Method for quantification of Ca, Cu, Fe, Mg, Sr and Zn in low

concentrated whole blood samples using ICP-AES

Magnus Mortensen
June 12th 2019

Degree Project C in Chemistry, 1KB010

Bachelor Program in Chemistry
Department of Chemistry BMC – Uppsala University

Supervisor: Jean Pettersson, Uppsala University

Abstract
This report is the start of a study in the possibility of using the Ca, Cu, Fe, Mg, Se, Sr and Zn
composition in human blood platelets (thrombocytes) as possible biomarkers for Alzheimer’s,
Parkinson’s disease and Prostate Cancer. The aim is to find a method for the quantification of
Ca, Cu, Fe, Mg, Se, Sr and Zn, in small platelet samples (~25 mg), this with the use of ICP-
AES and ICP-MS. For the study of suitable sample preparation methods, they are tested on
reference whole blood samples analysed with ICP-AES.
Two types of sample preparation methods are tested out in the report. One alkaline dilution
method and an acid digestion method.
Both sample preparation methods show promising results for the quantification of Fe, Mg and
Zn with the use of ICP-AES, where there are determined within the 95% CI of the reference
whole blood. The methods do not show good alignment with the reference values for Ca, Cu,
Sr and Se, this due to sensitivity issues for Cu, Sr and Se using ICP-AES for quantification.
Both methods show systematically low quantification for Ca in whole blood, where the
reasons behind this is discussed in the report but the source of error not pinpointed.

2
Table of content
1 Abbreviations.......................................................................................................................4
2 Introduction..........................................................................................................................5
3 Theory...................................................................................................................................5
3.1 ICP-AES.................................................................................................................5
3.2 Sample preparation.................................................................................................7
3.3 Internal standard.....................................................................................................8
4 Materials and Method.........................................................................................................8
4.1 Chemicals ..............................................................................................................8
4.2 Standard preparation...............................................................................................8
4.3 Whole blood...........................................................................................................8
4.4 Alkaline dilution.....................................................................................................8
4.5 Acid digestion.........................................................................................................9
4.6 Sample preparation for Flame Atomic Absorption Spectrometry (FAAS)............9
4.7 ICP-AES measurements.........................................................................................9
4.8 FAAS measurements..............................................................................................10
5 Result and Discussion..........................................................................................................10
6 Conclusion............................................................................................................................15
7 Acknowledgement................................................................................................................16
8 References.............................................................................................................................17
9 Appendix ..............................................................................................................................18

3
1 Abbreviations
95% CI 95 % Confidence Interval
CCD Charge Coupled Devise
FAAS Flame Atomic Absorption Spectroscopy
ICP-AES Inductive Coupled Plasma Atomic Emission Spectroscopy
ICP-MS Inductive Coupled Plasma Mass Spectrometry
ICP-OES Inductive Coupled Plasma Optical Emission Spectroscopy
ICP-SFMS Inductive Coupled Plasma Sector Field Mass Spectrometry
LOD Limit of Detection
LOQ Limit of Quantification
R2 Coefficient of determination
RF Radiofrequency
RSD Relative Standard Deviation
SD Standard Deviation

4
2 Introduction
The aim of this project is to develop a method for the determination of Ca, Cu, Fe, Mg, Se, Sr
and Zn in human blood platelets. The platelets elemental composition could possibly be used
as biomarkers for Alzheimer’s, Parkinson’s disease and Prostate Cancer1,2. The quantification
of the elements is to be determined with inductively coupled plasma (ICP) both with mass
spectrometry (MS) and atomic emission spectroscopy (AES) for detection. ICP-MS is the
most sensitive of the two instrumentations, but the Ar from the ICP interferes with the signals
from Ca, Fe and Se. This is due to the similar mass of Ar and Ca, same for ArO and Fe. Se
signal is disturbed by Ar2. This can be overcome with the use of a collision cell, that uses He
to collide with ions, stopping polyatomic ions from entering the MS. This does not fully solve
the disturbance of Ca. That’s why ICP-AES can be used as the instrumentation for
determination of Ca, Fe and Se. The drawback with ICP-AES is the high detection limit in
comparison with ICP-MS, which can be a problem for trace elements as Se and Sr.
Trace element analysis of blood platelets, also called thrombocytes, have been investigated in
earlier studies3,4,13,5–12. Results are in some cases ambiguous, that can be explained by the
different techniques used and the problem with determining when the sample is purely
platelets. A problem in the determination of trace elements in blood platelets is the separation
and purification of the platelets. The main problem is after separation, when the platelets
needs to be washed to remove blood plasma that has been adhered to the surface of the
platelets. This leads to the problem, when are the platelets washed enough to be deemed as
pure platelets, and how to perform this without leaching out analytes from the platelets and
not contaminating the sample. The differences in the separation and washing of the platelets
means that direct comparison between previous studies is not always possible3.
To perform a comprehensive study in to the use of Ca, Cu, Fe, Mg, Se, Sr and Zn as possible
biomarkers for Alzheimer, Parkinson and Prostate Cancer there is a need to use the same
sample preparation procedure for the platelets, to be followed both for samples of healthy
patients and from patients with the diseases of interest.
This report focuses on sample preparation procedures for the determination of elements in
reference whole blood, which has been chosen because its similarity to platelets, and for being
a reference sample with known concentration of the elements so the methods can be tested.
The reference whole blood is human blood that comes from a standard blood donation,
meaning the there is no additives to the blood.
Two main sample preparation methods have been tested before the ICP-AES measurement;
one method where the whole blood is diluted in an alkaline solution and one method with an
acid digestion sample preparation.
The initial testing has been performed on ICP-AES due to Ca, Fe and Se, have signal
interferences on ICP-MS. Later the same samples are to be used for the determination of Cu,
Mg, Sr and Zn on ICP-MS.

3 Theory
3.1 ICP-AES
ICP-AES also known as inductively coupled plasma optical emission spectroscopy (ICP-
OES) is an instrumentation used for multi-elemental analysis. It works by determining the
light intensity at elemental specific wavelengths. That comes from emission of exited atoms
5
and ions produced by an Ar plasma. With ICP-AES about 70 elements can be determined
simultaneous, giving ICP-AES a big advantage from previously used atomic absorption
spectroscopy (AAS) methods14.

The heart of the ICP is the torch, which is a three-part fused-silica tube, see figure 1. An inner
tube where the sample is introduced to the plasma, an intermediate tube transporting Ar(g) for
the plasma. Then also an outer tube that is transporting coolant gas to the torch.
The top of the torch is surrounded by copper coil that is connected to a radiofrequency (RF)
generator, that creates an altering current oscillating in the coil, that in turn creates an
oscillating electric and magnetic field in the top of the torch. The plasma is started by a
discharge from a Tesla coil, that produces electrons and ions in the Argon gas. These are
called seed electrons and ions, that are trapped by the magnetic field. Where they are
accelerated until they have enough energy to collide with other Argon atoms and ionize them.
This proceeds as a chain reaction creating the plasma.

The created plasma takes on a droplet shape, that has a temperature of 10 000 K at its base
and decreases down to 6000 K at the tip of the droplet shape, this can be observed in Figure
115.

Figure 1.
ICP torch, illustrates the temperature drop-off further out in the plasma15.

Samples introduced into the plasma can come in all phases (solid, liquid or gas phase), but
liquid samples are the most common. The liquid sample is pumped in to a nebulizer, where it
is forced through a small opening with the help of Ar(g), this creates an aerosol. That in turn
enters a spray chamber, where the larger droplets go to waste and only the smallest ones
continue to the plasma. The aerosol enters the plasma where it is atomized sometimes ionized
and exited, before going back to it’s ground state and emitting light. The energy of the emitted
photons corresponds to the energy levels for specific atoms and ions. By this knowledge each
wavelength can be paired up with the correct element.

The about 70 elements that can be determined with ICP-AES give rise to 70 000 emission
lines in the range 200-600 nm15. This calls for a high resolving power to separate out the
emission lines. The detector for the ICP-AES used in this report has a range of 125-800 nm.
Axial view was used for the measurements, this means that the emission is viewed
horizontally from the plasma, i.e. viewing it from the tip of the plasma. This view type gives a
better sensitivity with the drawback of higher risk for spectral and matrix interferences15.

6
The emitted light passes through a slit into the detector side of the ICP-AES. Where the light
hits what is called a Rowland´s circle. That is a circular setup of a concave grating on one side
and a series of 19 charge-coupled device (CCD) detectors in a half circle on the opposite side
of the grating. The electric signal from the CCD’s is amplified and a spectrum is created by
software.

3.2 Sample preparation

Acid digestion is one of most common sample preparation techniques for digestion organic
samples. The goal is to break down the solids in the matrix, giving a complete solution of
analytes16. This is done by an oxidizing acid breaking down the organic molecules in the
sample for example proteins. Therefore, releasing the analytes that are bound to the proteins,
so that they are free in the solution. This leads to more reliable atomization in the analysis
instrument and eliminates the risk of clogging the nebulizer. For example, in one of the
methods in this report HNO3 is used as the acid for digestion. The HNO3 reacts with carbon
chains in the organic molecules following this general formula16:
(CH2)n + 2 HNO3 → CO2 + 2 NO + 2 H2O
One common added reagent is H2O2 which increases the oxidizing power of the acid. H2O2 by
itself is a strong oxidizer. In the case of HNO3 this is done by H2O2 breaking down to H2O and
O2. Where the O2 re-oxidizes the NOx in to NO3- that in turn can react again with a new CH2
group16. The use of H2O2 together with HNO3 gives the added benefit of eliminating the NOx
in the solution, giving a clear solution when all solids are digested, with NOx still present in
the solution it will take on a yellow colour. For the digestion of whole blood, the digestion
can clearly be followed due to whole blood having a distinct red colour from the heme group
in mainly Hemoglobin. A full digestion of the Hemoglobin lead to the solution becoming
clear, indicating that the Fe ions in the heme groups are free in solution.
To speed up the process of acid digestion the temperature can be increased. A higher
temperature leads to higher reaction rates following the Arrhenius equation. The limitation of
the temperature increase is the boiling point of the solution, this can be circumvented by
heating the solution in a closed vessel. This increases the pressure of the system leading to
higher temperatures can be reached. In this report the use of microwave digestion takes
benefit of this. The samples are loaded into closed Teflon vessels called bombs, the solution is
then heated up by microwaves. That does not directly heat up the bombs, only the solution.
Meaning that the solution is heated from inside out, giving an even temperature distribution.
Teflon bombs are used due to the high bond strength of the CF bonds, that can stand up to the
harsh environment. One added benefit from the bombs is that the closed system stops volatile
analytes from leaving the system, for example Se.
The other sample preparation examined in this report, is the use of an alkali solution that
breaks the cell membranes in the whole blood and also prevents precipitation17. This method
does not completely break down the proteins, which is observed by the samples still having a
red colour from the Hemoglobin. Leaving a risk of the sample clogging the nebulizer, which
leads to fluctuating sample introduction to the plasma. This method also puts a higher stress
on the plasma to atomize the sample, with the risk that the plasma turns off.

7
3.3 Internal standard
An internal standard is a known amount of a compound that is added to the sample14. This
gives a fixed rate between the internal standard and the analyte. This is done at the start of the
sample preparation when there is a risk of sample lost during the sample preparation. The
outcome will still be that the analyte-internal standard ratio is the same. Internal standard also
corrects for differences in instrument response between runs, due to drift and variations in
sample introduction14,18. It can also correct for matrix effects18. There are a few things to
consider when choosing internal standard, it should not be present in the samples, it needs to
be chemically stable in the matrix and it should be chemically similar to the analyte18. In this
report Sc is chosen which is a rare earth metal that should not be present in whole blood and is
one of the most common internal standards for ICP-AES analysis18.

4 Materials and Method

4.1 Chemicals
The following chemicals were used for preparation of standards and samples: nitric acid for
analysis (65%, Merck KGaA, Germany), ethylenediaminetetra-acetic acid (EDTA) analytical
reagent (VWR Chemicals, Belgium), hydrogen peroxide 30 % for traces analysis (VWR
Chemicals, France), lanthanum chloride (>99 %, BDH Chemicals Ltd Poole England), 2-
Butanol Pro Analysis (>99.5 %, Merck KGaA, Germany), ammonia solution for analysis (25
%, Merck KGaA, Germany) and Triton X-100 (t-Octylphrnoxypolyethoxyethanol)
SigmaUltra (Sigma-Aldrich, Germany). All solutions used are diluted with Milli-Q purified
water (Millipore, Billerica, Ma, USA), henceforth referred as MQ. The reference whole blood
used was of the type Sernorm Trace Elements Whole Blood L-1 (ref. 201505, lot. MR4206,
SERO, Norway, expiration date 2009.08).

4.2 Standard preparation

For Ca, Cu, Mg, Sr and Zn 100 mg/L standard were prepared from 1000 mg/L single element
standards, see appendix Table A1, both in an alkaline solution and 10 (v/v) % nitric acid
solution. For most of the experiment’s multielement calibration standards with concentrations
0; 0.1; 0.2; 0.5 and 1.0 mg/L for Ca, Cu, Mg, Se, Sr and Zn were made from the 100 mg/L
standards, also containing Fe in the ranges of 0-2 mg/L up to 0-20 mg/L dependent on the
expected concentration in the samples. All calibration standards were made with 5 mg/L Sc as
internal standard.
Dependent on the sample matrix, calibration standards were matrix matched with alkaline
solution or 10 % HNO3 solution.
4.3 Whole blood
Sernorm Trace Element Whole Blood L-1 was prepared by weighing in 5.00 mL of MQ into a
vial containing freeze-dried whole blood. Then adding 12 pcs of pellets (lead) to help with the
homogenisation, before shaking the vial and storing it at <8°C.
4.4 Alkaline dilution
Alkaline solution were prepared with 1 % (w/v) ammonia (25 % NH4OH), 2 % (w/v)
2-butanol, 0.05 % (w/v) Triton X-100, 0.05 (w/v) ethylenediaminetetraacetic acid (EDTA)
and diluted with MQ17. Samples were prepared with 25/50 µL aliquots of whole blood, 3.5

8
mL alkaline solution and Sc standard all weighed in the stated order, to stop the acid in the Sc
standard from coagulate the whole blood. Blanks were made following the same procedure
using MQ from a test tube containing lead pellets. For recovery experiment 50 µL whole
blood samples were spiked with analytes from single element standards, to give a in solution
concentration approximately twice the concentration of un-spiked samples.
4.5 Acid digestion
Aliquots of 25/200 µL whole blood were added directly to the bombs with differential
weighing of the pipette on a non-closed 3 decimal point scale. Sc standard were added to give
a final concentration of 5 mg/L. HNO3 and H2O2 were added to give a concentration of 10
(v/v) % respectively. The samples were then diluted with MQ to a total volume of 6 mL.
Blanks following the same procedure were prepared with MQ containing lead pellets.
Microwave digestion were performed on a Perkin Elmer Microwave sample preparation
system Titan MPS following the program in Table 1.

Table 1.
Microwave digestion program, power decreased by 10 % for every number of samples lower
than 16, with a minimum of 40 %.
Step Target temp. (°C) Pressure max (Bar) Ramp time (min) Hold time (min) Power (%)
1 160 30 5 5 80
2 190 30 1 10 90
3 50 30 1 10 0

For recovery test a spike solution was added containing all analytes to give a sample
concentration about twice the un-spiked concentration, with the dilution factor of the whole
blood staying the same.

4.6 Sample preparation for Flame Atomic Adsorption Spectrometry (FAAS)

Samples were prepared following the sample preparation as in the acid digestion method.
Before measuring aliquots of samples, blanks and calibration standards were diluted two
times with a 2000 mg/L LaCl3 solution.

4.7 ICP-AES measurements

ICP-AES measurements were done on a Spectro Ciros CCD. Samples were injected with a
peristatic pump with a flowrate of 1 mL/min, giving a minimum sample volume needed of 3
mL. Between each sample the pump tube was moved to a container of MQ-water. Before
measurement the system was flushed 45 s with sample and then measured 3*25 s for one
complete measurement and giving a mean value of the replicates. Wavelength and
background were manually optimized using both samples and calibration standards. Coolant
flow of 14 L/min, auxiliary flow and nebulizer flow of 0.9 L/min was always applied.
Mean intensity of the replicates are in all presented data corrected with internal standard
intensity, by calculating the ratio. The wavelength used for the measurements were chosen by
taking into regard the LOD of the wavelength and the coefficient of determination (R2) of the
calibration curve at the wavelength. The chosen wavelength’s and the R2 for the calibration
curves are presented in Table 2.

9
Table 2.
ICP-AES wavelengths for each method and the R2 for the calibration curve at the wavelength.
Element Alkaline dilution Acid digestion
2
Wavelength (nm) R Wavelength (nm) R2
Ca 317.933 0.9998 396.847 0.9995
Cu 324.754 0.9999 327.396 0.9999
Fe 238.204 0.9999 238.204 0.9997
Mg 285.213 0.9999 279.553 0.9999
Sr 421.552 0.9999 407.771 0.9999
Zn 206.191 0.9999 213.856 0.9999

4.8 FAAS measurements

A Perkin Elmer Atomic Absorption Spectrometer 3100 with a Perkin Elmer Ca Intensitron
Lamp, with 10 mA of current, were used for the FAAS measurements. Settings: slit 0.7 and a
lean air-acetylene flame with a 4.3 to 1 relation. Measured at 422.7 nm with linear range of
5.0 mg/L and sensitivity of 92 µg/L.

5 Results and discussion

As the purpose of the method development is to determine trace elements in blood platelets,
the focus has been on miniaturization of the sample preparation. The blood platelet samples
that are at hand to be analysed have an approximate mass of 25 mg. Therefore, the use of 25
µL (≈25 mg) whole blood have been used. With a minimum sample volume of 3 mL for the
ICP-AES measurement, the sample volume has been kept higher than 3 mL. The minimum
sample volume for the microwave digestion bombs that has been at hand is 5 mL, which has
led to a higher dilution factor for the acid digestion method. The dilution factor of the alkaline
dilution method is 140 and for the acid digestion method the dilution factor is 240. The
element concentration of the whole blood in the samples with this dilution factors are stated in
Table 3.

Table 3.
Analytical values for Sernorm Trace Element Whole Blood L-1 and recalculated for method
dilution, 140 times for alkaline dilution and 240 times for acid digestion.
Element Analytical value Conc. in solution
mean ± CI Alkaline dilution Acid digestion
Ca 14.2 ± 0.8 (mg/L) 101 (µg/L) 59 (µg/L)
Cu 564 ± 33 (µg/L) 4 (µg/L) 2 (µg/L)
Fe 432 ± 28 (mg/L) 3.08 (mg/L) 1.80 (mg/L)
Mg 19.6 ± 1.1 (mg/L) 140 (µg/L) 82 (µg/L)
Sr 27.8 ± 1.7 (µg/L) 0.2 (µg/L) 0.1 (µg/L)
Zn 5.5 ± 0.3 (mg/L) 39 (µg/L) 23 (µg/L)

10
The limit of determination (LOD) and the limit of quantification (LOQ) is determined for
both methods, with two different techniques. In the first one they are calculated with the
standard deviation (SD) of the measured element concentration in blanks. In the second one
they are calculated from the standard error of the regression and the slope of the curve. The
LOD and LOQ of both methods are presented in Table 4.

Table 4.
Limit of determination (LOD), limit of quantification (LOQ) in measured solution for alkaline
dilution and acid digestion.
Element LOD LOD LOQ LOQ
Alkaline dilution Acid digestion Alkaline dilution Acid digestion
3.3SDa 3.3Sx/y/kb 3.3SDa 3.3Sx/y/kb 10SDa 10Sx/y/kb 10SDa 10Sx/y/kb
(n=10) (n=6) (n=10) (n=6)
Ca (µg/L) 9.3 10.6 30.4 35.1 28.1 47.8 92.1 106.4
Cu (µg/L) 2.7 2.9 5.5 15.5 8.2 8.9 16.8 46.9
Fe (µg/L) 7.4 42.2 6.5 76.4 22.6 128 19.8 231
Mg (µg/L) 1.2 10.6 3.3 8.8 3.8 32.2 10.0 26.6
Sr (µg/L) 2.2 5.9 0.1 11.0 6.8 18.1 0.3 33.4
Zn (µg/L) 6.8 7.1 3.9 11.4 20.8 21.6 12.0 34.6
a
LOD determined as 3.3 times the standard deviation (SD) of the blanks, and LOQ as 10 times.
b
LOD determined as 3.3 times the standard error of the regression (Sx/y) divided by the slope (k), and LOQ as 10
times.

The different estimates of LOD and LOQ give a large difference for several of the elements,
where the LOD and LOQ calculated from the calibration curve are on the high side.
By comparison of Table 2 and Table 3, the Ca, Fe, Mg and Zn concentration is over the LOQ
for the alkaline dilution method. While Cu and Sr are under the LOQ of the method and Sr
even under the LOD.
For the acid digestion method, Fe and Mg are over the LOQ. While Ca, Cu, Sr, and Zn are
below the LOQ, determined from the calibration curves. Ca, Cu and Sr concentrations are also
lower than the LOQ determined from the blanks. Zn stands out with the concentration 23
µg/L being between the calculated LOQ’s of 12 and 34 µg/L. Ca is in the range between LOD
and LOQ (for both calculations), while Cu and Sr is below the LOD.

Alkaline dilution and acid digestion methods precision is determined and compared in Table
5. The analytical values from Sernorm Trace Element Whole Blood L-1 have been
determined with inductively coupled plasma sector field mass spectrometry (ICP-SFMS) from
at least 12 replicates. Both alkaline dilution and acid digestion precision is determined from a
series of 12 samples. All samples in one series is made from the same whole blood vial, the
two different method series are made from different whole blood vials.

11
Table 5.
Analytical value for Sernorm Trace Element Whole Blood L-1, and measured value for
alkaline dilution and acid digestion method both with 25 µL whole blood. (n=12).
Element Analytical value Alkaline dilution Acid digestion
mean ± CI mean ± SD mean ± SD
Ca (mg/L) 14.2 ± 0.8 11.7 ± 0.7 9.8 ± 3.3
Cu (µg/L) 564 ± 33 631 ± 75 668 ± 192
Fe (mg/L) 432 ± 28 445 ± 24 417 ± 13
Mg (mg/L) 19.6 ± 1.1 19.5 ± 1.0 19.0 ± 0.6
Sr (µg/L) 27.8 ± 1.7 21.6 ± 1.8 19.2 ± 6.2
Zn (mg/L) 5.5 ± 0.3 5.8 ± 0.3 5.5 ± 0.2

For both methods mean values for Fe, Mg and Zn are within the 95 % confidence interval
(CI) of the analytical value, this although Zn concentration is between the LOQ’s determined
for the acid digestion method. It’s to be noted that the calibration for Fe in the acid digestion
(Table 2) does not have a perfect regression (R2<0.9999), decreasing the reliability of the
results.
Ca and Sr show lower concentrations than the analytical value, while Cu shows higher
concentrations for both methods, both Cu and Sr concentrations are under the LOQ’s. Ca is
over the LOQ for the alkaline dilution method, indicating interference for Ca giving low
results. This can be chemical interference from Ca forming non-volatile compound with SO42-
and PO43-, that reacts in the plasma leading to lower measured values14. EDTA is added to
alkaline solution to stop this by chelating the Ca2+ ions, stopping them from forming the non-
volatile compounds 14,17. This means that there can be a pH problem where Ca precipitates
out, the alkaline solution has a pH of 10, previous reports have stated that the optimal pH is 8-
917.
The whole blood has a concentration of 1223 mg/L S and 239 mg/L P, this high concentration
indicates a likelihood of a presence of a high level of SO42- and PO43-.
In the acid digestion the Ca concentration is below the LOQ, and therefore not a completely
reliable determination. But in the acidic matrix Ca behaves similarly with low measured
values. This can be from the same reason with the creation of non-volatile compounds.
Also, to be noted is the R2 is <0.9999 for Ca in both methods, but the error from the not
perfectly linear response should not give rise to this large of an error in the quantification.
In alkaline dilution Cu is measured close to LOD, meaning that the noise to signal ratio is
high and leads to a high relative standard deviation (RSD) of 12 %. This determination of the
Cu concentration is not reliable. Same for the acid digestion method, where the measurement
is done under the LOD, giving an even higher RSD of 29 %.
For Sr the problem is the same with measurements done under the methods LOD´s, leading to
none reliable determinations.

Measurements done on samples with a higher concentration of whole blood are displayed in
Table 6. Where the in-sample concentration for the alkaline dilution method is twice that of
Table 3 and for acid digestion the concentration is four times that of Table 3. In this
experiment Ca, Fe, Mg and Zn concentrations are over the LOQ. While Cu and Sr still are
under the LOQ´s, giving misleading values for Cu. For Sr the acid digestion method shows a
high RSD of 47 %, while measured value for the alkaline dilution method is within the 95 %

12
CI and with a low RSD of 7 %. This measurement is still under the LOD of the method, so the
measured value is not reliable.

Table 6.
Measured concentrations for alkaline dilution and acid digestion methods at a lower dilution
factor. Alkaline dilution method with 50 µL whole blood (n=12) and acid digestion with 200
µL whole blood(n=5).
Element Analytical value Alkaline dilution Acid digestion
mean ± CI mean ± SD mean ± SD
Ca (mg/L) 14.2 ± 0.8 12.0 ± 0.4 12.1 ± 1.6
Cu (µg/L) 564 ± 33 630 ± 38 742 ± 64
Fe (mg/L) 432 ± 28 435 ± 3.0 421 ± 5.5
Mg (mg/L) 19.6 ± 1.1 19.3 ± 0.2 19.6 ± 0.3
Sr (µg/L) 27.8 ± 1.7 27.1 ± 1.9 33.8 ± 16.0
Zn (mg/L) 5.5 ± 0.3 5.6 ± 0.1 5.8 ± 0.2

For Ca, even double concentration in sample from the alkaline dilution method, the measured
value is lower than the analytical value, indicating that previous stated interference for Ca is
likely.
For the acid digestion a higher concentration of whole blood gives a somewhat higher value
for Ca, which aligns more with the measured values for the alkaline dilution method, but still
showing systematically low measurements. The measurement is done at a concentration two
times the magnitude of LOQ, eliminating any risk of a high noise to signal rate giving false
readings. This shows a systematically low determination of Ca for both methods, that is not
dependent on the dilution factor.

Results from recovery experiments for both methods are presented in Table 7. The recovery
values are tested for significance with a student t-test, with the null-hypnotises that the
recovery is 100 % (p<0.05)19.

Table 7.
Recovery of spiked samples for alkaline dilution (n=3) and acid digestion (n=4) method.
* indicates significant deviation for a student t-test, H0: recovery=100%, (p<0.05).
Element Alkaline dilution Acid digestion
Recovery ± SD (%) Recovery ± SD (%)
Ca 100 ± 8 108 ± 5*
Cu 77 ± 7* 128 ± 52
Fe 36 ± 4* 91 ± 4*
Mg 120 ± 1* 98 ± 4
Sr 108 ± 10 140 ± 5*
Zn -7 ± 1* 110 ± 4*

Ca and Sr indicate no significant deviation in the alkaline dilution method for the student t-
test. This does not necessarily mean that it is no systematic error for Ca and Sr in the
samples 20.

13
For Cu, Fe, Mg and Zn the recovery test indicates a systematic error for the alkaline dilution
method. Irons low recovery comes from the spike added sedimented in the matrix, which was
observed as yellow particles in the solution. The negative recovery of Zn is most likely traced
back to human error in the form of no Zn spike added.
The student t-test for the recovery in the acid digestion method, indicates no significant
deviation for Cu and Mg. For Cu this is not reliable due to the high RSD of 40 %. For Mg it
indicates no systematic error, as stated before is not a guaranty that no systematic error is
present, even though Ca, Fe and Zn give a recovery within ±10 % the statistic test indicates a
significant difference, they can still be deemed as acceptable recoveries. Sr has a recovery of
140 %, which can be expected due to the measurement is done in a range where the noise to
signal ratio is high.
The recovery test gives the same indications as previous measurements where Fe, Mg and Zn
give reliable readings, Cu and Sr still have a sensitivity issue. While Ca gives a good recovery
for both methods, indicating that spiked Ca does not have any chemical interference and
creates non-volatile salts.
The Ca added from the spike may not be in the same chemical state as the Ca in the whole
blood, where there is a high level of Ca2+ ions1. Meaning that the spiked Ca does not
necessarily behave in the same manner as the Ca in the matrix. So, although the recovery test
does not indicate a high level of systematic error, previous discussed data indicates
systematically low readings.

A series of 5 samples measured by ICP-AES and then diluted according to the FAAS method
and measured, are displayed in Table 8. Paired t-test performed on the data set (H0: µd=0,
p<0.05) indicates no significant deviation between the methods.

Table 8.
ICP-AES and FAAS measurements on the same sample series (n=5). Analytical value is
presented as mean ± CI. ICP-AES and FAAS measurements presented as mean ± SD.
Element Analytical value ICP-AES FAAS
Ca (mg/L) 14.2 ± 0.8 11.1 ± 0.3 10.9 ± 0.8

This result indicates that there is no difference in systematic error between the methods. But
there seems to be a systematic error in the sample preparation, where Ca does not behave as
expected. La3+ is added to the FAAS samples to stop Ca2+ from binding to SO42- and/or PO43-
in the flame. This indicate a different kind of chemical interference or other interference being
present, there is a need for further studies to determine the reason behind the systematically
low values. It shall also be noted that the reference whole blood used is ten years past its
expiration date, but this should not affect the Ca concentration in the whole blood. As
previously stated there is a linearity issue for Ca, that may lead to the lower measured values.
To determine if this is the problem further studies are needed.

No results from measurements of Se has been presented, this due to problem with spectral
overlap of the Se signal and sensitivity issues.

14
6 Conclusion

From previously stated data the conclusion can be drawn that both alkaline dilution method
and the acid digestion method give good agreement i.e. measured values are within the 95 %
CI of the analytical value for Sernorm Trace Element Whole Blood L-1, for Fe, Mg and Zn.
The measured values have low SD, but note that this is SD for within run measurements.
Meaning that there is a need for a more extensive determination with between run
determinations. There is a sensitivity issue for the determination of Sr and Cu with the use of
ICP-AES. This is not troubling due the goal of the project is to also determine the element
concentrations with ICP-MS, that has a better sensitivity for Sr and Cu.
The main problem with the presented methods is the systematic low measurements of Ca in
whole blood, where there is a need for further studies to solve the interference issue. The
recovery experiment did not indicate a systematic error, which indicates that the use of
standard addition for Ca determination might be a more suitable method. Another experiment
to find the reason behind the low values, is to spike the samples with SO42- and/or PO43-, to
see how the measured Ca concentration reacts. There is also a need for further studies in
solving the linearity issue for Ca, to test if this is the source of the systematic error.
Another problem may be with digestion in a low acid concentration. The use of a lower acid
concentration in the digestion step is due to the interest in testing out a less aggressive method
on the samples, following the green chemistry initiative with the use of lower amounts of
chemicals. Also, to be able to measure directly on the digestion solution, to lower the dilution
factor for the whole blood. A more standard acid digestion for whole blood with a higher acid
concentration for digestion and with a dilution step after the digestion, could give different
results.

For the alkaline dilution method, the Ca problem may arise from a pH problem, where
previous reports have stated that the optimal pH is 8-9. The alkaline solution used in this
report had a pH of 10 before dilution with whole blood and internal standard.

15
7 Acknowledgment

I want to take this opportunity to thank my supervisor Jean Pettersson at Department of

Chemistry BMC-Uppsala University for all the help with the project. Also want to thank
Marcus Korvela and Marit Andersson at Department of Chemistry BMC-Uppsala University
for all the helpful discussions.

16
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17
9 Appendix

Table A1: Single-element standards.

Element: Conc. (µg/mL): SD: [HNO3] (v/v%): Density (g/mL):

Ca 998 2 0.1 1,003
Cu 1003 5 3 1,016
Fe 1002 5 2 1,009
Mg 1003 4 0.1 1,003
Se 100.21 0.68 2 1,009
Sr 1004 5 0.1 1,001
Zn 1006 3 2 1,011
Sc 996 3 7 1,039

18