£%	United States
Environmental Protection
kl # % Agency
Office of Water	EPA 821 R 16 010
www.epa.gov	December 2016
Validation of SPE Products
and Associated Procedures
with EPA Method 625.1

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Please address questions or comments to:
Clean Water Act Methods Team
Engineering and Analytical Support Branch/EAD (4303T)
Office of Science and Technology
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue, NW
Washington, DC 20460
www.epa.gov/cwa-methods/forms/contact-us-about-cwa-analytical-methods

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Validation of SPE Products and Associated Procedures with Method 625.1
December 2016
Validation of SPE Products and Associated Procedures with EPA Method 625.1
This document summarizes the requirements that EPA has developed in order for laboratories to utilize
solid-phase extraction (SPE) as an extraction technique in EPA Method 625.1. It also provides the
requirements that vendors of SPE products and equipment would have to meet in order to minimize the
effort required of each individual laboratory customer as described in EPA Method 625.1, Section
8.1.2.1.1. The document is divided into two sections, based on whether the product is to be applied to
analytes in Method 625.1 that have existing QC acceptance criteria, that is, those analytes listed in Tables
1 and 2 and any of the analytes listed in Table 3 for which QC acceptance criteria are specified in Table 6,
or if it is to be applied to analytes in Method 625.1 without existing QC acceptance criteria.
Note: Disk-based solid-phase extraction procedures developed by the 3M Corporation and approved by
EPA as an ATP are included in EPA Method 608.3 and are approved for use in measurement of
the pesticide compounds in Table 1 of that method as well as the PCB Aroclors listed in Table 2
of that method. Although validation requirements for the use of SPE products and their
associated procedures other than those described in Method 608.3, or for use with the additional
compounds listed in Table 2 of the method, are not discussed in this document, similar concepts
would apply.
For Measurement of the Analytes for which QC Acceptance Criteria are Specified in Table 6
Requirements for an Individual Laboratory
Laboratories wishing to use a vendor's SPE product and associated procedures for which a vendor has not
supplied a validation package would need to generate validation data from a Tier 1, single-laboratory
study on matrix type(s) (one, or additional matrix types) that represent all incoming samples at that
laboratory. A nine-matrix-type study would be required for a laboratory that wishes to apply the SPE
product and associated procedures to all matrix types. A laboratory wanting to use a vendor's SPE
product and associated procedures on a specific discharge (matrix type) or any matrix type must perform
an initial demonstration of capability (DOC) as specified in Section 8.2 of Method 625.1. The laboratory
must also analyze a background sample, and MS/MSD pairs on each different matrix type that they will
be analyzing up to a maximum of nine different matrix types. Note that DOC samples are also known as
laboratory control (LCS) samples in Method 625.1 or as initial precision and recovery (IPR) samples for
the purposes of this document.
The laboratory must fortify and analyze the IPR, and MS/MSD samples with all of the analytes that they
will be measuring using the method. All samples must also be fortified with surrogates. Additionally, the
laboratory must analyze a proficiency test (PT) sample containing all analytes that they will be measuring
using the method (see footnote 3 to Table A).
If they are measuring only a select list of analytes from Table 1 and 2 of Method 625.1 or any of the
analytes from Table 3 for which QC acceptance criteria are specified in Table 6, then they only need to
demonstrate that they are able to achieve acceptable performance for those specific analytes. However, if
they are running the full list of compounds in Tables 1 and 2 or any of the analytes from Table 3 for
which QC acceptance criteria are specified in Table 6, they must fortify all samples with the full list of
analytes in Tables 1 and 2 and any of the additional analytes from Table 3 for which QC acceptance
criteria are specified in Table 6 that will be included in the analyses.
A laboratory wanting to apply a vendor's SPE product and associated procedures to samples from all
matrix types (nationwide use) must prepare and analyze a background sample and MS/MSDs from nine
different matrix types as described in Table C. They must demonstrate acceptable performance for all
compounds on all matrix types tested. The testing of the SPE product and procedures only needs to be
performed once for a specific vendor's SPE product and associated procedures to enable that product and
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Validation of SPE Products and Associated Procedures with Method 625.1
December 2016
procedures to be used when reporting results from Method 625.1 for CWA compliance monitoring.
However, the test results may not be applied to products or procedures from another vendor or to
measurement of analytes that were not tested. The laboratory must continue performing the routine QC
testing as described in EPA Method 625.1, Section 8 when analyzing samples on an ongoing basis.
Requirements for a SPE Vendor
Alternatively, a vendor may test their SPE product and associated procedures by performing a DOC and
analyzing a background sample, and MS/MSD pairs on nine different matrix types as specified above.
They must also analyze a PT sample fortified with all analytes that they are claiming that their product
and associated procedures may be used to measure when performing Method 625.1. A vendor's study
should focus on the analytes in Tables 1 and 2. They may also choose to include any of the additional
analytes in Table 3 for which QC acceptance criteria are specified in Table 6, if desired. If the specific
product and procedures being tested fail to yield acceptable performance for any of those analytes, then
the product and associated procedures should be clearly labeled as applicable only to the select list of
analytes for Method 625.1 for which acceptable performance was demonstrated. See Table C, "Matrix
Types Recommended for Multiple Matrix Validation Studies" at the end of this document for the matrix
types required for use when validating a vendor's SPE product and associated procedures for use in all
matrix types. The vendor must provide afull data package, as described in Section 8.1.2.2.5 ofMethod
625.1, to each laboratory that will be using their product and associated procedures to report results from
Method 625.1 for CWA compliance monitoring. The laboratory must have the study results (from either
their own or a vendor-performed study) available for review upon request as described in EPA Method
625.1, Section 8.1.2.1.1. The laboratory must also continue to perform all routine QC testing as described
in the method when analyzing samples using the SPE product and procedures with Method 625.1.
Table A. Summary of Validation Approaches for SPE Products and Associated Procedures with
EPA Method 625.1 for Measurement of Analytes for which QC Acceptance Criteria are Specified
in Table 6 <1>
Method Application
Number of
Number of Analyses
Labs
Matrix
types
Back-
ground
Analysis
IPR-
Reagent
Water(2)
PT
Sample(3)
MS/
MSD <4>
MDL<5>
Total
Tier 1: Single-lab
First matrix type
1
1
1
4
1
2
7
15
Each additional
matrix type (8 max.)
1
1-8
1-8
0 <6>
0
2P)
(16 max)
0<6>
3
(24 max)
Vendor-Performed
Study: All labs, all
matrix types
1
9
9
4
1
18(7)
7
39
Notes:
(1)	Numbers of analyses in this table do not include additional QC tests such as calibration, blanks, etc. Nine is the
maximum number of matrix types needed to validate the use of SPE products and associated procedures with
Method 625.1 .for measurement of the analytes in for which QC acceptance criteria are specified in Table 6 in
wastewater. Additional matrix types will be required if the method is to be applied to analysis of sewage sludge
or ocean water.
(2)	Initial precision and recovery (IPR) reagent water analyses are used to demonstrate that the SPE product and
associated procedures can achieve acceptable method performance in reagent water.
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Validation of SPE Products and Associated Procedures with Method 625.1
December 2016
(3)	The proficiency testing (PT) sample should be obtained from a third party vendor and must contain all analytes
in Tables 1 and 2 and may include any of the additional analytes in Table 3 for which QC acceptance criteria
are specified in Table 6, if desired. If either sewage sludge or ocean water are matrices of interest, PT samples
for those matrices are required as well.
(4)	The matrix spike/matrix spike duplicate (MS/MSD) test would demonstrate that the EPA-approved method
MS/MSD QC acceptance criteria have been met when using the SPE product and associated procedures.
(5)	A method detection limit (MDL) test would be performed by the laboratory performing the study, using the SPE
product and associated procedures. 40 CFR Part 136 Appendix B requires a minimum of seven analyses per
laboratory to determine an MDL. If Appendix B is modified at a later date, then the validation study must
conform to the most recent MDL requirements.
(6)	The MDL and reagent water IPR tests do not have to be repeated after the first matrix type is validated.
(7)	The MS/MSD analyses would demonstrate that MS/MSD recovery and precision criteria associated with the
EPA-approved reference method have been met. The number of MS/MSD analyses is two times the number of
matrix types tested (i.e., one MS/MSD pair per laboratory).
For Measurement of Additional Analytes without QC Acceptance Criteria (Analytes in Table 3 for
which no QC Acceptance Criteria are Specified in Table 6)
Requirements for an Individual Laboratory
Laboratories wishing to use a vendor's SPE product and associated procedures for which a vendor has not
supplied a validation package for measurement of the additional analytes for which no QC acceptance
criteria have been developed by EPA (those analytes listed in Table 3) must generate QC acceptance
criteria for those compounds by performing a Tier 1, single-laboratory study, on matrix types (one, or
additional matrix types) that represent all incoming samples at that laboratory. A nine-matrix-type study
would be required for the laboratory that wishes to apply the SPE product and associated procedures to all
matrix types for measurement of these additional analytes. A laboratory wanting to use a vendor's SPE
product and associated procedures for measurement of these additional analytes in a specific discharge
(matrix type) or in any matrix type, must perform an initial demonstration of capability (DOC) as
specified in Section 8.2 of Method 625.1. The laboratory must also analyze a background sample, and
MS/MSD pairs on each different matrix type that they will be analyzing up to a maximum of nine
different matrix types.
The laboratory must fortify and analyze the IPR, and MS/MSD samples with all of the analytes that they
will be measuring using the method, including any of the additional analytes listed in Table 3. All
samples must also be fortified with surrogates. Additionally, the laboratory must analyze a PT sample
containing all analytes that they will be measuring using the method, including any of the analytes in
Table 3. If they are measuring only a select list of analytes from Table 3, then they only need to
demonstrate that they are able to achieve acceptable performance on those specific analytes.
If the laboratory wants to apply the use of a vendor's SPE product and associated procedures to all
samples from all matrix types (nationwide use), they must prepare and analyze MS/MSDs from nine
different matrix types as described in Table C. They must demonstrate acceptable performance for all
compounds for which QC acceptance criteria exist (the analytes in Tables 1 & 2) on all matrix types that
will be analyzed, up to a maximum of nine different matrix types. They must also use the results of the
validation study to develop QC acceptance criteria for any of the additional analytes in Table 3 using the
procedures in Section 3.1 of Appendix G to "Protocol for Review and Validation of New Methods for
Regulated Organic and Inorganic Analytes in Wastewater Under EPA's Alternate Test Procedure
Program" (EPA 821-B-16-001, February 2016). This document is available at: www.epa.gov/cwa-
methods/alternate-test-procedure-documents . The testing of the SPE product and procedures only needs
to be performed once for a specific vendor's SPE product and associated procedures to enable that
product and procedures to be used when reporting results from Method 625.1 for CWA compliance
3

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Validation of SPE Products and Associated Procedures with Method 625.1
December 2016
monitoring. However, the test results may not be applied to products or procedures from another vendor
or to measurement of analytes that were not tested. The laboratory must continue performing the routine
QC testing required by the method when analyzing samples on an ongoing basis.
Requirements for a SPE Vendor
Alternatively, a vendor may conduct a Tier 3, nine-laboratory, nine-matrix type (multi-lab, all matrix
types) validation study using their SPE product and associated procedures. The IPR samples used in the
DOC tests, and the MS/MSD samples analyzed in each of the nine laboratories must be fortified with all
of the analytes that the vendor is claiming that their product and associated procedures may be used to
measure when performing Method 625.1, including any additional analytes from Table 3. Additionally,
each laboratory in the vendor's study must analyze a PT sample fortified with all analytes that they are
claiming that their product and associated procedures may be used to measure when performing Method
625.1 including any additional analytes from Table 3 (see footnote 4 to Table B). The vendor must use
the results from the study to develop QC acceptance criteria for any of the additional analytes in Table 3
using the procedures in Section 3.3 of Appendix G to "Protocol for Review and Validation of New
Methods for Regulated Organic and Inorganic Analytes in Wastewater Under EPA's Alternate Test
Procedure Program" (EPA 821-B-16-001, February 2016). This document is available at:
www.epa.gov/cwa-metliods/alternate-test-procedure-dociiments. See Table C, "Matrix Types
Recommended for Multiple Matrix Validation Studies" for matrix types required for use when validating
a vendor's SPE product and associated procedures for use in all matrix types. The vendor must provide a
full data package, as described in the method, to each laboratory that will be using their product and
associated procedures to report results from Method 625.1 for CWA compliance monitoring. The
laboratory must have the study results (from either their own or a vendor-performed study) available for
review upon request as described in EPA Method 625.1, Section 8.1.2.1.1. The laboratory must also
continue to perform all routine QC testing as described in the method when analyzing samples using the
SPE product and procedures with Method 625.1.
Table B. Summary of Validation Approaches for SPE Products and Associated Procedures with
EPA Method 625.1 for Measurement of Analytes in Table 3 for which no QC Acceptance Criteria
are Specified in Table 6 (1)
Method
Application
Number of
Number of Analyses
Labs
Matrix
types
Back-
ground
Analysis
IPR-
reagent
water(2)
IPR-
sample
matrix(3)
PT
Sample
(4)
MS/
MSD
MDL
(5)
Total
Tier 1: Single-lab
First matrix type
1
1
1
4
4
1
0
7
17
Each additional
matrix type
(8 max.)
1
1-8
1-8
0 <6>
0
0
2 U)
(16 max)
0 <6>
3
(24 max)
Tier 3: Multi-lab,
all matrix types
9
9
9
36
0
9
18 (7)
63
135
Notes:
(1)	Numbers of analyses in this table do not include additional QC tests such as calibration, blanks, etc. Nine is the
maximum number of matrix types required for validation of use of SPE with Method 625.1 at Tier 1 or Tier 3.
(2)	Initial precision and recovery (IPR) reagent water analyses are used to validate a method modification. The
number of IPR analyses is four times the number of laboratories used to validate the use of SPE because each
laboratory performs a four-replicate IPR test.
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Validation of SPE Products and Associated Procedures with Method 625.1
December 2016
(3)	IPR sample matrix analyses are used to establish QC acceptance criteria for matrix spike/matrix spike duplicate
(MS/MSD) recovery and precision for a Tier 1 new method only. IPR sample matrix analyses are not needed
for validation of the use of SPE with Method 625.1 by vendors at Tier 3 because this variability data would be
obtained from MS/MSD tests in multiple labs.
(4)	The proficiency testing (PT) sample should be obtained from a third-party vendor and should be analyzed by
each laboratory participating in the study. If sewage sludge or ocean water are matrices of interest, PT samples
for those matrices are required as well. The PT samples must contain all analytes to which use of SPE will be
applied when performing Method 625.1 including any additional analytes from Table 3.
(5)	A method detection limit (MDL) test would be performed in each laboratory, using the SPE product and
associated procedures. 40 CFR Part 136 Appendix B requires a minimum of seven analyses per laboratory to
determine an MDL. If Appendix B is modified at a later date, then the validation study must conform to the
most recent MDL requirements.
(6)	The MDL and reagent water IPR tests do not have to be repeated after the first matrix type is validated.
(7)	The MS/MSD analyses would be used to establish MS/MSD acceptance criteria for recovery and precision for
the additional analytes in Table 3 to which the use of the SPE product and associated procedures with Method
625.1 will be applied. The number of MS/MSD analyses is two times the number of matrix types tested (i.e.,
one MS/MSD pair per laboratory).
Table C. Matrix Types Recommended for Multiple Matrix Type (Nationwide Use) Validation
	Studies	
1.
Effluent from a POTW
2.
ASTM D 5905 - 98 (Reapproved 2013), Standard Specification for Substitute Wastewater
3.
Sewage sludge, if sludge will be in the permit
4.
ASTM D 1141 - 98 (Reapproved 2013), Standard Specification for Substitute Ocean Water, if
ocean water will be in the permit
5.
Untreated and treated wastewaters up to a total of nine matrix types
(See www.epa.gov/eg/industrial-effluent-guidelines for a list of industrial categories with
existing Effluent Guideline regulations)
At least one of the above wastewater matrix types should have at least one of the following
characteristics:
•	Total suspended solids (TSS) greater than 40 mg/L
•	Total dissolved solids (TDS) greater than 100 mg/L
•	Oil and grease greater than 20 mg/L
•	NaCI greater than 120 mg/L
•	CaCCb greater than 140 mg/L
5

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