Challenges of Ultra-Trace Elemental Analysis by ICP-MS
Richard Zywicki, Ryan Connelly and Darryl Sullivan
Covance Laboratories Inc., Madison, Wisconsin
Abstract
Purity of Chemicals and Reagents
While very low levels of certain metals are believed to provide nutritional benefits, others are
known to be detrimental to humans, animals and the environment. The significance or impact of
an error in quantification of these very low levels is often misunderstood. When the variance in a
result of less than a part per billion can make the difference between meeting a regulatory
requirement or a product’s beneficial nutrient fortification, accurate determination is of the utmost
importance. The ability to accurately quantify ultra-trace concentrations of these elements poses a
number of challenges. In order to ensure accurate data, several important aspects should be
considered including: cleanliness of the laboratory environment, purity of the chemicals and
reagents, sample handling and preparation and instrument maintenance and analysis. When the
issues surrounding these important factors are properly managed, inductively coupled
plasma–mass spectrometry (ICP-MS) can easily provide the sub part per billion detection limits
necessary to produce quality data. This poster provides an overview of each challenge and
includes suggestions to minimize, avoid or overcome these challenges to ensure an accurate
result. Examples will include suggestions for optimization of a technique during method
development and examples of challenges that can occur during routine analysis.
As with the quality of the lab space, the chemicals used for the digestion and dissolution of
samples can also affect their measured metal concentrations. It is important to use the highest
quality reagents in order to minimize these contributions. This is most apparent with purified water
as it makes up the bulk of digested sample (up to 95%). For the lowest backgrounds, ultrapure
water (18.2 MΩ) should be:
▶ Deionized by ion exchange
▶ Filtered by reverse osmosis
▶ Refilled daily
▶ Tested often for capacitance and metal contamination
What is an ultra-trace element? This term first made an appearance in the literature in the early
1980s.1 Estimated dietary requirements for the elements that fall into this category typically are
less than 1 ppm or less than 50 ppb on a dry basis.2 It has been suggested that 21 to 23
elements (aluminum, arsenic, boron, bromine, cadmium, chromium, copper, fluorine, germanium,
iodine, iron, lead, lithium, molybdenum, nickel, rubidium, selenium, silicon, tin, vanadium, zinc and
possibly cobalt and manganese) may fall under this definition.1,3 While some of these elements
are known as essential nutrients in humans (colbalt, copper, iodine, iron, molybdenum, selenium
and zinc)3, others have been proposed as being nutritionally beneficial. Deficiencies of cobalt (as
vitamin B12), iodine, iron and zinc in humans raise concerns with health care professionals.3
Certain elements (e.g. selenium), while shown to be nutritionally important at appropriate levels3,
become toxic at elevated levels.4 Due to the essential nature of some and toxicity of others,
accurate quantification of each of these elements at ultra-trace levels is paramount. This poster
will expound several unique challenges surrounding ultra-trace elemental analysis by ICP-MS.
Cleanliness of the Laboratory Environment
The cleanliness of the general lab environment and instrument rooms is of great importance to
ICP-MS analysis, more so than most other analyses. Due to the to low level of analyte
concentrations, the quality of the workspace will directly impact the accuracy of data generated.
Some ways to minimize airborne and laboratory contamination are as follows:
▶ Work in a “clean” room with a HEPA filter to eliminate particles greater than 0.3 μm
▶ Use a laminar flow fume hood for sample handling to minimize risk of contamination
▶ Use plastic weighing utensils and vessels as well as Teflon® digestion vessels
▶ Store purified water in polypropylene (PP) containers
Even with a clean room environment, accurate quantification of certain elements, such as iron
and zinc, is still very easily compromised by water sources and latent chemical interactions. By
monitoring zinc blank concentrations of nitric acid solutions contained within sealed plastic tubes,
it was determined that zinc was being leached from their walls. In order to determine the extent of
this leaching, data was collected to investigate the relationship between concentration with
respect to time using varying acid concentrations (Table 1). This lead to several conclusions:
1. A significant amount of zinc can leach out of plasticware under acidic conditions
2. The amount of leachable zinc can vary between tubes
3. This zinc can be leached quickly (within approximately 24 hours) with 10% nitric acid
Not only will leached zinc negatively affect results but it makes blank correction impossible due to
inconsistency. Therefore, to guarantee accurate low level zinc determination, leaching
experiments must be performed to ascertain the possibility of contamination. Afterward a leaching
process should be implemented to ensure zinc-free vessels are available for use.
0 Hours
(ppb)
24 Hours
(ppb)
96 Hours
(ppb)
Purified water
<PLOQ
<PLOQ
<PLOQ
3% HNO3 in purified water
<PLOQ
0.54
0.67
5% HNO3 in purified water
<PLOQ
0.10
0.16
10% HNO3 in purified water
<PLOQ
0.45
0.50
6% HNO3/2% HCl in purified water
<PLOQ
0.40
0.44
Note: All data was generated using a PerkinElmer ELAN® DRC II ICP-MS in standard mode.
PLOQ = Practical limit of quantitation.
Presented at AOAC INTERNATIONAL 2014
▶ The vacuum and cooling systems’ oil/fluid must be changed
▶ Replace air filters regularly
Optimization of instrument parameters involves adjusting gas flows, the lens system, mass
spectrometer and detector electronics. These procedures have several broad purposes:
1. To optimize ion transmission across a large mass range while minimizing interfering species
and noise
2. To accurately select for mass with the desired resolution
Table 2. Replicate Chromium Analysis in Infant
Formula Using Reagents Differing in Purity
3. To ensure linearity in signal response across several orders of magnitude
Infant formula Cr content
(high purity H2O2)
Digest blank Cr content
(high purity H2O2)
Infant formula Cr content
(low purity H2O2)
Digest blank Cr content
(low purity H2O2)
207 ppb
0.029 ppb
233 ppb
0.127 ppb
Note: All data was generated using a Thermo iCAP Qc ICP-MS (Figure 1) in collision mode using He.
It is also very important to match matrix additions across solutions. The calibration standard
matrix should be matched as closely as possible to the final sample solution’s matrix. There are
several reasons to do so:
Special attention should also be given to the sample introduction system. The valve especially is
easily contaminated. Prior to operation:
▶ Flush tubing/valves with purified water
▶ Rinse stations should be flushed with an application-specific solution
▶ Analyze several post-analysis blank solutions
▶ Flush all tubing/valves with purified water and prepare for next analysis
▶ If contamination persists, a thorough cleaning may be required
To perform a specific analysis, the instrument must be set
up to accommodate it. Several steps should be taken to
ensure consistently accurate results:
▶ Ensures most similar analyte response
▶ Background contribution remains consistent across solutions
▶ Different matrices solubilize metals at different rates (e.g. Sn, Ag)
▶ Matrix components can enhance signal artificially (e.g. Se, S)
▶ Harsh matrix can suppress signal greatly or alter ion beam characteristics
1. Calibrate and monitor autosampler movements to
prevent misinjections
2. Condition the sample flow path to
analytical conditions
3. Condition the collision/reaction cell with
appropriate gas
Sample Handling and Preparation
Once a sample is received, any special initial preparation instructions should be followed as
requested by the client. These may include:
▶ Number of packages to be used for compositing/homogenizing
▶ How to prepare (grind/blend) individual packages or units for content uniformity testing
(e.g. single serving of a finished product, single supplement tablets, capsules, caplets)
▶ For nutritional supplements, combine 30 tablets (20 minimum) in order to ensure an adequate
representation
The next phase after sample collection is ensuring thorough and complete homogenization.
Several considerations for sample processing should be taken into account:
1. Particle size and distribution should be normalized using blenders, grinders and mixers
2. Contamination from homogenization apparatus should be assessed
3. Avoid excessive heat during grinding and/or use liquid nitrogen to prevent clumping
▶ Wear powder-free gloves to protect the analyst and to avoid contaminating the sample
▶ Thoroughly shake or mix sample container on an undulating roller
▶ Use disposable weighing utensils
▶ Digest by appropriate method
▶ Closed vessel microwave digestion with Teflon® vessels is preferable for many samples due to
low probability of contamination
▶ Wet or dry ash techniques may be required for difficult samples. These use porcelain, Vycor® or
quartz crucibles which are more prone to contamination
▶ Transfer digested material to single use Class A PP vessels with 18.2 MΩ water
It is important to note that a sample’s homogeneity is not always easy to ascertain. Evidence of a
homogeneity problem may not reveal itself until analysis is finished. This could be caused by poor
sampling procedure, source of fortification material (e.g. Cr salts) or striation of metal ions. In
these situations, representative samples can be taken using these techniques:
▶ Oscillating mixer
▶ Finer grinding
▶ Reconstitution in purified water
▶ Acidifiy and apply heat to aid in solvation
Table 3. Analysis of Selenium - Calibration Accuracy
Using Different Conditioning
Measured concentration –
Cell conditioned
only (ppb)
Percent
recovery
(%)
Measured concentration –
Cell and sample path
conditioned (ppb)
Percent
recovery
(%)
2.00 (Calibration Verification)
2.05
0.00 (Undigested Blank)
0.023
103
2.00
100
NA
0.001
12.0 test solution
NA
12.4
103
12.0
100
4.00 test solution
4.13
103
4.00
100
0.600 test solution
0.642
107
0.605
101
0.200 test solution
0.234
117
0.206
103
2.00 (Calibration Verification)
2.06
103
1.98
99.0
0.00 (Undigested Blank)
0.027
NA
0.001
NA
Test Solution
Concentration (ppb)
NA = not applicable.
Note: All data was generated using a Thermo iCAP Qc ICP-MS (Figure 1) in collision mode using H2.
Conclusion
It should be noted that constant assessment of irregularities and sources of contamination is an
important part ultra-trace elemental analysis by ICP-MS. No matter how well an instrument may
perform, all of the sample handling and preparation up to and including digestion play a
significant role in the accuracy of the results. Each analysis presents different problems that can
be overcome by adhering to several guidelines:
▶ Maintain the quality of the laboratory environment and instrument
▶ Use highest purity of chemicals and reagents
▶ Sample handling and instrument setup procedures should be tailored to the application
▶ Irregularities should be studied to aid in troubleshooting
▶ Perform root cause analysis to implement solutions and improve quality of results
4. Set up method and autosampler locations
In addition to the cleanliness of the laboratory environment, it is imperative to keep in mind that
any surface a sample comes in contact with is a potential source of contamination so it is
extremely important to minimize contact. The laboratory should define processes for obtaining
appropriate sample amounts to represent the entire batch, ensuring the integrity of original
containers and creating a representative composite of material.
The final phase in preparing a sample for analysis is the digestion. During this process:
Solution Composition
▶ Position of torch in relation to the RF coil and interface should be optimized
▶ The lens system must be cleaned/maintained (if applicable)
4. Inspect particle size under magnification
Table 1. Changes in Zinc Concentration in
PP Containers with Respect to Time
In order to benefit from the sampling procedures, the ICP-MS instrument’s performance must be
preserved. This is accomplished through regular instrument maintenance and optimization of
instrument parameters prior to analysis. Below are some of the most common subsystem
maintenance procedures prioritized in the order by which they should be performed*:
incompatible solvents react unfavorably. Mixing acidic and basic solvents within the valve will
create pools of liquid that compromises the sample flow path. These reservoirs solvate metals to
create a source of fluctuating background noise. This will make low level determination of many
transition metals difficult, if not impossible, requiring the valve to be cleaned. In many cases,
valve maintenance can be avoided by preventing these types of interactions with adequate
rinsing between analyses.
▶ Interface cones should be cleaned and tubing replaced as needed based on the requirements
of the instrument
In addition to water purity, the purity of digestion reagents can alter sample concentrations as
well. Replicate chromium analyses of infant formula differing only in the purity of H2O2 added are
presented (Table 2). These results are significantly different (11.8%). Based on their respective
undigested blank concentrations, this bias can be attributed to the H2O2 reagent and corrected.
Using the dilution/weight scheme (dilution factor of 222), a blank-corrected value of 205 ppb is
ascertained, yielding a difference of 0.5%.
Introduction
Instrument Maintenance and Analysis
5. Monitor run for irregularities
* Maintenance requirements differ depending on
make/model of ICP-MS
Acknowledgements
The authors wish to thank Marni Peterson of Covance and Stephanie Trimble for their significant
contributions to the content and presentation of this poster.
Figure 1. Thermo iCAP™ Q ICP-MS.
Discussion
Ultra-trace analysis is inherently difficult in nature and presents many challenges. This poster
attempts to summarize these challenges but due to the limited space available, many aspects
important to successful analysis were only touched upon or omitted entirely. Some applicationspecific examples will now be discussed in greater detail to illuminate possible solutions.
The nature and amount of conditioning required for an application can be difficult to ascertain.
These will change depending on the parameters of the method, analytes of interest, interferences
present, etc. This is apparent in the case of selenium analysis in which the main interference is
caused by the argon dimer (ArAr+). This interference is commonly mitigated with either a reactive
gas (CH4) or a collision gas (H2) in order to achieve adequate sensitivity. Data comparing the
calibration accuracy in two separate selenium analyses is displayed (Table 3). Two different
conditioning methods were employed:
1) The collision cell was conditioned with H2 for 30 minutes while blank matrix flowed through the
sample path.
2) The collision cell was conditioned with H2 for 30 minutes while sample matrix was injected.
There is a striking difference in the accuracy of the two curves, however, both yield acceptable
calibration verification and blank results (based on a low standard of 0.2 ppb). This is because the
verification solution does not fall on the affected part of the curve. This is most notable near the
limit of detection where the percent recovery increases dramatically in the 0.2 and 0.6 ppb test
solutions. When the sample path is conditioned as well, results are dramatically improved across
the entire calibration curve. A likely cause can be assigned to this phenomenon: the cell’s charge
transfer efficiency decreases as sample matrix is introduced to the cell. As efficiency decreases,
less ArAr+ will be removed by the cell and selenium response will be falsely enhanced.
In addition to tailoring instrument setup to the analysis, special care should be taken to prevent
adverse interactions between applications. While sampling valves allow for rapid analysis of liquid
samples, they can be easily compromised, especially in labs with varied analyses. While this
could be caused by samples with high metal content, it more commonly occurs when two
References
1) Nielsen, F. H., “Ultratrace Elements in Nutrition: Current Knowledge and Speculation,”
Annual Review of Nutrition Vol. 4: 21-41 (Volume publication date July 1984).
2) Nielsen, F. H., “Ultratrace Elements in Nutrition,” Annual Review of Nutrition Vol. 4: 21-41 (Volume
publication date July 1984).
3) Nielsen, F. H., “Ultratrace Elements of Possible Importance for Human Health: An Update,” In:
Essential and Toxic Trace Elements in Human Health and Disease: An Update, Wiley-Liss, Inc.,
355-376 (1993).
4) Sullivan, D., Zywicki, R., Yancey, M., “Method for the Determination of Total Selenium in a Wide Variety
of Foods Using Inductively Coupled Plasma/Mass Spectrometry,” Journal of the AOAC
INTERNATIONAL, 96 (4): 786-794 (2013).
5) Hua, T., “Available Options for the Determination of Ultra Trace Elements in Pristine Environments,”
ALS Laboratory Group, Environmental Division, Burnaby, B.C. (2009).
6) Namieśnik, J., “Trace Analysis – Challenges and Problems,” Critical Reviews in Analytical Chemistry,
32(4):271-300 (2002).
7) Methods of Analysis of AOAC INTERNATIONAL (2012) 19th Ed., AOAC INTERNATIONAL,
Gaithersburg, MD, USA Method 2012.15.
8) Sullivan, D., Zywicki, R., “Determination of Total Iodine in Foods and Dietary Supplements Using
Inductively Coupled Plasma-Mass Spectrometry,” Journal of the AOAC INTERNATIONAL, 95 (1):195202 (2012).
9) Ellingson, D., Zywicki, R., Sullivan, D., “Analytical Method for the Determination of Various Arsenic
Species in Rice, Rice Food Products, Apple Juice and Other Juices by IC-ICP-MS,” Journal of the
AOAC INTERNATIONAL, In press, (2014).