Sampling, preparation

and analysis of Heavy Metal in

Foods

Wee Siew Moi

Chemical Contaminants Expert
Nestlé Quality Assurance Center, Asia Oceania Africa

CII, 11th Food Safety & Quality Summit 6-7 December 2016
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

2
Heavy metals (Toxic metals)
Metal are elements that are typically hard, opaque, shiny, and has good electrical and
thermal conductivity. Metals are generally malleable, as well as fusible.
About 91 of the 118 elements in the periodic table are metals (some elements appear in
both metallic and non-metallic forms).

In food/feed, metals are usually

found in ionic or covalent form (not
metallic).

The species (oxididation degree,

inorganic versus organic) may
impact the toxicity, e.g.
 Cr (III) versus Cr (VI)
 As inorganic versus As organic
Toxic metals - food point of view

Metals can be

 Essential macronutrients (Ca, Mg, K, Na)

 Essential micronutrients (Cr, Co, Cu, Ni, Se)

 Toxic metals

But some essential metals may become toxic!

«Heavy metals» is often used to describe toxic metals.

Strictly speaking it should be used only for element > 200Da.
In practice it refers to metals with high gravity and which have high attraction for
biological tissues.
Heavy Metal Analysis Critical Points
• Avoid contamination when
Sampling and preparing a test portion
Sample • Obtain homogenized
Preparation representative test sample

• Use the appropriate digestion

program according to the type and
amount of sample.
• Avoid contamination of acids and dust
Digestion • Carry out blank test in parallel by the
Heavy same procedure
Metals
Analysis • Check if digestion is complete
Preparation of • Prepare the test solution with similar
Test solutions HNO3, HCl, Au and IPA concentrations as
those of calibrants depending on ICP-MS
technologies.

Determination • ICP-MS instrument set-up

Calibration • Check ICP-MS performance

5
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

6
 B. Types of Equipment (Atomic Spectroscopy)
Graphite
Flame AAS
Furnace AAS

AAS instruments can be flame only, furnace only, or combined (switchable)

ICP-OES ICP-MS

Photos taken from Agilent Technologies as illustration purposes

7
 Decision criterions in what to choose

8
 Decision criterions in what to choose
Number of Analytes vs Detection Limits

9
ICP-MS versus Atomic Absorption Spectroscopy(AAS)
For higher sample throughput

Figure 2 shows that ICP-MS is selected as the technique) than Flame

(FAA)
or Graphite Furnace (GFAA), and (Hydride Generation) Inductively
Coupled Plasma–Optical Emission Spectroscopy ((HG)-ICP-OES).
- Allow samples with varying analyte concentrations to be
analyzed together
due to its wide analytical working range (9 orders of magnitude):

Combination of wide analytical working range and excellent

sensitivity:
 Provides short ICP-MS analysis times.
 Can reduce sample-handling requirements.
 Minimizes potential analytical errors.
 Avoids to frequently recalibrate the system of choice to:
- Get better instrumental detection limits in solution (ng/L

Major limitations to use ICP-MS equipment is the high initial

investment and cost of consumable supplies / Gases / Power
compared to other atomic spectroscopic techniques

Figure 2. ICP-MS detection limit ranges and orders of magnitude of

signal intensity compared to other atomic spectroscopic techniques

10
What is ICP-MS? Determination
ICP-MS stands for Inductively Coupled Plasma Mass Spectrometry.
ICP-MS Instrument comprises five basic analytical parts as shown in Figure 1:
Sample introduction generating an aerosol of the liquid (or solid) sample
Plasma source ionizing the aerosol
Sampling interface extracting ions from ICP
Ion optics and mass spectrometer focusing and separating ions
Ion detector converting ions into an electronic signal processed by
the data handling system

Figure 1- Analytical parts of ICP-MS instrumentation

11
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

12
C. GLP for sample preparation
Sampling and
Sample
Preparation

Food and beverage samples should be stored in their typical commercial

storage conditions (either frozen, refrigerated, or at room temperature)
until analysis. Samples should be analyzed within 6 months of
preparation.

If food or beverage samples are subsampled from their original storage

containers, ensure that containers are free from contamination for the
elements of concern.

Well-homogenized samples and small reproducible aliquots help

minimize interferences. Particle size and distribution should be
normalized using blenders, grinders and mixers.

13
C. GLP for sample preparation
Sampling and
Sample
Preparation

• Management of analytical blanks (Cleanliness of laboratory environment)

During sample preparations for trace element analysis by ICP-MS, the main requirement
is the management of analytical blanks.

Analytical blank s the measure of all external sources of elemental contamination and is used
to make a correction to the measured sample correction.

Contamination for trace analysis can occur from the:

• Laboratory environment (clean air facilities, air filters and flow, hoods and bench tops)
• Materials (plastic ware for storage and handling)
• Reagents (standard solutions, water, acids, hydrogen peroxide, TMAH as alkaline buffer,
enzymes, isopropanol as carbon buffer)
• Apparatus (auto-sampler, plastic ware, nebulizer, spray chamber and torch, peristaltic,
transfer and drain tubings, sampler and skimmer cones and lens)
• Analyst (bare hand, cosmetics, hair, wearing jewels and watches)

14
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

15
D. Digestion techniques Digestion

For accurate decomposition (destruction of organic matter) of food sample.

Why decomposition is required?
• Conversion from solids into liquids Microwave
• Destruction of matrix Digestion
• Separation of interfering substances
• Isoformation of sample and standard
• Homogenization
High
• Preconcentration of analytes
Pressure
Pros: Cons: Asher

Leads to a representative sample Labor intensive cost factor

Reduces problems in the Bottle neck in analytical process
measurement step Risk of contamination or losses of
Easy to standardize analyte

16
D. Digestion techniques Digestion

Decomposition may be safe

1. Analytically accurate
2. Economically efficient
3. Safe and easy to perform Safety
Economically efficient Low amounts of
Analytical accurate hazardous chemicals
Low consumption of
No contamination chemicals Simple handling
No loss of elements Ease of handling Spontaneous reactions
Complete decomposition Low investment/operating Reduce operator error
costs
Reliable equipment Instrument safety
Automation (design/Manufacture)

Reproducible Reduce cost of faulty Safety First

analytical results analytical results

17
 Preparation of sample solution Preparation
of Test
solutions
Reagent Blank The blank test must be carried out in parallel with the
determination by the same procedure but omitting the test
portion
Preparation of digested Check if the digestion of test portion is complete. Prepare the
samples test solution by diluting the digested solution with ultrapure
water to a known volume.
After diluting to volume, the test solution should be clear and
colorless to slightly yellow.
Turbidity and/or a deep color usually indicate an incomplete
digestion
Reagents High-purity reagents should always be used. Each reagent
lot should be tested and certified to be low in the elements of
interest before use.
Standard (Stock/Internal) Elements must be compatible and stable in solutions
together. Concentrations need to be verified before use.
For analysis of As, Cd, Pb, and Hg in food matrices, internal
15.09.2013

standard solution of rhodium (Rh), indium (In), and thulium

(Tm) is recommended
Calibration standard Fresh calibration standards should be prepared every day, or
as needed.
18
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

19
 ICP-MS Instrument Setup/Optimization Determination

(1) Instrument startup

Tune ICP-MS instrument using suitable optimizing / tuning solutions and
according to manufacturer’s instructions
Note : Tuning of the ICP-MS instrument (i.e. optimization for best performance)
usually while aspirating a prescribed element solution called tuning solution is
started using default or recommended settings by manufacturer for plasma power,
sampling depth, gas flow rates and sample introduction rate (plasma gas flow rate
is set at a fixed value and not optimized at all). Ensure that ICPMS passes all
check criteria of tuning after stabilization of plasma during 30 min.

(2) Optimization
Check mass resolution, mass calibration, sensitivity and stability of the system
- Adjust ICP-MS instrument daily with an optimizing solution to achieve
maximum ion signals and both low oxide rates (e.g. < 2 %) and low rates of
doubly charged ions (e.g. < 2 %).
Note: The optimizing solution should contain elements that cover the whole mass
15.09.2013

range giving a high rate of oxides and doubly charged ions. The solutions
recommended by the manufacturer of the ICP-MS instrument may be used.

20
 Calibration Calibration

A set of at least seven different calibration solutions including blank solution

(i.e. zero member compensation) with evenly spaced concentrations must be
used for external standard calibration so that the concentration range should
be chosen with respect to the concentrations expected in samples and with
respect to the linear dynamic range. It is important that the concentration of
acids and IPA in the calibration solutions and in the sample solutions are the
same.
 Prepare calibration solutions with similar HNO3 ,IPA, HCl (or Au) concentrations
as those of sample digests depending on ICP-MS / digestion system.

 R2 must be > 0.995 using recommended weighted least squares

regression
15.09.2013

21
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

22
 Quality
Quality Control Control
• The correlation coefficients of the weighted-linear calibration curves
for each element must be ≥0.995 to proceed with sample analysis.
• The percent recovery of the ICV standard should be 90-110% for each
element being determined.
• Perform instrument rinses after any samples suspected to be high in
metals, and before any method blanks, to ensure baseline sensitivity
has been achieved.
• Run rinses between all samples in the batch to ensure a consistent
sampling method.
• Each analytical or digestion batch must have at least three
preparation (or method) blanks associated with it if method blank
correction is to be performed. The blanks are treated the same as the
samples and must go through all of the preparative steps. If method
blank correction is being used, all of the samples in the batch should
be corrected using the mean concentration of these blanks.
15.09.2013

QC samples (certified, P-test, in-house reference samples or spiked

samples) must be regularly included and analysed in duplicate.

23
 Summary of quality control samples Quality
(Ref: AOAC Official Method 2015.01 Heavy Metals in Food) Control
15.09.2013

24
 Summary of quality control samples Quality
(Ref: AOAC Official Method 2015.01 Heavy Metals in Food) Control
15.09.2013

25
Outline of lecture
A. Heavy (Toxic) metals analysis
B. Types of Equipment (Décision criterions)
C. Good Laboratory Practice (GLP) for sample preparation
D. Sample preparation
- digestion techniques/preparation of test solutions
E. Détermination
F . Quality Control
G. Method Performance

26
Method Performance (Ref: AOAC Official Method 2015.01 Heavy Metals in Food)
Performance Definition
Characteristics
Linearity A coefficient of determination R2 ≥ 0.995 should be generally obtained for ten
standards using weighted linear regression

Limit of Detection (LoD)
Limit of Quantification (LoQ)
Limit of detection (LOD) and LOQ were determined through the analysis
method blanks. LOD was calculated as 3 times the SD of the results of the
blanks, and LOQ was calculated as 2 times the value of the LOD, except
where the resulting LOQ would be less than the lowest calibration point, in
which case LOQ was elevated and set at the lowest calibration point and LOD
was calculated as 1/3 of the LOQ. All LOQs achieved are ≤10 μg/kg for all
food matrices and ≤8 μg/kg for liquid matrices, such as infant formula.

Repeatability The absolute difference between two independent single test results obtained
using the same method on identical test material in the same laboratory by the
same operator using the same equipment within a short interval of time and
calculated as should not be greater than 20 % (25 % for values close to
PLOQ) which corresponds to the repeatability limit, r, at 95 % confidence
level.

Intermediate reproducibility The absolute difference between two independent single test results obtained
using the same method, on identical test material by different operators using
different equipments at different days for intermediate reproducibility test and
calculated as should not be greater than 35 % (40 % for values close to
PLOQ) which corresponds to the reproducibility limit, iR, at 95 % confidence
level.
27
Method Performance (Ref: AOAC Official Method 2015.01 Heavy Metals in Food)
Sample-specific LOQs for several matrices, based on LOQs determined by
the default method, and adjusted for changes in sample mass for particular
samples, are shown. Values have been rounded up to the nearest
part-per-billion.
Sample-specific LOQs
Sample LOQ, µg/kg As Cd Pb Hg
Infant Formula 2 1 4 3
Chocolate 4 2 8 6
Rice Flour 4 2 8 6
Fruit juice 1 1 2 2

28
In summary Critical Points
• Avoid contamination when
Sampling and preparing a test portion
Sample • Obtain homogenized
Preparation representative test sample

• Use the appropriate digestion

program according to the type and
amount of sample.
• Avoid contamination of acids and dust
Digestion • Carry out blank test in parallel by the
Heavy same procedure
Metals
Analysis • Check if digestion is complete
Preparation of • Prepare the test solution with similar
Test solutions HNO3, HCl, Au and IPA concentrations as
those of calibrants depending on ICP-MS
technologies.

Determination • ICP-MS instrument set-up

Calibration • Check ICP-MS performance

29
 Thank you for your attention

30
31