Analysis of Perchlorate in Infant Formula byIon Chromatography

Application
Note: 533
Analysis of Perchlorate in Infant Formula by
Ion Chromatography-Electrospray-Tandem
Mass Spectrometry (IC-ESI-MS/MS)
Charles Yang1, Leo Wang2 , and William Schnute2
1
Thermo Fisher Scientific, San Jose, CA; 2Thermo Fisher Scientific, Sunnyvale, CA
Introduction
Key Words
•TSQ Quantum
Access
•Ion
chromatography
•Food safety
•Sensitivity
Ion chromatography-mass spectrometry (IC-MS/MS)
can be used to detect, quantify, and confirm a variety of
analytes in environmental and food matrices, including
haloacetic acids, bromate, and percholorate. Perchlorate, a
naturally occurring and man-made contaminant, is widely
found in the environment in surface water, groundwater,
and soil. Through environmental contamination, perchlorate has entered the food supply chain and has been
detected in a wide variety of foods including eggs, milk,
vegetables, and fruits. In humans, perchlorate interferes
with the ability of the thyroid gland to take up iodine,
which is needed to produce hormones that regulate many
body functions after they are released into the blood.
Because these thyroid hormones are essential for normal
growth and development, infants and children could more
likely be affected by perchlorate than adults.
A recent study by the Centers for Disease Control and
Prevention examined various types of commercially available powdered infant formulas to determine if perchlorate
could be found at measurable levels.1 All of the powdered
infant formulas tested contained perchlorate. Significantly
higher concentrations of perchlorate were found in cow’s
milk-based formulas with lactose than in other types.
A simple and ultra-sensitive IC-MS/MS technique
for the quantitation of perchlorate in powdered infant
formula, liquid infant formula, and milk is described here.
Unlike conventional detection methods that require labor
intensive solid phase extraction (SPE) sample enrichment,
only a simple sample preparation is necessary.
Goal
To develop a simple and ultra-sensitive IC-MS/MS method
to separate and quantitate perchlorate without time-consuming sample preparation.
Experimental Conditions
Sample Preparation
Samples of commercially available liquid infant formula,
reconstituted powdered infant formula, and milk were
prepared for analysis. A 4-mL sample of formula or milk
was mixed with 4 mL of ethanol (pre-cooled at 4 ºC) and
0.4 mL of 3% acetic acid. The sample was spiked with
40 µL (100 ng/mL) isotope-labeled internal standard. The
mixture was vortexed and then centrifuged at 5000 rpm
for 30 minutes under refrigeration (-5 ºC). The supernatant was run through a syringe filter that had been preconditioned with 5 mL ethanol and 15 mL of deionized
water. The filtrate was collected in 10-mL plastic autosampler vials and readied for IC-MS/MS analysis.
Ion Chromatography
IC analysis was performed on a Thermo Scientific Dionex
ICS-3000 Reagent-Free ion chromatography (RFIC) system. The IC conditions were as follows:
Column set: Thermo Scientific Dionex AG16 / AS16 hydroxide
selective anion exchange columns
Suppressor: Thermo Scientific Dionex ASRS 300 self-regenerated
suppressor (external water mode)
Column temperature: 30 °C
Injection volume: 100 μL
Eluent: Isocratic 45 mM hydroxide
Eluent Source: EGC III KOH
Solvent: 150 µL/min acetonitrile delivered by an AXP-MS pump
Flow rate: 300 μL/min
Detection: First detector: Suppressed conductivity
Second detector: Thermo Scientific TSQ Quantum Access mass spectrometer
Mass Spectrometry
MS analysis was carried out on a TSQ Quantum Access™
triple stage quadrupole mass spectrometer with an electrospray ionization (ESI) source. The MS conditions were as
follows:
Ion source polarity: Negative ion mode
Spray voltage: 4000 V
Sheath gas pressure: 40 arbitrary units
Ion sweep gas pressure: 15 arbitrary units
Auxiliary gas pressure: 5 arbitrary units
Capillary temperature: 300 °C
Collision gas pressure: 1.8 mTorr
Scan mode: Selected reaction monitoring (SRM)
Selected reaction monitoring allowed the following fragmentation patterns to be observed:
m/z 99 (35ClO4-) → m/z 83 (35ClO3-) primary transition for native perchlorate (quantitative)
m/z 101 (37ClO4-) → m/z 85 (37Cl18O3-) secondary transition for native perchlorate (confirmative)
m/z 107 (35Cl18O4-) → m/z 89 (35Cl18O3-) primary transition for labeled IS
(quantitative)
Results and Discussion
Figure 1 shows a representative SRM chromatogram for
a perchlorate standard of 20 pg/mL at the low end of the
calibration range. Even with such a low concentration,
perchlorate shows a well-defined peak that can be accurately quantified.
practical limit of detection (LOD) was determined by
AmtBlank + 3 × S0 = 28.9 pg/mL. The systematic factor
AmtBlank was calculated by running deionized water
instead of real sample through all sample preparation and
instrument analysis procedures.
The IC-MS/MS system provides excellent chromatographic performance and allows separation of perchlorate
from complex matrix components. Figure 3 displays the
SRM chromatograms for perchlorate in unspiked infant
formula and milk samples.
The recovery was evaluated by comparing the
differences between unspiked and spiked (5 ng/mL)
samples (n=2, n=3). Excellent recoveries were achieved as
shown in Table 1.
Table 1. Recovery of perchlorate in infant formula and milk
RT: 11.69
AA: 36716
SN: 179
100
m/z 99
80
(35ClO4-)
→ m/z 83
(35ClO3-)
NL: 1.74E3
TIC F: - c ESI
SRM ms2 99.102
[83.012-83.312]
MS Genesis
Cal_07-28_01
60
Unspiked
Spiked
Recovery
Liquid Infant Formula
1.74 (2.68)
6.91 (4.95)
103%
Milk
2.21 (2.34)
7.93 (2.71)
114%†
40
†Reported amounts are in the units of ng/mL with %RSD included in
parenthesis.
20
0
RT: 11.70
AA: 11103
SN: 164
NL: 6.41E2
TIC F: - c ESI
SRM ms2
101.120
[84.988-85.288]
MS Genesis
Cal_07-28_01
RT: 11.65
AA: 826966
SN: 31142
NL: 4.05E4
TIC F: - c ESI
SRM ms2
107.120
[89.020-89.320]
MS Genesis
Cal_07-28_01
Relative Abundance
100
m/z 101 (37ClO4-) → m/z 85 (37ClO3-)
80
60
The instrument accuracy and precision were evaluated by
repeat injections of standards at three levels, as shown in
Table 2.
40
Table 2. Method performance – Accuracy and precision
20
0
100
m/z 107 (35Cl18O4-) → m/z 89 (35Cl18O3-)
80
Specified
Amount
100 pg/mL
Quantified
Amount
%RSD
%Deviation
%RSDR.T.
98.18 pg/mL
3.05
1.82
0.13
60
1 ng/mL
1.04 ng/mL
1.62
3.52
0.08
40
10 ng/mL
10.74 ng/mL
1.45
7.36
0.11
20
0
0
2
4
6
8
10
12
14
Time (min)
Figure 1: SRM chromatograms of a perchlorate standard at 20 pg/mL
Calibration curves generated on the TSQ Quantum
Access mass spectrometer show excellent linearity
(Figure 2). For quantitative analysis, the 99 → 83 SRM
transition of perchlorate was used (ClO4- Quan), and
for qualitative analysis, the 101 → 85 SRM transition
of perchlorate was used (ClO4- Qual). The labeled IS
was used with the quantitative and qualitative ions to
calculate the squared correlation coefficients (r2) of the
99 → 83 and 101 → 85 SRM transitions of perchlorate,
which were were 0.9996 and 0.9998, respectively. The
data was weighted by 1/X to ensure better quantification
accuracy for low level samples. The calibration range was
20–10,000 pg/mL.
The instrument lower detection limit was determined
to be 5 pg/mL with S/N > 10, which is significantly
below the lowest reported value (30 pg/mL)1. The
upper calibration limit was set at 10 ng/mL, which
covered the highest reported value (5.05 ng/mL)1. The
Perchlorate was detected in every sample tested in this
study. The method precision, evaluated by repeat assays of
each unknown sample, was excellent as shown in Table 3.
The powdered infant formula sample was quantified
at 2.44 ng/mL, and the milk sample was quantified at
4.64 ng/mL. No interference was detected for any of the
samples analyzed.
Table 3. Method performance – Real samples
Sample
Quantified Amount (ng/mL)
%RSD
LIF-1
1.74
2.68 (n=3)
LIF-2
2.21
2.34 (n=3)
LIF-3
1.05
3.63 (n=7)
PIF-1
2.44
1.74 (n=3)
MLK
4.64
1.96 (n=7)
LIF: Liquid infant formula; PIF: Powdered infant formula; MLK: Milk
ClO4- Qual
Y = 0.0043091+0.000449722*X R^2 = 0.9998 W: 1/X
ClO4- Quan
Y = 0.0122265+0.0012984*X R^2 = 0.9996 W: 1/X
14
4.5
12
4.0
ClO4- Quan
Y = 0.0122265+0.0012984*X R^2 = 0.9996 W: 1/X
6
4
0.5
0.4
0.3
3.0
0.25
2.5
0.20
Area Ratio
0.6
ClO4- Qual
Y = 0.0043091+0.000449722*X R^2 = 0.9998 W: 1/X
3.5
Area Ratio
0.7
8
Area Ratio
Area Ratio
10
2.0
1.5
0.15
0.10
0.2
0.1
2
0.0
0
200
0
0
pg/mL
400
1.0
0.05
0.5
0.00
0
200
0.0
5000
pg/mL
400
5000
pg/mL
0
10000
pg/mL
10000
Figure 2. Calibration curves for quantitative and qualitative analysis of perchlorate
RT: 11.60
AA: 740997
SN: 5299
100
Infant Formula
80
NL: 3.76E4
TIC F: - c ESI SRM
ms2 99.102
[83.012-83.312] MS
Genesis
Sample_E_3
100
RT: 11.46
AA: 640329
SN: 5954
NL: 3.09E4
TIC F: - c ESI
SRM ms2 101.120
[84.988-85.288]
MS Genesis
Milk_Sample_5
RT: 11.44
AA: 312353
SN: 39213
NL: 1.46E4
TIC F: - c ESI
SRM ms2 107.120
[89.020-89.320]
MS Genesis
Milk_Sample_5
60
40
m/z 99 (35ClO4-) → m/z 83 (35ClO3-)
40
20
m/z 99 (35ClO4-) → m/z 83 (35ClO3-)
Perchlorate by 99
20
Perchlorate by 99
0
0
80
NL: 1.18E4
TIC F: - c ESI SRM
ms2 101.120
[84.988-85.288] MS
Genesis
Sample_E_3
60
40
m/z 101 ( ClO ) → m/z 85 ( ClO )
37
20
4
37
3
Perchlorate by 101
0
100
Relative Abundance
RT: 11.58
AA: 235803
SN: 6226
100
Relative Abundance
NL: 8.67E4
TIC F: - c ESI
SRM ms2 99.102
[83.012-83.312]
MS Genesis
Milk_Sample_5
Milk
80
60
RT: 11.48
AA: 1843099
SN: 3627
80
60
40
m/z 101 (37ClO4-) → m/z 85 (37ClO3-)
20
Perchlorate by 101
0
RT: 11.59
AA: 227857
SN: 21147
100
80
NL: 1.15E4
TIC F: - c ESI SRM
ms2 107.120
[89.020-89.320] MS
Genesis
Sample_E_3
60
100
80
60
40
m/z 107 (35Cl18O4-) → m/z 89 (35Cl18O3-)
40
m/z 107 (35Cl18O4-) → m/z 89 (35Cl18O3-)
20
Internal Standard
20
Internal Standard
0
0
2
4
8
6
Time (min)
10
12
14
0
0
2
4
6
8
Time (min)
10
12
14
Figure 3. Perchlorate in unspiked infant formula and milk samples. The calculated concentrations are shown in Table 3.
Conclusion
References
A selective and ultra-sensitive IC-MS/MS method has
been successfully applied for the quantification of
perchlorate in infant formula and milk. Because the
simple sample preparation was deployed and not the
long SPE enrichment method, several hours of sample
preparation time was saved. The wide linear range covers
the reported perchlorate levels in infant formula. Excellent
reproducibility (%RSD=3.63, n=7), accuracy, and
precision were achieved.
1. Schier, J.G.; Wolkin, A.F.; Valentin-Blasini, L.; Belson, M.G.; Kieszak, S.M.;
Rubin C.S.; and Blount, B.C. “Perchlorate exposure from infant formula
and comparisons with the perchlorate reference dose”; Journal of Exposure
Science and Environmental Epidemiology, advance online publication, 18
March 2009; doi:10.1038/jes.2009.18.
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