Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324

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Spectrochimica Acta Part B: Atomic Spectroscopy

journal homepage: www.elsevier.com/locate/sab

Determination of lead in blood by graphite furnace atomic absorption

spectrometry with Zeeman background correction: Improving a
well-established method to support a lower blood lead reference value
for children
Emily J. Pacer a, b, Christopher D. Palmer a, b, Patrick J. Parsons a, b, *
a
Laboratory of Inorganic and Nuclear Chemistry, Division of Environmental Health Sciences, Wadsworth Center, New York State Department of Health, Empire State
Plaza, Albany, NY 12237, USA
b
Department of Environmental Health Sciences, University at Albany, Rensselaer, NY 12144, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Lead is ubiquitous in the environment and exposure can have adverse health effects even at low concentrations,
Graphite furnace particularly in young children. Blood Pb levels are the best indicator of recent Pb exposure. The Centers for
Permanent modifier Disease Control and Prevention (CDC) recently lowered the blood Pb reference value from 5 μg/dL to 3.5 μg/dL,
Childhood lead poisoning, blood lead reference
based on data calculated from the 97.5th percentile of children 1–5 years old in the National Health and
value
Nutrition Examination Survey (NHANES). The established method for determining Pb in blood by GFAAS has an
estimated detection limit (LOD) of ≈1 μg/dL. This presents an analytical challenge for its clinical use in the US,
given that the current population mean blood Pb level is 0.82 μg/dL. Accuracy and precision may be insufficient
to quantitate low blood Pb levels in the 1–5 μg/dL range, which is essential now that the CDC blood Pb reference
value is 3.5 μg/dL.
The original GFAAS method for blood Pb published in 1993 has been significantly improved to achieve a lower
LOD of 0.2 μg/dL, with the ability to quantitate blood Pb levels down to 1 μg/dL. This method is fit for the
purpose of detecting blood Pb levels currently, with excellent accuracy and precision in the 1–5 μg/dL range.
Results from analyzing SRMs, PT materials and human blood samples were in good agreement between two
different GFAAS platforms, as well as with results obtained by ICP-MS. The improvements provide evidence of an
GFAAS method capable of achieving lower LODs with improved accuracy and precision at lower blood Pb levels,
which can support the lower blood Pb reference value for children at 3.5 μg/dL.

1. Introduction assessing exposure to lead. At that time, a biochemical test based on

In 1993, when our rapid method for the determination of Pb in blood
by graphite furnace atomic absorption spectrometry (GFAAS) with
Zeeman background correction was first published in this journal [1] the
US Centers for Disease Control and Prevention (CDC) had just recom­
mended lowering the blood Pb action level for young children from 25
μg/dL (1.21 μmol/L) to 10 μg/dL1 (0.48 μmol/L) [2]. This landmark
change in environmental health policy was adopted in other countries.
Many laboratories were forced to reconsider their routine method for
erythrocyte protoporphyrin was widely used to screen children for Pb
exposure. However, this biomarker of Pb exposure was not sufficiently
sensitive to identify children with blood Pb concentrations exceeding 10
μg/dL [3]. Consequently, direct measurements for blood Pb became
necessary for both screening and diagnostic purposes, and on small
blood volumes obtained from a capillary fingerstick. The GFAAS method
published in this journal in 1993 was developed for a transversely-
heated graphite atomizer (THGA) equipped with longitudinal Zeeman
background correction and had an estimated limit of detection (LOD) of

* Corresponding author at: Laboratory of Inorganic and Nuclear Chemistry, Division of Environmental Health Sciences, Wadsworth Center, New York State
Department of Health, Empire State Plaza, Albany, NY 12237, USA.
E-mail address: patrick.parsons@health.ny.gov (P.J. Parsons).
1
In the US and elsewhere, blood Pb levels are typically reported in units of micrograms per deciliter (μg/dL). To convert μg/dL to μmol/L divide the former by
20.72.

https://doi.org/10.1016/j.sab.2021.106324
Received 10 September 2021; Received in revised form 5 November 2021; Accepted 7 November 2021
Available online 9 November 2021
0584-8547/© 2021 Wadsworth Center, NYS DOH. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
E.J. Pacer et al.
1 μg/dL. The method was modified slightly for a conventional GFAAS
system equipped with continuum background correction [4] and was
also successfully transferred to other manufacturers’ platforms. For
almost 30 years, this method was widely recommended as the estab­
lished GFAAS protocol in clinical laboratory medicine [5,6].
Over the past 5 decades, extensive efforts have succeeded in reducing
Pb exposures and further help lower population blood Pb levels. Envi­
ronmental regulations and public health interventions have resulted in
the US geometric mean blood Pb level dropping from 12.8 μg/dL in
1976–1980 to 0.82 μg/dL in 2015–2016 [7,8]. This trend has been
observed in other countries too. Despite the drop in population blood Pb
levels across the globe, Pb still remains a significant environmental risk
to young children and there is increasing evidence that adverse cogni­
tive effects are associated with even lower blood Pb levels than previ­
ously realized [9–11]. In 2012 after more than 20 years, the CDC
recommended revisiting the definition of an elevated blood Pb level
preferring to use a “reference value” that was based on the 97.5th
percentile of the distribution of US children aged 1 to 5 years old. In
2012, the blood Pb reference value was established as 5 μg/dL, and has
since been adopted by many public health agencies in the US and else­
where. However in late 2021, the CDC lowered the blood lead reference
value yet further, from 5 μg/dL to 3.5 μg/dL (0.17 μmol/L) [12] based
on the most up to date 97.5th percentile of the distribution of US chil­
dren’s blood Pb levels [8]. This lower blood lead reference value now
raises the question, is the GFAAS method developed in the 1990s for an
elevated blood Pb level at 10 μg/dL still fit for purpose in 2021 to sup­
port measurements at 3.5 μg/dL?
In this report, we describe our efforts to re-visit and improve the
original GFAAS method proposed in 1993. We explore various ap­
proaches to achieving a lower LOD, and therefore a lower limit of
quantitation (LOQ) to support reporting blood Pb measurements below
3.5 μg/dL. The improved method also takes into account the diminishing
prevalence of blood Pb levels above 20 μg/dL, which allows us to focus
on better performance at lower concentrations. New validation data for
the improved method are included and are more focused on perfor­
mance at lower blood Pb levels. As the focus shifts toward blood Pb
concentrations at 3.5 μg/dL and below, the contribution of background
contamination becomes much more significant and needs special
attention.
2. Methods
2.1. Instrumentation
Two atomic absorption instruments were used throughout this study:
the Perkin Elmer (PE) AAnalyst 600 and the PE PinAAcle 900Z (Perki­
nElmer Inc., Shelton, CT, USA). Both instruments are equipped with a
THGA design, longitudinal Zeeman background correction, and facili­
tate the implementation of methods based on STPF technology. The
basic instrumental parameters (line source, analytical wavelength, etc.)
were adapted from the original method published in 1993. Sample ali­
quots were delivered using an AS-800/900 autosampler.
2.2. Reagents and consumables
In the original method, a conventional modifier was used consisting
of a diluent reagent comprising 0.5% (w/w) ammonium dihydrogen
phosphate, 0.2% (v/v) HNO3 and 0.5% (v/v) Triton X-100 [1]. In this
study, we also use a W-Rh permanent modifier, reagent solutions and the
coating process that are described in detail elsewhere [13,14]. In brief,
the W solution was prepared by dissolving 0.1 g of Na2WO4• 2H2O (Alfa
Aesar®, Ward Hill, MA, USA) in 100 mL of double deionized (DI) water.
The Rh solution was prepared by dissolving 0.1 g of (NH4)2[RhCl6] •
xH2O (Alfa Aesar®, Ward Hill, MA, USA) in 10 mL of 10% HCl and 90
mL of DI water, which gives an orange-red colour to the solution. The
procedure for coating the L’vov platform with a permanent W-Rh
2
Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324
modifier consists of depositing 250 μg of W and 200 μg of Rh on
pyrolytically-coated graphite tubes in a series of drying steps, which
takes approximately 45 min to perform [13].
Samples are prepared in 0.5 mL acid-washed autosampler sample
cups with a diluent that consists of 0.5% (v/v) Triton X-100 and 0.2%
(v/v) HNO3. All reference materials, standard reference materials
(SRMs), quality control materials (QCs), proficiency test (PT) samples,
and human specimens are treated in a same manner, i.e., diluted 1 + 9
with the Triton X-100/HNO3 diluent. A 10 mg/L intermediate Pb stock
solution is prepared to make weekly stock solutions, by pipetting 10 mL
of a 1000 μg/mL Pb standard solution (High-Purity Standards, North
Charleston, SC, USA) in 1% HNO3. The Pb standard solution used should
be traceable to SI units via a certificate issued by a National Metrological
Institute, for example the National Institute of Standards and Technol­
ogy (NIST, Gaithersburg, MD USA).
In contrast to the original published method, matrix matched (i.e.,
blood-based) calibration standards are now mandatory for reliable
work. They are prepared daily as a 1 + 9 dilution of a 50 μL Pb cali­
bration working stock solution with 50 μL of “base” blood, and 400 μL of
Triton X-100/HNO3 diluent. Ideally, a “base” blood should be confirmed
as having no more than 0.5 μg/dL endogenous Pb. For this study, the
base blood (BB005) was obtained from a caprine source available to the
New York State Department of Health (NYS DOH), but any whole blood
source (human, caprine or bovine) should suffice.
2.3. Certified reference materials, proficiency testing samples and human
blood specimens
Standard reference materials (SRM) 955c Toxic Metals in Caprine
Blood and 955d Toxic Metals and Metabolites in Frozen Human Blood
were obtained from NIST. The certified Pb values of these two SRMs
were established by Isotope Dilution Inductively Coupled Plasma Mass
Spectrometry (ID-ICP-MS). Archived blood Pb proficiency test (PT)
samples were obtained from the NYS DOH Biomonitoring PT Program
and from the Centre de Toxicologie du Québec (CTQ), which operates
the Québec Multielement External Quality Assessment Scheme (QME­
QAS). The PT material target values were assigned by the respective PT
program providers after the conclusion of each event. De-identified
human blood samples (n = 30) were available from the Wadsworth
Center’s Trace Elements Laboratory. The archived human samples had
been previously analyzed by ICP-MS and the range of blood Pb con­
centrations varied from 1 μg/dL to 22 μg/dL [15].
3. Results and discussion
3.1. Method optimization studies
3.1.1. Calibration
The method established previously recommended calibration stan­
dards in the range of 10 μg/L to 60 μg/L, equivalent to a blood Pb range
from 10 μg/dL to 60 μg/dL after correcting for the dilution factor [1].
Given that the current (2015–2016) geometric mean blood Pb level of
the US population has fallen to 0.82 μg/dL, and the blood Pb reference
value has been lowered to 3.5 μg/dL in 2021, the calibration range was
adjusted to fit the current population blood Pb levels. The dilutions the
weekly stock solutions from the intermediate Pb stock solution are
described in Table S1 (Appendix).
The daily calibration standards ranged from 1 to 40 μg/L, (equivalent
to 1–40 μg/dL blood Pb after correcting for the dilution factor) consis­
tent with CLSI recommendations [6]. Shifting the lowest calibrator to 1
μg/L more appropriately reflects the current geometric mean blood Pb
level. Given the levels in the current population, a top calibrator of 40
μg/L is better suited for clinical assessment and biomonitoring purposes
[6].
While aqueous Pb calibration standards were used in the original
method [1], matrix matched calibration was mandatory as the
E.J. Pacer et al.
atomization profiles of aqueous standards consistently showed double
peaks with a W-Rh permanent modifier. Double or multiple peaks may
be due to some of the analyte being transferred to the tube walls during
the drying steps. After visual inspection of the sample during the drying
steps and increasing the drying time by >30 s, double peaks were still
observed. Differences in atomization profile for (a) aqueous standards
compared to a blood (b) matrix-matched Pb standard are shown in
Fig. 1. The calibration of Pb in both standards was adjusted to 40 μg/L,
equivalent to 40 μg/dL blood Pb.
Significant differences in atomization profile, i.e., peak shapes can be
observed for the two calibrators when using a permanent W-Rh modi­
fier. A previous study noted a shoulder on the peak profiles of Pb when
W was used as the permanent modifier, but in the presence of Rh, well-
defined peaks were always observed for water-based reference materials
[13]. Other authors have reported good experience for blood Pb using a
Mo-coated L’vov platform [16]. Resano et al. successfully used a Pt
modifier with solid-sampling GFAAS to analyze dried blood spots for Pb
content [17] and consistent with our studies, they reported the necessity
to use matrix-matched calibration standards. Tsalev et al. reported that
one of the limitations of using permanent modifiers can be the presence
of double or multiple peaks [18]. In our study, double peaks were
consistently observed with aqueous calibration standards when using W
with Rh. Since matrix-matched standards showed no double peaks with
W-Rh permanent modifier, their use was considered mandatory.
3.1.2. Injection volume
Increasing the volume of diluted blood deposited on the platform
was explored as this increases the amount of analyte available for at­
omization, but it also increases the amount of blood matrix that should
be removed during pyrolysis. Any improvement in performance would
depend on how much the signal increases relative to the noise from the
matrix [19]. There is a limit to the maximum sample volume that can be
deposited onto the L’vov platform, typically, 50 μL or less [20]. Other
methods that describe the determination of Pb in biological matrices
have recommended injection volumes ranging from 10 μL to 20 μL
[21,22]. The 1993 blood Pb method recommended an injection volume
of 12 μL of 1 + 9 diluted blood equivalent to 1.2 μL of whole blood
deposited on a L’vov platform [1]. In this study, we compared injection
0.15
0.10
Absorbance
0.05
0.00
0 1
Fig. 1. Absorption profile from (a) aqueous Pb standard (blue, Ai = 0.0636 s) and (b) blood matrix-matched Pb standard (red, Ai = 0.0653 s), equivalent to 40 μg/L
using the W-Rh permanent modifier. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324
volumes of 16 μL, 18 μL, and 24 μL for 1 + 9 diluted blood, thus
increasing the amount of whole blood deposited up to 2.4 μL. Fig. 2a
shows the atomization profiles obtained for different injection volumes
of a matrix-matched calibration standard diluted 1 + 9, i.e., to 40-μg/L.
As expected, as more sample is deposited on the platform, the
recorded Ai increases too: 0.0653 s, 0.0810 s, 0.0862 s, and 0.1127 s, for
injection volumes of 12 μL, 16 μL, 18 μL, and 24 μL, respectively.
However, the magnitude of the increase in Ai is not proportionate to the
volume (i.e., analyte mass) deposited. The observed increase in Ai was
less than expected: ≈93% for 16 μL; ≈88% for 18 μL; and ≈86% for 24
μL. Clearly, atomization efficiency (and therefore sensitivity) decreases
as the volume of diluted blood deposited increases. This could be due to
the effect of the increased blood matrix on atomization. The absolute
sensitivity or characteristic mass (m0) for volume deposited was 32.3 pg
for 12 μL, 34.8 pg for 16 μL, 36.7 pg for 18 μL, and 37.5 pg for 24 μL.
While increased sensitivity is desirable, increasing the amount of
(blood) matrix on the platform can adversely affect measurement pre­
cision, and thus the LOD. Increasing the injection volume requires an
adjustment to drying, pyrolysis, and clean-out times. For example, a 24-
μL injection volume required an additional 25 s per replicate to achieve
complete drying. LODs were calculated by analyzing NIST SRM 955c
Level 1 over 7 independent runs (3SD). Increasing the injection volume
from 12 μL to 16 μL lowered the LOD from 0.27 μg/dL to 0.16 μg/dL, and
16 μL was then adopted.
3.1.3. Blood dilution factor
An alternative approach to depositing more analyte on the platform
is to change the blood sample dilution factor. The established method
used a 1 + 9 dilution factor, wherein 50 μL of whole blood was diluted
with 450 μL of the Triton X-100/HNO3 diluent with phosphate modifier
[1]. In this study, we explored using a 1 + 4 dilution factor while
maintaining the injection volume of 12 μL, equivalent to doubling the
amount of analyte deposited. For a 40-μg/L Pb standard, the mass of
analyte deposited onto the platform increased from 480 pg to 960 pg.
Fig. 2b shows the comparison of atomization profiles for 12 μL of a 40-
μg/L matrix-matched calibration standard diluted 1 + 9 (red) and one
diluted 1 + 4 (black).
The 1 + 9 dilution yielded an Ai of 0.0653 s, while the Ai for 1 + 4
(b) Matrix-Matched Pb Standard-AA
(b) Matrix-Matched Pb Standard-BG
(a) Aqueous Pb Standard-AA
(a) Aqueous Pb Standard-BG
2 3 4
Time (s)
3
E.J. Pacer et al. Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324

0.3 modifier, m0 ranged from 35 to 40 pg [1]. As previously found the

optimal m0 for Pb in blood using a permanent W-Rh modifier was 31 pg
(a) 24 µL-AA [14]. We found m0 ranged from 32 to 36 pg using the W-Rh modifier,
24 µL-BG similar to values previously reported.
The change from a conventional phosphate modifier to a permanent
18 µL-AA
0.2 W-Rh modifier also necessitated calibration using blood-based, i.e.,
18 µL-BG
matrix-matched Pb standards. The calibration range was limited to
Absorbance

16 µL-AA 1–40 μg/L, which better reflects current population blood Pb levels. Fig.
16 µL-BG S1 (Appendix) shows the typical calibration curves using the final
12 µL-AA optimized parameters for the two different THGA platforms.
0.1 12 µL-BG The LOD for the improved blood Pb method on two THGA platforms
was calculated based on the IUPAC/ISO harmonized protocol, i.e., 3 x
SD for n = 7 measurements, i.e., collected over 7 independent analytical
runs, and using NIST SRM 955c Level 1 [24]. The LOQ was calculated as
10 x SD per laboratory policy. The LOD with the method improvements
0.0 on the PE AAnalyst 600 was calculated as 0.16 μg/dL and the LOQ as 0.5
0 1 2 3 4
μg/dL. On the PE PinAAcle 900Z, the LOD was calculated as 0.21 μg/dL
Time (s)
and the LOQ as 0.7 μg/dL.
0.3
A lower LOD of ≈0.2 μg/dL was achieved on the two THGA platforms
(b) using the improved blood Pb method with a W-Rh permanent modifier
and matrix matched calibration. This also provides a lower blood Pb
LOQ of ≈0.6 μg/dL, which is certainly fit-for-purpose. Now that a lower
12 µL (1+4)-AA CDC blood Pb reference value of 3.5 μg/dL has been adopted, this is
0.2 12 µL (1+4)-BG uncomfortably close to the previously established method LOQ of 2.5–3
Absorbance

12 µL (1+9)-AA
12 µL (1+9)-BG
0.1
0.0
0 1 2 3
Time (s)
Fig. 2. (a) Typical atomization profiles for injection volumes of 12 μL (red, Ai
= 0.0653 s), 16 μL (blue, Ai = 0.0810 s), 18 uL (black, Ai = 0.0862 s) and 24 μL
(green, Ai = 0.1127 s) of a matrix-matched calibration standard equivalent to
40 μg/L. (b) Comparison of atomization profiles of a 12 μL injection of a 40-μg/
L matrix-matched Pb standard diluted 1 + 9 (red, Ai = 0.0653 s) and diluted 1
+ 4 (black, Ai = 0.1080 s). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
dilution only increased 0.1080 s, or ≈82.7% of the expected increase.
The corresponding sensitivities (m0) were 32.3 pg for a 1 + 9 dilution
and 39.1 pg for a 1 + 4 dilution, i.e., ≈21% poorer sensitivity. An
additional complication was observed with 1 + 4 diluted blood wherein
it was more difficult to clean the autosampler tip between injections.
The poorer sensitivity and increased maintenance issues with 1 + 4
diluted blood indicated a 1 + 9 dilution was preferable.
3.2. Final optimized parameters
The optimized THGA heating program shown in Table S2 (Appendix)
was modified from the original method, which called for a conventional
modifier. The updated method calls for a permanent W-Rh modifier,
which is applied as a coating to the L’vov platform prior to calibration
and sample analysis. Injection aliquots of 16 μL with a 1 + 9 dilution
were selected as optimal based on the development studies reported in
Sections 3.1.2 and 3.1.3 above.
The characteristic mass (m0), or absolute sensitivity is a fundamental
parameter in GFAAS that is useful as a day-to-day indicator of method
performance, and is defined as the mass of analyte that absorbs 1% of
transmitted light i.e., equivalent to an integrated absorbance value of
0.0044 s [23]. For the established GFAAS method using a phosphate
μg/dL, such that quantitating blood Pb levels below 2–3 μg/dL are
subject to larger degree of relative uncertainty. After implementing the
method improvements, blood Pb quantitation down to 1 μg/dL and even
lower is now feasible. The improved blood Pb method would extend the
application of GFAAS technology within small laboratory settings where
ICP-MS is neither cost effective nor feasible.
The W-Rh coating is stable for approximately 350 firings, approxi­
mately equivalent to an 8-h workday, consistent with that reported in
other studies [13,14]. The W-Rh can be recoated to extend THGA tube
lifetime up to 1000 firings [13,14], compared to using the established
4
GFAAS method in which, the tube lifetime using the conventional
modifier was approximately 350 firings [1]. Biological matrices such as
blood, or high concentrations of Triton X-100, have been known to cause
carbonaceous residue build-up with a decrease in graphite tube life [25].
After 250 firings, carbonaceous residues were visible on the L’vov
platform. This is consistent with other findings, that found an excessive
build-up after 250 firings using the W-Rh modifier compared to the
conventional phosphate modifier [14]. However, the carbonaceous
build up did not obstruct the optical beam, therefore measurements
were unaffected, but it is possible that this could lead to higher back­
ground levels and low accuracy over multiple firings. One study found
that the use of hydrogen peroxide could reduce the carbonaceous res­
idue build up from analyzing biological matrices [26].
3.3. Validation studies
Numerous human and caprine blood certified reference materials
were analyzed to assess the method accuracy and precision on two
THGA platforms. The results are shown in Table 1.
The mean and expanded uncertainty (U) were calculated from n = 12
measurements. Uncertainties of the measured SRMs were calculated
according to the GUM and Eurolab guidelines [27,28]. The expanded
uncertainty (U) was calculated using a coverage factor of 2:
UMEAS = 2uc (1)
For each certified reference material, the combined standard un­
certainty (uc) was defined as:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅
uc = s2 + b2 (2)
where s = standard deviation for each respective THGA platform and b
was defined as:

4
E.J. Pacer et al. Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324

Table 1
Analysis of NIST SRMs for blood Pb on the PE AAnalyst 600 and PE PinAAcle 900Z.
Standard reference material Certified value (μg/dL ± USRM)a AAnalyst 600 mean (μg/dL ± UMEAS)b PinAAcle 900Z mean (μg/dL ± UMEAS)b

NIST 955c- Level 1 0.424 ± 0.0011 0.4 ± 0.3 0.5 ± 0.2

NIST 955c- Level 2 13.950 ± 0.080 13.8 ± 1.0 14.1 ± 0.9
NIST 955c- Level 3 27.76 ± 0.16 26.8 ± 2.5 27.4 ± 2.0
NIST 955d- Level 1 1.480 ± 0.026 1.4 ± 0.3 1.6 ± 0.4
NIST 955d- Level 2 4.947 ± 0.085 4.7 ± 0.6 5.1 ± 0.4
a
USRM = expanded measurement uncertainty from the SRM certificate (k = 2).
b
UMEAS = expanded measurement uncertainty of the measured SRM (k = 2).

√̅̅̅̅̅̅
b= ∆2 + U 2SRM (3) 3.4. Human samples

Eq. (3) defines b as the square root of the sum of squares of both ∆
representing the bias between the certified and found values, and USRM
is the uncertainty of the SRM, given on the SRM certificate. The results
with the method improvements are in good agreement between both
THGA platforms, as well as with the certified values provided by NIST.
Participation in PT programs provides beneficial information
regarding laboratory performance via circulation of well characterized
blood Pb samples [29]. The current United States PT regulatory limits
for blood Pb were set more than 25 years ago, at ±4 μg/dL or 10% of the
target value, whichever is greater [30]. Most laboratories can achieve a
performance of at least ±2 μg/dL at low blood Pb levels [31], and there
have been calls over many years to tighten these PT limits. The current
US criteria mean a blood Pb PT sample of 5 μg/dL could be reported
within a range from 1 to 9 μg/dL, and still be considered acceptable. This
makes interpreting patient blood Pb test results uncertain at low levels.
In addition to the NIST SRMs, archived blood Pb PT materials from the
NYS DOH Biomonitoring PT program and the CTQ QMEQAS programs
were also analyzed on the two THGA platforms (Table 2).
The mean and standard uncertainty (u) were calculated based on the
analysis of 12 measurements. Standard uncertainty (uMEAS) of the
measured PT samples were calculated according to the GUM and
Eurolab guidelines [27,28], as:
s
uMEAS = √̅̅̅
n pathogens [33]. Non-commutability for a reference material is not a
where s = standard deviation and n = number of analyses for each
respective THGA platform.
The results obtained with the improved GFAAS method are in good
agreement between both instrument platforms, as well as with the
assigned target values from the PT program provider. All values were
within ±2 μg/dL of the assigned value, the NYS DOH program’s
acceptable range. All but one of the values were within the CTQ pro­
gram’s z-score defined range. The materials that were available pre­
cluded blind analysis.
Human blood samples were also analyzed using the improved
method as part of the assessment. The results of the 30 samples were
compared with those obtained using a well- established ICP-MS method
[15]. Results in Fig. 3 show the blood Pb data obtained from the
AAnalyst 600 and PinAAcle 900Z, plotted against values obtained by the
ICP-MS method. For comparison purposes, the solid line represents y = x
and the dashed lines represents ±2 μg/dL.
The results indicate excellent agreement between the GFAAS and
ICP-MS method over the 1–5 μg/dL blood Pb concentration range. All
the human blood Pb values measured by GFAAS were within ±1 μg/dL
of the ICP-MS value, providing further validation of the proposed
method.
3.5. Commutability
A reference material that is commutable will have the same numeric
result that is expected for an equivalent human sample containing the
same quantity of analyte [32]. Since caprine SRMs were used in method
development process to assess the improvements for blood Pb mea­
surements, it is important to address whether the caprine blood refer­
ence materials are commutable to human samples. While using caprine
blood may have advantages, such as being able to endogenously dose
(4) animals with desired levels of Pb, it may be less likely to carry human
deficiency of the method, especially those that produce acceptable re­
sults for samples. Rather, it suggests that a specific reference material
may not be appropriate for use to calibrate or verify accuracy for that
particular measurement due to matrix effects [34].
To assess commutability, human samples and variety of SRMs and PT
program materials discussed previously in Section 2.3 were analyzed for
Pb. All of the blood materials analyzed were compared using the
improved GFAAS method with the target values from ICP-MS instru­
mentation or certified values from the NIST certificate. Results are
shown in Fig. 4 for the AAnalyst 600 and PinAAcle 900Z, plotted against
values found with ICP-MS. The SRMs and PT program are portrayed by
different symbols for each respective material, while the human samples
are shown as black circles, as described in the figure legend. For

Table 2
Analysis of archived blood Pb proficiency testing materials on the PE AAnalyst 600 and PE PinAAcle 900Z.
PT samples Target value Acceptable range for program AAnalyst 600 mean (μg/dL ± uMEAS)b PinAAcle 900Z mean (μg/dL ± uMEAS)b
(μg/dL ± uPT)a

CTQ QMEQAS
QM-B-Q2010 1.36 ± 0.017 1.05–1.67 1.4 ± 0.03 1.8 ± 0.1
QM-B-Q2011 10.6 ± 0.110 9.08–12.1 11.2 ± 0.1 11.3 ± 0.1
QM-B-Q2012 6.64 ± 0.067 5.64–7.64 6.8 ± 0.05 7.2 ± 0.1
NYS Biomonitoring
BE20-12 1.94 ± 0.05 0.0–3.94 1.8 ± 0.04 2.2 ± 0.03
BE20-13 8.4 ± 0.2 6.4–10.4 8.9 ± 0.1 9.1 ± 0.1
BE20-14 1.19 ± 0.03 0.0–3.19 1.2 ± 0.03 1.4 ± 0.03
BE20-15 14.0 ± 0.3 12.0–16.0 14.5 ± 0.1 15.0 ± 0.2
a
uPT = standard measurement uncertainty from the PT provider report.
b
uMEAS = standard measurement uncertainty of the measured PT sample.

5
E.J. Pacer et al. Spectrochimica Acta Part B: Atomic Spectroscopy 190 (2022) 106324

25 30
AAnalyst 600
AAnalyst 600
25
20
GFAAS, µg/dL

20

GFAAS, µg/dL
15

15
10

10

5
5

0
0 5 10 15 20 25 0
0 5 10 15 20 25 30
ICP-MS, µg/dL
ICP-MS, µg/dL
30
25
PinAAcle 900Z
PinAAcle 900Z
25
20

20

GFAAS, µg/dL
GFAAS, µg/dL

15
15

10
10

5 5

0 0
0 5 10 15 20 25 0 5 10 15 20 25 30

ICP-MS, µg/dL ICP-MS, µg/dL

NIST 955c CTQ QMEQAS Human Samples

Fig. 3. Analysis of 30 human blood samples for Pb using the improved GFAAS
method: (top panel) AAnalyst 600 and (bottom panel) PinAAcle 900Z. All
values are plotted against values obtained using an established ICP-MS method
[15]. For comparison purposes, the solid line represents y = x and the dashed
lines represents ±2 μg/dL.
comparison purposed, the solid line represents y = x and the dashed
lines represent the limits ±2 μg/dL.
The caprine and human SRMs and PT materials align with the human
samples within acceptable criteria on both GFAAS platforms. This sug­
gests the caprine blood materials are commutable to human blood ma­
terials. The patient samples were limited to a small range with the
highest concentration at 22 μg/dL. Therefore, the SRM material above
the highest patient sample could be commutable, but with less confi­
dence than those below the highest human sample.
4. Conclusion
Throughout the past 5 decades, extensive efforts have been suc­
cessful in reducing Pb exposures to further help lower population blood
Pb levels. Even though blood Pb levels have fallen dramatically, Pb still
remains a significant environmental risk to young children. A new
reference value of 3.5 μg/dL was adopted by the CDC in 2021, based on
the 97.5th percentile of the distribution of US children’s blood Pb levels.
The GFAAS method developed in the 1993 for a blood Pb action level at
10 μg/dL has now been improved to ensure it is fit for purpose for
current population blood Pb levels.
We explored various approaches to achieving a lower LOD, and
therefore a lower LOQ to support blood Pb measurements below 3.5 μg/
NIST 955d NYS PT
Fig. 4. Analysis of 30 human blood samples (black circles) and for Pb using the
proposed GFAAS method improvements on the (top panel) AAnalyst 600 and
(bottom panel) PinAAcle 900Z. Caprine and human SRMs and PT materials are
represented by different shapes indicated in the legend. All values are plotted
against target or certified values established by ICP-MS. The solid line y = x is
shown for comparison purposes and the dashed lines represent ±2 μg/dL.
dL. Newer instrumentation, increased sample injection volume and
different calibration strategies were all combined to improve the
method. The improved GFAAS method achieved a detection limit of 0.2
μg/dL, that can quantitate blood Pb levels at 1 μg/dL. This provides
justification of an GFAAS method that could feasibly operate with a
lower blood Pb reference value, and improved accuracy and precision of
measurements in the 1–5 μg/dL concentration range. Additionally,
analysis of all SRMs, PT materials, and human samples were all well
within ±2 μg/dL of the values established by ICP-MS instrumentation.
Therefore, the improved GFAAS method could also operate within
narrower PT acceptable limits of ±2 μg/dL, compared to the current US
federal limit of ±4 μg/dL.
As reliability of blood Pb measurements improve, fewer false posi­
tives and false negatives of patient samples are an added benefit. This
could ease the burden of reporting patient blood Pb results, and benefit
public health officials who initiate expensive environmental in­
vestigations at higher blood Pb levels. The improved method could
benefit hospitals and smaller laboratories in terms of keeping pace with

6
E.J. Pacer et al.
the goals of detecting and quantifying lower blood Pb levels using
GFAAS instrumentation, especially if an ICP-MS instrument is not
available. The method improvements discussed provide evidence that
mature techniques, such as GFAAS, can still provide results that are fit
for purpose given the current population blood Pb levels.
Funding
This work was supported in part by funding provided by the Centers
for Disease Control and Prevention (5 NU88EH001329-03-00) and by
the Wadsworth Center, New York State Department of Health.
Author statement
This statement certifies that all authors have seen and approved the
final version of the manuscript being submitted. The authors warrant
that the article is the authors’ original work, hasn’t received prior
publication and isn’t under consideration for publication elsewhere.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This paper is dedicated to the memory of Walter Slavin (1927–2021),
a former editor of Spectrochimica Acta, Part B Atomic Spectroscopy, and a
leader in the design and development of atomic absorption spectrome­
ters. Walter was a mentor to many of us, and his numerous contributions
to graphite furnace atomic absorption spectrometry included the
determination of lead in blood.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.sab.2021.106324.
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