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A high-throughput method for Saccharomyces cerevisiae (yeast) ionomics†
John M. C. Danku,
a
Luke Gumaelius,
b
Ivan Baxter
b
and David E. Salt*
ab
Received 29th February 2008, Accepted 22nd August 2008
First published as an Advance Article on the web 21st October 2008
DOI: 10.1039/b803529f
Reliable and rapid analytical methods are the backbone for generating data and deciphering gene
functions in the post-genomics era. We describe here a high-throughput method for the rapid profiling
of fourteen elements in the 5153 strain gene deletion collection of Saccharomyces cerevisiae. Samples
were grown and processed in standard 96-well plate format followed by inductively coupled plasma
mass spectrometry (ICP-MS) analysis. Optical densities of the yeast were measured prior to ICP-MS
analysis and used for the normalization of the data. The elemental profiling data are stored in an online
database for later bioinformatics analysis. This method has the capacity to run 288 yeast samples per
day on a single ICP-MS, and has allowed the quantification of the ionome in four replicate cultures of
approximately 240 yeast deletion strains per week, along with appropriate wild-type and positive
control strains. We identified 400 strains that were outliers from the overall deletion collection in at
least one element out of the fourteen that were monitored.
Introduction
Ionomics is the ‘‘quantitative and simultaneous measurement of
the elemental composition of living organisms, and changes in
this composition in response to physiological stimuli, develop-
mental state and genetic modification’’.
1
The ionome is an
expansion on the previous concept of the ‘‘metallome’’
2,3
to
include metalloids and non-metals.
4
Since the ionome is involved
in a host of biologically important phenomena such as osmo-
regulation, transport, signalling, enzymology and electrophysi-
ology,
5
an understanding of its genetic basis, and how it interacts
with other cellular systems is paramount. This in turn is predi-
cated on developing reliable and rapid analytical tools capable of
handling the large numbers of biological samples required for
such an analysis.
A recent study characterized the ionome of 4385 Saccharo-
myces cerevisiae (yeast) mutants strains (homozygous diploid
collection) which represent a subset of the complete yeast
knockout collection including only non-essential genes.
6
However, given the limited sample throughput of the method
used by Eide and co-workers only single cultures of the majority
of the 4385 yeast mutants were analyzed. Such a limited sampling
density for each mutant reduces significantly the resolution of the
yeast ionome study published by Eide and co-workers.
6
To
improve both the sampling density, and to complete analysis of
the full collection of yeast knockouts (5153 strains, MATa
haploid collection
7,8
) it is essential to streamline and optimize the
ionomic method, to allow the efficient processing of significantly
more samples without the loss of analytical precision.
We describe here a high-throughput elemental profiling
methodology for the analysis of the yeast ionome that employs
a 96-well plate format integrated into the process from sample
growth through to the final ICP-MS analysis. This method has
the capacity to analyze 288 yeast cultures per day on a single
ICP-MS. Application of this methodology has allowed the
analysis of the complete yeast gene deletion collection of 5153
strains with four replicate cultures per mutant strain. To enhance
downstream data normalization each 96-well plate also con-
tained the same set of four different control lines. Analysis of the
complete knockout collection took 6 months and represents the
processing of approximately 30 000 yeast cultures. This repre-
sents a 10-fold increase in throughput over the previous study
6
while maintaining a high level of precision and improved
analytical sensitivity. The availability of such a high-throughput
methodology now opens up the possibility of performing other
genome-wide ionomics analyses in yeast, including the screening
of the complete ORF (open reading frame) over expression
collection, and cDNA (complementary DNA) expression
libraries from heterologous genomes and metagenomes.
Experimental section
Instrumentation
A quadrupole inductively coupled plasma mass spectrometry,
ICP-MS, (Elan DRC II, Perkin Elmer, Shelton, CT, USA)
coupled with an SC-2 autosampler and an Apex Q sample
introduction system (Elemental Scientific Inc., Omaha, NE,
USA) was used for analysis of the yeast cultures. A liquid
handling robot, MultiPROBE II PLUS HT EX, (Perkin Elmer)
was used to perform the initial yeast inoculation into 96-well
square deep-well plates. A shaking incubator (Shel Lab SI6,
Sheldon Mfg. Inc., Cornelius, OR, USA), which was custom
fitted to accommodate up to 15 deep-well plates (Metalhead
LLC, Salem, IN, USA), was employed for yeast cultivation.
Yeast optical densities (OD) were measured using an OpsysMR
plate reader (DYNEX Technologies, Chantilly, VA, USA).
a
Center for Plant Environmental Stress Physiology, 1165 Horticulture
Building, Purdue University, West Lafayette, IN 47907, USA. E-mail:
dsalt@purdue.edu
b
Bindley Bioscience Center, Discovery Park, Purdue University, West
Lafayette, IN 47907, USA
† Presented at the 2008 Winter Conference on Plasma Spectrochemistry,
Temecula, CA, USA, January 7–12, 2008.
This journal is ªThe Royal Society of Chemistry 2009 J. Anal. At. Spectrom., 2009, 24, 103–107 | 103
TECHNICAL NOTE www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry
Digestion of yeast samples was performed using a multi-block
heater (Lab Line Instruments, Melrose Park, IL, USA).
Materials
Custom made AcroPrep 96 PVDF (polyvinylidene fluoride) filter
membrane (0.45 mm, 350 mL) micro-well plates (Pall Life
Sciences, Ann Arbor, MI, USA) were used for processing yeast
samples. Clear View micro-plates were used for yeast optical
density measurements. 96-well square deep-well (2 mL) plates
(Axygen Scientific, Union City, CA, USA) were used both for
yeast growth and after processing for ICP-MS analysis (using the
SC-2 autosampler). Polypropylene lids (Axygen) were used to
cover the deep-well plates during sample storage and digestion,
while Axymat (Axygen) chemically resistant and flexible lids
were used to cover the plates during mixing. Adhesive breathable
sealing film (AeroSeal, Dot Scientific Inc., Burton, MI, USA)
was used to seal the deep-well plates during yeast growth. Three
multi-channel pipettors with volume ranges of 2 to 20 mL, 20 to
300 mL and 100 to 1200 mL were used to dispense yeast cultures,
reagents and solutions.
Standards and reagents
AR Select grade concentrated nitric acid from Mallinckrodt
(Phillipsburg, NJ, USA) was used for sample digestion. Single
element standard stock solutions for the calibration procedure
and for spiking yeast growth media were obtained from ULTRA
Scientific (Kingstown, RI, USA). Deionized water (18 MU) for
all dilutions was from a NANOpure Diamond (Barnstead
International, Dubuque, IA, USA) water purifier. Triton X-100
was obtained from Sigma (St. Louis, MO, USA), and was added
to both the processed samples and calibration standards to
enable smooth self aspiration up the micro-nebulizer. Sodium
chloride, sodium ethylenediaminetetraacetate (EDTA), meth-
anol and ethanol were purchased from Mallinckrodt (Phillips-
burg, NJ, USA). Synthetic defined minimal medium components
for yeast culture were from the following vendors: Yeast
Nitrogen Base with nitrogen (YNB) and CSM-Ura (complete
supplement mixture minus uracil), Sunrise Science Products
(obtained from MIDSCI, St. Louis, MO, USA); uracil from BIO
101 (Vista, CA, USA), and D-Glucose monohydrate from
Research Products International (Mt. Prospect, IL, USA).
Sample preparation
The yeast knock-out (YKO) collection used in this work was
from the yeast MATacollection generated from the BY4741
background—MATa his3O1 leu2O0 met15O0 ura3O0.
7,8
Most of the collection was obtained from Dr Tony Hazbun of
Purdue University and the rest purchased from Open Biosystems
(Huntsville, AL, USA). The stock YKO lines came in 96-well
micro-plates and were maintained at 80 C.
Growth of yeast was carried out in two stages. The first stage,
preliminary growth or pre-growth, involved bulking up from the
stock collection; the second, growth for analysis or simply
growth, was for both the ICP-MS analysis and the corresponding
optical density measurement. The yeast culture medium used
was synthetic defined minimal (YNB + CSM Ura + uracil)
medium. The minimal growth medium was supplemented with
the following elements for the second stage of growth: 2 ppb Co,
20 ppb Cd, 50 ppb Mo, 100 ppb Ni and 200 ppm Na. This was to
either compensate for the elements lacking (Co, Cd, Ni) in the
synthetic medium or else to increase the levels (Mo, Na) for
better ICP-MS detection. Note that the same lots or mix of lots,
of medium components were used throughout this work to
ensure consistent growth conditions. In all cases growth was
carried out in 96-well square deep-well plates.
For pre-growth 5 mL of starter yeast stock (inoculate) was
added to 500 mL of medium per well. The plate was sealed with
breathable sealing film and incubated at 30 C and 400 rpm for
48 h. Usually two and a half 96-well plates of the primary yeast
collection were utilized per week for analysis, along with back-
ground, wild-type and positive controls strains. During the
growth stage 20 mL of yeast inoculate was added to 750 mLof
supplemented medium per well. This stage required the use of
a liquid handling robot. A total of twelve 96 deep-well plates
were usually generated from the two and a half bulked pre-
growth yeast plates. Each plate had 20 yeast lines transferred
from a pre-growth plate with four replicates per line. These
covered 10 of the 12 columns of the plate (two lines per column)
with the background and wild-type, and the two positive control
lines occupying the other two columns. The plates were sealed as
before. Three plates per day were grown with the rest kept in the
refrigerator (4 C) and grown on successive days. The incubation
conditions in this case were 30 C and 400 rpm for 36 h. The yeast
cells are at the post-diauxic growth period before harvesting for
both of these growth conditions. The cells grow rather slowly
during this growth phase.
9
Eide and co-workers
6
harvested yeast
cells at a similar growth phase.
Sample processing for ICP-MS analysis, until the final step,
was performed in AcroPrep 96 PVDF filter membrane micro-
plates. The hydrophobic membrane of the plate was wetted with
methanol and then rinsed with deionized water. Yeast cultures
(200 mL well
1
) were transferred from the growth plates into filter
plates using multi-channel pipettes. The same amounts were
concurrently transferred into Clear View microtiter plates and
the optical densities measured with a plate reader. The cells in
the filter plates were washed and rinsed in situ, respectively, with
EDTA (1 mM, pH 8) and deionized water, using a vacuum
manifold. Four separate washes and rinses were performed
(350 mL well
1
each). The filter plates were dried (88 C for 2 h)
to restore the membranes hydrophobicity. Washed yeast cells
were digested directly inside the filter plates (100 mL well
1
nitric
acid, 88 C for 40–45 min) using a heating block. The yeast
digests were drawn through the filter and into 96 deep-well
collection plates containing Triton X-100 (0.025% v/v, 300 mL
well
1
) with Ga (6.67 ppb) internal standard solution. The final
dilution volume was 1.6 mL well
1
including Ga (5 ppb) internal
standard and Triton X-100 (0.005% v/v).
ICP-MS analysis
The processed yeast samples were run on an Elan DRC II ICP-
MS equipped with ESI SC-2 autosampler unit that could
accommodate 96 deep-well plates, and an Apex Q sample
introduction system. Triton X-100 (0.005% v/v) was also added
to the calibration standards as well as the wash solution to reduce
surface tension and enable smoother self aspiration of the PFA
104 | J. Anal. At. Spectrom., 2009, 24, 103–107 This journal is ªThe Royal Society of Chemistry 2009
micro-nebulizer. Fourteen elements (Na, Mg, P, S, K, Ca, Mn,
Fe, Co, Ni, Cu, Zn, Mo, Cd) were monitored in the yeast
samples.
A flowchart representation of the yeast ionomics high-
throughput workflow is shown in Fig. 1.
Results and discussion
The method reported here is a low cost, rapid, robust and
sensitive method for the elemental analysis of yeast, allowing for
the precise analysis of many thousands of samples with the
capacity to resolve small ionomic differences between samples.
Three 96-well plates of yeast cultures can be processed per day
using a single ICP-MS instrument. Currently, we analyze
approximately 240 yeast deletion strains per week, each in
replicates of four cultures, along with background, wild-type and
positive control strains. This translates into a throughput of
about 30 000 samples in 6 months. In comparison Eide and
co-workers
6
analyzed approximately 10 000 samples in 24
months. Fourteen biologically essential or potentially toxic
elements (Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd)
are quantified in the yeast samples, and the elemental profiling
data stored in an online ionomics database, based on the Purdue
Ionomics Information Management System (PIIMS).
10
Boron,
lithium, arsenic and selenium were also monitored during the
preliminary stages but later dropped due to low detection of these
elements in the yeast samples. Even after supplementation of the
culture medium with these elements there was not an appreciable
uptake by the yeast cells. ICP-MS is needed for the method
described here. However, ICP-OES (optical emission spectros-
copy) could also be used but would require minor modifications
of the protocol. For instance, a larger amount of yeast culture
would need to be digested to increase the elemental concentrations
in the sample, or a larger flow PFA micro-nebulizer could be
used with the Apex sample introduction system in order to
increase the amount of sample reaching the plasma. Assuming one
already has the necessary equipment and personnel in place, the
cost for sample analysis in the method reported here is under
US$200 per 96-well plate (or $2 per sample; 96 samples per
plate), making this a truly low cost, high throughput method.
In order to develop a high-throughput ionomic method that
not only can rapidly characterize many thousands of yeast
strains, but can do so in a robust, routine and analytically precise
manner, several factors have to be taken into account. Chief
among these is to use standardized instrumentation, equipment
and materials. The 96-well plate sample format was deemed
suitable for this purpose. It allows for parallel handling of large
numbers of yeast strains and can fit nicely into most liquid
handling robotic systems, and some specially configured ICP-MS
autosampler units. The method reported here was therefore
designed to allow the use of a 96-well plate format from yeast
cultivation through to ICP-MS analysis.
Yeast culture in a 96-well plate format requires the correct
amount of agitation, media volume and headspace to allow
adequate aeration for uniformity of yeast growth in all wells. The
initial conditions were adapted from work performed on bacte-
rial strains.
11
Preliminary experiments were performed to estab-
lish the ideal orbital shaking frequency of 400 rpm. The media
volumes used for the pre-growth and growth stages were selected
based on the amount needed to adequately carry out the yeast
culture processing, and the fact that there is some evaporative
loss during the cultivation. Evaporative loss was found to be
practically identical in all wells, and independent of position in
the incubator.
The custom ordered AcroPrep 96-well plate with poly-
vinylidene fluoride (PVDF) hydrophobic filter membrane was
chosen because the hydrophobic/hydrophilic nature of the
membrane can be switched by wetting with solvent, drying or
acid digestion. Once yeast cells had been dispensed into the wells,
this membrane property means that all the various sample
processing stages, including washing and digestion, can occur in
the same plate, minimizing the need for repeated transfers
between plates, reducing sample loss associated with transfers,
and the potential for contamination.
As part of this high-throughput yeast ionomic methodology,
we include 4 control yeast strains in each 96-well plate. These
control strains are the parental line for the knockout collection
YDL227c, which is derived from BY4741, along with two
previously identified yeast ionomic mutants
6
YLR396c (deletion
of VPS33 involved in vacuolar function) and YPR065w (deletion
of ROX1 involved in mineral nutrient homeostasis). YPR065w
and YLR396c, known to have elevated or depressed concentra-
tions of several elements (including Na, Mg, S, K, Mn, Co),
6
are
used as positive controls to confirm that for each 96-well plate
analyzed the analytical methodology is working correctly. The
ionomic profiles for these positive control lines are displayed in
Fig. 2A and B. Here the elements monitored by ICP-MS are
plotted against their z-scores, which represent the number of
standard deviations away from the mean of the control parental
line (YDL227c) grown in the same 96-well plate. Arabidopsis
Fig. 1 Yeast ionomics high-throughput method workflow. OD—optical
density.
This journal is ªThe Royal Society of Chemistry 2009 J. Anal. At. Spectrom., 2009, 24, 103–107 | 105
thaliana shoot ionomic data was previously displayed in a similar
manner.
4
It is clear from this type of z-score plot that both
positive control lines, YLR396c and YPR065w, show major
differences in several elements when compared to the YDL227c
background. YLR396c has elevated levels of Mg, S, Co, Ni and
Cd, and reduced Na, K, Mn, Cu and Mo levels. Whereas
YPR065w has elevated levels of K, Co, Ni and Cd, and reduced
Na, Mg, Ca and Mo levels.
In any high-throughput analysis platform, where comparisons
need to be made between samples, analytical precision is critical.
A precise measurement is one that when made repeatedly on the
same sample will give the same answer. Similarly, repeated
ionomic analyses of two different yeast strains, if made in
a precise way, would be expected to produce two sets of similar
ionomic profiles. Such profiles, if analyzed using Principal
Component Analysis (PCA) would form two clear clusters of
points associated with each of the two yeast strains. Using this
approach we show that the four control yeast strains BY4741,
YDL227c, YLR396c and YPR065w, included in all of the
96-well plates analyzed in this screen, form discrete clusters based
on a PCA of their ionomic profiles (Fig. 3). Such clear clustering
of strains, based on their ionomic profile measured repeated
across 356 96-well plates, clearly establishes the high precision of
our method; from yeast cultivation through ICP-MS analysis.
In this screen the first 850 yeast deletion strains were run
with eight replicate cultures, and eight deletion strains per plate.
Statistical simulations based on this initial data set established
that the high precision of the method would allow the reduction
of the number of replicate yeast cultures needed in the analysis.
The difference in means between the eight replicates and
a randomly selected set of four replicates was calculated for each
element across the 850 lines. All of the elements except Ca had
a difference <10% for 95% of the strains, whereas the mean
difference for Ca of <22%. This was attributed to the low levels
of Ca in the wild-type yeast. Given the only minor differences in
mean differences between eight and four replicate cultures we
adopted four replicate cultures for the rest of the screen. This
reduced level of replication allowed an increase in the number of
yeast strains that could be analyzed per 96-well plate, producing
a significant increase in throughput without loss of sensitivity to
detect ionomic differences between yeast strains.
High sensitivity, or the ability to detect small ionomic differ-
ences between yeast strains, is another important parameter of
this methodology. A major factor limiting sensitivity is the
reproducibility or precision of the measuring device.
12
Here we
express the sensitivity as the average percent relative standard
deviation (%RSD) for each element, based on each line across the
356 96-well plates analyzed in the screen. That is, if the %RSD is
small then we would expect to be able to detect small differences
between yeast strains. Table 1 shows the %RSD for each line
analyzed in every 96-well plate run in this screen (n¼6888
individual samples) at different confidence levels. It is apparent
from Table 1 that most of the elements monitored have 95% of
their %RSD values < 12% (Mg, P, K, Mn, Co, Ni, Cu, Zn and
Cd). The corresponding values for Na, Mo, S and Fe are <
20%RSD. Again, Ca shows the highest value of 32%RSD, due to
its rather low levels in yeast. Overall, the %RSD values measured
across the complete screen are low, allowing detection of small
differences in the ionome of the yeast strains in the deletion
collection. The %RSDs obtained from yeast are generally lower
than those previously observed in a genomic-scale ionomic
Fig. 2 Ion profile data for yeast mutant lines. Standard deviations (s.d.)
from the mean for (A) positive control yeast, YLR396c, and (B) positive
control yeast, YPR065w. Mean and standard deviations are calculated
for each element from the background yeast, YDL227c (n¼4), and used
to calculate the number of standard deviations each strain is distant from
the mean background value for each element. Same scaling on ordinate
used to highlight differences in mutant magnitude.
Fig. 3 Principal component analysis of the yeast ionome of the four
yeast control strains included in the 356 96-well plates analyzed. Black—
BY4741, blue—YDL227c, red—YPR065w, light grey—YLR396c.
106 | J. Anal. At. Spectrom., 2009, 24, 103–107 This journal is ªThe Royal Society of Chemistry 2009
screen of A. thaliana,
4
likely due to the more reproducible
method of cultivation and sample analysis afforded by yeast.
A preliminary analysis of the ionomic data set from the yeast
gene deletion collection identified 400 strains that display
differences from the overall average of the complete deletion set,
with at least one element exceeding three z-scores (out of a total
of 4952 lines, excluding repeat lines). Eide et al.
6
identified 233
strains using the same criterion (out of 4385 yeast mutants).
Conclusions
In the present study, a low cost, robust, precise, sensitive and
high-throughput method for profiling the yeast ionome was
developed. The method involves a novel integration of 96-well
plate sample formats for yeast cultivation, sample preparation
and ICP-MS analysis, with the power of simultaneous determi-
nation of a broad range of elements using ICP-MS. This analysis
platform was coupled to an online ionomics database (Yeast-
PIIMS) for data management. The value of this system has
been established by using it to profile fourteen elements across
the entire set of 5153 mutant strains in the yeast gene deletion
collection, representing the analysis of approximately 30 000
yeast cultures in a 6 month period. Bioinformatics analysis of this
dataset is currently ongoing to identify the genes and gene
networks involved in regulating the yeast ionome.
Acknowledgements
We thank Brett Lahner for developing the original spreadsheet
for analyzing the raw ICP-MS data. We are grateful to Tony
Hazbun for supplying most of the stock yeast for this work and
his helpful discussion concerning growing of the yeast, and we
thank Brad Kennedy of the Purdue University Discovery Park
Cyber Center for help developing the YeastPIIMS information
management system. The study was supported by the National
Institutes of Health (4 R33 DK070290-02).
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Table 1 Percent relative standard deviation (%RSD) for each element calculated across all yeast strains analyzed (n¼6888) at different confidence
levels
Confidence level Na Mg P S K Ca Mn Fe Co Ni Cu Zn Mo Cd
50% 6.0 4.3 3.8 5.4 5.1 8.5 4.5 8.2 3.9 4.6 4.4 4.2 7.3 3.8
75% 8.9 6.1 5.4 7.9 7.2 13.9 6.1 11.8 5.2 6.6 6.2 5.9 10.7 5.1
90% 13.2 8.4 7.6 12.2 9.5 22.4 8.2 16.2 6.6 9.3 8.1 7.7 14.8 6.7
95% 17.6 10.8 10.8 17.4 11.3 32.1 9.7 19.3 7.8 11.7 9.7 9.2 17.6 7.8
This journal is ªThe Royal Society of Chemistry 2009 J. Anal. At. Spectrom., 2009, 24, 103–107 | 107
