ASTM D7485-23

Standard Test Method for Determination of Nonylphenol, <emph type="bdit">p-tert</emph >-Octylphenol, Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate in Environmental Waters by Liquid Chromatography/Tandem Mass Spectrometry

SIGNIFICANCE AND USE
5.1 NP and OP have been shown to have toxic effects in aquatic organisms. The source of NP and OP is prominently from the use of common commercial surfactants. The most widely used surfactant is nonylphenol ethoxylate (NPEO) which has an average ethoxylate chain length of nine. The ethoxylate chain is readily biodegraded to form NP1EO, NP2EO, nonylphenol carboxylate (NPEC) and, under anaerobic conditions, NP. NP will also biodegrade, but may be released into environmental waters directly at trace levels. This method has been investigated and is applicable for environmental waters, including seawater.
SCOPE
1.1 This test method covers the determination of nonylphenol (NP), nonylphenol ethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), and octylphenol (OP), extracted from water utilizing solid phase extraction (SPE), separated using liquid chromatography (LC) and detected with tandem mass spectrometry (MS/MS). These compounds are qualitatively and quantitatively determined by this method. This method adheres to single reaction monitoring (SRM) mass spectrometry.  
1.2 The method detection limit (MDL) and reporting limit (RL) for NP, NP1EO, NP2EO, and OP are listed in Table 1.    
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 14-Apr-2023
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: D19 - Water
• Drafting Committee: D19.06 - Methods for Analysis for Organic Substances in Water

Relations

• Refers: ASTM D3694-96(2024) - Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• Effective Date: 01-Apr-2024
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D5905-98(2013) - Standard Practice for the Preparation of Substitute Wastewater
• Effective Date: 01-Jan-2013
• Refers: ASTM D2777-12 - Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• Effective Date: 15-Jun-2012
• Refers: ASTM D3856-11 - Standard Guide for Management Systems in Laboratories Engaged in Analysis of Water
• Effective Date: 15-Nov-2011
• Refers: ASTM D3694-96(2011) - Standard Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
• Effective Date: 01-May-2011
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D5905-98(2008) - Standard Practice for the Preparation of Substitute Wastewater
• Effective Date: 15-Jul-2008
• Refers: ASTM D2777-08 - Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• Effective Date: 15-Jan-2008
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D2777-06 - Standard Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• Effective Date: 15-Aug-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D3856-95(2006) - Standard Guide for Good Laboratory Practices in Laboratories Engaged in Sampling and Analysis of Water
• Effective Date: 15-Feb-2006

Overview

ASTM D7485-23 is the internationally recognized standard test method for the determination of nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), and p-tert-octylphenol (OP) in environmental waters. This standard utilizes liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to provide both qualitative and quantitative analysis of these compounds, which are frequently encountered as residues from widely-used commercial surfactants.

The test method is significant in environmental monitoring because NP and OP, along with their ethoxylate derivatives, have been shown to exhibit toxic effects in aquatic organisms. Their determination at trace levels in freshwater, seawater, and wastewater is crucial for environmental protection and regulatory compliance.

Key Topics

• Target Compounds: This standard covers the determination of:

  • Nonylphenol (NP)
  • Nonylphenol monoethoxylate (NP1EO)
  • Nonylphenol diethoxylate (NP2EO)
  • p-tert-Octylphenol (OP)

• Analytical Technique:

  • Solid-Phase Extraction (SPE): Extraction of analytes from water samples.
  • Liquid Chromatography (LC): Separation of compounds for analysis.
  • Tandem Mass Spectrometry (MS/MS): Detection and quantification using single reaction monitoring (SRM).

• Detection Limits: Method detection limits (MDLs) for NP, NP1EO, NP2EO, and OP are in the low nanogram per liter (ng/L) range, supporting trace level detection in environmental samples.

• Sample Handling:

  • Acidification of water samples at collection.
  • Strict sample preservation and storage requirements to prevent loss or degradation of target analytes.
  • Use of amber glass bottles and pre-cleaned labware to minimize contamination and analyte adsorption.

• Quality Assurance:

  • Regular calibration and use of surrogate standards.
  • Implementation of lab control samples, method blanks, and matrix spikes to ensure data reliability.
  • Comprehensive procedures for calculating recovery and precision.

Applications

ASTM D7485-23 is widely applicable in scenarios including:

• Environmental Monitoring: Assessing contamination in rivers, lakes, seawater, and wastewater effluents.
• Regulatory Compliance: Supporting agencies and industries in meeting water quality standards for hazardous organic pollutants.
• Wastewater Treatment Evaluation: Monitoring removal efficiency of surfactant-derived compounds in treatment processes.
• Research Studies: Tracing the environmental fate and impact of alkylphenol compounds and their degradation products.
• Risk Assessment: Providing data on the presence and concentration of endocrine-disrupting chemicals to inform environmental and public health decisions.

Related Standards

ASTM D7485-23 references and aligns with several other standards and regulations to ensure consistency and reliability in water analysis:

• ASTM D1129 - Terminology Relating to Water
• ASTM D1193 - Specification for Reagent Water
• ASTM D2777 - Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
• ASTM D3694 - Practices for Preparation of Sample Containers and Preservation of Organic Constituents
• ASTM D3856 - Guide for Management Systems in Laboratories Engaged in Analysis of Water
• ASTM D5847 - Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
• Code of Federal Regulations, 40 CFR Part 136 - Determination of Method Detection Limit

ASTM D7485-23 provides a robust framework for laboratories and organizations analyzing trace levels of nonylphenol and related contaminants in environmental waters, supporting improved water quality, environmental safety, and effective regulatory compliance.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D7485 − 23
Standard Test Method for
Determination of Nonylphenol, p-tert-Octylphenol,
Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate
in Environmental Waters by Liquid Chromatography/Tandem
Mass Spectrometry
This standard is issued under the fixed designation D7485; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of
1.1 This test method covers the determination of nonylphe-
Applicable Test Methods of Committee D19 on Water
nol (NP), nonylphenol ethoxylate (NP1EO), nonylphenol di-
D3694 Practices for Preparation of Sample Containers and
ethoxylate (NP2EO), and octylphenol (OP), extracted from
for Preservation of Organic Constituents
water utilizing solid phase extraction (SPE), separated using
D3856 Guide for Management Systems in Laboratories
liquid chromatography (LC) and detected with tandem mass
Engaged in Analysis of Water
spectrometry (MS/MS). These compounds are qualitatively
D5847 Practice for Writing Quality Control Specifications
and quantitatively determined by this method. This method
for Standard Test Methods for Water Analysis
adheres to single reaction monitoring (SRM) mass spectrom-
D5905 Practice for the Preparation of Substitute Wastewater
etry.
2.2 Other Documents:
1.2 The method detection limit (MDL) and reporting limit
40 CFR Part 136, Appendix B Definition and Procedure for
(RL) for NP, NP1EO, NP2EO, and OP are listed in Table 1.
the Determination of the Method Detection Limit
1.3 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3. Terminology
standard.
3.1 Definitions:
1.4 This standard does not purport to address all of the
3.1.1 For definitions of terms used in this standard refer to
safety concerns, if any, associated with its use. It is the
Terminology D1129.
responsibility of the user of this standard to establish appro-
3.2 Definitions of Terms Specific to This Standard:
priate safety, health, and environmental practices and deter-
3.2.1 environmental water, n—shall refer to water tested
mine the applicability of regulatory limitations prior to use.
using this method; see Section 5.
1.5 This international standard was developed in accor-
3.2.2 nonylphenol, NP, n—is a mixture of branched
dance with internationally recognized principles on standard-
p-nonylphenol isomers; commercial NP is produced by the
ization established in the Decision on Principles for the
reaction of phenol with commercial nonene; commercial non-
Development of International Standards, Guides and Recom-
ene is not simply a linear C9H18 alpha olefin; it is a complex
mendations issued by the World Trade Organization Technical
mixture of predominantly nine-carbon olefins, called propylene
Barriers to Trade (TBT) Committee.
trimer, containing no linear isomers; this synthesis results in a
2. Referenced Documents
mixture of various branched nonylphenol isomers rather than a
discrete chemical structure; the branched nonyl group is
2.1 ASTM Standards:
positioned predominantly in the para position on the phenol
D1129 Terminology Relating to Water
ring.
This test method is under the jurisdiction of ASTM Committee D19 on Water
3.2.3 octylphenol, OP, n—commercial octylphenol is pro-
and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for
duced by the reaction of phenol and diisobutylene to produce
Organic Substances in Water.
predominantly the 4-(1,1,3,3-tetramethylbutyl)phenol isomer.
Current edition approved April 15, 2023. Published May 2023. Originally
approved in 2009. Last previous edition approved in 2016 as D7485 – 16. DOI:
10.1520/D7485-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Available from U.S. Government Printing Office Superintendent of Documents,
Standards volume information, refer to the standard’s Document Summary page on 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
the ASTM website. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7485 − 23
TABLE 1 MDL and Reporting Limits
3.3.26 TC, n—Target Compound
A B
-6
MDL Reporting Range
Analyte 3.3.27 μM, n—micromolar, 1 × 10 moles/L
(ng/L) (ng/L)
3.3.28 VOA, n—Volatile Organic Analysis
NP 33 100–2000
NP1EO 9 100–2000
NP2EO 9 100–2000
4. Summary of Test Method
OP 24 100–2000
4.1 This is a performance-based method and modifications
A
MDL determined following The Code of Federal Regulations, 40 CFR Part 136,
Appendix B. are allowed to improve performance.
B
Lowest point of the reporting range is calculated from the LV 1 concentration
4.2 For NP, NP1EO, NP2EO, and OP analysis, solid phase
calibration standard in Table 4.
extraction is used to extract water samples.
4.2.1 Solid Phase Extraction—250 mL volume of sample
3.2.4 independent reference material, IRM, n—a material of adjusted to pH 2 is extracted using a solid phase extraction
cartridge. The acetonitrile/water extract is concentrated to a
known purity and concentration obtained either from the
National Institute of Standards and Technology (NIST) or other volume of 1.0 mL, and then analyzed by LC/MS/MS operated
in the multiple reaction monitoring (MRM) mode.
reputable supplier; the IRM must be obtained from a different
lot of material than is used for calibration.
4.3 The target compounds are identified by retention time
and SRM transition and are quantitated using the SRM
3.3 Acronyms:
transition of the target compounds utilizing external calibra-
3.3.1 CCC, n—Continuing Calibration Check
tion. The final report issued for each sample lists the concen-
3.3.2 IC, n—Initial Calibration
tration of NP, NP1EO, NP2EO, and OP.
3.3.3 LC, n—Liquid Chromatography
5. Significance and Use
3.3.4 LCS/LCSD, n—Laboratory Control Sample/
Laboratory Control Sample Duplicate
5.1 NP and OP have been shown to have toxic effects in
3.3.5 MDL, n—Method Detection Limit aquatic organisms. The source of NP and OP is prominently
from the use of common commercial surfactants. The most
3.3.6 MeOH, n—Methanol
widely used surfactant is nonylphenol ethoxylate (NPEO)
-3
3.3.7 mM, n—millimolar, 1 × 10 moles/L
which has an average ethoxylate chain length of nine. The
3.3.8 MRM, n—Multiple Reaction Monitoring
ethoxylate chain is readily biodegraded to form NP1EO,
3.3.9 MS/MSD, n—Matrix Spike/Matrix Spike Duplicate NP2EO, nonylphenol carboxylate (NPEC) and, under anaero-
bic conditions, NP. NP will also biodegrade, but may be
3.3.10 NA, adj—Not Available
released into environmental waters directly at trace levels. This
3.3.11 ND, n—non-detect
method has been investigated and is applicable for environ-
3.3.12 NP1EO, n—represents branched nonylphenol mono-
mental waters, including seawater.
ethoxylate.
6. Interferences
3.3.13 NP2EO, n—represents branched nonylphenol di-
ethoxylate.
6.1 Method interferences may be caused by contaminants in
solvents, reagents, glassware and other apparatus producing
3.3.14 n-NP2EO, n—represents normal straight chain non-
discrete artifacts or elevated baselines. All of these materials
ylphenol diethoxylate. n-NP2EO is used in this method as a
are routinely demonstrated to be free from interferences by
surrogate. It is not produced commercially and is not expected
analyzing laboratory reagent blanks under the same conditions
to be found in environmental waters.
as the samples.
3.3.15 P&A, n—Precision and Accuracy
6.2 All glassware is washed in hot water with detergent,
3.3.16 PPB, n—parts per billion
rinsed in hot water and rinsed with distilled water. The
3.3.17 PPT, n—parts per trillion
glassware is then dried and heated in an oven at 250 °C for
3.3.18 QA, adj—Quality Assurance
15 min to 30 min. All glassware is subsequently cleaned with
acetone and methanol. Detergents containing alkylphenolic
3.3.19 QC, adj—Quality Control
compounds must not be used.
3.3.20 RL, n—Reporting Limit
6.3 All reagents and solvents should be of pesticide residue
3.3.21 RSD, n—Relative Standard Deviation
purity or higher to minimize interference problems.
3.3.22 RT, n—Retention Time
6.4 Matrix interferences may be caused by contaminants
3.3.23 SDS, n—Safety Data Sheets
that are co-extracted from the sample. The extent of matrix
3.3.24 SRM, n—Single Reaction Monitoring
interferences can vary considerably from sample source to
3.3.25 SS, n—Surrogate Standard sample source, depending on variations of the sample matrix.
D7485 − 23
7. Apparatus 8.8 Branched nonylphenol monoethoxylate (NP1EO) avail-
able as a high purity custom standard.
7.1 LC/MS/MS System:
7.1.1 Liquid Chromatography System—A complete LC sys- 8.9 Branched nonylphenol diethoxylate (NP2EO) available
tem is needed in order to analyze samples. This should include as a high purity custom standard.
a sample injection system, a solvent pumping system capable
8.10 Nonylphenol, NP, >95 % para isomer (CAS # 84852-
of mixing solvents, a sample compartment capable of main-
15-3).
taining required temperature and a temperature controlled
8.11 Octylphenol, OP, 99 + % 4-(1,1,3,3-
column compartment. A system that is capable of performing at
tetramethylbutyl)phenol (CAS # 140-66-9).
the flows, pressures, controlled temperatures, sample volumes
and requirements of the standard may be used. 8.12 Concentrated HCl (CAS # 7647-01-0).
7.1.2 Analytical Column —A LC analytical column with
8.13 Ammonium Acetate (CAS # 631-61-8) (ACS Reagent
the ability to separate alkylphenols or equivalent.
Grade or Better).
7.1.3 Tandem Mass Spectrometer (MS/MS) System—A
8.14 n-Nonylphenol diethoxylate (n-NP2EO).
MS/MS system capable of MRM analysis. A system that is
capable of performing at the requirements in this standard may
8.15 n-Nonylphenol (suggested alternate surrogate, if
be used.
needed).
7.2 SPE Vacuum Manifold System—A solid phase extraction
8.16 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP).
vacuum manifold or similar may be utilized.
8.16.1 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-
OP) is used in this method as a surrogate. It is not produced
7.3 Organic solvent evaporation device.
commercially and is not expected to be found in environmental
8. Reagents and Materials
waters. It was reported that compounds in highly chlorinated
bromide rich wastewaters could potentially interfere with the
8.1 Purity of Reagents—High Performance Liquid Chroma-
Br-OP surrogate. If this interference is encountered
tography (HPLC) pesticide residue analysis and spectropho-
n-nonylphenol is suggested as an alternative surrogate.
tometry grade chemicals must be used in all tests. Unless
indicated otherwise, it is intended that all reagents must
8.17 Solid Phase Extraction Cartridges —An SPE extrac-
conform to the specifications of the Committee on Analytical tion cartridge that will separate alkylphenols from the matrix or
Reagents of the American Chemical Society. Other reagent
equivalent.
NOTE 1—Alkylphenols have been found in SPE cartridges therefore it
grades may be used provided it is first ascertained that they are
is advisable that the cartridges be lot certified alkylphenol free. Glass
of sufficiently high purity to permit their use without affecting
6 cartridges should have a much lower risk of alkylphenol contamination.
the accuracy of the measurement.
8.2 Purity of Water—Unless otherwise indicated, references
9. Hazards
to water must be understood to mean reagent water conforming
9.1 Normal laboratory safety applies to this method. Ana-
to Type I of Specification D1193. It must be demonstrated that
lysts should wear safety glasses, gloves and lab coats when
this water does not contain contaminants at concentrations
working with acids. Analysts should review the Safety Data
sufficient to interfere with the analysis.
Sheets (SDS) for all reagents used in this method.
8.3 Gases—Ultrapure nitrogen and argon.
10. Sample Collection, Preservation, and Storage
8.4 Acetonitrile (CAS # 75-05-8).
10.1 Sampling—Grab samples must be collected in amber
8.5 Methanol (CAS # 67-56-1).
glass bottles, minimum size of 250 mL. This must be done in
8.6 Isopropanol (CAS # 67-63-0).
order to allow for the rinsing of the bottle with acidified water
8.7 Acetone (CAS # 67-64-1).
and acidified 10 % methanol/water in order to get complete
transfer of the sample into the SPE cartridge and extraction
process. Alkylphenols tend to adsorb to glassware and rinsing
A Waters ACQUITY UPLC (a trademark of the Waters Corporation, Milford,
MA) HSS T3, 1.8 μm, 2.1 mm × 50 mm column was used, if you are aware of an will allow optimum recoveries. Conventional sampling prac-
alternative column that meets the performance of the standard, please provide this
tices should be followed. Refer to Guide D3856 and Practices
information to ASTM International Headquarters. Your comments will receive
D3694. Automatic sampling equipment should be as free as
careful consideration at a meeting of the responsible technical committee, which
possible of alkylphenolic-containing tubing and other potential
you may attend.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and sources of contamination.
Standard-Grade Reference Materials, American Chemical Society, Washington,
NOTE 2—Pre-cleaned bottles demonstrated to be free of interferences
DC. For suggestions on the testing of reagents not listed by the American Chemical
may be used.
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
copeial Convention, Inc. (USPC), Rockville, MD.
Two sources of the alkylphenol standards are: Cambridge Isotope Laboratories,
50 Frontage Road, Andover, MA 01810-5413 and Accustandard, Inc., 125 Market A Sep-Pak (a trademark of the Waters Corporation, Milford, MA) Vac (500 mg)
Street, New Haven, CT 06513. If you are aware of alternative suppliers, please tC18 Cartridges was used, if you are aware of an alternative cartridge that meets the
provide this information to ASTM International Headquarters. Your comments will performance of the standard, please provide this information to ASTM International
receive careful consideration at a meeting of the responsible technical committee, Headquarters. Your comments will receive careful consideration at a meeting of the
which you may attend. responsible technical committee, which you may attend.
D7485 − 23
10.2 Preservation—Adjust sample to pH 2 with concen-
The instrument is set in the Electrospray source setting.
Capillary Voltage: 3.5 kV
trated HCl at time of collection. Store samples between above
Cone: Variable depending on analyte (Table 3)
freezing and 6 °C from the time of collection until extraction.
Extractor: 2 Volts
Extract the sample within 14 days of collection and completely
RF Lens: 0.1 Volts
Source Temperature: 120 °C
analyze within 14 days of extraction.
Desolvation Temperature: 300 °C
Desolvation Gas Flow: 900 L/h
10.3 Sample extracts may be stored in sealed glass contain-
Cone Gas Flow: 300 L/h
ers at <0 °C indefinitely.
Low Mass Resolution 1: 14
High Mass Resolution 1: 14
Ion Energy 1: 0.5
11. Preparation of LC/MS/MS
Entrance Energy: –1
Collision Energy: Variable depending on analyte (Table 3)
11.1 LC Chromatograph Operating Conditions:
Exit Energy: 2
11.1.1 Injections of all calibration standards and samples are
Low Mass Resolution 2: 14
High Mass resolution 2: 14
made at a 50 μL volume using a full loop injection. If a 50 μL
Ion Energy 2: 0.5
volume loop is installed in the LC, a “full loop” mode is the
Multiplier: 650
-3
preferred technique when performing fast, qualitative analyses.
Collision Cell Pirani Gauge: 7 × 10 Torr
-5
Analyser Penning Gauge : 3 × 10 Torr
This mode should be used whenever accuracy and precision are
Inter-Channel Delay : 0.02 s
the primary concerns. The first sample analyzed after the
Inter-Scan Delay: 0.1 s
calibration curve is a blank to ensure there is no carry-over. The Repeats: 1
Span: 0 Daltons
gradient conditions for the liquid chromatograph are shown in
Dwell: 0.1 s
Table 2.
11.3.3 In order to acquire the maximum number of data
11.2 LC Auto Sampler Conditions:
points per MRM channel, the above scan, delay and dwell
11.2.1 Wash Solvents—Weak wash is 1.2 mL of 95 %
times may be changed and optimized according to the instru-
water/5 % acetonitrile, Strong wash is 1 mL of 30 %
ment. Fig. 1 displays a SRM chromatogram of each analyte and
acetonitrile, 30 % methanol, 30 % isopropyl alcohol, 10 %
the number of scans per peak which data was generated. Each
water. The strong wash solvent is needed to eliminate carry-
peak requires at least 10 scans per peak for adequate quanti-
over between injections of alkylphenol samples. The weak
tation. This standard contains only 4 target compounds and 2
wash is used to remove the strong wash solvent. Specific
surrogates which can be broken up into MRM experiment
instrument manufacturer specifications should be followed in
windows in order to optimize the number of scans and
order to eliminate sample carry-over in the analysis of al-
sensitivity of the instrument. For details regarding retention
kylphenols.
times and SRM transitions cone and collision energies refer to
11.2.2 Temperatures—Column, 30 °C; Sample
Table 3. If the instrument can only acquire in one mode
compartment, 15 °C.
effectively, two analyses will need to be performed, one in
11.2.3 Seal Wash—5 min. electrospray ionization (ESI) negative and one in ESI positive
mode in order to optimize speed and sensitivity.
11.3 Mass Spectrometer Parameters:
11.3.1 The instrument may require different settings.
12. Calibration and Standardization
11.3.2 Variable parameters depending on analyte are shown
12.1 In order to be certain that analytical values obtained
in Table 3.
using this test method are valid and accurate within the
confidence limits of the test, the following procedures must be
followed when performing the test method.
TABLE 2 Gradient Conditions for Liquid Chromatography
12.2 Calibration and Standardization—To calibrate the
Percent Percent instrument, analyze eight calibration standards containing the
Time Flow
95 % CH CN/5 % Water 95 % Water/5 % CH CN
3 3
eight concentration levels of NP, NP1EO, NP2EO, OP,
(min) (μL/min)
2 mmolar NH OAc 2 mmolar NH OAc
4 4
n-NP2EO, and Br-OP prior to analysis as shown in Table 4. A
0 300 0 100
calibration stock standard solution is prepared from standard
1 300 0 100
3 300 50 50 materials or purchased as certified solutions. Stock standard
4 300 60 40
solution A (Level 8) containing NP, NP1EO, NP2EO, OP,
6 300 70 30
n-NP2EO and Br-OP is prepared at Level 8 concentration and
7 300 70 30
9 300 100 0 aliquots of that solution are diluted to prepare Levels 1 through
13 300 100 0
7. The following steps will produce standards with the concen-
14 300 0 100
trations values shown in Table 4. The analyst is responsible for
16 300 0 100
recording initial component weights carefully when working
D7485 − 23
TABLE 3 Retention Times, MRM Ions, and Analyte-Specific Mass Spectrometer Parameters
SRM
Retention Time Cone Voltage Collision Energy
Analyte ESI Mode Mass Transition
(min) (Volts) (eV)
(Parent > Product)
Octylphenol neg 6.77 40 25 205.2 > 133
NP2EO pos 7.71 20 12 326.3 > 183.2
NP1EO pos 7.78 15 10 282.3 > 127.1
Nonylphenol neg 7.82 40 30 219.2 > 133
Br-OP (surrogate) neg 7.73 35 25 283.1 > 78.8
n-NP2EO (surrogate) pos 8.62 20 17 326.3 > 88.9
FIG. 1 Example SRM Chromatograms and Number of Scans per Peak
TABLE 4 Concentrations of Calibration Standards (PPB)
Analyte/Surrogate LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7 LV 8
NP 25 50 75 125 200 250 350 500
NP1EO 25 50 75 125 200 250 350 500
NP2EO 25 50 75 125 200 250 350 500
Octylphenol 25 50 75 125 200 250 350 500
Br-OP 25 50 75 125 200 250 350 500
n-NP2EO 25 50 75 125 200 250 350 500
with the pure materials, and correctly carrying the weights individual laboratory. Depending on the stock concentrations
through the dilution calculations. prepared, the solubility at that concentration will have to be
12.2.1 Prepare stock standard Solution A (Level 8) by ensured.
adding to a 100 mL volumetric flask individual acetonitrile/ 12.2.2 Aliquots of Solution A are then diluted with 75 %
methanol solutions of the following: 20 μL of NP, OP, NP1EO, water/25 % acetonitrile to prepare the desired calibration levels
NP2EO, Br-OP and n-NP2EO each at 2500 ppm, dilute to in 2 mL amber LC vials. The calibration vials must be used
100 mL with 75 % water ⁄25 % acetonitrile. The 2500 ppm within 24 h to ensure optimum results. Stock calibration
individual stock solutions are made in predominantly acetoni- standards are routinely replaced every six months if not
trile with methanol added to ensure solubility. Nonylphenol previously discarded for QC criteria failure.
2500 ppm stock may require up to a 50 % concentration of 12.2.3 Inject each standard and obtain a chromatogram for
methanol, the OP, NP1EO, NP2EO, Br-OP and n-NP2EO are each one. An external calibration is used monitoring the SRM
predominantly in acetonitrile. The preparation of the Level 8 transition of each analyte. Calibration software is utilized to
standard can be accomplished using different volumes and conduct the quantitation of the target analytes and surrogates.
concentrations of stock solutions as is accustomed in the The SRM transition of each analyte is used for quantitation and
D7485 − 23
confirmation. This gives confirmation by isolating the parent 12.3.3 This study should be repeated until the single opera-
ion, f
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D7485 − 16 D7485 − 23
Standard Test Method for
Determination of Nonylphenol, p-tert-Octylphenol,
Nonylphenol Monoethoxylate and Nonylphenol Diethoxylate
in Environmental Waters by Liquid Chromatography/Tandem
Mass Spectrometry
This standard is issued under the fixed designation D7485; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method covers the determination of nonylphenol (NP), nonylphenol ethoxylate (NP1EO), nonylphenol diethoxylate
(NP2EO), and octylphenol (OP), extracted from water utilizing solid phase extraction (SPE), separated using liquid chromatog-
raphy (LC) and detected with tandem mass spectrometry (MS/MS). These compounds are qualitatively and quantitatively
determined by this method. This method adheres to single reaction monitoring (SRM) mass spectrometry.
1.2 The method detection limit (MDL) and reporting limit (RL) for NP, NP1EO, NP2EO, and OP are listed in Table 1.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.5 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D2777 Practice for Determination of Precision and Bias of Applicable Test Methods of Committee D19 on Water
D3694 Practices for Preparation of Sample Containers and for Preservation of Organic Constituents
D3856 Guide for Management Systems in Laboratories Engaged in Analysis of Water
D5847 Practice for Writing Quality Control Specifications for Standard Test Methods for Water Analysis
D5905 Practice for the Preparation of Substitute Wastewater
This test method is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.06 on Methods for Analysis for
Organic Substances in Water.
Current edition approved Feb. 1, 2016April 15, 2023. Published March 2016May 2023. Originally approved in 2009. Last previous edition approved in 20092016 as
D7485D7485 – 16. – 09. DOI: 10.1520/D7485-16.DOI: 10.1520/D7485-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D7485 − 23
TABLE 1 MDL and Reporting Limits
A B
MDL Reporting Range
Analyte
(ng/L) (ng/L)
NP 33 100–2000
NP1EO 9 100–2000
NP2EO 9 100–2000
OP 24 100–2000
A
MDL Determined Followingdetermined following The Code of Federal
Regulations, 40 CFR Part 136, Appendix B.
B
Lowest Pointpoint of the Reporting Range is Calculatedreporting range is
calculated from the LV 1 Concentration Calibration Standardconcentration calibra-
tion standard in Table 4.
2.2 Other Documents:
The Code of Federal Regulations40 CFR Part 136, Appendix B 40 CFR Part 136, Appendix BDefinition and Procedure for the
Determination of the Method Detection Limit
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this standard refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 environmental water, n—shall refer to water tested using this method. Seemethod; see Section 5.
3.2.2 nonylphenol, NP, n—is a mixture of branched p-nonylphenol isomers. Commercialisomers; commercial NP is produced by
the reaction of phenol with commercial nonene. Commercialnonene; commercial nonene is not simply a linear C9H18 alpha olefin;
it is a complex mixture of predominantly nine-carbon olefins, called propylene trimer, containing no linear isomers. Thisisomers;
this synthesis results in a mixture of various branched nonylphenol isomers rather than a discrete chemical structure. Thestructure;
the branched nonyl group is positioned predominantly in the para position on the phenol ring.
3.2.3 octylphenol, OP, n—commercial octylphenol is produced by the reaction of phenol and diisobutylene to produce
predominantly the 4-(1,1,3,3-tetramethylbutyl)phenol isomer.
3.2.4 independent reference material, IRM, n—a material of known purity and concentration obtained either from the National
Institute of Standards and Technology (NIST) or other reputable supplier. The IRM shallsupplier; the IRM must be obtained from
a different lot of material than is used for calibration.
3.3 Acronyms:
3.3.1 CCC, n—Continuing Calibration Check
3.3.2 IC, n—Initial Calibration
3.3.3 LC, n—Liquid Chromatography
3.3.4 LCS/LCSD, n—Laboratory Control Sample/Laboratory Control Sample Duplicate
3.3.5 MDL, n—Method Detection Limit
3.3.6 MeOH, n—Methanol
-3
3.3.7 mM, n—millimolar, 1 × 10 moles/L
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
D7485 − 23
3.3.8 MRM, n—Multiple Reaction Monitoring
3.3.9 MS/MSD, n—Matrix Spike/Matrix Spike Duplicate
3.3.10 NA, adj—Not Available
3.3.11 ND, n—non-detect
3.3.12 NP1EO, n—represents branched nonylphenol monoethoxylate.
3.3.13 NP2EO, n—represents branched nonylphenol diethoxylate.
3.3.14 n-NP2EO, n—represents normal straight chain nonylphenol diethoxylate. n-NP2EO is used in this method as a surrogate.
It is not produced commercially and is not expected to be found in environmental waters.
3.3.15 P&A, n—Precision and Accuracy
3.3.16 PPB, n—parts per billion
3.3.17 PPT, n—parts per trillion
3.3.18 QA, adj—Quality Assurance
3.3.19 QC, adj—Quality Control
3.3.20 RL, n—Reporting Limit
3.3.21 RSD, n—Relative Standard Deviation
3.3.22 RT, n—Retention Time
3.3.23 SDS, n—Safety Data Sheets
3.3.24 SRM, n—Single Reaction Monitoring
3.3.25 SS, n—Surrogate Standard
3.3.26 TC, n—Target Compound
-6
3.3.27 μM, n—micromolar, 1 × 10 moles/L
3.3.28 VOA, n—Volatile Organic Analysis
4. Summary of Test Method
4.1 This is a performance-based method and modifications are allowed to improve performance.
4.2 For NP, NP1EO, NP2EO, and OP analysis, solid phase extraction is used to extract water samples.
4.2.1 Solid Phase Extraction—250 milliliter 250 mL volume of sample adjusted to pH 2 is extracted using a solid phase extraction
cartridge. The acetonitrile/water extract is concentrated to a volume of 1.0 mL, 1.0 mL, and then analyzed by LC/MS/MS operated
in the multiple reaction monitoring (MRM) mode.
D7485 − 23
4.3 The target compounds are identified by retention time and SRM transition and are quantitated using the SRM transition of the
target compounds utilizing external calibration. The final report issued for each sample lists the concentration of NP, NP1EO,
NP2EO, and OP.
5. Significance and Use
5.1 NP and OP have been shown to have toxic effects in aquatic organisms. The source of NP and OP is prominently from the
use of common commercial surfactants. The most widely used surfactant is nonylphenol ethoxylate (NPEO) which has an average
ethoxylate chain length of nine. The ethoxylate chain is readily biodegraded to form NP1EO, NP2EO, nonylphenol carboxylate
(NPEC) and, under anaerobic conditions, NP. NP will also biodegrade, but may be released into environmental waters directly at
trace levels. This method has been investigated and is applicable for environmental waters, including seawater.
6. Interferences
6.1 Method interferences may be caused by contaminants in solvents, reagents, glassware and other apparatus producing discrete
artifacts or elevated baselines. All of these materials are routinely demonstrated to be free from interferences by analyzing
laboratory reagent blanks under the same conditions as the samples.
6.2 All glassware is washed in hot water with detergent such as powdered Alconox, Deto-Jet, Luminox, or Citrojet, detergent,
rinsed in hot water and rinsed with distilled water. The glassware is then dried and heated in an oven at 250°C for 15 to 30
minutes.250 °C for 15 min to 30 min. All glassware is subsequently cleaned with acetone and methanol. Detergents containing
alkylphenolic compounds must not be used.
6.3 All reagents and solvents should be of pesticide residue purity or higher to minimize interference problems.
6.4 Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences
can vary considerably from sample source to sample source, depending on variations of the sample matrix.
D7485 − 23
7. Apparatus
7.1 LC/MS/MS System:
7.1.1 Liquid Chromatography System—A complete LC system is needed in order to analyze samples. This should include a sample
injection system, a solvent pumping system capable of mixing solvents, a sample compartment capable of maintaining required
temperature and a temperature controlled column compartment. A system that is capable of performing at the flows, pressures,
controlled temperatures, sample volumes and requirements of the standard may be used.
7.1.2 Analytical Column —A LC analytical column with the ability to separate alkylphenols or equivalent.
7.1.3 Tandem Mass Spectrometer (MS/MS) System—A MS/MS system capable of MRM analysis. A system that is capable of
performing at the requirements in this standard may be used.
7.2 SPE Vacuum Manifold SystemSystem——A solid phase extraction vacuum manifold or similar may be utilized.
7.3 Organic solvent evaporation device.
8. Reagents and Materials
8.1 Purity of Reagents—High Performance Liquid Chromatography (HPLC) pesticide residue analysis and spectrophotometry
grade chemicals shallmust be used in all tests. Unless indicated otherwise, it is intended that all reagents shallmust conform to the
specifications of the Committee on Analytical Reagents of the American Chemical Society. Other reagent grades may be used
provided it is first ascertained that they are of sufficiently high purity to permit their use without affecting the accuracy of the
measurement.
8.2 Purity of Water—Unless otherwise indicated, references to water shallmust be understood to mean reagent water conforming
to Type I of Specification D1193. It must be demonstrated that this water does not contain contaminants at concentrations sufficient
to interfere with the analysis.
8.3 Gases—Ultrapure nitrogen and argon.
8.4 Acetonitrile (CAS # 75-05-8).
8.5 Methanol (CAS # 67-56-1).
8.6 Isopropanol (CAS # 67-63-0).
8.7 Acetone (CAS # 67-64-1).
8.8 Branched nonylphenol monoethoxylate (NP1EO) available as a high purity custom standard.
8.9 Branched nonylphenol diethoxylate (NP2EO) available as a high purity custom standard.
A Waters ACQUITY UPLC (a trademark of the Waters Corporation, Milford, MA) HSS T3, 1.8 μm, 2.12.1 mm × 50 mm column, or equivalent, was found suitable for
use.column was used, if you are aware of an alternative column that meets the performance of the standard, please provide this information to ASTM International
Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
A Waters Quattro Premier (a trademark of the Waters Corporation, Milford, MA) mass spectrometer, or equivalent, was found suitable for use.
A Supelco Visiprep (a trademark of Sigma-Aldrich Co., LLC, St. Louis, MO) was found suitable to use, any SPE extraction manifold may be used.
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For Suggestionssuggestions on the testing of reagents not listed by the American Chemical Society, see
AnnualAnalar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial
Convention, Inc. (USPC), Rockville, MD.
Two sources of the alkylphenol standards are: Cambridge Isotope Laboratories, 50 Frontage Road, Andover, MA 01810-5413 and Accustandard, Inc., 125 Market Street,
New Haven, CT 06513. If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful
consideration at a meeting of the responsible technical committee, which you may attend.
D7485 − 23
8.10 Nonylphenol, NP, >95 % para isomer (CAS # 84852-15-3).
8.11 Octylphenol, OP, 99 + % 99 + % 4-(1,1,3,3-tetramethylbutyl)phenol (CAS # 140-66-9).
8.12 Concentrated HCl (CAS # 7647-01-0).
8.13 Ammonium Acetate (CAS # 631-61-8) (ACS Reagent Grade or Better).
8.14 n-Nonylphenol diethoxylate (n-NP2EO).
8.15 n-Nonylphenol (suggested alternate surrogate, if needed).
8.16 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP).
8.16.1 2-Bromo-4-(1,1,3,3-tetramethylbutyl)phenol (Br-OP) is used in this method as a surrogate. It is not produced commercially
and is not expected to be found in environmental waters. It was reported that compounds in highly chlorinated bromide rich
wastewaters could potentially interfere with the Br-OP surrogate. If this interference is encountered nnonylphenoln-nonylphenol
is suggested as an alternative surrogatesurrogate.
8.17 Solid Phase Extraction Cartridges —An SPE extraction cartridge that will separate alkypphenolsalkylphenols from the
matrix or equivalent.
NOTE 1—Alkylphenols have been found in SPE cartridges therefore it is advisable that the cartridges be lot certified alkylphenol free. Glass cartridges
should have a much lower risk of alkylphenol contamination.
9. Hazards
9.1 Normal laboratory safety applies to this method. Analysts should wear safety glasses, gloves and lab coats when working with
acids. Analysts should review the Safety Data Sheets (SDS) for all reagents used in this method.
10. Sample Collection, Preservation, and Storage
10.1 Sampling—Grab samples must be collected in 250 mL amber glass bottles. amber glass bottles, minimum size of 250 mL.
This must be done in order to allow for the rinsing of the bottle with acidified water and acidified 10 % methanol/water in order
to get complete transfer of the sample into the SPE cartridge and extraction process. Alkylphenols tend to adsorb to glassware and
rinsing will allow optimum recoveries. Conventional sampling practices should be followed. Refer to Guide D3856 and Practices
D3694. Automatic sampling equipment should be as free as possible of Tygonalkylphenolic-containing tubing and other potential
sources of contamination.
NOTE 2—Pre-cleaned bottles demonstrated to be free of interferences may be used.
10.2 Preservation—Adjust sample to pH 2 with concentrated HCl at time of collection. Store samples between 0ºC and 6ºCabove
freezing and 6 °C from the time of collection until extraction. Extract the sample within 14 days of collection and completely
analyze within 14 days of extraction.
10.3 Sample extracts may be stored in sealed glass containers at <0ºC<0 °C indefinitely.
11. Preparation of LC/MS/MS
11.1 LC Chromatograph Operating Conditions:
A Sep-Pak (a trademark of the Waters Corporation, Milford, MA) Vac (500 mg) tC18 Cartridges or equivalent were found suitable for use.was used, if you are aware
of an alternative cartridge that meets the performance of the standard, please provide this information to ASTM International Headquarters. Your comments will receive careful
consideration at a meeting of the responsible technical committee, which you may attend.
D7485 − 23
11.1.1 Injections of all calibration standards and samples are made at a 50 μL volume using a full loop injection. If a 50 μL volume
loop is installed in yourthe LC, a “full loop” mode is the preferred technique when performing fast, qualitative analyses. This mode
should be used whenever accuracy and precision are the primary concerns. The first sample analyzed after the calibration curve
is a blank to ensure there is no carry-over. The gradient conditions for the liquid chromatograph are shown in Table 2.
11.2 LC Auto Sampler Conditions:
11.2.1 Wash Solvents—Weak wash is 1.2 mL of 95 % water/5 % acetonitrile, Strong wash is 1 mL of 30 % acetonitrile, 30 %
methanol, 30 % isopropyl alcohol, 10 % water. The strong wash solvent is needed to eliminate carry-over between injections of
alkylphenol samples. The weak wash is used to remove the strong wash solvent. Specific instrument manufacturer specifications
should be followed in order to eliminate sample carry-over in the analysis of alkylphenols.
11.2.2 Temperatures—Column, 30°C;30 °C; Sample compartment, 15°C.15 °C.
11.2.3 Seal Wash—5 minutes.min.
11.3 Mass Spectrometer Parameters:
11.3.1 YourThe instrument may require different settings.
11.3.2 Variable parameters depending on analyte are shown in Table 3.
The instrument is set in the Electrospray source setting.
Capillary Voltage: 3.5 kV
Cone: Variable depending on analyte (Table 3)
Extractor: 2 Volts
RF Lens: 0.1 Volts
Source Temperature: 120°C
Source Temperature: 120 °C
Desolvation Temperature: 300°C
Desolvation Temperature: 300 °C
Desolvation Gas Flow: 900 L/hr
Desolvation Gas Flow: 900 L/h
Cone Gas Flow: 300 L/hr
Cone Gas Flow: 300 L/h
Low Mass Resolution 1: 14
High Mass Resolution 1: 14
Ion Energy 1: 0.5
Entrance Energy: –1
Collision Energy: Variable depending on analyte (Table 3)
Exit Energy: 2
Low Mass Resolution 2: 14
High Mass resolution 2: 14
Ion Energy 2: 0.5
Multiplier: 650
-3
Collision Cell Pirani Gauge: 7 × 10 Torr
-5
Analyser Penning Gauge : 3 × 10 Torr
Inter-Channel Delay : 0.02 seconds
Inter-Channel Delay : 0.02 s
Inter-Scan Delay: 0.1 seconds
TABLE 2 Gradient Conditions for Liquid Chromatography
Percent Percent
Time Flow
95 % CH CN/5 % Water 95 % Water/5 % CH CN
3 3
(min) (μL/min)
2 mmolar NH OAc 2 mmolar NH OAc
4 4
0 300 0 100
1 300 0 100
3 300 50 50
4 300 60 40
6 300 70 30
7 300 70 30
9 300 100 0
13 300 100 0
14 300 0 100
16 300 0 100
D7485 − 23
TABLE 3 Retention Times, MRM Ions, and Analyte-Specific Mass Spectrometer Parameters
SRM
Retention Time Cone Voltage Collision Energy
Analyte ESI Mode Mass Transition
(min) (Volts) (eV)
(Parent > Product)
Octylphenol neg 6.77 40 25 205.2 > 133
NP2EO pos 7.71 20 12 326.3 > 183.2
NP1EO pos 7.78 15 10 282.3 > 127.1
Nonylphenol neg 7.82 40 30 219.2 > 133
Br-OP (surrogate) neg 7.73 35 25 283.1 > 78.8
n-NP2EO (surrogate) pos 8.62 20 17 326.3 > 88.9
Inter-Scan Delay: 0.1 s
Repeats: 1
Span: 0 Daltons
Dwell: 0.1 Seconds
Dwell: 0.1 s
11.3.3 In order to acquire the maximum number of data points per MRM channel, the above scan, delay and dwell times may be
changed and optimized according to yourthe instrument. Fig. 1 displays a SRM chromatogram of each analyte and the number of
scans per peak which data was generated. Each peak requires at least 10 scans per peak for adequate quantitation. This standard
contains only 4 target compounds and 2 surrogates which can be broken up into MRM experiment windows in order to optimize
the number of scans and sensitivity of yourthe instrument. For details regarding retention times and SRM transitions cone and
collision energies refer to Table 3. If the instrument can only acquire in one mode effectively, two analyses will need to be
performed, one in electrospray ionization (ESI) negative and one in ESI positive mode in order to optimize speed and sensitivity.
12. Calibration and Standardization
12.1 In order to be certain that analytical values obtained using this test method are valid and accurate within the confidence limits
of the test, the following procedures must be followed when performing the test method.
12.2 Calibration and Standardization—To calibrate the instrument, analyze eight calibration standards containing the eight
concentration levels of NP, NP1EO, NP2EO, OP, n-NP2EO, and Br-OP prior to analysis as shown in Table 4. A calibration stock
standard solution is prepared from standard materials or purchased as certified solutions. Stock standard solution A (Level 8)
FIG. 1 Example SRM Chromatograms and Number of Scans per Peak
D7485 − 23
TABLE 4 Concentrations of Calibration Standards (PPB)
Analyte/Surrogate LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7 LV 8
NP 25 50 75 125 200 250 350 500
NP1EO 25 50 75 125 200 250 350 500
NP2EO 25 50 75 125 200 250 350 500
Octylphenol 25 50 75 125 200 250 350 500
Br-OP 25 50 75 125 200 250 350 500
n-NP2EO 25 50 75 125 200 250 350 500
containing NP, NP1EO, NP2EO, OP, n-NP2EO and Br-OP is prepared at Level 8 concentration and aliquots of that solution are
diluted to prepare Levels 1 through 7. The following steps will produce standards with the concentrations values shown in Table
4. The analyst is responsible for recording initial component weights carefully when working with the pure materials, and correctly
carrying the weights through the dilution calculations.
12.2.1 Prepare stock standard Solution A (Level 8) by adding to a 100 mL 100 mL volumetric flask individual acetonitrile/
methanol solutions of the following: 20 μL of NP, OP, NP1EO, NP2EO, Br-OP and n-NP2EO each at 2500 ppm, dilute to 100 mL
100 mL with 75 % water/25 %water ⁄25 % acetonitrile. The 2500 ppm individual stock solutions are made in predominantly
acetonitrile with methanol added to ensure solubility. Nonylphenol 2500 ppm 2500 ppm stock may require up to a 50 %
concentration of methanol, the OP, NP1EO, NP2EO, Br-OP and n-NP2EO are predominantly in acetonitrile. The preparation of
the Level 8 standard can be accomplished using different volumes and concentrations of stock solutions as is accustomed in the
individual laboratory. Depending on the stock concentrations prepared, the solubility at that concentration will have to be ensured.
12.2.2 Aliquots of Solution A are then diluted with 75 % water/25 % acetonitrile to prepare the desired calibration levels in 2 mL
amber LC vials. The calibration vials must be used within 24 hoursh to ensure optimum results. Stock calibration standards are
routinely replaced every six months if not previously discarded for QC criteria failure.
12.2.3 Inject each standard and obtain a chromatogram for each one. An external calibration is used monitoring the SRM transition
of each analyte. Calibration software is utilized to conduct the quantitation of the target analytes and surrogates. The SRM
transition of each analyte is used for quantitation and confirmation. This gives confirmation by isolating the parent ion, fragmenting
it to the product ion fragment, and also relating it to the retention time in the calibra
...