Apex Application Report
APEX-ACM
Ca Ratios
Rapid and precise calcium isotope ratio
determinations using the Apex-ACM desolvating
inlet system with sector-field ICP-MS in low
resolution
Abstract
High resolution ICP-MS is used to evaluate the APEX-ACM membrane desolvator for the
determination of 42Ca/44Ca and 43Ca/44Ca isotope ratios using low resolution. The APEX-ACM
has a rapid uptake and wash and lower Ca blanks than a conventional spray chamber. The
APEX-ACM also reduces the ArH2+ interference on 42Ca by more than 100x, lowers total
polyatomic interferences and blanks to <0.5% of the Ca signal, and provides excellent short term
and run to run precision on Ca isotope ratios.
Introduction
The determination of three calcium isotopes in
biological samples is necessary for ICP-MS
applications in human metabolic isotopic tracer
studies. The mass spectrum obtained from an ICPMS is subject to a number of plasma, water and
matrix based interferences. This is particularly
problematic in the low mass range were these
interference are both numerous and significant.
As a result, it is impossible to determine 3
interference free calcium isotopes in low
resolution on solutions aspirated using a
conventional
spray
chamber.
Polyatomic
interferences can be reduced sufficiently by using cool plasma 1 and hexapole 2 or resolved using
HR-ICP-MS 3,4,5 however precision is typically not better than 0.25% (1-std). The
implementation of desolvation results in excellent precision for Ca isotoperatio determination in
low resolution on single collector (<0.1%,1-std 6) and multi-collector (0.005%,1-std 7) ICP-MS.
Here we use oxalate precipitation of urinary calcium and the APEX-ACM sample introduction
system for low-resolution determination of the 42, 43 and 44 isotopes of calcium. Stability and
short term precision is excellent (<0.04% 1-std) and remained better than (<0.10% 1-std) during
long runs when a 50% reduction in sensitivity due to cone deposition is observed. Spectral
interference reductions with the APEX desolvating inlet system were investigated using medium
resolution on the ELEMENT-1. The APEX-ACM desolvation system reduced the ArH2+
plasma/water based interference by 100x, less than <0.5% of 42Ca signal in samples.
Apex Application Report
APEX-ACM
Nebulizer gas
Nitrogen gas
Addition
gas
Figure 1: Apex internal flow path
The Apex is a fully-integrated inlet system that connects directly to the torch injector and
incorporates ESI’s MicroFlow PFA nebulizer technology. Liquid samples are nebulized into a
spray chamber and desolvation system where the sample aerosol is conditioned to produce
uniform aerosol that is transported to the ICP. For additional desolvation, the Actively Cooled
Membrane (ACM) module is placed inline between the APEX and the injector. The ACM is a
cooled Nafion® fluoropolymer membrane desolvation module that reduces the solvent load in
the plasma. Solvent vapors pass into the Nafion® membrane and are removed by a counter
current sweep flow of a dry gas such as nitrogen or argon. The sweep gas does not pass through
the membrane or enter the sample aerosol stream, it simply serves to receive solvent vapor
molecules that pass through the membrane.
Figure 2: Nafion membrane
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Apex Application Report
Operating Parameters
Table 1: Operating parameters
The most precise isotope ratios are obtained on a
single collector ICP-MS when a combination of stable
sample introduction and rapid scanning is used to
minimize plasma noise. The APEX-ACM with a free
aspirating µFlow100 nebulizer optimizes signal
stability be eliminating the peristaltic pump and
reducing the water load in the plasma. The adverse
affect of plasma noise is further compensated on the
ELEMENT-1 by settling the magnet at the start mass
(42) and electro-statically scanning rapidly the
isotopes of interest without moving the magnet.
Further operating parameters are outlined in Table 1.
Sample Preparation
ELEMENT-1 with CD-1
Gas Flow
Sample
Auxillary
Cool
RF
L min-1
0.8
1.00
16.0
1350W
Sample uptake
Analysis time
Sample wash
Sample flow rate
Resolution
Detector mode
60 sec
120 sec
60 sec
200 µL min-1
300
Analog
Urine was prepared for analysis by precipitation as Apex-Q
2º C (2)
Condenser
calcium oxalate. The procedure for the precipitation of
140º C (2)
Heater
calcium oxalate from urine is as follows:
on
MFC
1) Pipette 5.0 mL urine in acid washed pyrex test Gas Flows
tube.
Nitrogen Flow
8.8 mL min-1
Additional Ar Flow
150 mL min-1
2) In a fume hood, add 3 drops of 25% NH4OH.
3) Add 3 mL of saturated ammonium oxalate solution (1g in 20mL of Milli-Q water).
4) Precipitate in a fume hood overnight.
5) Decant off liquid then centrifuge for 15 min @ 2000 rpm.
6) Wash with 1 mL of a 1:4 dilution of saturated ammonium oxalate solution then vortex.
7) Centrifuge at 2500 RPM for 10 min.
8) Decant off liquid then ash in furnace at 500ºC for 4 hours.
9) Cool and dissolve in 100 µL of 3% HNO3.
10) Dilute sample to approximately 5ppm (100X).
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Apex Application Report
Uptake and Wash
8.E+07
60 sec Uptake
60 sec Wash
7.E+07
1) basline <200000 cps
2) probe moved to sample
3) signal plateaues (40 sec)
4) probe moved to wash
5) air bubble
6) signal reaches baseline
<200000 cps (< 50 sec)
7) stable baseline
Ca (cps)
6.E+07
42
Apex
Uptake and Wash
5.E+07
(3) (4)
4.E+07
3.E+07
2.E+07
(2)
1.E+07 (1)
(5)
(6)
(7)
1.E+03
0
20
40
60
80 100 120 140 160 180 200 220 240 260
Time (seconds)
Figure 3
The uptake and wash times are determined by monitoring the 42Ca signal. The sample probe,
originally in a blank solution is placed in the sample solution at time 2 and returned to the blank
solution again at time 4. The signal plateaus at time 3 and return to baseline again at time 6.
Note that wash out is complete and no signal spiking is observed (7). Based on these
observations 60 second was determined to be a sufficient uptake and wash time.
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Apex Application Report
Interferences and Blanks
8.E+05
42
2.E+03
Ca (100 ppb Ca)
R=4300
2.E+03
Intensity
6.E+05
4.E+05
42
Ca blank
+
ArH2
40
1.E+03
5.E+02
> than 100x reduction
in signal relative
to interference
0.E+00
41.94
41.96
41.98
Mass
Conventional
Spray Chamber
2.E+05
42
APEX
Ca
14
+
N3
0.E+00
41.94
41.96
41.98
42
42.02
Mass
Figure 4
2.5E+04
43 Ca (100 ppb Ca)
R=4300
2.0E+04
1.5E+03
Ca blank
1.0E+03
43
Ca
5.0E+02
Intensity
43
Similar
blank
1.5E+04
APEX
4x increase
in sensitivity
1.0E+04
5.0E+03
0.0E+00
42.92
Conventional
Spray Chamber
0.0E+00
42.92
42.94
42.97
43.02
Mass
14
15
+
N2 N
42.96
42.98
Mass
Figure 5
5
43.00
43.02
Apex Application Report
4.E+05
44
3.E+05
8.E+04
Ca (100 ppb)
R=4300
44
Ca
6.E+04
4.E+04
3.E+05
2.E+04
Intensity
APEX
2.E+05
44
Ca blank
0.E+00
43.90
43.95
44.00
44.05
2.E+05
14
16
+
15
14
+
N2 O
1.E+05
5.E+04
N2 N
Conventional
Spray Chamber
12
28
16
+
Si O
0.E+00
43.90
16
+
C O2
43.95
44.00
44.05
Mass
Figure 6
Spectral interferences originate from doubly charged 86Sr++ on 43Ca, 88Sr++ on 44Ca and
polyatomic ions (ref). The doubly charged ions are corrected by measuring 87Sr++ at half mass
43.5, but polyatomic interferences are more significant and numerous and cannot be easily
corrected at the required level of precision. Most polyatomic interferences on Ca isotopes are
water based (oxides and hydrides) and are significantly reduced when using the APEX -ACM
desolvation unit. Using the APEX-ACM for sample introduction, interferences and blanks are
evaluated at a resolution of 4300 on the ELEMENT-1 in dilute acid and a 100 ppb Ca solution.
At similar sample flow rates Ca sensitivity is increased by 4X when using APEX-ACM relative
to a standard Scott spray chamber, but Ca blanks and interferences on 43Ca and 44Ca remain
similar. These data indicate the APEX-ACM provides a 4-fold increase in Ca to
blank/interference for 43Ca and 44Ca. The most important and significant improvement is
realized in the 100 fold reduction in the ArH2+ interference on 42Ca isotope. Without the
reduction of ArH2+ the determination of 42Ca in low resolution would not be possible.
The APEX-ACM provides a significant reduction in blank and interferences relative to
Ca signal when compared to a conventional Scott spray chamber. Ca isotope analyses are
performed on approximately 10 ppm Ca solutions. In low resolution the APEX-ACM reduces
the combined blank and interference contribution to less than 0.5% of the sample signal. For best
precision these ratios can now be determined in low resolution (R=300) with minimal correction
for blank and interferences.
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Apex Application Report
Short-term Precision
0.298
0.0640
Short-term precision
0.297
0.0638
average = 0.2958 + 0.032% (1-sigma)
0.0636
Ca/44Ca
0.0634
43
0.295
42
Ca/44Ca
0.296
average = 0.06328 + 0.038% (1-sigma)
0.294
0.0632
Error bars represent
1-standard deviation
internal precison
0.293
0.292
0.0630
0
1
2
3
4
5
6
7
8
9
Replicate number (n)
Figure 7
On a signal collector ICP-MS plasma noise contributes significantly to isotope ratio precision.
As discussed above this is minimized by, 1) rapidly scanning the mass spectrum and 2) reducing
instabilities associated with sample introduction. The ELEMENT-1 is configured for rapid
scanning and free aspirating micro flow nebulization combined with the APEX-ACM reduces
pump periodicity and plasma noise associated with variable sized water droplets. The short-term
precision is determined for eight two-minute acquisitions (n=8) of Ca isotope ratios. Error bars
represent the precision (1-std) of each analysis, dashed and solid lines indicate the 1 and 2
standard deviations of the data set. The data indicate that better than 0.04% (1-std) precision is
obtained for data collected over 15 min.
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Apex Application Report
Effect of Salt Deposition on Cones on Signal and Isotope Ratio Stability
1.2E+08
0.300
(1)
42
8.0E+07
Sensitivity
recovered
after cleaning
cones
0.296
44
1.4% drift in
isotope ratio
6.0E+07
0.298
0.294
4.0E+07
(4)
0.292
2.0E+07
0.0E+00
0.290
0
1
2
3
4
5
6
Time (hrs)
Figure 8 – Isotope Ratio Drift Corrected by Sample/Standard Bracketing
1) start of run 1 (24 unknowns)
4) end of run 2
2) end of run 1
5) clean cones, start of run 3
3) start of run 2 (24 unknowns)
Figure 9
8
7
8
42
Ca sensitivity (cps)
(2) (3)
(5)
Ca/ Ca ratio
52% drift in
sensitivity
due to cone
deposition
1.0E+08
Apex Application Report
During long runs, required for high sample through put, both signal intensity and isotope ratios
drift with time. With this sample type this is often due to either cone deposition. Ca forms
oxides that deposit on the cones and result in a gradual, but constant reduction in sensitivity.
When analyzing samples containing high ppm levels of Ca this is significant and can be as much
as 50% over the course of 6 to 8 hours. Two runs consisting of 24 unknowns with standards
every 6th unknown indicates that isotope ratios drift only slightly (1.4%). The drift is very
smooth and usually less than 0.1% between standards (every 28 min) requiring a small samplesample correction (<0.02%). Furthermore, the precision of individual analysis remains less than
0.1% (1-std). These factors combined with the near complete recover of sensitivity (to within
10%) after cleaning the cones indicate that the APEX-ACM is a very stable sample introduction
system and that drift is due to cone deposition.
Run-to-Run Precision and Accuracy
To determine run-to-run reproducibility one Ca oxalate
precipitated urine sample was analyzed at the beginning
and end of three runs over two days. The precision,
based on the reproducibility of this sample is better
than 0.1% (1-std) between runs. The sample is from a
person not involved in enrichment studies and should
therefore exhibit a natural isotopic signature. The
determined ratio for the unspiked individual is within 2
standard deviations of the expected natural ratio 8.
These data indicate that the APEX-ACM produces both
precise and accurate data for Ca isotope ratios using
low resolution ICP-MS.
Table 2: Run to run precision and
accuracy
n=6
42
43
start of run 1
end of run 1
start of run 2
end of run 2
start of run 3
end of run 3
0.3119
0.3115
0.3116
0.3117
0.3116
0.3120
0.06486
0.06472
0.06469
0.06474
0.06476
0.06473
average
%RSD
0.3117
0.06
0.06475
0.09
8
0.3121
The percent enrichment of urinary Ca isotope ratios is natural
%
difference
0.12
simply determined by comparing the post spiking
isotopic ratio to a baseline sample collected before spiking of the patient.
0.06486
0.17
Determination of Percent Enrichment
Ca/44Ca
Ca/44Ca
Summary
High resolution ICP-MS is used to evaluate the APEX-ACM for the determination of 42Ca/44Ca
and 43Ca/44Ca isotope ratios in low resolution. We find that the APEX-ACM
- Has a rapid uptake and wash (<50 seconds).
- Exhibits Ca blanks 4x lower than a conventional spray chamber.
- Reduces the ArH2+ interference on 42Ca by more than 100x.
- Lowers total polyatomic interferences and blanks to <0.5% of the Ca signal.
- Provides excellent short term precision <0.04% (1-std) on Ca isotope ratios.
- Provides excellent run to run precision <0.1% (1-std) on Ca isotope ratios.
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Apex Application Report
Conclusion
The APEX-ACM is a stable sample introduction system that reduces interferences on the 42 43
and 44 isotopes of Ca. The reduction of interferences combined with signal stability allows for
the precise determination of Ca isotope ratios in low resolution. These results indicate that this
method should be easily adapted to the precise determination of Ca isotopes on low-resolution
ICP-MS instruments.
References
1) K. Y. Patterson, C. Veillon, A. D. Hill, P. B. MoserVeillon and T. C. O'Haver, J. Anal. At.
Spectrom., 1999, 14, 1673
2) S. F. Boulyga and J. S. Becker, Fresenius J. Anal. Chem., 2001, 370, 618.
3) S. Sturup, M. Hansen and C. Molgaard, J. Anal. At. Spectrom., 1997, 12, 919.
4) S. Sturup, J. Anal. At. Spectrom., 2002, 17, 1.
5) Z. Chen, I. J. Griffin, Y.L. Kriseman, L.K. Liang, S.A. Abrams, Clin. Chem., 2003, 49, 2050.
6) M. P. Field, S. Shapses, M. Cifuentes, and R.M. Sherrell J. Anal. At. Spectrom., 2003, 18, 727.
8) W. A. Russell, D. A. Papanastassiou and T. A. Tombrello, Geochim. Cosmochim. Acta., 1978,
42, 1075.
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