September 2009
Environmental Technology
Verification Report
ABRAXIS 17p-EsTRADioi_ (E2) MAGNETIC
PARTICLE ENZYME-LINKED IMMUNOSORBENT
ASSAY (ELISA) TEST KITS
Prepared by
Battelle
Baiteiie
The Business of Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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September 2009
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
ABRAXIS 17p-EsTRADioi_ (E2) MAGNETIC
PARTICLE ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA) TEST KITS
By
Stephanie Buehler, Zachary Willenberg, Amy Dindal, Battelle
Eric Kleiner, Michelle Henderson, and John McKernan, U.S. EPA
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Notice
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed, or partially funded and collaborated in, the research described herein. It
has been subjected to the Agency's peer and administrative review and has been approved for
publication. Any opinions expressed in this report are those of the author (s) and do not
necessarily reflect the views of the Agency, therefore, no official endorsement should be inferred.
Any mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Foreword
The EPA is charged by Congress with protecting the nation's air, water, and land resources.
Under a mandate of national environmental laws, the Agency strives to formulate and implement
actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, the EPA's Office of Research and
Development provides data and science support that can be used to solve environmental
problems and to build the scientific knowledge base needed to manage our ecological resources
wisely, to understand how pollutants affect our health, and to prevent or reduce environmental
risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at
http ://www. epa.gov/etv/centers/centerl .html.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We sincerely appreciate the
involvement and support of all staff from the participating laboratories who conducted testing as
part of this verification test. In particular, we would like to thank the following staff for their
contribution in conducting this verification at their respective laboratories: Mark Mills and Scott
Jacobs, U.S. EPANRMRL, Cincinnati, OH; Jennifer Gundersen, Dave Russell, Ronald Landy,
Annie Hilliard, John Curry, and Martin Lazarus, U.S. EPA Region 3 Fort Meade, MD; Dennis
Wesolowski, Larry Zintek, and Charles Steiner, U.S. EPA Region 5 Chicago, IL; Mike Meyer,
Keith Loftin, Larry Barber, and James Gray, USGS, Kansas; Jim Lazorchak, Tirumuru Reddy,
and Dan Bender, U.S. EPANERL, Cincinnati, OH; and Jeanette Van Emon, U.S. EPA NERL
Las Vegas, NV. Finally, we would like to thank Lisa Olsen, USGS; Paul Pennington, NOAA,
and Marion Kelly, U.S. EPA, for their review of this verification report.
IV
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
Chapter 1 Background 1
Chapter 2 Technology Description 2
Chapter 3 Test Design and Procedures 4
3.1 Introduction 4
3.2 Test Facilities 5
3.3 Test Procedures 6
3.3.1 Test Sample Collection and Preparation 7
3.3.2 Test Sample Analysis Procedure 8
Chapter 4 Quality Assurance/Quality Control 10
4.1 Quality Control Samples 10
4.1.1 GC-MS Method Blank and Surrogate Spike Results 10
4.1.2 Test Kit Method Blanks 11
4.2 Audits 11
4.2.1 Performance Evaluation Audit 11
4.2.2 Technical Systems Audit 12
4.2.3 Data Quality Audit 13
4.3 QA/QC Reporting 13
4.4 Data Review 13
Chapters Statistical Methods 15
5.1 Precision 15
5.2 Percent Bias 16
5.3 Matrix Effects 16
5.4 Operational Factors 17
Chapter 6 Test Results 18
6.1 Precision 18
6.2 Percent Bias 20
6.4 Matrix Effects 21
6.5 Operational Factors 23
Chapter 7 Performance Summary 24
Chapters References 26
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Figures
Figure 2-1. Abraxis 17|3-Estradiol (E2) magnetic particle ELISA Test Kit 2
Tables
Table 3-1. Target Analyte 5
Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory 5
Table 4-1. PE Audit Sample Results 12
Table 4-2. Summary of Data Recording Process 14
Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD)
Results 19
Table 6-2. Expected Concentrations for each Phase 19
Table 6-3. ELISA Test Kit Percent Bias vs. GC-MS 20
Table 6-4. GC-MS Average Concentration, RSD, and Percent Bias Results 21
Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration 21
Table 6-5. Percent Recovery 22
Table 6-6. Concentrations of Cross-Reactive Compounds 23
VI
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List of Abbreviations
AMS
°C
coc
DI
DIR
El
E2
E3
EDC
EE2
ELISA
EPA
ETV
GC-MS
OFF
HPLC
L
LC-MS
MB
MDL
NERL
ng
nm
NP
NRMRL
ORD
PE
ppt
QA
QC
QMP
rpm
RSD
Advanced Monitoring Systems
degrees Celsius
chain of custody
deionized
direct
estrone
17-p-estradiol
estriol
endocrine-disrupting compound
17-a-ethynylestradiol
enzyme-linked immunosorbent assay
U.S. Environmental Protection Agency
Environmental Technology Verification
gas chromatography-mass spectrometry
glass fiber filter
high performance liquid chromatography
liter
liquid chromatography-mass spectrometry
method blank
method detection limit
microliter
micron
EPA ORD National Exposure Research Laboratory
nanogram
nanometer
nonylphenol
EPA ORD National Risk Management Research Laboratory
EPA Office of Research and Development
performance evaluation
parts per trillion
quality assurance
quality control
quality management plan
revolutions per minute
relative standard deviation
vn
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S standard deviation
SOP standard operating procedure
SPE solid phase extraction
TSA technical systems audit
USGS United States Geological Survey
WWTP wastewater treatment plant
Vlll
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification Program (ETV) to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The Program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders,
conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and
that the results are defensible.
The EPA's National Risk Management Research Laboratory (NRMRL) and its verification
organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under
ETV. The AMS Center recently evaluated the performance of the Abraxis 17|3-estradiol (E2)
magnetic particle enzyme-linked immunosorbent assay (ELISA) test kit for determining
endocrine-disrupting compounds (EDCs) in water.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This report provides results for
the verification testing of the Abraxis 17|3-estradiol (E2) magnetic particle ELISA test kit. The
following is a description of the test kit, based on information provided by the vendor.
The E2 magnetic particle ELISA Kit applies the principle of ELISA to determine 17|3-estradiol
in water samples. The E2 ELISA kit uses a colorimetric procedure to detect 17|3-estradiol.
The sample to be tested is added to a disposable test tube, and pre-incubated for 30 minutes with
paramagnetic particles attached with antibodies specific to 17|3-estradiol followed by the
addition of an enzyme labeled estradiol conjugate. At this point, a competitive reaction occurs
for a finite number of antibody binding sites between the estradiol which may be in the sample
and the enzyme labeled estradiol. The reaction is allowed to continue for ninety (90) minutes.
At the end of the incubation period, a magnetic field is applied to retain the para-magnetic
particles (with estradiol and labeled estradiol bound to the antibodies on the particles, in
proportion to their original concentration) in the test tube, and allow the unbound reagents to be
decanted. After decanting, the particles are washed with Washing Solution. A substrate is then
added and enzymatically converted from a colorless to a blue solution. After an incubation
period, the reaction is stopped by the addition
of diluted acid. The estradiol concentration
is determined by measuring the absorbance
of the sample solution with a photometer
(450 nm) and comparing it to the absorbance
of standards.
The E2 magnetic particle ELISA Kit (Figure
2-1) contains a 65 mL bottle of estradiol
antibody (rabbit anti-estradiol covalently
bound to paramagnetic particles suspended in
a buffered solution with preservatives and
stabilizers), a 35 mL bottle of horseradish
peroxidase-labeled estradiol analog diluted in
a buffered solution with preservative and
stabilizers, three 2.0 mL vials of estradiol
standard concentrations of 2.5, 7.5, 25.0 parts
per trillion (ppt) with preservatives and stabilizers, a 2.0 mL vial of estradiol (10 + 2 ppb) control
with preservative and stabilizers, a 35 mL bottle of estradiol-free solution is used as zero
Figure 2-1. Abraxis 17|3-Estradiol (E2) magnetic
particle ELISA Test Kit
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standard, a 65 mL bottle of hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine solution in an
organic base, a 60 mL bottle of diluted acid, a 250 mL bottle of preserved deionized water, and 3
boxes of 33 polystyrene tubes.
The E2 magnetic particle ELISA Kit measures 14 by 6 Vi by 3 1A inches. Final results and
calibration curves are printed from the photometric analyzer or sent directly to a laboratory
computer. List price is $350 for a 100-test kit. Other materials that are required but are not
provided with the E2 magnetic particle ELISA Kit are pipettes (including a repeating pipette for
the addition of reagents), a vortex mixer, a magnetic separation system, and a photometer
capable of reading at 450 nanometer (nm). These materials can be purchased separately or
rented.
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Chapter 3
Test Design and Procedures
3.1 Introduction
This verification test was conducted according to procedures specified in the Test/QA Plan for
Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits for the Quantitative
Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples1
Deviations to the test/QA plan were made due to unanticipated circumstances. As such, the test
procedures described in this chapter are a complete description of the actual test conditions.
Because of their potential to interfere with human, domestic animal, and wildlife reproduction,
EDCs are of increasing concern throughout the country. Several EPA Regions have undertaken
activities to monitor for these compounds, and several states are considering including
monitoring for EDCs in their regulatory programs. Presently, gas chromatography-mass
spectrometry (GC-MS), high performance liquid chromatography (HPLC), and liquid
chromatography-mass spectrometry (LC-MS) are being used for detecting these compounds.
However, immunoassay techniques, particularly ELISA, are becoming increasingly popular in
the field of environmental analysis due to their high sensitivity, ease of use, short analysis time,
and cost-effectiveness.
Immunoassay analytical detection is based on the capability of antibodies to specifically
recognize and form stable complexes with antigens. Immunoassays employ antibodies as
analytical reagents. In ELISA test kits, an enzyme conjugate competes with the chemical in the
sample for a limited number of binding sites on the antibody coated plate or particles. The extent
of color development is inversely proportional to the amount of chemical in the sample or
standard. The higher the concentration of a specific steroid or other EDC in the sample, the less
color reaction produced and recorded using a plate reader or tube photometer.
Testing was conducted with multiple collaborating laboratories, specifically the EPA Office of
Research and Development (ORD) NRMRL, EPA Region 3, and the United States Geological
Survey (USGS) Organic Geochemistry Research Laboratory in Kansas. The laboratory
participation was coordinated by EPA NRMRL, in collaboration with Battelle. Laboratory
names are removed, and simply stated as "Laboratory (or Lab) 1, 2, and 3" in the test results
section, since inter-laboratory comparison was not an objective of this report.
This verification test was conducted in four phases to evaluate the ability of the Abraxis E2
magnetic particle ELISA test kit to quantitate 17-p-estradiol (E2) in four different water matrices
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(see 3.3 Test Procedures), per the manufacturer protocols. More detailed information on the EDC
tested is provided in Table 3-1. EPA and USGS laboratories used the Abraxis E2 magnetic
particle ELISA kit (according to Table 3-2) to quantitate triplicate spiked samples for hormones
(E2), which were prepared and shipped by EPA NRMRL. As the more established method for
detecting these compounds, GC-MS served as the reference method2 for this test.
Table 3-1. Target Analyte
Analyte
Synonyms
CAS#
Use
(17p)-Estra-l,3,5(10)-
triene-3,17-diol
17-p-Estradiol (E2)
50-28-2 Naturally occurring
hormone
Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory
Responsibility
Sample Collection, Processing and Distribution
Test Kit Evaluation - E2 magnetic particle ELISA
Reference Measurement - E2 GC-MS
NRMRL
A/
A/
A/
Region 3
A/
USGS-KS
A/
The E2 magnetic particle ELISA test kit was verified by evaluating the following parameters:
• Precision
• Percent bias
• Matrix effects
• Operational factors.
Verification of the system was conducted from June to September 2008. Precision was
determined by measuring the relative standard deviation of average concentration values as
reported by the test kit. Percent bias was determined as positive or negative, with positive values
indicating that ELISA concentration was higher than the reference method and negative values
indicating that it was lower. Matrix effects were determined by comparing the percent bias
measurements for Phase I deionized (DI) water samples to the percent bias measurements for the
Phase II through IV matrix-water samples.
Operational factors were determined based on documented observations of the testing staff and
the Verification Test Coordinator. Operational factors were described qualitatively, not
quantitatively; therefore, no statistical approaches were applied to the operational factors.
3.2 Test Facilities
Laboratory analyses of the Abraxis E2 magnetic particle ELISA test kit were conducted in three
different collaborating laboratories by the laboratory staff. These laboratories were: EPA ORD
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NRMRL laboratory in Cincinnati, OH; EPA Region 3; and USGS - Kansas. Reference
measurements for E2 were preformed at EPA ORD NRMRL in Cincinnati.
3.3 Test Procedures
This verification test was conducted in four phases. Phase I consisted of a clean water sample
(DI water) spiked with a single concentration of E2, split into single samples, and submitted to
the ELIS A kit users in each collaborating laboratory to measure the concentration, in triplicate.
The split sample, as well as the un-spiked, matrix background sample, were also simultaneously
sent for reference GC-MS analysis of E2 and for various compounds which are known to cause
cross-reactivity with some ELISA kits. Phase II consisted of environmental surface water
samples subjected to the same spiking and splitting process as Phase I. Phase III consisted of a
complex matrix of wastewater treatment plant (WWTP) effluent samples subjected to the same
spiking and splitting process as Phase I and II. Phase IV consisted of a complex matrix of
WWTP influent samples, spiked and split as in previous phases. Details on the sample matrices,
spiking levels, and spiking procedures for each phase are provided in Section 3.3.1. All E2 spike
concentrations used in each phase of this verification test were based on real-world
concentrations found in environmental samples, per the procedure described in the test/QA plan.1
Background concentrations of E2 were measured for each matrix for each phase. These GC-MS
measurements were made to determine if any measureable amounts of E2 might exist in the
sample matrix prior to the addition of any sample spikes. If a detectable concentration was
found, this concentration was then added to the spiked amount of E2 to calculate the total
concentration for all spiked samples of a particular phase. Specific concentrations of E2, as
presented in Section 3.3.1, were spiked into the sample matrix for each phase, regardless of any
background concentrations of these compounds that may have been present in the collected
water. For Phase III and Phase IV, 4.03 ng/L and 4.00 ng/L of E2, respectively, were found in
the background matrix samples. The nominal concentration of each sample was then calculated
using the measured background concentration and the expected spiked concentrations for each
phase. The ELISA kit results from the various laboratories for the Abraxis E2 magnetic particle
test kit were compared to each other and compared to GC-MS results.
The E2 magnetic particle ELISA test kit was tested only under laboratory controlled conditions,
as opposed to field conditions which would have been more variable. The analyses were
performed according to the vendor's recommended procedures as described in the user's manual.
Simple cleanup procedures, as directed by the manufacturer of the test kit, were used for the four
different matrices. Each sample was analyzed both directly (prior to cleanup) and after solid
phase extraction (SPE) cleanup using the procedure detailed in the kit instructions and provided
in Section 3.3.2. Each sample for ELISA analysis was filtered through a 1 micron (um) glass
fiber filter prior to direct analysis and SPE. Calibration and maintenance of the technology
reader (i.e., photometer) was performed as specified by the vendor.
A US EPA NRMRL GC-MS standard operating procedure (SOP) was followed for reference
measurements. The GC-MS method for estrone (El), E2, estriol (E3), EE2, testosterone,
dihydrotestosterone, androstenedione, and progesterone operated within a concentration range of
2-50 ng/L. Samples for the GC-MS methods went through an extraction step to concentrate (or
dilute, depending upon the sample) to ensure the samples were within the method's analytical
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range.2 The procedures for preparing, storing, and analyzing the test samples are provided
below.
3.3.1 Test Sample Collection and Preparation
All sample bottles and glassware associated with hormone samples, including the glass carboy,
were cleaned and silanized using a procedure included in the test/QA plan.l All samples were
thoroughly mixed and were thus assumed to contain the same concentration. Samples were
spiked with E2 as one large stock solution and then split into smaller sub-samples in bottles. All
sample bottles were amber glass to prevent photodegradation of the analytes. All samples were
prepared and shipped by NRMRL, immediately after being made, in coolers on ice or freezer
packs to maintain a 4 degrees Celsius (°C) temperature. When samples were received by each
laboratory, the condition of the samples, i.e., temperature, broken bottles etc., was noted by the
receiving laboratory operator and the samples were then immediately placed in a refrigerator at
4°C until analyzed. Holding times of hormone samples are currently unknown; therefore, all
samples were either analyzed or solvent exchanged within 24 hours of receipt to reduce error
associated with analyte degradation during sample holding. All laboratories performing
quantitative analysis, ELISA or GC-MS, received split samples from the same bulk sample.
Each laboratory that participated in the ELISA analysis received one 2.5 L spiked sample plus
one 500 mL DI water method blank. The laboratory that performed the reference analysis
received one 4L spiked sample and one 1 L DI water method blank to be processed by the GC-
MS method.
3.3.1.1 Phase I Samples
A sample of DI water was collected in a cleaned, 20 L, glass carboy from the USEPA laboratory
in Cincinnati, Ohio. The water was spiked with E2 to a concentration of 10 ng/L. This
concentration was selected because it is on the higher end of the range of concentrations
expected to be encountered in a real-world situation and is representative of the anticipated mid-
range of the test kit. The carboy was thoroughly mixed, by inserting a stir bar and stirring on a
stir plate at 300 revolutions per minute (rpm) for 2 hours, to ensure homogeneous concentrations
of the analyte throughout the carboy. One 2.5L spiked sample was collected for each
participating laboratory as well as one 4L sample for each reference laboratory. DI water blanks
were also prepared and shipped in separate 500 mL bottles. The blank samples were analyzed
both directly (DIR) and after SPE but only in two wells (or test tubes) on the kits as opposed to
three wells (or test tubes) for all other samples. Before spiking, the DI water was sampled and
analyzed by GC-MS to confirm the background levels of E2. Samples of the spiked mixtures
were taken and the concentrations of these samples and blank were determined using the Abraxis
E2 magnetic particle ELISA test kit and GC-MS.
3.3.1.2 Phase II Samples
Grab samples of stream water were collected in three, clean, five gallon buckets from the South
Hasha Tributary to Eastfork Lake in Clermont County, Ohio. The tributary was accessed from
where it crosses Williamsburg-Bantam Road. Before the stream water was spiked, a single
sample of the collected stream water was taken, split into triplicate aliquots, and analyzed by
GC-MS to confirm the background levels of E2. Background levels of E2 measured in the
samples were added to the spiked concentration of E2 once results were obtained. Next, a
7
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cleaned, 20 L, glass carboy was used to collect 20 L of the stream water, which was then spiked
to contain a 10 ng/L concentration of E2. The carboy was thoroughly mixed by inserting a stir
bar and stirring on a stir plate at 300 rpm for 2 hours, to ensure homogeneous concentration of
the analyte throughout the carboy. Split samples were taken, as noted for Phase I. DI water
method blanks were filled with DI water at the same time as the stream water in the 20 L
carboys.
3.3.1.3 Phase III Samples
Grab samples of final effluent wastewater were collected in three, clean, five gallon buckets from
the Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the
sample was transported back to the NRMRL laboratory, the effluent was measured and then
transferred into a clean, 20 L carboy. Before spiking, a single sample of the effluent was taken,
split into triplicate aliquots, and analyzed by GC-MS to confirm the background levels of E2. In
a cleaned, 20 L, glass carboy, 20 L of WWTP effluent was prepared containing 10 ng/L of E2.
The carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300 rpm for
2 hours to ensure homogeneous concentration of the analyte throughout the carboy. Split
samples were collected as noted for Phase I.
3.3.1.4 Phase IV Samples
Grab samples of influent wastewater were collected in three, clean, five gallon buckets from the
Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the sample
was transported back to the NRMRL laboratory, the influent was measured and transferred into a
20 L carboy. Before spiking, a single sample of the influent was taken, split into triplicate
aliquots, and analyzed by GC-MS to confirm the background levels of E2. In a cleaned, 20 L,
glass carboy, 20 L of WWTP influent was prepared containing 10 ng/L concentration of E2. The
carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300 rpm for 2
hours to ensure homogeneous concentration of the analyte throughout the carboy. Split samples
were collected as noted in Phase I.
3.3.2 Test Sample Analysis Procedure
The ELISA test kit users followed simple cleanup procedures as directed in the vendor's
instructions. The 2.5 L sample was split into three 500 mL aliquots. Each of the three aliquots
was analyzed by direct analysis utilizing only glass fiber filter (GFF) cleanup and by utilizing
GFF cleanup and SPE. Each aliquot sample was transferred in triplicate to the ELISA kits for
quantification, per the test kit protocols. The cleanup procedures are described below.
Each sample for ELISA analysis was filtered through a l|Jm GFF prior to direct analysis on the
E2 magnetic particle ELISA test kit and for SPE clean-up. After filtering, one 1600 |JL aliquot
was removed and transferred to three tubes (250 |JL for each tube) in the E2 magnetic particle
test kit. After all aliquots were removed for direct analysis, three 500 mL aliquots were removed
from the filtered sample for SPE. These three aliquots were treated as three independent
samples. SPE directions entitled "Extractions for EE2 from Water Sample for ELISA", which
were based on the vendor's protocols and summarized by EPA NRMRL, were followed.1 The
SPE protocol consists of the following steps:
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1. Filter 500 mL of the sample, or the remainder of liquid in the sample bottle noting the volume
for later calculation, through l|j,m glass fiber filter.
2. Rinse a CIS cartridge with 5 mL of methanol and then 10 mL of distilled water at a flow rate
not exceeding 20 mL/min (preconditioning).
3. Pour the filtered sample through the CIS SPE cartridge at a flow rate, no faster than 20
mL/min.
4. Wash the cartridge with 5mL of distilled water (up to 20 mL/min). Keep suctioning for about
a minute to dry the cartridge.
5. Wash the cartridge with 5mL of hexane (up to 20 mL/min).
6. Elute the analyte with 5mL of dichloromethane at a rate, no faster than 3 mL/min.
7. Evaporate the solvent with nitrogen gas to dryness.
8. Add ImL of 100% methanol to the residue and stir the mixture with a vortex mixer. To adjust
the content to 10% methanol (volume/volume) add 9 mL of distilled water for a total volume of
lOmL.
After the SPE column, the E2 sample is concentrated 50 times that of the original spike
concentration. Samples were reconstituted with lOmL of a 10% methanol solution. From this
reconstitution, 20 microliter (|jL) was removed and added to 1980 |jL of DI water. For the
spiked samples, this process effectively reduced the spiked concentration by a factor of 100,
down to an expected level of 5.05 ng/L. All reconstituted samples were transferred to three tubes
(250 |jL for each tube) according to the manufacturer's instructions. Samples were quantified by
reading at 450 nm using a tube style spectrophotometer following the manufacturer's
instructions. The general steps for operating the ELISA test kit that were followed during this
verification test are provided below.
The Abraxis ELISA E2 magnetic particle kit assay procedure consists of the following steps:
1. Add 250 |jL of the appropriate standard, control, or sample. Each standard will be added to
two tubes while each sample will be added to four tubes.
2. Mix the E2 antibody coupled paramagnetic particles thoroughly and add 500 |jL to each tube.
3. Vortex for 1 to 2 seconds minimizing foaming.
4. Incubate for 30 minutes at room temperature.
5. Add 250 |jL of estradiol enzyme conjugate to each tube.
6. Vortex for 1 to 2 seconds minimizing foaming.
7. Incubate for 90 minutes at room temperature.
8. Separate in the magnetic separation system for 2 minutes.
9. Decant and gently blot all tubes briefly in a consistent manner.
10. Add ImL of washing solution to each tube and allow them to remain in the magnetic
separation unit for 2 minutes.
11. Decant and gently blot all tubes briefly in a consistent manner.
12. Repeat steps 10 and 11 an additional time.
13. Remove the rack from the separator and add 500 (jL of color solution to each tube.
14. Vortex for 1 to 2 seconds minimizing foaming.
15. Incubate for 20 minutes at room temperature.
16. Add 500|jL of stopping solution to each tube.
17. Add ImL washing solution to a clean test tube. Use as a blank in step 18.
18. Read results at 450 nm within 15 minutes after adding the stopping solution.
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Chapter 4
Quality Assurance/Quality Control
QA/Quality Control (QC) procedures were performed in accordance with the quality
management plan (QMP) for the AMS Center3 and the test/QA plan for this verification test.1
Test procedures were as stated in the test/QA plan1; however deviations to the test/QA plan were
made due to unanticipated circumstances. As such, the test procedures described in Chapter 3 are
a complete description of the actual test conditions. The statistical calculations intended for
analysis of the test kit results were also changed. This deviation is further described in Chapter
5. This change had no impact on the quality of the results. QA/QC procedures and results are
described below.
4.1 Quality Control Samples
Steps taken to maintain the quality of data collected during this verification test included
analyzing specific quality control samples for both the reference method (GC-MS) and the test
kits.
4.1.1 GC-MS Method Blank and Surrogate Spike Results
This verification test included a comparison of the Abraxis E2 magnetic particle ELISA test kit
results to those of the GC-MS reference method for E2. Samples analyzed for each phase
included performance evaluation (PE) samples, test samples, background samples, and blank
samples. The quality of the reference measurements was evaluated by adherence to the
requirements of the GC-MS method for this compound, including requirements for method
blanks (MBs), instrument solvent blanks, and surrogate spikes, as indicated in the test/QA plan1.
Method blank samples were analyzed to ensure that no sources of contamination were present. If
the analysis of a method blank sample indicated a concentration above five times the method
detection limit, contamination was suspected. Any contamination source(s) were corrected and
samples were reanalyzed or flagged before proceeding with the analyses. Surrogate spikes were
also included in each sample. Average acceptable recoveries for these samples were between 60
and 140%. Samples outside of the acceptable range were generally flagged and/or reanalyzed.
D4-EE2 was used as a surrogate standard for the GC-MS analysis of E2 in the samples. No levels
of E2 were detected in any of the reference method blank samples.
Surrogate recoveries in Phase I - IV samples varied across phases. Phase I surrogate recoveries
ranged from 59 - 96% and averaged 85 ± 10% across 13 samples. All recoveries were
considered in the acceptable range. Phase II recoveries ranged from 77 to 155% and averaged
10
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132 ± 25% over 11 samples. Surrogate recoveries for six of the samples were outside of the
acceptable range. Compared to the surrogates, the peak shapes for the target analytes were good
and the baselines were clean in the chromatogram. Phase III surrogate recoveries ranged from
154% to 197% and averaged 176 ± 14% over 8 samples. Surrogate recoveries for all samples
were outside of the acceptable range for Phase III. Phase IV surrogate recoveries ranged from
61% to 93% and averaged 76 ± 10% over 11 samples. Surrogate recoveries for all Phase IV
samples were within the acceptable range.
4.1.2 Test Kit Method Blanks
Method blank samples were run in duplicate using both direct analysis and after SPE clean-up
with each set of samples for all four phases. Method blank samples were unspiked DI water.
Because concentrations for samples analyzed with the test kit are calculated based on the
interpolations from a curve constructed from the standards run with each batch of samples, it is
possible to obtain concentration values for all samples. However, the E2 magnetic particle test
kit has a stated method detection limit (MDL) of 1.5 ng/L. Based on this MDL, it is assumed
that sample concentrations lower than this level cannot be reliably determined or reported. Thus,
any samples, including method blank samples, with concentrations lower than the
manufacturer's stated MDL were considered non-detects.
The E2 magnetic particle test kit was evaluated by three laboratories (see Table 3-2).
Concentrations of E2 were not detected in any of the method blank samples from two of the
participating laboratories. For the Region 3 results, levels of E2 above the MDL were not
detected in Phase II - IV method blanks. However, during the analysis of Phase I samples,
concentrations of E2 above the MDL were found for both sets of duplicate method blanks for
one test kit. Two Abraxis E2 magnetic particle ELISA test kits were evaluated by this laboratory
for each phase of testing. The second test kit operated during Phase I did not show any
detectable levels of E2 in any of the method blanks analyzed. All method blanks for this phase
came from the same initial sample. Phase I sample concentration values were on average twice
as high for one kit versus the other between the two kits operated by Region 3. This appears to
be related to differences in the standard curve generated for each kit, which could also impact the
results for the method blanks. No signs of contamination were apparent in the results.
4.2 Audits
Three types of audits were performed during the verification test: a PE audit of the reference
method measurements (GC-MS analyses), a technical systems audit (TSA) of the verification
test performance, and a data quality audit. Audit procedures are described further below.
4.2.1 Performance Evaluation Audit
A PE audit was conducted to assess the quality of the reference method measurements (GC-MS
analyses) made in this verification test. The reference method PE audit was performed by
supplying an independent second standard solution of E2 prepared from a different source than
that used in verification testing. The PE audit samples were analyzed in the same manner as all
other samples and the analytical results for the PE audit samples were compared to the nominal
concentration. The target criterion for this PE audit was agreement of the analytical result within
11
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30% of the expected concentration. This audit was performed once during each phase of testing.
Table 4-1 shows the percent error results for the PE samples for each phase. The percent error
was calculated based on the difference between the actual and expected E2 concentrations
divided by the expected concentration. The E2 PE audit samples were within 30% of the
expected concentration for Phases I, II and IV, while Phase III PE audit samples were outside of
this agreement range (94% error). The PE results were consistent with the surrogate results
presented in Section 4.1.1, where Phases I and IV were within specifications; Phase II was
slightly outside, and Phase III did not meet requirements. The PE audit sample results were also
similar to the GC-MS sample results for Phase III. It is not certain what caused the poor
performance of the Phase III PE samples; however, these results suggest that the pre-treatment
method for GC-MS analysis may have been insufficient and that some matrix effects may have
been present. This could have impacted the comparison of the ELISA test kit results to the GC-
MS data for Phase III. No adjustments were made to the standards nor were PE audit samples
reanalyzed based on these results. It does not appear that the reference laboratory results for
Phase II and III were overall of lower quality than Phases I and IV, since reference results were
fairly consistent within phases for the actual samples (e.g., see Table 6-2). However, the test kit
results were more comparable to the expected spiked concentrations (Table 6-3 and Table 6-4)
than to the GC-MS results (Table 6-2).
Table 4-1. PE Audit Sample Results
Phase
1
II
III
IV
Expected
Concentration (ng/L)
10
10
10
10
Actual
Concentration (ng/L)
9.03
7.43
19.4
8.17
% Error
-10
-26
94
-18
4.2.2 Technical Systems Audit
The Battelle Quality Manager performed a TSA twice during this verification test. Because the
testing was taking place in multiple laboratories across the country, Battelle's Quality Manager
visited only two laboratories for in-person TSAs. Battelle conducted TSAs at the Cincinnati, OH
facility on July 23-24, 2008 and at the Fort Meade, MD facility on July 31, 2008. All TSA
findings were reported to the Verification Test Coordinator.
The purpose of this audit was to ensure that the verification test was being performed in
accordance with the AMS Center QMP,3 the test/QA plan,1 and the GC-MS SOP2 used during
this verification test. In the TSA, the Battelle Quality Manager reviewed the reference methods
used, compared actual test procedures to those specified or referenced in test/QA plan, and
reviewed data acquisition and handling procedures. The Battelle Quality Manager also toured the
laboratory where verification and reference testing were taking place, inspected sample chain of
custody (COC) documentation, reviewed technology-specific record books, checked standard
certifications and technology data acquisition procedures, and conferred with technical staff. A
TSA report was prepared, including a statement of findings and the actions taken to address any
12
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adverse findings, and a copy of Battelle's ISA report was sent to the EPA AMS Center QA
Manager. No adverse findings were reported. The TSA findings were communicated to
technical staff at the time of the audit.
4.2.3 Data Quality Audit
At least 10% of the data acquired during the verification test were audited. Battelle's Quality
Manager traced the data from the initial acquisition, through reduction and statistical analysis, to
final reporting to ensure the integrity of the reported results. All calculations performed on the
data undergoing the audit were checked. Minor transcription errors and errors due to rounding
were identified and corrected before the results were used for the calculations described in
Chapter 5.
4.3 QA/QC Reporting
Each audit was documented in accordance with Section 3.3.4 of the AMS Center QMP.3 Once
the audit reports were prepared, the Battelle Verification Test Coordinator ensured that a
response was provided for each adverse finding or potential problem and implemented any
necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up
corrective action was taken. The results of the TSA were submitted to the EPA.
4.4 Data Review
Records generated in the verification test received an independent internal review before these
records were used to calculate, evaluate, or report verification results. Table 4-2 summarizes the
types of data recorded. Data were reviewed by a Battelle technical staff member involved in the
verification test. The person performing the review added his/her initials and the date to a hard
copy of the record being reviewed.
13
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Table 4-2. Summary of Data Recording Process
Data Recorded
Dates times and
details of test
events
Technology
calibration
information
Technology
readings
Sample collection
and reference
method analysis
procedures,
calibrations, etc.
Reference method
results
Where Recorded
Laboratory record
books or data
recording forms, or
electronically
Laboratory record
books, data
recording forms, or
electronically
Recorded
electronically or
manually by the
operator or
electronically by
the technology
reader, as
appropriate
Laboratory record
books, chain-of-
custody,
electronically, or
other data
recording forms
Electronically from
reference
measurement
technology
How Often
Recorded
Start/end of test
procedure, and at
each change of a
test parameter or
change of
technology status
At technology
reader calibration
or recalibration, as
applicable
Each sample and
QC analysis
Throughout
sampling and
analysis processes
Every sample or
QC analysis
By Whom
Participating
laboratories
Participating
laboratories
Participating
laboratories
Participating
laboratories
Participating
laboratories
Disposition of
Data
Used to organize
and check test
results; manually
incorporated into
data spreadsheets
as necessary
Incorporated into
verification report
as necessary
Converted to or
manually entered
into spreadsheets
for statistical
analysis or
comparisons
Retained as
documentation of
sample collection
or reference
method
performance
Transferred to
spreadsheets for
calculation of
results and
statistical analysis
or comparisons
14
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Chapter 5
Statistical Methods
The statistical methods used to evaluate the quantitative performance factors listed in Section 3.1
are presented in this chapter. Qualitative observations were also used to evaluate verification test
data.
Per the test/QA plan,l repeatability and reproducibility were intended to be calculated as
performance parameters for this verification test. However, after further discussion with EPA,
and in agreement with EPA, it was determined that higher level summary statistics provided a
better synopsis of the test kit results. Thus, the mean and relative standard deviations (precision)
were calculated for the test kit results.
5.1 Precision
The standard deviation (S) of the results for the replicate analyses of the same sample was
calculated as follows:
(1)
/ * A ^ = |
where n is the number of replicate samples, M is the ELISA test kit measurement for the k
K.
sample, and Mis the average ELISA test kit measurement of the replicate samples. The precision
for each sample is reported in terms of the relative standard deviation (RSD), which was
calculated as follows:
RSD(%) =
S
M
xlOO
(2)
The RSD was calculated for each laboratory that participated in the verification test and for each
test kit that was tested. The RSD was also calculated across all laboratories and test kits for each
phase of testing.
15
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5.2 Percent Bias
Percent bias was calculated as a percentage for each measurement in each phase for each kit
using Equation 3 :
%Bias = [ — -1] xlOO (3)
where y = 1, 2, 3 denotes the laboratory, / = 1, 2 denotes the ELISA test kit within laboratory, n =
1, 2 denotes the reference method, Xjt is the ELISA concentration for the/ laboratory and the i'
test kit, yn is the concentration of the reference method GC-MS or the concentration of the spike.
Ideally percent bias results will be within ±25%.
5.3 Matrix Effects
Matrix effects were examined by comparing the percent bias measurements for the Phase IDI
water samples to the percent bias measurements for the Phase II - IV samples. Percent bias was
determined as described in Section 5.2.
General observations of potential matrix effects, such as false negatives, if observed, were
documented but were not used in statistical calculations. False negatives were defined as a
negative (zero) response in a sample that is spiked with contaminant at a detectable
concentration.
General observations on potential cross-reactivity were documented. Blank samples of each
matrix were evaluated by GC-MS to determine background levels of the compounds with which
the kits have cross-reactivity, as stated by the vendor.
Percent recovery results were calculated on a per-sample and per-phase basis and were based on
the expected spiked concentration of the analyte in each sample matrix. Percent recovery was
calculated using the Equation 4:
^
% Recovery = — x 100 (4)
E
Where A is the actual ELISA test kit measurement and E is the expected concentration. The
expected concentration includes the known spike concentration as well as any detected
background levels of E2 in the matrix water (see Section 3.3). Percent recovery results are
presented to provide another measure of test kit performance to the end user. Ideal percent
recovery values are near 100%.
A comparison of the ELISA results generated with and without the use of SPE cleanup was also
performed. This evaluated whether the use of the more involved SPE cleanup procedure was
necessary/warranted with the ELISA test kits. Percent bias calculations based on the actual and
expected spike concentrations and a t-test were used to evaluate these results.
16
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5.4 Operational Factors
Operational factors were determined based on documented observations of the testing staff.
Operational factors are described qualitatively, not quantitatively; therefore, no statistical
approaches were applied to the operational factors.
17
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Chapter 6
Test Results
The results of the verification tests of the Abraxis E2 magnetic particle ELISA test kit are
presented below for each of the performance parameters.
6.1 Precision
The relative standard deviation (RSD) is used as a means of evaluating the precision of the
ELISA test kit. Three laboratories operated the E2 magnetic particle ELISA test kit. Two
laboratories (Lab 1 and Lab 2) ran identical samples on two separate test kits (kit "a" and kit
"b"). Lab 3 ran a single kit. Table 6-1 presents the resulting RSD for each participating
laboratory and test kit along with the overall average concentrations per phase of E2 found using
the E2 magnetic particle ELISA test kit for all analyses. RSD values are also presented across all
results for each phase. Expected concentrations for both direct and SPE analysis are presented
for each phase in Table 6-2.
RSDs ranged from 5 to 33% for direct analysis and 3 to 92% for SPE analysis. The RSDs across
all analyses were similar for Phase IV using both direct and SPE analysis (13% and 16%,
respectively). For both the direct and SPE analysis, the RSD for Phase I was greater than that in
Phase IV. For both direct and SPE analysis, Phase I (which was performed with DI water)
samples had the highest overall RSD. Average concentrations were similar between Phases I
and II using direct analysis and between Phases III and IV. Resulting concentration relationships
between phases were the same for SPE analysis, though average concentrations for Phases III
(24.24 ng/L) and IV (17.77 ng/L) samples differed by approximately 7 ng/L.
18
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Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD)
Results"
Phase I
Phase II
Phase
Phase IV
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Avg
Cone (ng/L)
10.10
13.64
15.96
8.11
10.24
10.17
10.43
10.03
10.71
8.13
out of rangeb
out of rangeb
30.49
32.45
47.75
22.23C
18.84C
32.92
37.09
35.54
Direct
Overall Avg
RSD Cone (ng/L) RSD
17%
28%
12% 11.61 30%
14%
11%
12%
19%
10% 9.89 19%
10%
33%
8% 36.90 24%
11%
12%
NA
NA
7% 34.17 13%
5%
5%
Avg
Cone (ng/L)
4.61
5.40
8.04
3.32
4.81
5.45
5.46
5.58
6.44
1.42
22.09
22.64
22.34
23.33
29.62
17.44
18.02
16.21
16.16
21.08
SPE
Overall Avg
RSD Cone (ng/L)
22%
26%
40% 5.24
49%
37%
15%
12%
11% 4.86
7%
92%
9%
8%
13% 24.24
4%
12%
18%
20%
3% 17.77
5%
9%
RSD
47%
41%
16%
16%
The average concentration and RSD are based on all replicates within the detectable range of the test kit for direct
measurements (i.e., no cleanup) and for analyses which included SPE cleanup.
Test kit results were above the upper end of the test kit's range.
° Only one replicate was used for the average. All other results were above the upper end of the test kit's range.
Table 6-2. Expected Concentrations for Each Phase
Phase
I
II
III
IV
Expected Concentration
(ng/L)
Direct
10.09
10.09
14.12
14.09
SPE
5.05
5.05
7.06
7.05
19
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6.2 Percent Bias
Bias is a systematic error that causes measurements to err in one direction, either high or low.
For this section, percent bias was calculated relative to the GC-MS reference method results. A
positive percent bias indicates that the ELISA test kit concentration is higher than the reference
method, while a negative percent bias indicates that the ELISA test kit concentrations are lower
than the reference method. Table 6-3 presents the percent bias results.
Table 6-3. ELISA Test Kit Percent Bias vs. GC-MS
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Phase I
DIRECT
6
43
67
-15
7
SPE
-3
13
69
-30
1
Phase II
DIRECT
-20
-18
-21
-16
-36
SPE
-15
-14
-13
1
-78
Phase III
DIRECT
out of range3
out of range3
65
75
158
SPE
139
145
142
152
220
Phase IV
DIRECT
141
104
257
302
285
SPE
278
290
251
250
357
Test kit results were above the upper end of the test kit's range.
Phase I percent bias results varied for the direct and SPE analyses. All results for the Abraxis E2
magnetic particle ELISA test kit were biased high for Phase I except for those from Lab 2 kit b
and the SPE samples from Lab 1 kit a. The resulting percent biases for these samples were
negative, indicating that the test kits results were biased low in comparison to the reference
analysis results. All Phase II results were biased low except for the SPE analysis of the Lab 2 kit
b sample, which had a small positive percent bias. Phase III and IV results were biased high for
both direct and SPE analysis. Phase III SPE percent bias results were higher than those for direct
analysis of samples for that phase. Phase IV SPE percent bias results were also generally higher
than those for direct analysis. All percent bias results for Phase IV were >100%, indicating a
significant bias of the test kit results to be higher than the GC-MS results. The percent bias was
<5% in only three cases across all phases. All three of those cases were for SPE samples in
Phases I and II. For Phases II - IV, Lab 3 results had the highest percent bias in most cases.
Only the direct samples for Phase IV did not have the highest percent bias of the group. In
general, for direct and SPE analysis, the bias tended to increase from Phase I to Phase IV, with
Phase IV having the highest percent bias for all laboratories.
For comparison, average concentrations, RSD, and percent bias for the GC-MS measurements
with regard to the expected concentration are presented in Table 6-4 for each phase. RSD values
were less than 30% for all phases, and percent bias results were within ±35% of the expected
concentration. These results demonstrate that the GC-MS results were biased high (in Phases II
and III) and low (in Phases I and IV) as compared to the expected concentration.
20
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Table 6-4. GC-MS Average Concentration, RSD, and Percent Bias Results
Phase
1
II
III
IV
Average
Cone (ng/L)
9.53
12.76
18.50
9.26
RSD
10%
3%
1%
7%
% Bias
(vs. Expected Cone)
-6
27
31
-34
6.4 Matrix Effects
To understand how the matrix of each phase of testing might have affected the results, percent
bias and percent recovery were calculated for the test kit results in comparison to the expected
spiked concentration of E2. A positive percent bias indicates that the ELISA test kit
concentration is higher than the expected spike concentration, while a negative percent bias
indicates that the ELISA test kit concentrations are lower than the expected spike concentration.
Table 6-4 presents the percent bias results. No false negatives were observed during this
verification test.
Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Phase I
DIRECT
0
35
58
-20
1
SPE
-9
7
59
-34
-5
Phase II
DIRECT
1
3
-1
6
-19
SPE
8
8
11
28
-72
Phase III
DIRECT
out of range3
out of range3
116
130
238
SPE
213
221
216
230
320
Phase IV
DIRECT
58
34
134
163
152
SPE
148
156
130
129
199
Test kit results were above the upper end of the test kit's range.
Phase III and IV percent bias results are positive for both direct and SPE analysis across all
participating laboratories. These phases also generally have the highest bias of all of the phases.
Phase I and II have a mix of positive and negative bias. In some cases, the results from tests on
multiple test kits by the same laboratory produced opposing percent bias results. For example,
Phase I direct analysis results for Lab 2 kit a are positive, while those for kit b are negative. The
percent bias determined for the SPE samples tended to be higher than that for the direct analysis
in all phases. Laboratory 1 had positive bias across all phases and samples except for one
sample. These positive bias results varied across phases. The percent bias results for Phases III
and IV tend to be clustered closer together than those for Phases I and II. In comparing the
results of Table 6-4 to Table 6-3 where the test kit results were compared to the GC-MS results,
in general the test kits results were closer to the expected concentrations than they were to the
GC-MS results.
As another measure of accuracy, percent recovery results, comparing the test kit results against
the expected spiked concentration, were also calculated on a per sample and per phase average
basis. Table 6-5 presents these results.
21
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Table 6-5. Percent Recovery
%
Recovery
LAB
Lab 1 kit a
Lab 1 kit b
Lab 2 kit a
Lab 2 kit b
Lab 3
Average
Phase I
DIRECT
100
135
158
80
101
115
SPE
91
107
159
66
95
104
Phase II
DIRECT
101
103
99
106
81
98
SPE
108
108
111
128
28
97
Phase III
DIRECT
out of range3
out of range3
216
230
338
261
SPE
313
321
316
330
420
340
Phase IV
DIRECT
158
134
234
263
252
208
SPE
248
256
230
229
299
252
Test kit results were above the upper end of the test kit's range.
Percent recoveries averaged close to or well above 100% for all phases. For Phases III and IV,
average percent recoveries were >200% for each phase. Phases I and II recoveries were closer to
100%. For Phases III and IV, percent recoveries for samples evaluated using SPE clean-up were
larger than those found using direct analysis, though the difference was not statistically
significant. The percent recoveries for Phases I and II were within the range of acceptable
recoveries for the GC-MS reference method.
A comparison of the ELISA results generated with and without the use of SPE was performed.
Throughout this section, comparisons between results generated through direct analysis and with
the use of SPE have been explored using the percent bias and percent recovery results. For this
discussion, comparison of the two techniques will be determined through the use of t-tests. T-
tests were conducted using the percent bias spike results. T-tests were conducted on each phase.
In all cases, the t-test results indicated that the differences in biases found using results generated
with and without the use of SPE were not statistically significant (p > 0.05).
Some ELISA kits will react with compounds similar to the target compound, known as cross-
reactivity. The Abraxis E2 magnetic particle ELISA E2 magnetic particle test kit will react with
known percent reactivities to multiple hormones. During each phase of the study, some of the
compounds with which the test kit has cross-reactivity were measured alongside background
levels of the kit's target compound in that matrix by GC-MS. For some of the compounds for
which there is known cross-reactivity with the E2 magnetic particle ELISA test kit, there are no
established analytical methods available by GC-MS at these concentrations and in these
matrices. Therefore some error will have to be accepted from influence of cross-reactive
compounds that cannot be identified via GC-MS. According to the test kit instructions, there is
expected to be minimal error from these compounds compared to the primary target compound.
Table 6-6 lists concentrations found in each matrix blank sample from each phase along with the
known percent reactivities for the cross-reactive compounds to the E2 magnetic particle ELISA
test kit that were measured during this verification test. Unfortunately, matrix blank samples
used for background analysis were not analyzed on the Abraxis E2 magnetic particle ELISA test
kit. Because of this, the potential for cross-reactive compounds present in the matrix for each
phase to interfere with the test kit results cannot be truly evaluated. Significant amounts of
estrone were found in Phase III and IV samples. With a 50% cross reactivity, it is likely that the
presence of this compound could have substantially impacted the ELISA test kit results.
However, the full extent of this impact is not known.
22
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Table 6-6. Concentrations of Cross-Reactive Compounds11
Steroid Hormones
D i hyd rotestoste ro n e
Estrone (E1)
Testosterone (TEST)
Androstenedione (AND)
Estriol (E3)
Progesterone
Concentration (ng/L)
Phase I
ND
ND
ND
ND
ND
ND
Phase II
ND
1.4
ND
ND
ND
ND
Phase III
ND
44.9
1.7
2.9
1.4
ND
Phase IV
43.7
16.8
23.2
93.4
11.6
4.1
Cross-Reactivity
(%)
<0.01
50
<0.01
<0.01
0.3
<0.01
aND = not detected
6.5 Operational Factors
In general, training is needed to effectively and properly operate ELISA test kits. The vendor
trained staff on the operation of the test kit, but these trained staff were, in some cases, not
available for the verification test because of testing delays and staff turn over. Therefore, staff
that operated the test kits during the verification test may not have been trained by the vendor.
Operational concerns or issues were not reported from any of the three participating laboratories.
The test kit instructions were readily followed by each of the operators. Operation of the test kit
from the introduction of the sample until the reaction was stopped and the results were read, took
approximately 3-3.5 hours. Preparation time was required prior to the introduction of the sample
to allow all reagents time to come to room temperature before using them. Calibrated pipettes, a
vortex mixer, a magnetic separation system, and a photometer capable of reading at 450 nm are
required for the operation of the test kit, but are not supplied with the test kit. Any GFF or SPE
equipment used with the samples was also not supplied with the test kit. For at least one
laboratory, the concentration step after SPE was time consuming, requiring 1-5 hours.
Each purchased test kit is capable of conducting 100 tests and costs $350. For comparison, GC-
MS analyses of these samples are estimated to cost between $500 and $900 per sample.l
23
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Chapter 7
Performance Summary
The ability of the Abraxis E2 magnetic particle ELISA test kit to detect E2 in water was
evaluated using four different water matrices. The test kit was operated by three different
laboratories with and without the use of SPE. The test kit results were evaluated against the
expected spike concentrations and the reference measurements of the same samples made using
GC-MS.
Relative standard deviations (RSDs) ranged from 5 to 33% for direct analysis and 3 to 92% for
SPE analysis. The RSDs across all analyses were similar for Phase IV using both direct and SPE
analysis (13% and 16%, respectively). For both the direct and SPE analysis, the RSD for Phase I
was greater than that in Phase IV. For both direct and SPE analysis, Phase I (which was
performed with DI water) samples had the highest overall RSD. Average concentrations were
similar between Phases I and II using direct analysis and between Phases III and IV. Resulting
concentration relationships between phases were the same for SPE analysis, though average
concentrations for Phases III (24.24 ng/L) and IV (17.77 ng/L) samples differed by
approximately 7 ng/L.
Percent bias, as compared to the GC-MS reference analysis results, was varied for both sample
results using both direct and SPE analysis. There did not appear to be any consistent trend in
bias across phases or laboratories. Percent bias, as compared to the expected spiked E2
concentration, was also mixed with no clear trends. T-test results indicated that the bias found
using results generated with and without the use of SPE were not significantly different
(p > 0.05).
No false negatives were observed during this verification test. Average percent recoveries were
close to or well above 100% for all phases. For Phases III and IV, average percent recoveries
were >200% for each phase. Phases I and II recoveries were closer to 100%. The percent
recoveries for Phases I and II were within the range of acceptable recoveries for the GC-MS
reference method.
Operational concerns or issues were not reported from any of the three participating laboratories.
The test kit instructions were readily followed by each of the operators. Operation of the test kit
from the introduction of the sample until the reaction was stopped and the results were read took
approximately 3-3.5 hours. Preparation time was required prior to the introduction of the sample
to allow all reagents time to come to room temperature before using them. Calibrated pipettes, a
vortex mixer, a magnetic separation system, and a photometer capable of reading at 450 nm are
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required for the operation of the test kit but are not supplied with the test kit. OFF and SPE
equipment used with the samples was not supplied with the test kit. For at least one laboratory,
the concentration step after SPE was time consuming, requiring 1-5 hours. Each purchased test
kit is capable of conducting 100 tests and costs $350.
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Chapter 8
References
1. Test/QA Plan for Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits for
the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous
Phase Samples, Battelle, Columbus, Ohio, July 1, 2008.
2. U.S. EPA NRMRL Standard Operating Procedure (SOP) for the Analysis of Steroid
Hormones in Aqueous Samples, QA ID 503-P3-0, 09/29/05.
3. Quality Management Plan (QMP)for the ETV Advanced Monitoring Systems Center,
Version 6.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, November 2005.
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