EPA Document #: EPA/600/R-20/270
METHOD 559. DETERMINATION OF NONYLPHENOL AND 4-TERT-
OCTYLPHENOL IN DRINKING WATER BY SOLID PHASE
EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM
MASS SPECTROMETRY (LC/MS/MS)
Version 1.0
September 2020
D.R. Tettenhorst, US EPA, Office of Research and Development, Center for Environmental
Solutions and Emergency Response
J.A. Shoemaker, US EPA, Office of Research and Development, Center for Environmental
Solutions and Emergency Response
OFFICE OF RESEARCH AND DEVELOPMENT
CENTER FOR ENVIRONMENTAL SOLUTIONS AND EMERGENCY RESPONSE
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 559
DETERMINATION OF NONYLPHENOL AND 4-TERT-OCTYLPHENOL IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)
1. SCOPE AND APPLICATION
1.1. This is a solid phase extraction (SPE) liquid chromatography/tandem mass
spectrometry (LC/MS/MS) method for the determination of nonylphenol and 4-tert-
octylphenol in drinking water. Accuracy and precision data have been generated in
reagent water and drinking water for the compounds listed in the table below.
a This CASRN describes and this method reports technical nonylphenol, comprised
mostly of branched C9-alkyl phenols, and not linear nonylphenol (CASRN 104-40-5)
which is a laboratory generated chemical not typically found in the environment.
1.2.	Minimum Reporting Level (MRL) is the lowest analyte concentration that meets Data
Quality Objectives (DQOs) that are developed based on the intended use of this
method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true
concentration for which the future recovery is predicted to fall, with high confidence
(99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in this
method are 4.9 ng/L for 4-tert-octylphenol and 24 ng/L for nonylphenol and are listed
in Table 5. The procedure used to determine the LCMRL is described elsewhere.1
1.3.	Laboratories using this method will not be required to determine the LCMRL for this
method but will need to demonstrate that their laboratory MRL for this method meets
requirements described in Section 9.2.4.
1.4.	Determining the Detection Limit (DL) for analytes in this method is optional
(Sect. 9.2.6). Detection limit is defined as the statistically calculated minimum
concentration that can be measured with 99% confidence that the reported value is
greater than zero.2 The DL is compound dependent and is dependent on extraction
efficiency, sample matrix, fortification concentration, and instrument performance.
1.5.	This method is intended for use by analysts skilled in solid phase extractions, the
operation of LC/MS/MS instruments, and the interpretation of the associated data.
Chemical Abstract Services
Analyte
Nonylphenol
4-tert-Octylphenol
Acronym
NP
4-t-OP
Registry Number (CASRN)
84852-15-33
140-66-9
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1.6. METHOD FLEXIBILITY - In recognition of technological advances in analytical
systems and techniques, the laboratory is permitted to modify the separation technique,
LC column, mobile phase composition, LC conditions and MS and MS/MS conditions
(Sect. I , 1 I I, l" and 12 1). Changes may not be made to sample collection
and preservation (Sect. 8), the sample extraction steps (Sect. ), or to the
quality control requirements (Sect. 9). Method modifications should be considered
only to improve method performance. Modifications that are introduced in the interest
of reducing cost or sample processing time, but result in poorer method performance,
must not be used. In all cases where method modifications are proposed, the analyst
must perform the procedures outlined in the initial demonstration of capability (IDC,
Sect. 9.2). verify that all Quality Control (QC) acceptance criteria in this method
(Sect. 9) are met, and that acceptable method performance can be verified in a real
sample matrix (Sect. 9.3.6).
NOTE: The above method flexibility section is intended as an abbreviated summation
of method flexibility. Sections 4-12 provide detailed information of specific portions
of the method that may be modified. If there is any perceived conflict between the
general method flexibility statement in Section and specific information in
Sections 4-12, Sections 4-12 supersede Section L6.
2.	SUMMARY OF METHOD
A 100-250 mL water sample is fortified with surrogate and passed through a copolymeric
SPE cartridge to extract the method analytes and surrogate. The compounds are eluted from
the solid phase sorbent with a small amount of acetone. The extract is adjusted to a 5 mL
volume with acetone after addition of the internal standard. A 10 |iL injection is made into an
LC equipped with a Ci8 column that is interfaced to an MS/MS. The analytes are separated
and identified by comparing retention times and signals produced by unique mass transitions
to retention times and reference signals for calibration standards acquired under identical
LC/MS/MS conditions. The concentration of each analyte is determined by using the internal
standard technique. A surrogate analyte is added to all Field and QC Samples to monitor the
extraction efficiency of the method analytes.
3.	DEFINITIONS
3.1.	ANALYSIS BATCH - A set of samples that is analyzed on the same instrument
during a 24-hour period that begins and ends with the analysis of the appropriate
Continuing Calibration Check (CCC) standards. Additional CCCs may be required
depending on the length of the analysis batch and/or the number of Field Samples.
3.2.	CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution and/or stock standard solution, internal standard, and the surrogate.
The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
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3.3.	COLLISIONALLY ACTIVATED DISSOCIATION (CAD) - The process of
converting the precursor ion's translational energy into internal energy by collisions
with neutral gas molecules to bring about dissociation into product ions.
3.4.	CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing
the method analytes, internal standard and surrogate. The CCC is analyzed
periodically to verify the accuracy of the existing calibration for those analytes.
3.5.	DETECTION LIMIT (DL) - The minimum concentration of an analyte that can be
identified, measured, and reported with 99% confidence that the analyte concentration
is greater than zero. This is a statistical determination of precision (Sect. 9.2.6). and
accurate quantitation is not expected at this level.2
3.6.	EXTRACTION BATCH - A set of up to 20 Field Samples (not including QC
samples) extracted together by the same person(s) during a work-day using the same
lot of SPE devices, solvents, surrogate, internal standard and fortifying solutions.
Required QC samples include Laboratory Reagent Blank, Laboratory Fortified Blank,
Laboratory Fortified Sample Matrix, and either a Field Duplicate or Laboratory
Fortified Sample Matrix Duplicate.
3.7.	FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at the same
time and placed under identical circumstances and treated exactly the same throughout
field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the
precision associated with sample collection, preservation, and storage, as well as
laboratory procedures.
3.8.	FIELD REAGENT BLANK (FRB) - An aliquot of reagent water that is placed in a
sample container in the laboratory and treated as a sample in all respects, including
shipment to the sampling site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose of the FRB is to determine if
method analytes or other interferences are present in the field environment.
3.9.	INTERFERENCE CHECK STANDARD (ICS) - A standard injected to ensure
nonylphenoxy carboxylic acids do not overlap retention time and interfere with
nonylphenol quantitation.
3.10.	INTERFERENCE PRIMARY DILUTION STANDARD (IPDS) SOLUTION - A
solution containing the nonylphenoxy carboxylic acid interferents prepared in the
laboratory from stock standard solutions and diluted as needed to prepare the
interference check standard.
3.11.	INTERFERENCE STOCK STANDARD SOLUTION (ISSS) - A concentrated
solution containing one or more nonylphenoxy carboxylic acids for checking retention
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time interference. The standard is prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.12.	INTERNAL STANDARD (IS) - A pure chemical added to an extract or standard
solution in a known amount(s) and used to measure the relative response of other
method analytes and surrogate(s) that are components of the same solution. The
internal standard must be a chemical that is structurally similar to the method analytes,
has no potential to be present in water samples, and is not a method analyte.
3.13.	LABORATORY FORTIFIED BLANK (LFB) - A volume of reagent water or other
blank matrix to which known quantities of the method analytes and all the preservation
compounds are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the
laboratory is capable of making accurate and precise measurements.
3.14.	LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A preserved field
sample to which known quantities of the method analytes are added in the laboratory.
The LFSM is processed and analyzed exactly like a sample, and its purpose is to
determine whether the sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix must be determined in
a separate sample extraction and the measured values in the LFSM corrected for
background concentrations.
3.15.	LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A
duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified,
extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the
Field Duplicate to assess method precision when the occurrence of method analytes is
expected to be low.
3.16.	LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents and reagents, sample preservatives, internal standard, and
surrogate that are used in the analysis batch. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents,
or the apparatus.
3.17.	LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) - The
single laboratory LCMRL is the lowest true concentration for which a future recovery
is expected, with 99% confidence, to be between 50 and 150% recovery.1
3.18.	MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a method analyte in a sample following analysis.
This defined concentration can be no lower than the concentration of the lowest
calibration standard for that analyte and can only be used if acceptable QC criteria for
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this standard are met. A procedure for verifying a laboratory's MRL is provided in
Section 9.2.4.
3.19.	PRECURSOR ION - For the purpose of this method, the precursor ion is the
deprotonated molecule ([M-H]") of the method analyte. In MS/MS, the precursor ion is
mass selected and fragmented by collisionally activated dissociation to produce
distinctive product ions of smaller m/z.
3.20.	PRIMARY DILUTION STANDARD (PDS) SOLUTION - A solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as needed
to prepare calibration solutions and other needed analyte solutions.
3.21.	PRODUCT ION - For the purpose of this method, a product ion is one of the fragment
ions produced in MS/MS by collisionally activated dissociation of the precursor ion.
3.22.	QUALITY CONTROL SAMPLE (QCS) - A solution containing the method analytes
at a known concentration that is obtained from a source external to the laboratory and
different from the source of calibration standards. The purpose of the QCS is to verify
the accuracy of the primary calibration standards.
3.23.	SAFETY DATA SHEET (SDS) - Written information provided by vendors
concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.24.	STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one or
more method analytes prepared in the laboratory using assayed reference materials or
purchased from a reputable commercial source.
3.25.	SURROGATE ANALYTE (SUR) - A pure chemical which chemically resembles
method analytes and is extremely unlikely to be found in any sample. This chemical is
added to a sample aliquot in known amount(s) before processing and is measured with
the same procedures used to measure other method analytes. The purpose of the SUR
is to monitor method performance with each sample.
4. INTERFERENCES
4.1. All glassware must be meticulously cleaned. Wash glassware with detergent and tap
water, rinse with tap water, followed by a reagent water rinse. Non-volumetric
glassware can be heated in a muffle furnace at 400 °C for 2 h or solvent rinsed.
Volumetric glassware should be solvent rinsed and not be heated in an oven above
120 °C. Store clean glassware inverted or capped.
NOTE: Detergents could be a source of high NP or OP background. Laboratories
should examine their detergent(s) if having difficulty meeting LRB requirements.
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Alternate detergents may need to be evaluated to determine if background
contamination levels can be lowered.
4.2.	Method interferences may be caused by contaminants in detergents, solvents, reagents
(including reagent water), sample bottles and caps, and other sample processing
hardware that lead to discrete artifacts and/or elevated baselines in the chromatograms.
The analytes in this method can also be found in many common laboratory supplies
and equipment that include plastics. All items such as these must be routinely
demonstrated to be free from interferences (less than 1/3 the MRL for each method
analyte) under the conditions of the analysis by analyzing laboratory reagent blanks as
described in Section 9.3.1. Subtracting blank values from sample results is not
permitted.
4.2.1 There are many potential sources of nonylphenol contamination in the laboratory,
especially from plastics and chemicals that may have been stored in plastic
containers. Care must be taken to minimize sources of contamination, and the QC
criteria for LRBs must be met (Sect. 9.3.1). Special precautions must also be
taken when creating calibration curves for analytes consistently found in LRBs
(Sect. 10.2.6).
4.3.	Matrix interferences may be caused by contaminants that are co-extracted from the
sample. The extent of matrix interferences will vary considerably from source to
source, depending upon the nature of the water.
4.3.1	Two common nonylphenoxy carboxylic acids (nonylphenoxyethoxy acetic acid
and nonylphenoxy acetic acid) are known to interfere3 with nonylphenol because
they share the same transition (219 > 133). These two carboxylic acids were
tested during method development and found to be separated from nonylphenol
by five minutes. Severely compressing the gradient program and increasing flow
may allow these substances to interfere wth nonylphenol.
4.3.2	Humic and/or fulvic material can be co-extracted during SPE and high levels can
cause enhancement and/or suppression in the electrospray ionization source or
low recoveries on the SPE sorbent.4'5 Total organic carbon (TOC) is a good
indicator of humic content of the sample. Under the LC conditions used during
method development, matrix effects due to total organic carbon (TOC) were not
observed.
4.4.	Preservatives (Sect. 8.1.2) are added to sample bottles in large quantities, therefore the
potential exists for trace-level organic contaminants in these reagents. Interferences
from these sources should be monitored by analysis of laboratory reagent blanks
(Sect. 9.3.1). particularly when new lots of reagents are acquired.
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4.5. SPE cartridges can be a source of interferences or alkylphenol contamination. The
analysis of field and laboratory reagent blanks can provide important information
regarding the presence or absence of such interferences. Brands and lots of SPE
devices should be tested to ensure that contamination does not preclude analyte
identification and quantitation.
SAFETY
5.1.	The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined. Each chemical should be treated as a potential health hazard, and
exposure to these chemicals should be minimized. Each laboratory is responsible for
maintaining an awareness of OSHA regulations regarding safe handling of chemicals
used in this method. A reference file of SDSs should be made available to all
personnel involved in the chemical analysis. Additional references to laboratory safety
are available.6"8
5.2.	Nonylphenol and 4-t-octylphenol are harmful if ingested, can cause skin and serious
eye burns, and can cause reproductive harm. Pure standard materials and stock
standard solutions of these method analytes should be handled with suitable protection
to skin and eyes, and care should be taken not to breathe the vapors or ingest the
materials.
EQUIPMENT AND SUPPLIES
Brand names and/or catalog numbers are included for illustration only, and do not imply
endorsement of the product. Plastic materials must be avoided as they have the potential to
contaminate standards and/or samples with alkylphenols. The usage of Teflon™ is
acceptable.
6.1.	SAMPLE CONTAINERS - Amber glass bottles (125-250 mL) fitted with Teflon™-
faced screw caps.
6.2.	CONICAL CENTRIFUGE TUBES - 15 mL conical glass tubes (Corning Cat.
No.: 8082-15 or equivalent) or other glassware suitable for collection of the extracts.
6.3.	LOW VOLUME AUTOSAMPLER VIALS - Amber glass 2 mL autosampler vials
with 0.4 mL low volume insert (Thermo Fisher Cat. No.: C4000-LV2W or
equivalent).
6.4.	AUTOSAMPLER VIALS - Amber glass 2.0 mL autosamplers vials for calibration
standard preparation and storage (Thermo Fisher Cat. No.: C4000-2W or equivalent).
6.5.	AUTOSAMPLER VIAL CAPS - Vial caps with PTFE/Silicone septum (Thermo
Fisher Cat. No.: C5000-54B or equivalent).
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NOTE: Vial cap septa have the potential to introduce nonylphenol contaminants
once punctured, therefore vials can only be used once for an injection.
Autosampler injection needles cored vial cap septa with three layers
(PTFE/Silicone/PTFE) more easily than two layer septa (PTFE/Silicone). The
three layer septa introduced NP contamination into the vial as the sample was
injected and are not recommended. Pre-slit septa are not permitted because they
cause excess extract evaporation and have the potential to allow nonylphenol
from laboratory air to intrude into standards and extracts.
6.6.	GRADUATED CYLINDERS - Glass, suggested sizes include 25, 50, 100, 250 and
1000 mL cylinders.
6.7.	MICRO SYRINGES - Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and
1000 |iL syringes.
6.8.	VOLUMETRIC FLASKS - Suggested sizes include 5, 10, 25, 500, and 1000 mL.
6.9.	GLASS PIPETS -Borosilicate glass disposable pipets (Fisher Cat. No.: 13-678-20D
or equivalent).
6.10.	ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.11.	SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES -
Cartridges specified here were found to contain acceptable background levels of
nonylphenol. Alternate cartridges with equivalent sorbents need to be evaluated to
ensure absence of nonylphenol contamination prior to use with field samples.
6.11.1	SPE CARTRIDGES
6.11.1.1	Waters Oasis HLB, 150 mg, 6 cc (Waters Cat No.: 186003365 or
equivalent) - divinylbenzene-N-vinylpyrrolidone copolymer.
6.11.1.2	Phenomenex Strata-X, 100 mg, 6 cc (Phenomenex Cat No.: 8B-S100-ECH
or equivalent) - styrene divinylbenzene-N-vinylpyrrolidone copolymer.
6.11.2	VACUUM EXTRACTION MANIFOLD - A manual vacuum manifold with
Visiprep™ large volume sampler (Supelco Cat. No. 57030 and 57275 or
equivalent) for cartridge extractions, or an automatic/robotic sample preparation
system designed for use with SPE cartridges, may be used if all QC requirements
discussed in Section 9 are met.
6.11.3	SAMPLE DELIVERY SYSTEM - Use of a transfer tube system (Supelco
"Visiprep," Cat No.: 57275) which transfers the sample directly from the sample
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container to the SPE cartridge, is recommended, but not mandatory. Standard
extraction manifolds come equipped with PTFE transfer tube systems.
NOTE: Transfer tubes are preferred to limit potential nonylphenol
contamination, but plastic reservoirs may be used provided LRBs meet QC
criteria.
6.12.	LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of approximately 10 to 15 inches of mercury for extraction
cartridges.
6.13.	LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER
(MS/MS) WITH DATA SYSTEM
6.13.1	LC SYSTEM - Instrument capable of reproducibly injecting up to 10 |iL aliquots
and performing binary linear gradients at a constant flow rate near the flow rate
used for development of this method (0.3 mL/min). The usage of a column heater
and thermostated autosampler compartment are optional. Data in Section 17 were
collected with autosampler vials thermostated at 10 °C.
NOTE: During the course of method development, nonylphenol was
observed as a background peak in solvent blank injections. The source of this
background peak is likely components of the LC systems and solvents.
Analysts must evaluate their instrument background and determine if
additional action is required to lower contamination to an acceptable level. A
second LC column (Sect. 6.13.4.2) used to delay background nonylphenol
may be installed after the pump mixer but prior to the autosampler injector.
The delay column causes the system background contamination to elute later
in the chromatogram and separates it from the injected nonylphenol peak.
6.13.2	LC/TANDEM MASS SPECTROMETER - The LC/MS/MS must be capable of
negative ion electrospray ionization (ESI) near the suggested LC flow rate of
0.3 mL/min. The system must be capable of performing MS/MS to produce
unique product ions (Sect. 3.21) for the method analytes within specified retention
time segments. A minimum of 10 scans across the chromatographic peak is
required to ensure adequate precision. Data are presented in Tables 5-13 using a
triple quadrupole mass spectrometer (Thermo Scientific TSQ Endura).
6.13.3	DATA SYSTEM - An interfaced data system is required to acquire, store, reduce,
and output mass spectral data. The computer software should have the capability
of processing stored LC/MS/MS data by recognizing an LC peak within any given
retention time window. The software must allow integration of the ion abundance
of any specific ion within specified time or scan number limits. The software must
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be able to calculate relative response factors, construct linear regressions or
quadratic calibration curves, and calculate analyte concentrations.
6.13 .4 LC COLUMNS
6.13.4.1	ANALYTICAL COLUMN - An LC Ci8 column (2.1 x 50 mm) packed with
3 |am Ci8 solid phase particles (Thermo Fisher Cat No.: 25003-052130 or
equivalent) was used during method development. Any column that
provides adequate resolution, peak shape, capacity, accuracy, and precision
(Sect. 9) may be used.
NOTE: It is recommended the LC column be dedicated for use with only
ammonium fluoride due to potential deterioration of LC column
performance9 when switching between ammonium fluoride and other
mobile phase additives.
6.13.4.2	DELAY COLUMN (optional) - An LC Ci8 column (2.1 x 100 mm) packed
with 4 |am Cis solid phase particles (Thermo Fisher Cat No.: 74104-102130
or equivalent) used to retain nonylphenol contamination present in the LC
system and separate it from the injected nonylphenol. The delay column
should be installed after the binary pump mixer but prior to the autosampler
injector and be of sufficient length and particle size to move the delayed
nonylphenol peak so it is separated by a minimum of one minute from the
injected nonylphenol peak without significantly increasing the system back
pressure. The one minute peak separation is required due to multiple
isomers in technical nonylphenol causing significant tailing beyond the peak
elution time.
7. REAGENTS AND STANDARDS
7.1. GASES, REAGENTS, AND SOLVENTS - Reagent grade or better chemicals should
be used. Unless otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used, provided it
is first determined that the reagent is of sufficiently high purity to permit its use
without lessening the quality of the determination.
7.1.1 REAGENT WATER - Purified water which does not contain any measurable
quantities of any method analytes or interfering compounds greater than 1/3 the
MRL for each method analyte of interest. Prior to daily use, reagent water should
be flushed from the purification system to rinse out any build-up of analytes in the
system's tubing.
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7.1.2	METHANOL (CH3OH, CAS#: 67-56-1) - High purity, demonstrated to be free of
analytes and interferences (Fisher LC/MS grade or equivalent). Methanol used as
the mobile phase must be replaced every 48 hours if not using a delay column to
avoid increases in background contamination due to potential nonylphenol in the
laboratory air.
7.1.3	ACETONE [(CH3)2CO, CAS#: 67-64-1] - High purity, demonstrated to be free
of analytes and interferences (Fisher Optima grade or equivalent).
7.1.4	AMMONIUM FLUORIDE (NH4F, CAS#: 12125-01-8) - High purity,
demonstrated to be free of analytes and interferences (Sigma-Aldrich Cat.
No.: 216011 or equivalent).
7.1.4.1	10 mM AMMONIUM FLUORIDE STOCK SOLUTION - To prepare
100 mL, add 0.037 g ammonium fluoride to a 100 mL volumetric flask and
bring to volume with reagent water. This solution should be stored in the
refrigerator at 4 °C to minimize bacterial growth and replaced after one
month.
7.1.4.2	0.2 mM AMMONIUM FLUORIDE - To prepare 500 mL, add 10 mL of the
10 mM ammonium fluoride stock solution to a 500 mL volumetric flask and
bring to volume with reagent water. This solution is used as the LC aqueous
mobile phase. The mobile phase must be replaced every 48 hours if not
using a delay column to avoid increases in background contamination due to
potential nonylphenol in the laboratory air, otherwise replace weekly to
avoid microbial growth.
7.1.5	SODIUM BISULFATE (CAS# 7681-38-1) - Preserves the sample to a pH of 2 to
inhibit microbial growth and prevent analyte degradation (Fluka #71656 or
equivalent) (Sect. 0.
7.1.6	L-ASCORBIC ACID (CAS# 50-81-7) - Reduces free chlorine at the time of
sample collection (Sigma-Aldrich Cat. No.: 255564 or equivalent). (Sect. 8.1.2).
7.1.7	NITROGEN - Nitrogen aids in aerosol generation of the ESI liquid spray and is
used as collision gas in some MS/MS instruments. The nitrogen used should meet
or exceed instrument manufacturer's specifications.
7.1.8	ARGON - Used as collision gas in MS/MS instruments. Argon should meet or
exceed instrument manufacturer's specifications. Nitrogen gas may be used as the
collision gas provided sufficient sensitivity (product ion formation) is achieved.
7.2. STANDARD SOLUTIONS - When a compound purity is assayed to be 96% or
greater, the weight can be used without correction to calculate the concentration of the
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stock standard. Solution concentrations listed in this section were used to develop this
method and are included as an example. Alternate concentrations may be used as
necessary depending on instrument sensitivity and the calibration range used.
Standards for sample fortification generally should be prepared so the smallest volume
that can be accurately measured is used to minimize the addition of excess organic
solvent to aqueous samples. PDS and calibration standards were found to be stable for
at least one month during method development. Laboratories should use standard QC
practices to determine when standards need to be replaced. The target analyte
manufacturer's guidelines may be helpful when making the determination.
NOTE: Stock standards (Sect. 7.2. L L 7.2.2.1 and 7.2.3.1) and primary dilution
standards (Sect.7.2.1.2. 7.2.2.2 and 7.2.3.2) were stored at <6 °C. Standards should be
allowed to come to room temperature and mixed prior to use.
7.2.1 INTERNAL (IS) STOCK STANDARD SOLUTION - This method uses 4-(l,3-
dimethyl-l-ethylpentyl) phenol-13C6 as the IS compound. The IS compound was
carefully chosen during method development because it mimics the structure of
technical nonylphenol (mostly branched nonylphenols). Alternate IS compounds
may be used provided it is an isotopically labeled branched nonylphenol, however
the analyst must have documented reasons for using alternate IS compounds.
Alternate IS compounds must meet the QC requirements in Section 9.3.4.
NOTE: Isotopically labeled linear alkylphenol compounds do not elute at the
same retention time as the branched IS and would not sufficiently correct for
matrix suppression or enhancement of nonylphenol.
7.2.1.1	IS STOCK STANDARD SOLUTION (IS SSS) - The IS stock can be
obtained as an individual certified stock standard solution (Cambridge
Isotopes Cat. No.: CLM-8356-1.2) or neat material (Cambridge Isotopes
Cat. No.: CLM-8356-0; custom order). During development of this method,
the IS was prepared from neat material at 1000 |ag/mL in methanol. The IS
stock standard solution was stable for at least six months when stored at
6 °C or less in amber glass screw cap vials.
7.2.1.2	INTERNAL STANDARD PRIMARY DILUTION (IS PDS) STANDARD
(1 |ig/mL) - Prepare, or purchase commercially, the IS PDS at a suggested
concentration of 1.00 |ig/mL. If prepared from individual stock standard
solutions (Sect.	), the table below can be used as a guideline for
preparing the IS PDS although concentrations may need to be adjusted for
instrument sensitivity. The IS PDS used in these studies was prepared in
methanol. The IS PDS has been shown to be stable for at least six months
when stored at 6 °C or less in amber glass screw cap vials. Fifty |iL of this
1.00 |ig/mL IS PDS was used to fortify the final 5 mL extracts (Sect. 11.5).
This will yield a concentration of 10.0 |ig/L in 5 mL extracts.
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Cone, of IS
Stock (|ig/mL)
Vol. of IS Stock
(nL)
Final Vol. of IS
PDS (mL)
Final Cone, of IS
PDS (ng/mL)
1000
10
10
1.00
7.2.2 SURROGATE (SUR) STANDARD SOLUTION - The SUR for this method is 4-
(1,1,3,3-tetramethylbutyl) phenol-13C6 (4-tert-octylphenol-13C6). This isotopically
labeled SUR standard was carefully chosen during method development because
it is an isotopically labeled analogue of a target analyte and behaves similarly to
method analytes during SPE extraction. Although alternate SUR standards may be
used provided they are isotopically labeled branched compounds of a C8 or C9
branched alkylphenol, the analyst must have documented reasons for using
alternate SUR standards. In addition, any alternate SUR standard must meet the
QC requirements in Section 9.3.5.
NOTE: During method development it was discovered that linear alkyl phenols
do not elute efficiently from the sorbent during SPE extraction under the same
conditions as the branched alkyl phenols, resulting in lower recoveries. The
branched SUR used in this method was chosen to produce the best SUR recovery
and mimic the branched alkyl phenol target analytes of the method.
7.2.2.1	SUR STOCK STANDARD SOLUTION (SUR SSS) -During development
of this method, the SUR was prepared from neat material (Toronto Research
Chemicals Cat. No.: 0293782) at 1000 |ig/mL in methanol. The SUR stock
standard solution was stable for at least six months when stored at 6 °C or
less in amber glass screw cap vials.
7.2.2.2	SURROGATE PRIMARY DILUTION STANDARD (SUR PDS)
(1.25 |ig/mL) - Prepare the SUR PDS at a suggested concentration of 1.25
|ig/mL as shown in the table below. Use 50 |iL of this 1.25 |ag/m L solution
to fortify all QC and Field Samples. (Sect. 11.5). This will yield SUR
concentrations of 250 ng/L in 250 mL aqueous samples.
Cone, of SUR
Stock (|ig/mL)
Vol. of SUR
Stock (|iL)
Final Vol. of
SUR PDS (mL)
Final Cone, of
SUR PDS
(lig/mL)
1000
12.5
10
1.25
7.2.3 ANALYTE STANDARD SOLUTIONS - Analyte standards may be purchased
commercially as ampulized solutions or prepared from neat materials.
7.2.3 .1 ANALYTE STOCK STANDARD SOLUTION (SSS) - If preparing from
neat material, accurately weigh approximately 5 mg of pure material to the
nearest 0.1 mg and dilute to 5 mL with methanol for a final concentration of
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1000 |ig/mL, Repeat for each method analyte prepared from neat material.
Alternatively, purchase commercially available individual stock standards of
the analytes, preferably in methanol or acetonitrile, if available. For
development of this method, a commercially available stock standard of
1000 |ig/mL in methanol was purchased for 4-tert-octylphenol (Spex Cat.
No: S-4379). Technical grade nonylphenol (Sigma Cat. No: 290858) was
purchased as a neat material and prepared at 1000 jag/mL in methanol.
These stock standards were stable for at least six months when stored at
6 °C or less in amber glass screw cap vials.
7.2.3 .2 ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION
(1.0 |ig/mL) - The analyte PDS contains all method analytes in methanol.
The analyte PDS was prepared in methanol at a concentration of 1.0 |ig/mL.
The analyte PDS is prepared by diluting (10 |iL of each SSS into 10 mL of
methanol) the combined Analyte Stock Standard Solutions (Sect. )
and is used to prepare CAL standards, and fortify LFBs, LFSMs, and
LFSMDs with the method analytes. The analyte PDS has been shown to be
stable for at least six months when stored at 6 °C or less in amber glass
screw cap vials.
7.2.4 NONYLPHENOXY CABOXYLIC ACID INTERFERENCE SOLUTION - The
interference solution is prepared and analyzed only if the LC column and LC
conditions in Table 1 are changed from method suggested parameters (See
Section	).
7.2.4.1	INTERFERENCE STOCK STANDARD SOLUTIONS (ISSS) - For
development of this method, commercially available stock standards of
100 |ig/mL in methanol were purchased for nonylphenoxy ethoxy acetic
acid (CAS# 106807-78-7, Accustandard Cat. No: PEO-012S) and
nonylphenoxy acetic acid, (CAS# 3115-49-9, Accustandard Cat. No: PEO-
009S). These stock standards were stable for at least three months when
stored at 6 °C or less in amber glass screw cap vials.
7.2.4.2	INTERFERENCE PRIMARY DILUTION STANDARD (IPDS)
SOLUTION (5.00 |ig/mL) - The IPDS contains all method analytes in
methanol. The IPDS was prepared in methanol at a concentration of
5.00 |ig/mL. The IPDS is prepared by diluting (50 |iL of the ISSS into 1 mL
of methanol) the combined Interference Stock Standard Solutions
(Sect. 7.2.4.1). The IPDS has been shown to be stable for at least three
months when stored at 6 °C or less in amber glass screw cap vials.
7.2.4.3	INTERFERENCE CHECK STANDARD (ICS) - The ICS is prepared at
100 |ig/L and analyzed according to Section	. The ICS is prepared by
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diluting (20 |iL of IPDS into 1 mL of acetone) the Interference Primary
Dilution Standard (Sect. 7.2.4.2).
7.2.5 CALIBRATION STANDARDS (CAL) - At least five calibration concentrations
are required to prepare the initial calibration curve spanning a 20-fold
concentration range (Sect. 10.2). Larger concentration ranges will require more
calibration points. Prepare the CAL standards over the concentration range of
interest from dilutions of the analyte PDS in acetone. The suggested analyte
concentrations found in Tables 5-11 can be used as a starting point for
determining the calibration range. The IS and SUR are added to the CAL
standards at a constant concentration. During method development, the
concentration of the SUR was 12.5 |ig/L in the standard (250 ng/L in the aqueous
sample) and the IS concentration was 10.0 |ig/L. The lowest concentration CAL
standard must be at or below the MRL, which may depend on system sensitivity.
The CAL standards may also be used as CCCs (Sect. 9.3.2). CAL standards
should be stored at 6 °C or less. Calibration standards are valid for up to thirty
days if properly stored. CAL standards must be aliquoted in separate vials for
each injection so that no more than one injection occurs per vial. Example CAL
standard preparations are found below.
CAL STD
Prepared at
1 mL in
2 mL vial
Vol. of
1.00 |ig/mL
analyte PDS
(nL)
Vol. of
1.25 |ig/mL
SUR PDS
(HL)
Vol. of
1.00 |ig/mL
IS PDS
(HL)
Final Cone, of
Target Analytesa in
CAL standard
(ur/L)
CAL1
2.5
10
10
2.5
CAL2
5.0
10
10
5.0
CAL3
10.0
10
10
10.0
CAL4
15.0
10
10
15.0
CAL5
20.0
10
10
20.0
CAL6
40.0
10
10
40.0
CAL7
60.0
10
10
60.0
a Final standarc
concentration of SUR is 12.5 |ig/L and IS is 10.0 |ig/L.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1. SAMPLE BOTTLE PREPARATION
8.1.1 Collect samples in 250 mL (or smaller) amber glass bottles fitted with a Teflon™
lined screw-cap.
NOTE: Smaller sample volumes (100 mL minimum) can be collected if the
laboratory demonstrates acceptable performance in meeting the required MRLs (Sect.
9.2.4) using the smaller sample volume. However, the entire sample volume collected
must be processed (e.g., a 100 mL sample cannot be aliquoted from a 250 mL
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sample). The amount of added preservatives and surrogate/analyte fortification levels
must be adjusted accordingly.
8.1.2 The preservation reagents, listed in the table below, are added to each sample
bottle as solids prior to shipment to the field (or prior to sample collection).
Compound
Amount
Purpose
Sodium bisulfate (Sect. >)
1.0 g/L
Inhibits microbial growth
L-ascorbic acid (Sect. )
100 mg/L
Removes free chlorine
8.2.	SAMPLE COLLECTION
8.2.1	Open the tap and allow the system to flush until the water temperature has
stabilized (approximately 3 to 5 min). Collect samples from the flowing system.
8.2.2	Fill sample bottles, taking care not to flush out the sample preservation reagents.
Samples do not need to be collected headspace free.
8.2.3	After collecting the sample, cap the bottle and agitate by hand until preservatives
are dissolved. Keep the sample sealed from time of collection until extraction.
8.3.	FIELD REAGENT BLANKS (FRB)
8.3.1	An FRB must be handled along with each sample set. The sample set is composed
of samples collected from the same sample site and at the same time. At the
laboratory, fill the field blank sample bottle with reagent water, then seal, and ship
to the sampling site along with the sample bottles. For each FRB shipped, a
second FRB bottle containing only the preservative must also be shipped. At the
sampling site, the sampler must open the shipped FRB and pour the preserved
reagent water into the empty shipped sample bottle, seal and label this bottle as
the FRB. The FRB is shipped back to the laboratory along with the samples and
analyzed to ensure that nonylphenols were not introduced into the sample during
sample collection/handling.
8.3.2	The same batch of preservatives must be used for the FRBs as for the field
samples.
8.3.3	The reagent water used for the FRBs must be initially analyzed for method
analytes as an LRB (using the same lot of sample bottles as the field samples) and
must meet the LRB criteria in Section 9.3.1 prior to use. This requirement will
ensure samples are not being discarded due to contaminated reagent water or
sample bottles rather than contamination during sampling.
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8.4.	SAMPLE SHIPMENT AND STORAGE - Samples must be chilled during shipment
and must not exceed 10 °C during the first 48 hours after collection. Sample
temperature must be confirmed to be at or below 10 °C when the samples are received
at the laboratory. Samples stored in the lab must be held at or below 6 °C until
extraction but must not be frozen.
NOTE: Samples that are significantly above 10° C, at the time of collection, may need
to be iced or refrigerated for a period of time, in order to chill them prior to shipping.
This will allow them to be shipped with sufficient ice to meet the above requirements.
8.5.	SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage
stability study (Table 12) indicated that all compounds listed in this method have
adequate stability for 28 days when collected, preserved, shipped and stored as
described in Sections 8.1. 8.2. and 8,4. Therefore, water samples should be extracted
as soon as possible but must be extracted within 28 days. Extracts must be stored at or
below 6 °C and analyzed within 28 days after extraction. The extract storage stability
study data are presented in Table 13.
9. QUALITY CONTROL
9.1. QC requirements include the Initial Demonstration of Capability (IDC) and ongoing
QC requirements that must be met when preparing and analyzing Field Samples. This
section describes the QC parameters, their required frequencies, and the performance
criteria that must be met in order to meet EPA quality objectives. The QC criteria
discussed in the following sections are summarized in Tables 14 and 15. These QC
requirements are considered the minimum acceptable QC criteria. Laboratories are
encouraged to institute additional QC practices to meet their specific needs.
9.1.1 METHOD MODIFICATIONS - The analyst is permitted to modify LC columns,
LC conditions, evaporation techniques, internal standards or surrogate standards
(refer to cautions about substituting SUR and IS standards. Sections 7.2.1 and
7.2.2). and MS and MS/MS conditions. Each time such method modifications are
made, the analyst must repeat the procedures of the IDC. LC modifications to
shorten the analytical run will move method analyte peaks closer to matrix
interferences from humics/fulvics and nonylphenoxy carboxylic acids,
potentially increasing the probability of suppression/ enhancement effects.
9.1.1.1 DEMONSTRATION OF NON-INTERFERENCE FROM SELECT
NONYLPHENOXY CARBOXYLIC ACIDS - If the LC column and
conditions are modified from suggested parameters in Table 1, it must be
demonstrated that nonylphenoxy carboxylic acids do not co-elute with
nonylphenol. Prepare the interference check standard as specified in Section
7.2.4.3. Inject the ICS and search the chromatogram for nonylphenoxy
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carboxylic acid peaks at the same transition (m/z 219^-133) as nonylphenol.
Both nonylphenoxy carboxylic acids must be separated from the
nonylphenol peak by at least one minute to eliminate the possibility of
interference. Nonylphenoxy carboxylic acids peaks may tail under method
conditions and could interfere with nonylphenol since they share the same
MS/MS transition.
9.2. INITIAL DEMONSTRATION OF CAPABILITY - The IDC must be successfully
performed prior to analyzing any Field Samples. Prior to conducting the IDC, the
analyst must first generate an acceptable Initial Calibration following the procedure
outlined in Section . The IDC must be repeated if the laboratory changes the brand
of SPE cartridge.
9.2.1	INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Any time
a new lot of SPE cartridges, solvents, centrifuge tubes, disposable pipets, and
autosampler vials are used, it must be demonstrated that an LRB is reasonably
free of contamination and that the criteria in Section 9.3.1 are met. If an
automated extraction system is used, an LRB should be extracted on each port to
ensure that all the valves and tubing are free from potential nonylphenol
contamination.
9.2.2	INITIAL DEMONSTRATION OF PRECISION (IDP) - Prepare, extract, and
analyze four to seven replicate LFBs fortified near the midrange of the initial
calibration curve according to the procedure described in Sections 11.3 and 11.4.
Sample preservatives as described in Section 8.1.2 must be added to these
samples. The percent relative standard deviation (%RSD) of the results of the
replicate analyses must be less than or equal to 20%.
Standard Deviation of Measured Concentrations
%RSD = 	-			X 100
Average Concentration
9.2.3	INITIAL DEMONSTRATION OF ACCURACY (IDA) - Using the same set of
replicate data generated for Section 9.2.2. calculate average percent recovery
(%R). The average recovery of the replicate values must be within ± 30% of the
true value.
Average Measured Concentration
%R = 	5——	X 100
Fortified Concentration
9.2.4	MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Establish a target
concentration for the MRL based on the intended use of the method. The MRL
may be established by a laboratory for their specific purpose or may be set by a
regulatory agency. If there is a programmatic MRL requirement, the laboratory
MRL must be set at or below this level. Establish an Initial Calibration following
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the procedure outlined in Section 10.2. The lowest CAL standard used to establish
the Initial Calibration (as well as the low-level CCC, Section ) must be at or
below the concentration of the MRL. Establishing the MRL concentration too low
may cause repeated failure of ongoing QC requirements. Confirm the MRL
following the procedure outlined below.
NOTE: Setting an MRL for method analytes that are consistently present in the
background (e.g., nonylphenol) is particularly important so that false positive data
are not reported for Field Samples. See Sect. 9.3.1 for guidance in setting an MRL
for these analytes.
9.2.4.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL
concentration. These LFBs must contain all method preservatives described
in Section 8.1.2. Calculate the mean measured concentration (Mean) and
standard deviation for these replicates. Determine the Half Range for the
prediction interval of results (HRpir) using the equation below
HRpm = 3.963s
where
5 = the standard deviation
3.963 = a constant value for seven replicates.1
9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result
(PIR = Mean ± HRpir) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be <150% recovery.
Mean + HRpir
x 100% < 150%
Fortified Concentration
The Lower PIR Limit must be > 50% recovery.
Mean — HRpir
Fortified Concentration
x 100% > 50%
9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the
criteria described above (Sect. 9.2.4.2). If these criteria are not met, the
MRL has been set too low and must be determined again at a higher
concentration. Because background contamination can be a significant
problem, some MRLs may be background limited.
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9.2.5	CALIBRATION CONFIRMATION - Analyze a QCS as described in
Section to confirm the accuracy of the standards/calibration curve.
9.2.6	DETECTION LIMIT DETERMINATION (optional) - While DL determination
is not a specific requirement of this method, it may be required by various
regulatory bodies associated with compliance monitoring. It is the responsibility
of the laboratory to determine if DL determination is required based upon the
intended use of the data.
9.2.6.1	Replicate analyses for this procedure should be done over at least three days
(i.e., both the sample extraction and the LC/MS/MS analyses should be done
over at least three days). Prepare at least seven replicate LFBs at a
concentration estimated to be near the DL. This concentration may be
estimated by selecting a concentration at 2-5 times the noise level. The DLs
in Table 5 were calculated from LFBs fortified at various concentrations as
indicated in the table. The appropriate fortification concentrations will be
dependent upon the sensitivity of the LC/MS/MS system used. All
preservation reagents listed in Section 8.1.2 must also be added to these
samples. Analyze the seven replicates through all steps of Section 11.
NOTE: If an MRL confirmation data set meets these requirements, a DL
may be calculated from the MRL confirmation data, and no additional
analyses are necessary.
Calculate the DL using the following equation
DL = sx t^n_^ 1_Qr=099)
where
5 = standard deviation of replicate analyses
I o-i, i-a=o.99) = Student's t value for the 99% confidence
level with n-1 degrees of freedom
n = number of replicates.
NOTE: Do not subtract blank values when performing DL calculations. The
DL is a statistical determination of precision only.2 If the DL replicates are
fortified at a low enough concentration, it is likely that they will not meet
the precision and accuracy criteria for CCCs. Therefore, no precision and
accuracy criteria are specified.
9.2.6.2	If a laboratory is establishing their own MRL, the calculated DLs should not
be used as the MRL for analytes that commonly occur as background
contaminants. Method analytes that are seen in the background should be
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reported as present in Field Samples, only after careful evaluation of the
background levels. It is recommended that a MRL be established at the
mean LRB concentrations + 3a or 3 times the mean LRB concentration,
whichever is greater. This value should be calculated over a period of time,
to reflect variability in the blank measurements. It is recommended that this
value be used as an MRL in order to avoid reporting false positive results.
9.3. ONGOING QC REQUIREMENTS - This section summarizes the ongoing QC
criteria that must be followed when processing and analyzing Field Samples.
9.3.1	LABORATORY REAGENT BLANK (LRB) - An LRB is required with each
extraction batch (Sect. 3.6) to confirm that potential background contaminants are
not interfering with the identification or quantitation of method analytes. If more
than 20 Field Samples are collected, analyze an LRB for every 20 samples. If the
LRB produces a peak within the retention time window of any analyte that would
prevent the determination of that analyte, determine the source of contamination
and eliminate or reduce as much as possible the interference before processing
samples. Background contamination must be reduced to an acceptable level
before proceeding. Blank contamination is estimated by extrapolation, if the
concentration is below the lowest CAL standard. This extrapolation procedure is
not allowed for sample results as it may not meet data quality objectives.
Background concentrations of method analytes must be less than or equal to one-
third the MRL. If method analytes are detected in the LRB at concentrations
greater than this level, then all positive field sample results (i.e., results at or
above the MRL) for those analytes are invalid for all samples in the extraction
batch. Because background contamination may be a problem for nonylphenol,
maintaining a historical record of LRB data is highly recommended.
NOTE: It is extremely important to evaluate background values of analytes that
commonly occur in LRBs. The MRL must be set at a value greater than three
times the mean concentration observed in replicate LRBs. If LRB values are
highly variable, setting the MRL to a value greater than the mean LRB
concentration plus three times the standard deviation may provide a more realistic
MRL.
9.3.2	CONTINUING CALIBRATION CHECK (CCC) - CCC Standards are analyzed
at the beginning of each analysis batch, after every 10 Field Samples, and at the
end of the analysis batch. See Section 10.3 for concentration requirements and
acceptance criteria.
9.3.3	LABORATORY FORTIFIED BLANK (LFB) - An LFB is required with each
extraction batch (Sect. 3.6). The fortified concentration of the LFB must be
rotated between low, medium, and high concentrations from batch to batch. The
low concentration LFB must be as near as practical to, but no more than two
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times, the MRL. Similarly, the high concentration LFB should be near the high
end of the calibration range established during the initial calibration (Sect. 10.2).
Results of the low-level LFB analyses (within a factor of 2-times the MRL
concentration) must be 50-150% of the true value. Results of the medium and
high-level LFB analyses must be 70-130% of the true value. If the LFB results do
not meet these criteria for method analytes, then all data for the problem
analyte(s) must be considered invalid for all samples in the extraction batch.
9.3.4	INTERNAL STANDARDS (IS) - The analyst must monitor the peak areas of the
IS in all injections during each analysis day. The IS responses (peak areas) in any
chromatographic run must be within 70-130%) of the response in the most recent
CCC and must not deviate by more than 50% from the average area measured
during initial analyte calibration. If the IS areas in a chromatographic run do not
meet these criteria, inject a second aliquot of that extract placed into a new capped
autosampler vial.
9.3.4.1	If the reinjected aliquot produces an acceptable IS response, report results
for that aliquot.
9.3.4.2	If the reinjected extract fails again, the analyst should check the calibration
by reanalyzing the most recently acceptable CAL standard. If the CAL
standard fails the criteria of Section 10.3. recalibration is in order per
Section 10.2. If the CAL standard is acceptable, extraction of the sample
may need to be repeated provided the sample is still within the holding time.
Otherwise, report results obtained from the reinjected extract, but annotate
as suspect. Alternatively, collect a new sample and re-analyze.
9.3.5	SURROGATE RECOVERY - The SUR standard is fortified into all samples,
CCCs, LRBs, LFBs, LFSMs, LFSMDs, FDs, and FRBs prior to extraction. It is
also added to the CAL standards. The SUR is a means of assessing method
performance from extraction to final chromatographic measurement. Calculate the
recovery (%R) for the SUR using the following equation
9.3.5.1 SUR recovery must be in the range of 70-130%. When SUR recovery from
a sample, blank, or CCC is less than 70% or greater than 130%, check
1) calculations to locate possible errors, 2) standard solutions for
where
A = calculated SUR concentration for the QC or Field Sample
B = fortified concentration of the SUR.
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degradation, 3) contamination, and 4) instrument performance. Correct the
problem and reanalyze the extract.
9.3.5.2	If the extract reanalysis meets the SUR recovery criterion, report only data
for the reanalyzed extract.
9.3.5.3	If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by injecting the last CAL standard that passed.
If the CAL standard fails the criteria of Section 10.3. recalibration is in order
per Section 10.2. If the CAL standard is acceptable, extraction of the sample
should be repeated provided the sample is still within the holding time. If
the re-extracted sample also fails the recovery criterion, report all data for
that sample as suspect/SUR recovery to inform the data user that the results
are suspect due to SUR recovery. Alternatively, collect a new sample and
re-analyze.
9.3 .6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of an
LFSM is required in each extraction batch and is used to determine that the
sample matrix does not adversely affect method accuracy. Assessment of method
precision is accomplished by analysis of a Field Duplicate (FD) (Sect. 9.3.7);
however, infrequent occurrence of method analytes would hinder this assessment.
If the occurrence of method analytes in the samples is infrequent, or if historical
trends are unavailable, a second LFSM, or LFSMD, must be prepared, extracted,
and analyzed from a duplicate of the Field Sample. Extraction batches that
contain LFSMDs will not require the extraction of a FD. If a variety of different
sample matrices are analyzed regularly, for example, drinking water from ground
water and surface water sources, method performance should be established for
each. Over time, LFSM data should be documented by the laboratory for all
routine sample sources.
9.3.6.1	Within each extraction batch (Sect. 3.6), a minimum of one Field Sample is
fortified as an LFSM for every 20 Field Samples analyzed. The LFSM is
prepared by spiking a sample with an appropriate amount of the Analyte
PDS (Sect. 7.2.3.2). Select a spiking concentration that is greater than or
equal to the matrix background concentration, if known. Use historical data
and rotate through the low, mid and high concentrations when selecting a
fortifying concentration.
9.3.6.2	Calculate the percent recovery (%R) for each analyte using the equation
%r = (a~bK 100
C
where
A = measured concentration in the fortified sample
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B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.6.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or
above their native concentration, recoveries should range between 70-130%,
except for low-level fortification near or at the MRL (within a factor of
2-times the MRL concentration) where 50-150% recoveries are acceptable.
If the accuracy of any analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control in the
CCCs, the recovery is judged to be matrix biased. The result for that analyte
in the unfortified sample is labeled suspect/matrix to inform the data user
that the results are suspect due to matrix effects.
9.3.7 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX
DUPLICATE (FD or LFSMD) - Within each extraction batch (not to exceed 20
Field Samples, Sect. 3.6). a minimum of one FD or LFSMD must be analyzed.
Duplicates check the precision associated with sample collection, preservation,
storage, and laboratory procedures. If method analytes are not routinely observed
in Field Samples, an LFSMD should be analyzed rather than an FD.
9.3.7.1 Calculate the relative percent difference (RPD) for duplicate measurements
(I'DI and FD2) using the equation
9.3.7.2	RPDs for FDs should be <30%. Greater variability may be observed when
FDs have analyte concentrations that are within a factor of 2 of the MRL. At
these concentrations, FDs should have RPDs that are <50%. If the RPD of
any analyte falls outside the designated range, and the laboratory
performance for that analyte is shown to be in control in the CCC, the
recovery is judged to be matrix influenced. The result for that analyte in the
unfortified sample is labeled suspect/matrix to inform the data user that the
results are suspect due to matrix effects.
9.3.7.3	If an LFSMD is analyzed instead of a FD, calculate the relative percent
difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the
equation
NOTE: LFSMs and LFSMDs fortified at concentrations near the MRL,
where the associated Field Sample contains native analyte concentrations
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above the DL but below the MRL, should be corrected for the native levels
in order to obtain meaningful %R values. This example, and the LRB
extrapolation (Sect. ), are the only permitted uses of analyte results
below the MRL.
9.3.7.4 RPDs for duplicate LFSMs must be <30% for samples fortified at or above
their native concentration. Greater variability may be observed when
LFSMs are fortified at analyte concentrations that are within a factor of 2 of
the MRL. LFSMs fortified at these concentrations must have RPDs that are
<50% for samples fortified at or above their native concentration. If the
RPD of any analyte falls outside the designated range, and the laboratory
performance for that analyte is shown to be in control in the CCC, the
recovery is judged to be matrix biased. The result for that analyte in the
unfortified sample is labeled suspect/matrix to inform the data user that the
results are suspect due to matrix effects.
9.3 .8 FIELD REAGENT BLANK (FRB) - The purpose of the FRB is to ensure that
method analytes measured in the Field Samples were not inadvertently introduced
into the sample during sample collection/handling. Analysis of the FRB is
required only if a Field Sample contains a method analyte or analytes at or above
the MRL. The FRB is processed, extracted and analyzed in exactly the same
manner as a Field Sample. If the method analyte(s) found in the Field Sample is
present in the FRB at a concentration greater than 1/3 the MRL, then all samples
collected with that FRB are invalid and must be recollected and reanalyzed.
9.3 .9 QUALITY CONTROL SAMPLES (QCS) - As part of the IDC (Sect. 92), each
time a new Analyte PDS (Sect. 7.2.3.2) is prepared, and at least quarterly, analyze
a QCS sample from a source different from the source of the CAL standards. If a
second vendor is not available, then a different lot of the standard should be used.
The QCS should be prepared at a mid-level concentration and analyzed just like a
CCC. Acceptance criteria for the QCS are identical to the CCCs; the calculated
amount for each analyte must be ± 30% of the expected value. If measured
analyte concentrations are not of acceptable accuracy, check the entire analytical
procedure to locate and correct the problem.
10. CALIBRATION AND STANDARDIZATION
10.1. Demonstration and documentation of acceptable initial calibration is required before
any samples are analyzed. After the initial calibration is successful, a CCC is required
at the beginning and end of each period in which analyses are performed, and after
every tenth Field Sample.
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2. INITIAL CALIBRATION
10.2.1	ESI-MS/MS TUNE
10.2.1.1	Calibrate the mass scale of the MS with the calibration compounds and
procedures prescribed by the manufacturer.
10.2.1.2	Optimize the [M-H]" for each method analyte by infusing approximately
1.0 ng/mL of each analyte directly into the MS at the chosen LC mobile
phase flow rate (approximately 0.3 mL/min). The mobile phase conditions
may be varied to optimize sensitivity of the infused analytes(s). This tune
can be done on a mix of the method analytes. The MS parameters (voltages,
temperatures, gas flows, etc.) are varied until optimal analyte responses are
determined. The method analytes may have different optima requiring some
compromise between the optima. See Table 2 for ESI-MS conditions used in
method development.
10.2.1.3	Optimize the product ion (Sect. 3.21) for each analyte by infusing
approximately 1.0 ng/mL of each analyte directly into the MS at the chosen
LC mobile phase flow rate (approximately 0.3 mL/min). The mobile phase
conditions may be varied to optimize sensitivity of the infused analytes(s).
This tune can be done on a mix of the method analytes. The MS/MS
parameters (collision gas pressure, collision energy, etc.) are varied until
optimal analyte responses are determined. See Table 4 for MS/MS
conditions used in method development.
10.2.2	Establish LC operating parameters that optimize resolution and peak shape.
Ammonium fluoride was added to the aqueous phase at 0.2 mM to enhance
sensitivity and to increase stability from potential matrix interference competing
for ionization in the source. Suggested LC conditions can be found in Table 1.
The LC conditions listed in Table 1 may not be optimum for all LC systems and
may need to be optimized by the analyst.
Cautions: LC system components, as well as the mobile phase constituents,
may contain trace amounts of nonylphenol. Thus, nonylphenol will build up
on the head of the LC column during mobile phase equilibration. To
minimize the background nonylphenol peaks and to keep background levels
constant, the time the LC column is held at initial conditions must be kept
constant and as short as possible (while ensuring reproducible retention
times). In addition, prior to daily use, flush the column with 100% methanol
for at least 15 min before initiating a sequence. It may be necessary on some
systems to flush other LC components such as wash syringes, sample needles
or any other system components before daily use. Acetone was found to rinse
nonylphenol from syringes and valves better than methanol.
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10.2.3	Inject a mid-level CAL standard under LC/MS conditions to obtain the retention
times of each method analyte. Divide the chromatogram into retention time
windows each of which contains one or more chromatographic peaks. During
MS/MS analysis, fragment a small number of selected precursor ions ([M-H]";
Sect. ) for the analytes in each window and choose the most abundant product
ion. The product ions (also the quantitation ions) chosen during method
development are in Table 4, although these will be instrument dependent.
10.2.4	Inject a mid-level CAL standard under optimized LC/MS/MS conditions to ensure
that each method analyte is observed in its MS/MS window and that there are at
least 10 scans across the peak for optimum precision.
10.2.5	Prepare a set of at least five CAL standards as described in Section 7.2.5. The
lowest concentration CAL standard must be at or below the MRL, which may
depend on system sensitivity. It is recommended that at least four of the CAL
standards are at a concentration greater than or equal to the MRL.
10.2.6	The LC/MS/MS system is calibrated using the IS technique. Use the LC/MS/MS
data system software to generate a linear regression or quadratic calibration curve
for each of the analytes. This curve must always be forced through zero and may
be concentration weighted, if necessary. Forcing zero allows for a better estimate
of the background levels of method analytes.
10.2.7	CALIBRATION ACCEPTANCE CRITERIA - Validate the initial calibration by
calculating the concentration of each analyte as an unknown against its regression
equation. For calibration levels that are < MRL, the result for each analyte must
be within ± 50% of the true value. All other calibration points must calculate to be
within ± 30% of their true value. If these criteria cannot be met, the analyst will
have difficulty meeting ongoing QC criteria. It is recommended that corrective
action is taken to reanalyze the CAL standards, restrict the range of calibration, or
select an alternate method of calibration (forcing the curve through zero is still
required).
CAUTION: When acquiring MS/MS data, LC operating conditions must be
carefully reproduced for each analysis to provide reproducible retention times. If
this is not done, the correct ions will not be monitored at the appropriate times. As
a precautionary measure, the chromatographic peaks in each window must not
elute too close to the edge of the segment time window.
3. CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
verification is as follows. Verify the initial calibration at the beginning and end of each
group of analyses, and after every tenth sample during analyses. In this context, a
"sample" is considered to be a Field Sample. LRBs, CCCs, LFBs, LFSMs, FDs, FRBs
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and LFSMDs are not counted as samples. The beginning CCC of each analysis batch
must be at or below the MRL to verify instrument sensitivity prior to any analyses. If
standards have been prepared such that all low CAL points are not in the same CAL
solution, it may be necessary to analyze two CAL standards to meet this requirement.
Alternatively, the analyte concentrations in the analyte PDS may be customized to
meet these criteria. Subsequent CCCs should alternate between a medium and high
concentration CAL standard.
10.3.1	Inject an aliquot of the appropriate concentration CAL standard and analyze with
the same conditions used during the initial calibration.
10.3.2	Determine that the absolute areas of the quantitation ions of the IS(s) are within
70-130% of the areas measured in the most recent continuing calibration check,
and within 50-150% from the average areas measured during initial calibration. If
any of the IS areas has changed by more than these amounts, adjustments must be
made to restore system sensitivity. These adjustments may include cleaning of the
MS ion source, or other maintenance as indicated in Section . Major
instrument maintenance requires recalibration (Sect. 10.2) and verification of
sensitivity by analyzing a CCC at or below the MRL (Sect. 10.3). Control charts
are useful aids in documenting system sensitivity changes.
10.3.3	Calculate the concentration of each analyte and SUR in the CCC. The calculated
amount for each analyte and SUR for medium and high level CCCs must be
within ± 30%) of the true value. The calculated amount for the lowest calibration
point for each analyte must be within ± 50%> and the SUR must be within ± 30%>
of the true value. If these conditions do not exist, then all data for the problem
analyte must be considered invalid, and remedial action should be taken
(Sect. 10.3.4) which may require recalibration. Any Field or QC Samples that
have been analyzed since the last acceptable calibration verification that are still
within holding time must be reanalyzed after adequate calibration has been
restored, with the following exception. If the CCC fails because the calculated
concentration is greater than 130% (150% for the low-level CCC) for a
particular method analyte, and Field Sample extracts show no detection for
that method analyte, non-detects may be reported without re-analysis.
10.3.4	REMEDIAL ACTION - Failure to meet CCC QC performance criteria may
require remedial action. Major maintenance, such as cleaning the electrospray
probe, atmospheric pressure ionization source, cleaning the mass analyzer,
replacing the LC column, etc., requires recalibration (Sect. 10.2) and verification
of sensitivity by analyzing a CCC at or below the MRL (Sect. 10.3)
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11. PROCEDURE
11.1.	This procedure may be performed manually or in an automated mode using a robotic
or automatic sample preparation device. The data presented in Tables 5-13
demonstrate data collected by manual extraction. If an automated system is used to
prepare samples, follow the manufacturer's operating instructions, but all extraction
and elution steps must be the same as in the manual procedure. Extraction and/or
elution steps may not be changed or omitted to accommodate the use of an automated
system. If an automated system is used, the LRBs should be rotated among the ports to
ensure that all the valves and tubing meet the LRB requirements (Sect. 9.3.1).
11.2.	Nonylphenol may adsorb to surfaces. Therefore, the aqueous sample bottles must be
rinsed with the elution solvent whether extractions are performed manually or by
automation. The bottle rinse is passed through the cartridge to elute the method
analytes and is then collected (Sect. ). Due to this adsorption, the entire sample
volume collected, excluding sample used for pH verification and residual chlorine
testing must be extracted.
NOTE: The SPE cartridges described in this section are designed as single use items
and must be discarded after use. They may not be refurbished for reuse in subsequent
analyses.
11.3.	SAMPLE PREPARATION
11.3.1	Samples are preserved, collected and stored as presented in Section 8. All Field
and QC Samples, including the LRB, LFB and FRB, must contain the
preservatives listed in Section 8.1.2. Before extraction, verify that the sample pH
is <3. If pH is outside this range, the sample must be rejected. Determine sample
volume. An indirect measurement may be done in one of two ways: by marking
the level of the sample on the bottle or by weighing the sample and bottle to the
nearest 1 g. After extraction, proceed to Section for final volume
determination. Nonylphenol may adsorb to surfaces, thus the sample volume may
NOT be transferred to a graduated cylinder for volume measurement. The LRB,
LFB and FRB may be prepared by measuring 250 mL of reagent water with a
glass graduated cylinder or filling a 250 mL sample bottle to near the top.
11.3.2	Add an aliquot of the SUR PDS (Sect. 7.2.2.2) to each sample, cap and invert to
mix. During method development, a 50 |iL aliquot of the 1.25 |j,g/mL SUR PDS
was added to each 250 mL sample for a final aqueous sample concentration of
250 ng/L for 4-tert-octylphenol-13C6.
11.3.2.1 In addition to the SUR, dechlorination agent and antimicrobial, if the sample
is an LFB, LFSM, or LFSMD, add the necessary amount of analyte PDS
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(Sect. 7.2.3.2), taking into account the final extract volume is 5 mL. Cap and
invert each sample to mix.
11.4.	CARTRIDGE SPE PROCEDURE
11.4.1	CARTRIDGE CLEAN-UP AND CONDITIONING - DO NOT allow cartridge
packing material to go dry during any of the conditioning steps. Rinse each
cartridge with 15 mL of acetone. Next, rinse each cartridge with 15 mL of reagent
water, without allowing the water to drop below the top edge of the packing. If
the cartridge goes dry during the conditioning phase, the conditioning must be
started over. Add 2-3 mL of reagent water to each cartridge, attach the sample
transfer tubes (Sect. .), turn on the vacuum, and begin adding sample to the
cartridge.
11.4.2	SAMPLE EXTRACTION - Adjust the vacuum so that the approximate flow rate
is 10-15 mL/min. Do not allow the cartridge to go dry before all the sample has
passed through.
11.4.3	SAMPLE BOTTLE AND CARTRIDGE RINSE - After the entire sample has
passed through the cartridge, rinse the sample bottles with one 5 mL aliquot of
reagent water and draw each aliquot through the sample transfer tubes and the
cartridges. Draw air or nitrogen through the cartridge for 5 min at high vacuum
(10-15 in. Hg).
11.4.4	SAMPLE BOTTLE RINSE AND CARTRIDGE ELUTION - Turn off and
release the vacuum. Lift the extraction manifold top and insert a rack with
collection tubes into the extraction tank to collect the extracts as they are eluted
from the cartridges. Rinse the sample bottles with 2 mL of acetone and elute the
analytes from the cartridges by pulling the 2 mL of acetone through the sample
transfer tubes and the cartridges. Use a low vacuum such that the solvent exits the
cartridge in a dropwise fashion. Repeat sample bottle rinse and cartridge elution
with a second 2 mL aliquot of acetone.
11.5.	EXTRACT VOLUME ADJUSTMENT - Add IS PDS (50 |aL of the 1.00 |ig/mL IS
PDS for extract concentrations of 10.0 |ig/L were used for method development;
Sect. 7.2.1.2) to the collection vial and bring the volume to 5 mL with acetone and
vortex. Transfer a small aliquot with a glass pipet (Sect. 6.9) to a low volume
autosampler vial.
NOTE: It is recommended that a small aliquot be transferred to a single
autosampler vial because each vial can only be used for one injection due to the
potential of extract contamination from nonylphenol after septa puncture.
Extract aliquots can be stored in autosampler vials prior to puncture of the vial
cap. Alternatively, extracts can be stored in 15 mL centrifuge tubes (Sect. 6.2)
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with caps. Pre-slit septa are not permitted because they cause excess extract
evaporation and have the potential to allow nonylphenol from laboratory air to
intrude into the sample extract.
11.6.	SAMPLE VOLUME DETERMINATION - If the level of the sample was marked on
the sample bottle, use a graduated cylinder to measure the volume of water required to
fill the original sample bottle to the mark made prior to extraction. Determine to the
nearest 2 mL. If using weight to determine volume, weigh the empty bottle to the
nearest 1 g and determine the sample weight by subtraction of the empty bottle weight
from the original sample weight (Sect. 11.3.1V Assume a sample density of 1.0 g/mL.
In either case, the sample volume will be used in the final calculations of the analyte
concentration (Sect. 12.2).
11.7.	EXTRACT ANALYSIS
11.7.1	Establish operating conditions equivalent to those summarized in Tables 1-4 of
Section 17. Instrument conditions and columns should be optimized prior to the
initiation of the IDC.
11.7.2	Establish an appropriate retention time window for each analyte. This should be
based on measurements of actual retention time variation for each method analyte
in CAL standard solutions analyzed on the LC/MS/MS over the course of time. A
value of plus or minus three times the standard deviation of the retention time
obtained for each method analyte while establishing the initial calibration and
completing the IDC can be used to calculate a suggested window size. However,
the experience of the analyst should weigh heavily on the determination of the
appropriate retention window size.
11.7.3	Calibrate the system by either the analysis of calibration standards (Sect. 10.2) or
by confirming the initial calibration is still valid by analyzing a CCC as described
in Section . If establishing an initial calibration, complete the IDC as
described in Section 92.
11.7.4	Begin analyzing Field Samples, including QC samples, at their appropriate
frequency by injecting the same size aliquots (10 |oL was used in method
development), under the same conditions used to analyze the CAL standards.
11.7.5	At the conclusion of data acquisition, use the same software that was used in the
calibration procedure to identify peaks of interest in predetermined retention time
windows. Use the data system software to examine the ion abundances of the
peaks in the chromatogram. Identify an analyte by comparison of its retention
time with that of the corresponding method analyte peak in a reference standard.
Comparison of the MS/MS mass spectra is not particularly useful given the
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limited ±0.5 dalton mass range around a single product ion for each method
analyte.
11.7.6 The analyst must not extrapolate beyond the established calibration range. If an
analyte peak area exceeds the range of the initial calibration curve, the extract
must be diluted with acetone and the appropriate amount of IS added to match the
original concentration. Re-inject the diluted extract. Incorporate the dilution factor
into the final concentration calculations. Acceptable SUR performance
(Sect.	) should be determined from the undiluted sample extract. The
resulting data for the analyte peak that exceeded the initial calibration range must
be documented as a dilution and MRL adjusted accordingly.
12. DATA ANALYSIS AND CALCULATION
12.1.	Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations using MS/MS. In validating this method,
concentrations were calculated by measuring the product ions listed in Table 4. Other
ions may be selected at the discretion of the analyst.
12.2.	Calculate analyte and SUR concentrations using the multipoint calibration as
described in Section 10.2. Do not use daily calibration verification data to quantitate
analytes in samples. Adjust final analyte concentrations to reflect the actual sample
volume determined in Section 11.6.
12.3.	Prior to reporting the data, the chromatogram should be reviewed for any incorrect
peak identification or poor integration.
12.4.	Calculations must utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures (one
digit of uncertainty), typically two, and not more than three significant figures.
NOTE: Some data in Section 17 of this method are reported with more than two
significant figures. This is done to better illustrate the method performance.
13. METHOD PERFORMANCE
13.1.	PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS - Tables for
these data are presented in Section 17. LCMRLs and DLs for each method analyte are
presented in Table 5. Precision and accuracy are presented for three water matrices:
reagent water (Tables 6 and 7); chlorinated (finished) ground water (Tables 8 and 9);
chlorinated (finished) surface water (Tables 10 and 11).
13.2.	SAMPLE STORAGE STABILITY STUDIES - An analyte storage stability study was
conducted by fortifying the analytes into chlorinated surface water samples that were
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collected, preserved, and stored as described in Section 8. The precision and mean
recovery (n=4) of analyses, conducted on Days 0, 7, 14, 21, and 28 are presented in
Table 12.
13.3. EXTRACT STORAGE STABILITY STUDIES - Extract storage stability studies
were conducted on extracts obtained from a chlorinated surface water fortified with the
method analytes. The precision and mean recovery (n=4) of injections conducted on
Days 0, 7, 14, 21, and 28 are reported in Table 13.
13 .4. MULTI-LABORATORY DEMONSTRATION - The performance of this method
was demonstrated by multiple laboratories, with results reported in Section 17. The
authors wish to acknowledge the work of Bill Deckelmann and Katie Kohoutek at
American Water Central Laboratory (Belleville, IL), David Schiessel and Susann
Thomas at Babcock Laboratories, Inc. (Riverside, CA), William Lipps and Ali
Haghani at Eurofins Eaton Analytical, LLC (Monrovia, CA), Yongtao Li and Joshua
Whitaker at Eurofins Eaton Analytical, LLC (South Bend, IN).
14.	POLLUTION PREVENTION
14.1.	This method utilizes SPE to extract analytes from water. It requires the use of very
small volumes of organic solvent and very small quantities of pure analytes, thereby
minimizing the potential hazards to both the analyst and the environment as compared
to the use of large volumes of organic solvents in conventional liquid-liquid
extractions.
14.2.	For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Guide to Minimizing Waste in Laboratories"
available from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street N.W., Washington, D.C., 20036 or on-line at
https://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafety
/publications/1 ess-is-better.txlf (accessed November 2019).
15.	WASTE MANAGEMENT
The analytical procedures described in this method generate relatively small amounts of
waste since only small amounts of reagents and solvents are used. The matrices of concern
are finished drinking water. However, laboratory waste management practices must be
conducted consistent with all applicable rules and regulations, and that laboratories protect
the air, water, and land by minimizing and controlling all releases from fume hoods and
bench operations. Also, compliance is required with any sewage discharge permits and
regulations, particularly the hazardous waste identification rules and land disposal
restrictions.
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16. REFERENCES
1.	Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P.,
Hedrick, E. J., Krop, R. A. "Statistical Procedures for Determination and Verification of
Minimum Reporting Levels for Drinking Water Methods." Environ. Sci. Technol. 2006, 40,
281-288.
2.	Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for
Wastewaters." Environ. Sci. Technol. 1981, 15, 1426-1435.
3.	Petrovic, M., Barcelo, D., Diaz, A., Ventura, F. "Low nanogram per liter determination of
halogenated nonylphenols, nonylphenol carboxylates, and their non-halogenated precursors
in water and sludge by liquid chromatography electrospray tandem mass spectrometry." J.
Am. Soc. Mass Spectrom., 2003, 14, 516-527.
4.	Leenheer, J.A., Rostad, C.E., Gates, P.M., Furlong, E.T., Ferrer, I. "Molecular Resolution
and Fragmentation of Fulvic Acid by Electrospray Ionization/Multistage Tandem Mass
Spectrometry." Anal. Chem. 2001, 73, 1461-1471.
5.	Cahill, J.D., Furlong E.T., Burkhardt, M.R., Kolpin, D., Anderson, L.G. "Determination of
Pharmaceutical Compounds in Surface- and Ground-Water Samples by Solid-Phase
Extraction and High-Performance Liquid Chromatography Electrospray Ionization Mass
Spectrometry." J. Chromatogr. A, 2004, 1041, 171-180.
6.	"OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational Safety
and Health Administration, OSHA 2206, (Revised, July 1, 2001).
7.	"Carcinogens-Working with Carcinogens," Publication No. 77-206, Department of Health,
Education, and Welfare, Public Health Service, Center for Disease Control, National Institute
of Occupational Safety and Health, Atlanta, Georgia, August 1977.
8.	"Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 8th Edition. Information on obtaining a copy is available at
https://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafetv/public
ations/safetv-in-academic-chemistrv-laboratories-students.pdf (accessed November 2019).
9.	Pesek, J. J., Matyska, M. T. "Ammonium fluoride as a mobile phase additive in aqueous
normal phase chromatography." J. Chromatogr. A, 2015, 1401, 69-74.
10.	B. B. Potter and J. C. Wimsatt, U.S. EPA Method 415.3: Measurement of total organic
carbon, dissolved organic carbon and specific UV absorbance at 254 nm in source water and
drinking water (Revision 1.1),
https://cfpub.epa.gov/si/si public record report.cfrn?Lab NERL&dirEntryld 103917.
(accessed December 2019).
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17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. LC METHOD
CONDITIONS
'rime (mill)
% 0.2 m.M ;i in in oil in in
Chloride
% Melhsinol
Initial
90
10
1.0
90
10
15.1
5
95
19.0
5
95
19.1
90
10
23.0
90
10
10 |iL injection into a 20
|iL loop
Flow rate of 0.3 mL/min
Autosampler compartment
thermostated at 10 °C
Analytical Column:
Thermo Hypersil Gold
Ci8 2.1 x 50 mm packed
with 3.0 |im Ci8
stationary phase
thermostated to 30 °C
Delay Column:
Thermo Accucore Ci8
2.1 x 100 mm packed
with 4 |am Ci8
stationary phase
TABLE 2. ESI-MS METHOD CONDITIONS
KM Conditions
Soiling
Polarity
Negative ion
Capillary needle voltage
-2.8 kV
Sheath Gas
25 L/hr
Aux Gas
5 L/hr
Sweep Gas
1 L/hr
Ion Transfer Tube Temperature
325 °C
Vaporizer Temperature
300 °C
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TABLE 3. Arr.TTTOD ANAI.YTES AM) RETENTION TTA
rr.s (rtv

Peak #
KT
Ansilvlo
(lis- 1)
(mill)
4-t-OP
1
13.44
NP
2
14.08
4-tert-octylphenol-13C6 (SUR)
3
13.43
4-( 1,3-dimethyl-1-ethylpentyl) phenol-13C6 (IS)
4
14.08
aAn LC/MS/MS chromatogram of the analytes is shown in Figure 1.
TABLE 4. ATS/MS ATETTTOD COXDTTTOXS

Precursor Ion 11
Product Ion11-1'
Collision Knor«v'
Ul I ons
Ansilvlo
(in/:.)
(///.;:)
(v)
(v)
4-t-OP
205
133
23
99
NPd
219
133
29
93
4-t-OP-13C6 (SUR)
211
139
24
95
Branched-NP-13C6 (IS)
225
139
29
101
a Precursor and product ions listed in this table are nominal masses. During MS and MS/MS optimization,
the analyst should determine precursor and product ion masses to one decimal place by locating the apex
of the mass spectral peak place (e.g., m/z 219—>133 for NP). These precursor and product ion masses
(with at least one decimal place) should be used in the MS/MS method for all analyses.
b Ions used for quantitation purposes.
c Argon used as collision gas at 2 mTorr.
d Analyte has multiple unresolved chromatographic peaks due to multiple branched isomers. All peaks
summed as one peak for quantitation purposes.
TABLE 5. DLs A>
fl) LCMRLs IN REAGEN
T WATER
Ansilvlc
l-'orlificri (One. (n«/l.):l
1)1.h (ng/L)
I.CMUI.1 (n«/l.)
4-tert-OP
6.0
3.4
4.9
NP
24
6.2
24
a Spiking concentration used to determine DL.
b Detection limits were determined by analyzing seven replicates over three days according to
Section 9.2.6.
c LCMRLs were calculated according to the procedure in reference 1.
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TABLE 6. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
REAGENT WAT]
ER FORTIFIEI
> AT 600 ng/L (n=4)
Ansilvlc
lorlillcd
Cone.
(ng/L)
Osisis Mi l}
Menu
% Uccovcrv
Osisis Mi l}
% USD
Slrsitsi-\
Mcsiii
% Uccovcrv
Slr:ilsi-\
% USD
4-t-OP
600
99.2
2.1
98.5
1.5
NP
600
96.4
1.6
97.4
0.76
4-t-OP-13C6
250
99.0
2.2
99.5
1.6
TABLE 7. PRECISION ANE
REAGENT WAT]
ACCURACY]
ER FORTIFIEI
9ATA FOR METHOD ANALYTES IN
> AT 100 ng/L (n=4)
Ansilvlc
l-'orlificd
Cone.
(ng/L)
Oiisis lll.li
Mcsiii
% Uccovcrv
Osisis lll.li
% USD
S(rsitsi-\
Menu
% Uccovcrv
Si rjilii-\
% USD
4-t-OP
100
95.1
2.4
95.1
2.4
NP
100
100
3.8
97.4
4.5
4-t-OP-13C6
250
98.4
3.0
98.8
1.1
TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
DRINKING WATER FROM A GROUND WATER SOURCE FORTIFIED AT
	600 ng/L (n=4) 				
Ansilvtc
lorlillcd
Cone,
(ng/l.)
Oiisis lll.li
Mcsiii
% Uccovcrv
Osisis lll.li
% USD
Strsitsi-\
Mcsiii
% Uccovcrv
Strsilsi-\
% USD
4-t-OP
600
97.1
3.5
97.0
0.89
NP
600
96.0
3.4
93.9
0.59
4-t-OP-13C6
250
98.3
3.2
99.9
1.8
a TOC10 = 0.71 mg/L and hardness = 360 mg/L measured as calcium carbonate.
559-38

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TABLE 9. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
DRINKING WATER FROM A GROUND WATER SOURCE FORTIFIED AT
10C
ng/L (n=4)
Aiiiilvlc
lorlillcd
Cone.
(ng/L)
Oiisis Mi l}
Menu
% Recovery
Oiisis Mi l}
% USD
Slrsitsi-\
Menu
% Recovery
Slr:il!i-\
% USD
4-t-OP
100
101
3.8
101
2.0
NP
100
100
3.3
103
3.6
4-t-OP-13C6
250
99.0
1.0
99.1
1.6
a TOC10 = 0.71 mg/L and hardness = 360 mg/L measured as calcium carbonate.
TABLE 10. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
DRINKING WATER FROM A SURFACE WATER SOURCE FORTIFIED
AT 600 ng/ (n=^

Ansilvlc
lorliUcd
Cone.
(ng/L)
Oiisis lll.li
Menu
% Uecoverv
Oiisis lll.li
% USD
Slrsitsi-\
Menu
% Uecoverv
S(r:ilsi-\
% USD
4-t-OP
600
96.4
3.4
98.2
2.1
NP
600
97.2
1.5
96.6
2.1
4-t-OP-13C6
250
99.4
3.4
99.8
3.0
a TOC10 = 1.5 mg/L and hardness = 86 mg/L measured as calcium carbonate.
TABLE 11. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
DRINKING WATER FROM A SURFACE WATER SOURCE FORTIFIED
	AT 100 ng/L (n=4)				
Ansilvlc
l-'orlillcd
Cone,
(ng/l.)
Oiisis lll.li
Menu
% Uecoverv
Oiisis Mi l}
% USD
Striitii-\
Mciin
% Uecoverv
Striitii-\
% USD
4-t-OP
100
103
4.8
102
4.0
NP
100
102
1.2
101
3.7
4-t-OP-13C6
250
98.4
1.9
100
1.0
a TOC10 = 1.5 mg/L and hardness = 86 mg/L measured as calcium carbonate.
559-39

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TABLE 12. AQUEOUS SAMPLE HOLDING TIME DATA FOR TAP WATER SAMPLES FROM A SURFACE
WATER SOURCE3, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND
STORED ACCORDING TO SECTION (n=4)

l-'orlificri
Dsiv 0

Dsiv 7

Dsiv 14

Dsiv 21

Dsiv 28


Cone.
Menu
Dsiv 0
Mcsiii
Day 7
Mcsiii
Dsiv 14
Mcsiii
Dsiv 21
Mcsiii
Dsiv 28
Ansilvlc
(ng/L)
% Ucc
% USD
% Ucc
% USD
% Ucc
% USD
% Ucc
% USD
% Ucc
% USD
4-t-OP
600
96.1
1.0
96.8
1.1
95.6
2.8
92.7
2.1
96.7
1.9
NP
600
98.2
0.85
93.9
1.5
94.0
2.4
92.9
1.4
95.4
0.80
4-t-OP-13C6
250
100
1.9
99.5
0.78
99.2
2.0
95.3
1.5
100
2.0
TOC =1.0 mg/L and hardness = 86 mg/L measured as calcium carbonate.
TABLE 13. EXTRACT HOLDING TIME DATA FOR TAP WATER SAMPLES FROM A SURFACE WATER
SOURCE, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED
Ansilvlc
l-'orlificri
Cone,
(ng/l.)
Dsiv 0
Mcsiii
% Ucc
Dsiv 0
% USD
Dsiv 7
Mcsiii
% Ucc
Dsiv 7
% USD
Dsiv 14
Mcsiii
% Ucc
Dsiv 14
% USD
Dsiv 21
Mcsiii
% Ucc
Dsiv 21
% USD
Dsiv 28
Mcsiii
% Ucc
Dsiv 28
% USD
4-t-OP
600
96.1
1.0
99.5
1.6
98.9
1.3
97.7
1.4
100
2.5
NP
600
98.2
0.85
96.6
1.2
97.4
1.0
100
1.2
99.7
1.6
4-t-OP-13C6
250
100
1.9
99.3
1.4
101
1.2
97.3
1.5
99.2
1.5
559-40

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TABLE 14. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS
Method
Reference
Requirement
Speciliciition jiiuI l-requency
Acceptance Crileriii
Sect. 9.2.1.
Initial Demonstration of
Low System Background
Analyze LRB prior to any other IDC steps.
Demonstrate that all method analytes are less than or equal to
1/3 the MRL and that possible interferences from extraction
media do not prevent the identification and quantification of
method analytes.
Sect. 9.2.2
Initial Demonstration of
Precision (IDP)
Analyze four to seven replicate LFBs fortified near
the midrange calibration concentration.
%RSD must be <20%
Sect. 9.2.3
Initial Demonstration of
Accuracy (IDA)
Calculate average recovery for replicates used in
IDP.
Mean recovery + 30% of true value
Sect. 9.2.4
Minimum Reporting Limit
(MRL) Confirmation
Fortify, extract and analyze seven replicate LFBs
at the proposed MRL concentration. Calculate the
Mean and the Half Range (HR). Confirm that the
upper and lower limits for the Prediction Interval
of Result (Upper PIR, and Lower PIR, Sect.
9.2.4.2) meet the recovery criteria.
Upper PIR < 150%
Lower PIR > 50%
Sect. 9.2.5
and 9.3.9
Quality Control Sample
(QCS)
Analyze a standard from a second source, as
part of IDC.
Results must be within 70-130% of true value.
Sect. 9.2.6
Detection Limit (DL)
Determination (optional)
Over a period of three days, prepare a minimum of
seven replicate LFBs fortified at a concentration
estimated to be near the DL. Analyze the replicates
through all steps of the analysis. Calculate the DL
using the equation in Sect. 9.2.6.1.
Data from DL replicates are not reauired to meet method
precision and accuracy criteria. If the DL replicates are
fortified at a low enough concentration, it is likely that they
will not meet precision and accuracy criteria.
NOTE: Table 14 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text is needed.
In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.
559-41

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TABLE 15. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Modioli
Reference
Requirement
Spccil'iciilion ;mcl l-reqnency
Acceptance ("rilerisi
Sect. ,81 -
Sect. 8.5
Sample Holding Time
28 days with appropriate preservation and storage as
described in Sections 8.1.-8.5.
Sample results are valid only if samples are extracted within the
sample holding time.
Sect. 83
Extract Holding Time
28 days when stored at < 6 °C in glass centrifuge
tubes or vials with un-punctured septa.
Extract results are valid only if extracts are analyzed within the
extract holding time.
Sect. 9.3.1.
Laboratory Reagent Blank
(LRB)
One LRB with each extraction batch of up to 20
samples.
Demonstrate that all method analytes are less than or equal to 1/3 the
MRL and confirm that possible interferences do not prevent
quantification of method analytes. If analytes are >1/3 the MRL or if
interferences are present, any samples from the extraction batch for
the problem analyte(s) that yielded a positive result are invalid.
Sect. 9.3.3
Laboratory Fortified Blank
(LFB)
One LFB is required for each extraction batch of up
to 20 Field Samples. Rotate the fortified
concentrations between low, medium and high
amounts.
Results of LFB analyses must be 70-130% of the true value for each
method analyte for all fortified concentrations except the lowest
CAL point. Results of the LFBs corresponding to the lowest CAL
point for each method analyte must be 50-150% of the true value.
Sect. 9.3.4
Internal Standard (IS)
Internal standard, 4-( 1,3 -dimethyl- 1-ethylpentyl
phenol-13C6, is added to all standards and sample
extracts, including QC samples. Compare IS areas to
the average IS area in the initial calibration and to the
most recent CCC.
Peak area counts for IS in all injections must be within + 50% of the
average peak area calculated during the initial calibration and
70-130% from the most recent CCC. If ISs do not meet this criterion,
corresponding target results are invalid.
Sect. 9.3.5
Surrogate Standards
(SUR)
Surrogate standard, 4-tert-octylphenol-13C6, is added
to all CAL standards and samples, including QC
samples. Calculate SUR recovery.
SUR recoveries must be 70-130% of the true value. If a SUR fails
this criterion, report all results for sample as suspect/SUR recovery.
559-42

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TABLE 15. (Continued)
Method
Reference
Requirement
Specilicitlioii itiul l-requency
Acceptance Crileriii
Sect. 9.3.6
Laboratory Fortified
Sample Matrix (LFSM)
Analyze one LFSM per extraction batch (20
samples or less) fortified with method analytes at a
concentration close to but greater than the native
concentration, if known. Calculate LFSM
recoveries.
Recoveries at mid and high levels must be within 70-130%
and within 50-150% at the low-level fortified amount (near
the MRL). If these criteria are not met, results are labeled
suspect due to matrix effects.
Sect. 9.3.7
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD) or
Field Duplicates (FD)
Extract and analyze at least one FD or LFSMD
with each extraction batch (20 samples or less). A
LFSMD may be substituted for a FD when the
frequency of detects are low. Calculate RPDs.
Method analyte RPDs for the LFMD or FD must be
<30% at mid and high levels of fortification and <50% near
the MRL. If these criteria are not met, results are labeled
suspect due to matrix effects.
Sect. 9.3.8
Field Reagent Blank (FRB)
Analysis of the FRB is required only if a Field
Sample contains a method analyte or analytes at or
above the MRL. The FRB is processed, extracted
and analyzed in exactly the same manner as a Field
Sample.
If the method analyte(s) found in the Field Sample is present
in the FRB at a concentration greater than 1/3 the MRL, then
all samples collected with that FRB are invalid and must be
recollected and reanalyzed.
Sect. 9.3.9
Quality Control Sample
(QCS)
Analyze at least quarterly or when preparing new
standards, as well as during the IDC.
Results must be within 70-130% of true value.
Sect. 1.0.2 and
Sect. 9.3.2
Initial Calibration
Use IS calibration technique to generate a first or
second order calibration curve forced through zero.
Use at least five standard concentrations. Check
the calibration curve as described in Sect. 10.2.7.
When each CAL standard is calculated as an unknown using
the calibration curve, the analyte and SUR results must be
70-130% of the true value for all except the lowest standard,
which must be 50-150% of the true value. Recalibration is
recommended if these criteria are not met.
Sect. 9.3.2
and Sect. 1.0.3
Continuing Calibration
Check (CCC)
Verify initial calibration by analyzing a low level
(at the MRL or below) CCC prior to analyzing
samples. CCCs are then injected after every 10
samples and after the last sample, rotating
concentrations to cover the calibrated range of the
instrument.
Recovery for each analyte and SUR must be within 70-130%
of the true value for all but the lowest level of calibration.
Recovery for each analyte in the lowest CAL level CCC must
be within 50-150% of the true value and the SUR must be
within 70-130% of the true value.
NOTE: Table 15 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text is needed.
In all cases, the full text of the QC in Sections 8-10 supersedes any missing or conflicting information in this table.
559-43

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FIGURE 1. EXAMPLE CHROMATOGRAM OF A CALIBRATION STANDARD WITH METHOD 559 ANALYTES AT
CONCENTRATION LEVELS OF 5-12 jig/L. NUMBERED PEAKS ARE IDENTIFIED IN TABLE 3.
Time imm
559-44

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