LABORATORY ANALYSIS TECHNIQUES

FOR LEAD IN BLOOD

EXPERIENCE FROM
GCLA
Dar es Salaam, Tanzania

Prof. S.V. Manyele

Chief Government Chemist
Tanzania
Government Chemist Laboratory Agency,
Government Chemist Laboratory Agency, Dar es Salaam, Tanzania
Tanzania

A: Available Methods for Determining Lead in Blood

Atomic absorption spectrometry (AAS)
Flame AAS
Graphite Furnace AAS

Anodic stripping voltammetry (ASV)

Inductively coupled plasma OES TJH3
Detection to ppb level

Inductively coupled plasma mass spectrometry (ICPMS)

The coupled MS makes it very expensive
Can quantify trace metals at ppt level

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Slide 2
TJH3

please spell out

TEMPOWSKI, Joanna Helena, 12/08/2014

Anodic Stripping Voltameter .

Anodic Stripping Voltameter is useful for detecting
metal ions at the ppb level.
The pre-concentration step is key for achieving
these low detection limits.
When this pre-concentration step utilizes
electrochemical accumulation, the metal must be
reduced and form an amalgam in the mercury (drop
or film).
In a mercury electrode, the concentration of metals
is 100-1000 times that in solution.
It is, however, not widely used in Laboratories
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B: Choice Methods is determined by:

1. Analytical capacities (e.g. limits of
detection, accuracy),
2. Costs (e.g., purchase and maintenance
costs, laboratory infrastructure
required, reagents and supplies)
3. Technical requirements (e.g., sample
preparation, calibration, skilled
personnel)
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Required Laboratory Infrastructure

Requirements

ICP-OES

FAAS

GFAAS

1. Metal standards

A prerequisite

A prerequisite

A prerequisite

2. Gas

Argon/Nitrogen

3. Light source

Torch plasma

4. Cooling system

Air conditioned
room

5. Extractor

Fume hood
extractor

Flame type
Air/Acetylene
Nitrogen
Graphite furnace
Argon/Nitrogen
Cathode lamp for all Cathode lamp for all
elements
elements
Air conditioned room
Air conditioned
room

Fume hood extractor Fume hood extractor

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Resources required
S/No AAS
1

EOS -ICP

1. Acetylene gases and

1. Nitrogen gas and
Nitrogen white sport.
Argon gas.
2. Metal reference
2. Metal reference
standards
standards
3. Element Cathode lamp.

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Table 1. Overview of analytical methods for blood lead measurement

Key
Characteristics
Detection limit

ICP-MS

FAAS

GFAAS

Excellent: ~0.1 g/dl

Low: ~10 g/dl

Good: <1-2 g/dl

Good: ppb level

Sample size

Small: 50-100 l

Small: 50-100 l

Small: ?

5-10 ml

Spectral
interference
Lead atomization
temperatures

Relatively Few,
understood
High temperature
ionized gas

Relatively Few

Few

20003000C

Great potential,
more than FAAS
up to 3000C

Purchase &
Running costs
Multielement
capacity

High

Low

Moderate

Moderate

Multielement

??

Some
multielement

Analysis time

Very rapid

Longer

Operator skills
required

Very Highly skilled

Rapid
(without autosampler, 1
sample per run)
Moderate skills,
relative
simplicity of use

Multielement
(as many at
once)
Rapid (30 sec)
(auto-sampler
present)

High skills

ICP-OES

6000C for all

metals

Very high skills

Laboratory Agency,
Economics: Off-setting runningGovernment
costs byChemist
increasing
the number of samples per run
Tanzania

Determination of metal in blood

Sample preparation procedure
AAS

EOS -ICP

Graphite Furnace AAS.

1. 1ml of blood sample preserved
by EDTA or Oxalate
2. Dilute with 9ml of 1% triton X100, (200ppm ammonium dihydrogen phosphate and 750
ppm magnesium Nitrate)
3. Analyse with GFAAS

1. In 1ml of blood sample .

2. add 9ml of 1% aquilegias
solution(1:3, NH3:HCl
respectively)
3. Shaker vigorously and
left to stand.
4. Subject for ICP .

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Strengths and Weaknesses of different methods for lead in

blood
Method
Strengths
Flame atomic
Requires only basic laboratory
absorption
expertise
spectrometry (FAAS) Rapid analysis
Small sample size using Delves cup
(50100 l)
Low purchase and running costs
Relatively few interferences
Robust interface

Limitations
Relatively high detection limit
(~10 g/dl)
Time needed for sample
digestion/preconcentration if not
using Delves cup
Large sample size needed for
nebulization methods
Should not be left to run
unattended

Graphite furnace
atomic absorption
spectrometry
(GFAAS)

Good detection limit (<12 g/dl)

Small sample size
Moderate purchase and running
costs
Some multielement capacity
Relatively few interferences
(although more than with FAAS)
Widely used, available from multiple
vendors

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Longer analysis time

Requires some laboratory
expertise (more than FAAS)
Greater potential spectral
interference than with FAAS

Strengths and Weaknesses ..

Method
Laboratory anodic
stripping voltammetry
(ASV)

Inductively coupled
plasma mass Optical
Emission spectrometry
(ICP-OES )

Inductively coupled
plasma mass
spectrometry (ICP-MS)

Strengths

Limitations

Good detection limit (2-3 g/dl)

Low purchase and running costs
Rapid
Small sample size (~100 l)
Relative simplicity of equipment

Excellent method detection limit (~0.1 g/dl)

Rapid
Small sample size (5ml-10ml)
Relatively few, well-understood, spectral
interferences
Isotopic measurements possible
Economic if very large number of samples
Multielement capability
Excellent method detection limit (~0.1 g/dl)
Rapid
Small sample size (50100 l)
Relatively few, well-understood, spectral
interferences
Isotopic measurements possible
Economic if very large number of samples
Multielement capability
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Requires some laboratory

expertise (similar to GFAAS)
Sample pretreatment needed
Some factors might affect
measurement (e.g. presence
of copper)
Becoming less available
High purchase and running
costs
Highly skilled laboratory
operator required

High purchase and running

costs
Highly skilled laboratory
operator required

10

Spectra interference in ICP-OES and AAS

Spectral interferences are caused by presence of another
atomic absorption line or a molecular absorbance band close
to the spectral line of element of interest.
Most common spectral interferences are due to molecular
emissions from oxides of other elements in the sample.
The main cause of background absorption is presence of
undissociated molecules of matrix that have broad band
absorption spectra and tiny solid particles, unvaporized
solvent droplets or molecular species in the flame which may
scatter light over a wide wavelength region.
When this type of non-specific adsorption overlaps the atomic
absorption of the analyte, background absorption occurs.
The problem is overcome by measuring and subtracting the
background absorption from the total measured absorption to
determine the true atomic absorption.
Deterium lamp can be used in AAS and ICP can substracting
automatically.

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Spectra interference in ICP-OES and AAS

Post analysis

Elimination of
spectra
interference
Matrix modifier
reagents

Temperature
range

ICP-OES
Eliminated by sub-array software
which is used to subtract spectra
outside the normal p.d.f.
Matching of matrices is used to
eliminate interference
Not applicable

6000C, all elements are ashed

completely thus eliminating
interference
Gives both atomization and
ionization, eliminating interference
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AAS
AAS has no post analysis
to eliminate interference
Matching of matrices is
used to eliminate
interference
Allowed to eliminate
interference (using
specialized reagents)
At 2000-2500C, some
elements not ashed
completely leading to
interference
Gives only atomization
leading to interference
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Operational details
Operation

ICP-OES

Stabilization 15 minutes,
time
running with gas

throughout
High running
expense
Auto
Gives reading of
sampling
multielement in
single run
Gas costs in Argon: USD 625
Tanzania
/cyclinder of 50 kg

AAS

Stabilization required
only during lamp
change
Gas runs when needed
One element for each
run for few samples
and few elements
TJH8
USD 45/cyclinder
of 50
kg

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Slide 13
TJH8

$42
TEMPOWSKI, Joanna Helena, 12/08/2014

Experience from GCLA Tanzania

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Collection and submission of blood samples

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Metals analyzed using AAS or ICP

For biological samples: e.g., blood, urine,
Lead and mercury element are analyzed
more than other metals in both GFAAS and
ICP-OES becuase the metals are found in low
level (ppb) or (mg/dl) in blood or urine.
Numbers refers: the frequency of element
analyzed.
The frequency of metals analyzed depends
to the request of customer.
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A: Which Instrument is more Multielemental?

(frequencies)

Number of metals analyzed in

one year for AAS and ICP
Different sample types for ICP
presented
Lead analysis for blood and
other samples compared using
ICP
Numbers indicate requests by
clients for different samples
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Frequency analysis for metals analyzed

(multielemental) between AAS and ICP

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A: Sample Registration

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B: Sample Analysis

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C: Certified Reference Standard Concentration

(CRSC)1000 mg/L

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D: Instrument Description Lead in Blood

using ICP

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D: Instrument Description

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Procedure for determination of lead in blood

Potential Sources of Variability
Blood clots present in the sample may partly
block the sampling tube causing variations
in the sampled volume which causes wrong
results.
Contamination of sample during collection
Contamination during preparation of
sample in a non-dust-free environment, use
of not-acid washed lab-ware and use of not
talk free gloves.
Use of not-ultra pure reagents
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Procedure for determination of lead in blood

Potential Sources of intefrence
Presence of clots in the blood sample may
interference with the measurements.
Whole blood contains a variety of salts and
organic compounds, including phosphates and
alkali compounds.
Phosphates introduce spectral interference at
196.0 nm, which can be corrected using
DEUTERIUM background correction.
The complex chemical matrix of blood will result
in an excessively large background signal upon
atomization.
This allows ashing temperatures of 1300C
without analyte loss, while removing a large part
of the background matrix signal.
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Operator machine-use frequency analysis for testing

Lead in Blood

Names of staff members of GCLA

operating the ICP for testing lead
in blood
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Specimen Collection
Whole blood samples collected
in metal free Vacutainer tubes
containing EDTA
Stored in the refrigerator at <4oC
prior to analysis.
To assure proper mixing, tubes
pre-filled with EDTA, should be
inverted 8 times towards the
stopper before being kept for
further analysis steps.
Allthe tubes should be labeled
with the subject identification
code
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Observed range of values of Pb in blood

NB: N = 86 below detection level BDL

Detection level: 0.01 ppm (or 10 ppb) to saturation

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E: Reports

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 Pb in blood Plasma - ICP

Pb in water - Flame/Plasma (both AAS and
ICP were used)
Pb in cereal Plasma - ICP
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Instrument analysis based on gasmixture flame-types

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Which wavelength is used?

AAS

ICP

Wavelength not changed,

measurement mode is absorbance
Machine selects automatically, then
stabilizes
Machine is sensitive to l towards good
results
In case the frequency is out of range,
there will be no results
Must select wavelength to avoid
interference of metals
Uses triple (1,2, 3)
3 for comparison
of results
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Distribution of metals detection in water samples (Pb

detection only for blood and cereal samples)

101 is total number of blood

samples analysed for the
period July 2013- June 2014

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Distribution of samples analyzed in the ICPOES (2013-2014)

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QUALITY CONTROL MEASURES

1. In-house quality controls samples are prepared
using spiked volumes of working standard resulting
to concentration of analytes ranging from 0.00 to 80.
2. Recovery is computed with reference to known
concentration of diluted standards.
3. Using Micro pipette, One milliliters 1 ml of blood
sample are diluted with 9ml of 1% TritonX-100 (v/v)
in Polystyrene tubes covered with caps and mix
well in vortex mixer.
4. Then Samples and control are poured into auto
sampler vials and ready for analysis
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AAS for Pb measurements

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How is the AAS prepared for analysis?

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Wavelength range for different metal

detection in the AAS

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AAS use by Laboratory Technicians at GCLA

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Working standard concentration distribution

for metals (C)

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AAS Detection levels from reported metal

concentrations

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Thanks

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