:v) nitric technique, choice of the appropri-
ethically approved by the Research acid for at least one night prior to ate wavelength for the biological
Ethics Committee of the Medical each experimental work. matrix, calibration concentration
Faculty, Ankara University (Deci- range in accordance with element
Prior to analysis, the biological
sion Number:11-343-12/25.06.2012). concentration in real samples,
samples (except for urine) were
Each volunteer was given a written assessing the best furnace program
pre-treated using the acid digestion
informed consent form in accor- and establishing the linearity, were
procedure. One milliliter of each
dance with the principles as estab- the major criteria for developing
blood sample was taken into the
lished in The Declaration of and optimizing this atomic absorp-
Teflon® tubes. The microwave sys-
Helsinki (World Medical Associa- tion spectrometry method. Prelimi-
tem (CEM Mars Xpress) was utilized
tion, Declaration of Helsinki, 1964). nary studies were performed under
for digestion of the samples with
The blood, urine, and hair samples these subheadings to establish the
5 mL of 65% HNO3 solution. Simi-
were stored separately at 4 oC in best methodology for accurate mea-
larly, 100-mg amounts of hair sam-
vacutainer blood collection tubes, surements (22). The graphite fur-
ples were taken and washed with
polypropylene tubes, and polyeth- nace temperature program for
Triton®-X, rinsed, and left standing
ylene lock bags, respectively, until arsenic determination in biological
to air-dry. This microwave digestion
the day of analysis. samples is listed in Table III.
procedure was also applied to the
hair samples. For the urine samples, Detection was performed at the
TABLE I 1-mL amounts were mixed with 5 193.7-nm arsenic line. This wave-
Operating Parameters for mL of 65% HNO3 (21, 26). All bio- length was selected due to a higher
GFAAS System logical samples were diluted with signal-to-noise ratio in the spectrum
Element As ultra-pure water to 10 mL. The of the sample matrices than at the
Matrix Blood, Urine and Hair microwave temperature program is 197.2-nm and 189.0-nm lines. The
listed in Table II.
Instrument Zeeman
Concentration Unit µg/L, µg/kg TABLE II
Instrument Mode Absorbance Temperature Program For Microwave Digestion
Sampling Auto-Mix Max. Power Power Ramp Pressure Temperature Hold
Calibration Mode Concentration (W) (%) (min) (ºC) (min.)
Measurement Mode Integrated 1600 100 10:00 Maximum 210 10:00
Replicates Standard 3
Replicate Sample 3
TABLE III
Expansion Factor 1.0
Graphite Furnace Temperature Program for
Wavelength 193.7 nm Arsenic Determination in Biological Samples
Slit Width 0.5 nm
Step Temperature Time Flow Signal Reading
Gain 59% (oC) (s) (L/min) Collection
Current 10.0 mA
1 85 5 0.3 × No × No
Background correction ON
Standard 1 3.0 µg/L 2 95 40 0.3 × No × No
Standard 2 6.0 µg/L 3 120 10 0.3 × No × No
Standard 3 9.0 µg/L 4 800 5 0.3 × No × No
Standard 4 12.0 µg/L 5 800 1 0.3 × No × No
Standard 5 15.0 µg/L 6 800 2 0.0 √ Yes × No
Reslope Standard Standard 2 7 2450 0.9 0.0 √ Yes √ Yes
Recalibration Rate 50 8 2450 2 0.0 √ Yes √ Yes
Calibration Algorithm Linear 9 2450 2 0.3 √ Yes × No

173
proposed method showed linearity BCR-CRM 397 Human Hair Powder Limit of Detection and
in the range of 1–100 µg/L and and Seronorm™ Trace Elements Quantification
good repeatability not exceeding Whole Blood L-2 were analyzed for The limit of detection (LOD) and
3% for As. On the other hand, the arsenic. Each certified reference lowest limit of quantification (LOQ)
average arsenic levels in the real material was analyzed 10 times were determined based on the stan-
hair sample solutions without using with triplicate measurements. The dard deviation of the response and
dilution factors was measured results were compared with the the slope of the calibration curve,
roughly as 2 µg/kg. Hence, for cali- certified values for accuracy, preci- according to ICH guidelines (23,
bration purposes, five calibration sion, and reproducibility of the 24) (LOD=3.3σ/S, LOQ=10σ/S,
standards (namely, 3, 6, 9, 12, and method. The certified arsenic con- where σ is the standard deviation
15 µg/L) were prepared. The cali- tent of BCR-CRM 397 (Hair) was of the response and S is the slope
bration graph showed good linear- 0.31±0.02 mg/kg, while the mea- of the calibration curve). The LOD
ity in the concentration range sured value was 0.30±0.01 mg/kg, and LOQ values were calculated for
examined (Figure 3). The correla- with the successful recovery and arsenic in the blood samples and
tion coefficient and equation of relative standard deviation (RSD) as found as 0.37 µg/L and 1.1 µg/L,
the calibration curve were, respec- 96.77% and 3.97%, respectively. respectively.
tively, found to be r=0.9991 and Similarly, the certified arsenic con-
Abs=0.0071C+0.0018, where Abs tent of Seronorm™ Trace Elements RESULTS AND DISCUSSION
stands for integrated absorbance Whole Blood L-2 was 13.20±1.3 µg/L,
and C for the arsenic concentration while the measured value was Epidemiological studies have
in µg/L. 12.87±0.77 µg/L, with a satisfactory provided compelling evidence that
recovery and RSD as 97.50% and inorganic arsenic is carcinogenic to
Method Validation 5.98%, respectively. The analytical humans. Chronic ingestion of
In order to validate the method results of the certified reference arsenic increases the risk of devel-
in terms of accuracy and precision, materials are summarized in Table oping skin, lung, urinary bladder,
IV. and liver cancer (25).
The assessed arsenic levels of
the biological samples from the
metal workers ranged between
3.83 and 52.44 µg/L in blood;
1.26 and 27.54 µg/L in urine; 0.06
and 7.90 mg/kg As in hair. The
mean arsenic levels in the blood,
urine, and hair samples of the silver
metal workers were found as
21.25±12.47 µg/L, 6.43±4.99 µg/L,
1.81±1.79 mg/kg As, respectively,
while the acceptable arsenic levels
in human biological samples
(blood, urine, hair, and nail) were
Fig. 3. (Yüksel et al.) Calibration graph of arsenic, performed by graphite furnace below: 1 µg/L, 100 µg/L, 1 mg/kg,
atomic absorption spectrometry (GFAAS), equipped with Zeeman-effect back- and 1 mg/kg, respectively, and are
ground correction. listed in Table V (8).
TABLE IV According to the results
Analysis of Certified Reference Materials (CRMs) obtained from this toxicological
arsenic analysis, 43 of 95 individu-
CRMs Number of Certified Measured Recovery RSD
Analyses (n) Value Value
TABLE V
BCR-CRM 397 10 0.31±0.02 0.30±0.01 96.77 3.97
Normal Arsenic Levels in
(Hair) mg/kg mg/kg (%) (%)
Human Biological Samples (8)
Seronorm™ Trace 10 13.20±1.30 12.87±0.77 97.50 5.98 Blood Urine Hair Nail
Elements Whole µg/L µg/L (%) (%)
Blood L-2 (Blood) <1 µg/L <100 µg/L ≤1 mg/kg ≤1 mg/kg

174
 Vol. 36(4), July/August. 2015

als have hair-arsenic concentrations TABLE VI

above the safe limits. As for the Descriptive Statistics of Arsenic Levels
blood-arsenic and urine-arsenic lev- in Biological Samples of Metal Workers
els, all individuals have above nor- N=95 Age Body Exposure Blood Urine Hair
mal levels of blood-arsenic, but are (Years) Mass Time Arsenic Arsenic Arsenic
at safe limits for urine-arsenic levels Index (Year) Level Level Level
(21). The evaluated arsenic levels in (ppb) (ppb) (ppm)
the biological samples of the metal
Mean 33.22 25.45 3.49 21.25 6.43 1.81
workers are listed in Table VI.
Standard
CONCLUSION Deviation 8.08 4.22 2.09 12.47 4.99 1.79
A graphite furnace atomic Minimum 18 17.34 1.0 3.83 1.26 0.06
absorption spectrometry (GFAAS)
method, using Zeeman background Maximum 61 37.50 10 52.44 27.54 7.90
correction, was developed for the
determination of arsenic in human
blood, urine, and hair. Using the ACKNOWLEDGMENT 5. S.M. Cohen, L.L. Arnold,.M. Eldan,
Zeeman-effect for background cor- A.S. Lewis and B. D. Beck, Crit.
The authors wish to thank the Rev. Toxicol. 36, 99 (2006)
rection, a strong magnetic field is Ankara University Institute of
turned on and off in rapid sequence. 6. M.F. Hughes, B.D. Beck, Y. Chen,
Forensic Sciences, Directory of A.S. Lewis and D.J. Thomas, Toxi-
Total absorbance (element-specific Ankara Police Forensic Laboratory,
and non-specific background col. Sci. 123(2), 305–332 (2011).
Ankara Occupational Diseases Hos-
absorption) is measured with the pital, and Turkish Prime Ministry 7. J. Liu, Y. Lu, Q. Wu, R.A. Goyer and
magnetic field in OFF-position and State Planning Organization
M.P. Waalkes, J. Pharmacol. Exp.
the background absorption with Ther. 326(2), 363 (2008).
Research Fund, Grant Number:
the magnetic field in ON-position 2003K1201902, for financial sup- 8. Agency for Toxic Substances and
The difference of the two values port. Disease Registry (ATSDR), Toxico-
gives the corrected element-spe- logical profile for arsenic, Atlanta,
cific absorption. The advantages of GA: US Public Health Service
the Zeeman-effect technique are as (2007).
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