METHOD 539: DETERMINATION OF HORMONES IN DRINKING 

WATER BY SOLID PHASE EXTRACTION (SPE) AND LIQUID 

CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM 

MASS SPECTROMETRY (LC-ESI-MS/MS)

Office of Water (MLK 140) EPA Document No. 815-B-10-001 November 2010 http://water.epa.gov/drink/
539-1 

METHOD 539: DETERMINATION OF HORMONES IN DRINKING WATER BY SOLID 

PHASE EXTRACTION (SPE) AND LIQUID CHROMATOGRAPHY 

ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY 

(LC-ESI-MS/MS) 

Version 1.0 

November 2010 

Glynda A. Smith (U.S. EPA, Office of Ground Water and Drinking Water)

Alan D. Zaffiro, (Shaw Environmental, Inc.) 

M. L. Zimmerman (Shaw Environmental, Inc.)

D. J. Munch (U.S. EPA, Office of Ground Water and Drinking Water)

TECHNICAL SUPPORT CENTER

STANDARDS AND RISK MANAGEMENT DIVISION

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
539-2 

METHOD 539

DETERMINATION OF HORMONES IN DRINKING WATER BY SOLID PHASE 

EXTRACTION (SPE) AND LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION 

TANDEM MASS SPECTROMETRY (LC/ESI-MS/MS)

1. SCOPE AND APPLICATION

1.1 METHOD – Method 539 is a liquid chromatography, electrospray ionization, tandem mass 

spectrometry (LC-ESI-MS/MS) method for the determination of hormones in finished drinking water. Method 539 requires the use of MS/MS in Multiple Reaction Monitoring (MRM) 

mode to enhance selectivity. This method is intended for use by analysts skilled in the 

performance of solid phase extraction, the operation of LC-ESI-MS/MS instrumentation, and 

in the interpretation of the associated data. Method 539 is applicable for the measurement of 

the following analytes: 

Analyte

Chemical Abstracts Services 

Registry Number (CASRN)

16α-Hydroxyestradiol (Estriol) 50-27-1 

17β-Estradiol 50-28-2 

17α-Ethynylestradiol 57-63-6 

Testosterone 58-22-0 

Estrone 53-16-7 

4-Androstene-3,17-dione 63-05-8 

Equilin 474-86-2 

1.2 SUPPORTING DATA

1.2.1 Precision and accuracy data have been generated for the detection of the method analytes 

in reagent water and finished drinking water from both ground water and surface water 

sources (Sect. 17, Tables 6 to 9). 

1.2.2 Single laboratory lowest concentration minimum reporting levels (LCMRLs) for the analytes in this method ranged from 0.06 to 4.0 nanograms per liter (ng/L) (Section 17, Table 

5). The LCMRL is the lowest spiking concentration such that the probability of spike 

recovery in the 50% to 150% range is at least 99%. The procedure used to determine the 

LCMRL is described elsewhere.1 Laboratories using this method are not required to 

determine LCMRLs, but they must demonstrate that the Minimum Reporting Level 

(MRL) for each analyte meets the requirements described in Section 9.2.4.

1.2.3 Determining detection limits (DL) for the method analytes is optional (Sect. 9.2.6). The 

DL is defined as the statistically calculated minimum concentration that can be measured 

with 99% confidence that the reported value is greater than zero.2 DLs for method 

analytes fortified into reagent water ranged from 0.04 to 2.9 ng/L (Table 5). 
539-3 

 

1.3 METHOD FLEXIBILITY – The laboratory is permitted to change LC columns, LC 

conditions, internal standards or surrogate standards, and MS conditions different from those 

utilized to develop the method. Changes may not be made to sample collection and 

preservation (Sect. 8), the quality control (QC) requirements (Sect. 9), or the extraction and 

elution steps (Sect 11). Single quadrupole instruments are not permitted. 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, may not be used. Analytes should be adequately resolved 

chromatographically in order to permit the mass spectrometer to dwell on a minimum 

number of compounds eluting within a retention time window. Instrumental sensitivity can 

decrease if too many compounds are permitted to elute within a retention time window. 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 QC 

acceptance criteria in this method are met (Tables 11 and 12), and verify method 

performance in a real sample matrix (Sect. 9.4.2). 

2. SUMMARY OF METHOD

Samples are dechlorinated with sodium thiosulfate and protected from microbial degradation using 

2-mercaptopyridine-1-oxide sodium salt during sample collection. Samples are fortified with 

surrogates and passed through solid phase extraction (SPE) disks containing octadecyl (C18) 

functional groups in order to extract the method analytes and surrogates. The compounds are 

eluted from the solid phase with a small amount of methanol. The extract is concentrated to 

dryness with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with 50:50 

methanol:water after adding the internal standards. An aliquot of the sample is injected into an LC 

equipped with a C18 column that is interfaced to a MS/MS. The analytes are separated and 

identified by comparing the acquired mass spectra and retention times to reference spectra and 

retention times for calibration standards acquired under identical LC-MS/MS conditions. The 

concentration of each analyte is determined using the internal standard technique. 

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 the number of field samples. 

3.2 CALIBRATION STANDARD – A solution of the method analytes, surrogate analytes and 

internal standards prepared from the Primary Dilution Standards (Sect. 3.18). The calibration 

standards are used to calibrate the instrument response with respect to analyte concentration.

3.3 CONTINUING CALIBRATION CHECK (CCC) – A calibration standard that is analyzed 

periodically to verify the accuracy of the existing calibration. 
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3.4 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 (Sect. 9.2.6), and accurate quantitation is 

not expected at this level.

3.5 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 solid phase 

extraction devices, solvents, surrogate solution, and fortifying solutions. Required QC 

samples include a Laboratory Reagent Blank (Sect. 3.12), a Laboratory Fortified Blank (Sect. 

3.9), a Laboratory Fortified Sample Matrix (Sect. 3.10), and either a Field Duplicate (Sect. 

3.6) or a Laboratory Fortified Sample Matrix Duplicate (Sect. 3.11). 

3.6 FIELD DUPLICATES (FD) – Separate samples collected at the same time, shipped and 

stored under identical conditions. Method precision, including the contribution from sample 

collection procedures, is estimated from the analysis of FDs. For the purposes of this 

method, Field Duplicates are necessary to conduct repeat analyses if the original field sample 

is lost, or to conduct repeat analyses in the case of QC failures associated with the analysis of 

the original field sample. Field Duplicates are used to prepare Laboratory Fortified Sample 

Matrix (Sect. 3.10) and Laboratory Fortified Sample Matrix Duplicate (Sect. 3.11) QC 

samples.

3.7 INTERNAL STANDARD (IS) – A pure compound added to all standard solutions and 

sample extracts in a known amount and used to measure the relative response of other 

method analytes 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.

3.8 ION SUPPRESSION/ENHANCEMENT – An observable decrease or increase in analyte 

response in complex (field) samples as compared to the response obtained in standard 

solutions. 

3.9 LABORATORY FORTIFIED BLANK (LFB) – A volume of reagent water, containing 

method preservatives, to which known quantities of the method analytes are added. The LFB 

is used during the IDC (Sect. 9.2) to verify method performance for precision and accuracy. 

The LFB is also a required QC element with each extraction batch. The results of the 

extracted LFB verify method performance in the absence of sample matrix. 

3.10 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 as a sample, and its purpose is to determine whether the sample 

matrix contributes bias to the analytical results. 

3.11 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A Field 

Duplicate of the sample used to prepare the LFSM which is fortified and analyzed identically 

to the LFSM. The LFSMD is used instead of the Field Duplicate to assess method precision 
539-5 

when the method analytes are rarely found at concentrations greater than the MRL (Sect. 

3.15). 

3.12 LABORATORY REAGENT BLANK (LRB) – A volume of reagent water or other blank 

matrix that is processed exactly as a sample including exposure to all glassware, equipment, 

solvents and reagents, sample preservatives, surrogates and internal standards that are used in 

the extraction and analysis batches. The LRB is used to determine if the method analytes or 

other interferences are present in the laboratory environment, the reagents, or the apparatus.

3.13 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The single 

laboratory LCMRL is the lowest true concentration for which the future recovery is predicted 

to fall between 50% to 150% with 99% confidence.1

3.14 MATERIAL SAFETY DATA SHEETS (MSDS) – Written information provided by vendors 

concerning a chemical’s toxicity, health hazards, physical properties, fire and reactivity data, 

storage instructions, spill response procedures, and handling precautions. 

3.15 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be 

reported by a laboratory as a quantified value for the method analyte in a sample following 

analysis. This concentration must meet the criteria defined in Section 9.2.4 and must be no 

lower than the concentration of the lowest calibration standard for each method analyte. 

3.16 MULTIPLE REACTION MONITORING (MRM) – A mass spectrometric technique in 

which a precursor ion is first isolated, then subsequently fragmented into a product ion(s). 

Quantitation is accomplished by monitoring a specific product ion. As described in Section 

10.1.2, MS parameters must be optimized for each precursor ion (Sect. 3.17) and product ion 

(Sect. 3.19). 

3.17 PRECURSOR ION – The precursor ion is the gas-phase species corresponding to the method 

analyte produced in the ESI interface. In MS/MS, the precursor ion is mass selected and 

fragmented by collision-activated dissociation to produce distinctive product ions of smaller 

mass/charge (m/z) ratio.

3.18 PRIMARY DILUTION STANDARD (PDS) – A solution containing the method analytes, 

internal standards, or surrogate analytes, which is prepared from Stock Standard Solutions 

(Sect. 3.22). The PDS solutions are diluted to prepare calibration standards and sample 

fortification solutions.

3.19 PRODUCT-ION – For the purpose of this method, a product ion is one of the fragment ions 

produced in MS/MS by collision-activated dissociation of the precursor ion. 

3.20 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.
539-6 

3.21 REAGENT WATER (RW) – Purified water that does not contain any measurable quantity of 

the method analytes or interfering compounds at or above 1/3 the MRL. 

3.22 STOCK STANDARD SOLUTION (SSS) – A concentrated standard solution that is prepared 

in the laboratory using assayed reference materials or that is purchased from a commercial 

source with a certificate of analysis.

3.23 SURROGATE ANALYTE (SUR) – A pure chemical which is unlikely to be found in any 

sample, and which is added to a sample volume in a known amount before extraction. 

Surrogates are evaluated using the same procedures as other sample components. Because 

surrogates are present in every sample, they provide a means of assessing method 

performance for each sample extraction.

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap water, 

rinse with tap water, followed by reagent water. Non-volumetric glassware can be heated in 

a muffle furnace at 400 o

C for two hours or solvent rinsed. Volumetric glassware should be 

solvent rinsed and never heated in an oven above 120 oC.

4.2 Method interferences may be caused by contaminants in solvents, reagents (including reagent 

water), sample bottles and caps, and other sample processing hardware. These interferences 

may lead to discrete artifacts and/or elevated baselines in the chromatograms. All laboratory 

reagents and equipment must be routinely demonstrated to be free from interferences (less 

than 1/3 the MRL for the target analytes) under the conditions of the analysis. This may be 

accomplished by analyzing LRBs as described in Sections 3.12 and 9.3.1. Subtracting 

blank values from sample results is not permitted. 

4.3 Matrix interferences may be caused by contaminants that are co-extracted from the sample. 

Matrix components may directly interfere by producing a signal at or near the retention time 

of an analyte peak. The extent of matrix interferences will vary considerably from source to 

source, depending on the nature of the water. Humic and/or fulvic material in environmental 

samples may be co-extracted during SPE and can cause enhancement and/or suppression in 

the electrospray ionization source. Total organic carbon (TOC) is an indicator of the humic 

content of a sample. Analysis of LFSMs (Sect. 3.10 and 9.3.6) provides evidence for the 

presence (or absence) of matrix effects.

4.4 Solid phase extraction media may be a source of interferences. The analysis of LRBs can 

provide important information regarding the presence or absence of such interferences. Each 

brand and lot of SPE devices should be tested to ensure that contamination does not preclude 

analyte identification and quantitation. 

4.5 Depending on the source and purity, labeled analogs used as internal standards may contain a 

small percentage of the corresponding native analyte. Such a contribution may be significant 

when attempting to determine LCMRLs and DLs. The labeled internal standards must meet 

the purity requirements stated in the IDC (Section 9.2.1). 
539-7 

4.6 The method involves the extraction and concentration of trace levels of human hormones. 

As such, a potential source of interference lies with the analyst. Nitrile gloves should be 

worn at all times while handling clean glassware and throughout the extraction process.

4.7 Depending on the sampling site, it may be appropriate to include a Field Blank with the 

sampling bottles. At the lab, fill a sample bottle with reagent water and preservatives, seal, 

and ship it to the sampling site along with the other sample bottles. Also, include an empty 

sealed bottle. At the sampling site, open the bottle containing the preserved reagent water 

and pour it into the empty bottle (thus exposing it to the sampling environment). Seal and 

label as “Field Blank”. The Field Blank is shipped back to the lab along with the samples 

and analyzed along with the samples to ensure that no human hormones were introduced into 

the samples during the collection and handling process. In general, if the Field Blank shows 

analytes present at a level >1/3 the MRL, the samples should be flagged accordingly and new 

samples collected if possible.

5. SAFETY

5.1 The toxicity and 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.3

 A reference 

file of MSDSs should be made available to all personnel involved in the chemical analysis.

5.2 Pure standard materials and stock standard solutions of the method compounds should be 

handled with suitable protection for skin, eyes, etc.4

5.3 Concentrated ammonium hydroxide was used during method development as a pH modifier 

for the HPLC mobile phase. Concentrated ammonium hydroxide should only be handled in a 

chemical fume hood. 

6. EQUIPMENT AND SUPPLIES 

 

References to specific brands or catalog numbers are included as examples only and do not imply 

endorsement of the product. Such reference does not preclude the use of other vendors or 

suppliers. 

6.1 SAMPLE CONTAINERS – 1000-mL amber glass bottles fitted with polytetrafluoroethylene 

(PTFE) lined screw caps. (Alternate sample container volumes may be used as discussed in 

Section 8.1.1) 

6.2 AUTOSAMPLER VIALS – Amber glass vials with PTFE/silicone septa.

6.3 MICRO SYRINGES – Suggested sizes include 5, 10, 25, 50 and 100 microliters (μL). 

6.4 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 gram (g). 
539-8 

6.5 DISPOSABLE PASTEUR PIPETTES – 5 3⁄4-inch or 9-inch borosilicate glass, used to 

transfer samples to autosampler vials and for sample preparation (Fisher Cat. No. 13-678-

20B, 13-678-20C, or equivalent). 

6.6 DISPOSABLE SYRINGES (Optional) – 3-mL, polypropylene, Luer Lock syringes for use in 

filtering standards and extracts (Aldrich Cat. No. Z248002, Fisher Cat No. 14-817-27, or 

equivalent). 

6.7 FILTERS – 0.22 μm, 47 mm PVDF disk filters for filtering LC mobile phase components 

(Millipore Durapore Cat. No. GVWP04700, or equivalent).

6.8 SYRINGE FILTERS (Optional) – 13 mm, 0.2 μm pore size GHP filters (Waters Corp. Part 

No. WAT097962, or equivalent). 

6.9 SOLID PHASE EXTRACTION APPARATUS FOR USING SPE DISKS

6.9.1 SPE DISKS – 47 mm diameter, manufactured with octadecyl (C18) sorbent phase (Fisher 

Cat. No. 14-386-2, or equivalent). 

6.9.2 SPE DISK EXTRACTION GLASSWARE – Funnel, PTFE-coated support screen, PTFE 

gasket, base, and clamp used to support SPE disks and contain samples during extraction. 

May be purchased as a set (Fisher Cat. No. K971100-0047, or equivalent) or separately. 

6.9.3 VACUUM EXTRACTION MANIFOLD – Designed to accommodate extraction 

glassware and disks (Varian Cat. No. 1214-6001, or equivalent). 

6.9.4 GRADUATED COLLECTION TUBES – 50-mL conical glass tubes with beaded rim 

(Kimax #45165-50) or 40-mL glass tubes with 24-410 screw cap (Kimax #45200-40) 

suitable for collection of eluent from the solid phase disks. 

6.10 LABORATORY OR ASPIRATOR VACUUM SYSTEM – Sufficient capacity to maintain a 

vacuum of approximately 15 to 25 inches of mercury. 

6.11 EXTRACT CONCENTRATION SYSTEM – Extracts are concentrated by blowdown with 

nitrogen using a water bath set at 40 °C (N-Evap, Model 11155, Organomation Associates, 

Inc., or equivalent). 

6.12 LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM MASS 

SPECTROMETRY SYSTEM (LC-ESI-MS/MS) 

6.12.1 LC SYSTEM – The LC system must provide consistent sample injection volumes and be 

capable of performing binary linear gradients at a constant flow rate.

6.12.2 ANALYTICAL COLUMN – The method was developed using a Waters Xterra® MS 

C18 2.1 x 150 mm, 3.5 μm dp column (Waters Part No. 186000408). Any column 
539-9 

capable of tolerating basic conditions (mobile phase pH > 9.5), provides adequate 

resolution, peak shape, capacity, accuracy and precision (Sect. 9), and does not result in 

suppression or enhancement of analyte responses may be used.

6.12.3 ELECTROSPRAY IONIZATION-TANDEM MASS SPECTROMETER (ESI-MS/MS) 

– The mass spectrometer must be capable of rapid switching between negative ion and 

positive ion electrospray ionization modes. The system must be capable of performing 

MS/MS to produce unique product ions (Sect. 3.19) for the method analytes within 

specified retention time segments. At least 10 – 15 scans across the chromatographic 

peak are needed to ensure adequate precision.

6.12.4 MS/MS DATA SYSTEM – An interfaced data system is required to acquire, store and 

output MS data. The computer software must have the capability of processing stored 

data by recognizing a chromatographic peak within a given retention time window. The 

software must allow integration of the abundance of any specific ion between specified 

time or scan number limits. The software must be able to construct a linear regression or 

quadratic calibration curve and calculate analyte concentrations using the internal 

standard technique.

7. REAGENTS AND STANDARDS 

7.1 GASES, REAGENTS AND SOLVENTS – Reagent grade or better chemicals must be used. 

Unless otherwise indicated, all reagents will conform to the specifications of the Committee 

on Analytical Reagents of the American Chemical Society (ACS), where such specifications 

are available. Other grades may be used if the reagent is demonstrated to be free of analytes 

and interferences and all requirements of the IDC (Sect. 9.2) are met when using these 

reagents. 

7.1.1 ACETONITRILE (CH3CN, CASRN 75-05-8) – (HPLC-grade, Honeywell Burdick & 

Jackson Brand®, Catalog No. 015 or equivalent).

7.1.2 AMMONIUM HYDROXIDE - (NH4OH, CASRN 1336-21-6) – Mobile phase modifier 

(29% by weight, Fisher Cat. No. S93120A, or equivalent). 

7.1.3 COLLISION GAS - High purity compressed gas (e.g., nitrogen or argon) used in the 

collision cell of the mass spectrometer. The specific type of gas, purity and pressure 

requirements will depend on the instrument manufacturer’s specifications.

7.1.4 DESOLVATION GAS - High purity compressed gas (e.g., nitrogen or zero-air) used for 

desolvation in the mass spectrometer. The specific type of gas, purity and pressure 

requirements will depend on the instrument manufacturers’ specifications.

7.1.5 METHANOL – (CH3OH, CASRN 67-56-1) – (LC/MS grade, Fisher Optima®, Fisher 

Cat. No. A456, or equivalent). 
539-10 

7.1.6 REAGENT WATER – Purified water that does not contain any measurable quantities of 

any method analytes or interfering compounds greater than 1/3 the MRL for each method 

analyte of interst.

7.1.7 (2-MERCAPTOPYRIDINE-1-OXIDE, SODIUM SALT, “SODIUM OMADINE”, 

(C5H4NOSNa, CASRN 3811-73-2) – > 96% (Sigma Cat. No. H3261, or equivalent). 

Used to inhibit microbial growth in dechlorinated water samples.

7.1.8 SODIUM THIOSULFATE (Na2S2O3, CASRN 7772-98-7) – Certified, anhydrous (Fisher 

Cat. No. S446, or equivalent). Added to remove free chlorine in chlorinated finished 

waters.

7.2 STANDARD SOLUTIONS – When a compound’s purity is assayed to be 96 percent or 

greater, the weight can be used without correction to calculate the concentration of the stock 

standard. The solution concentrations listed in this section were used to develop this method 

and are included only as examples. Guidance on the storage stability of Primary Dilution 

Standards and calibration standards is provided in the applicable sections below. Although 

estimated stability times for standard solutions are given, laboratories should use standard 

QC practices to determine when standards need to be replaced.

7.2.1 INTERNAL STANDARDS – This method uses four isotopically enriched internal 

standards listed in the table below. Although alternate internal standards may be used, 

the analyst must ensure that the QC requirements defined in Section 9.3.4 are met. 

Internal Standard CASRNa Neat Materials Catalog No. Solution Standards , Cat. No.

16α-Hydroxyestradiol-d2

(Estriol-d2) 53866-32-3 C/D/N Isotopes, Cat. No. 

D-5279 N/A

13C6-Estradiol None None Cambridge Isotope Labs, 100 μg/mL in 

Methanol, Cat. No. CLM-7936-1.2

13C2-Ethynylestradiol None None Cambridge Isotope Labs, 100 μg/mL in 

Acetonitrile, Cat. No. CLM-3375-1.2

Testosterone-d3 77546-39-5 None Sigma Drug Std., 100 μg/mL in 

dimethoxyethane, Cat. No. T5536 a

 CASRN = Chemical Abstract Registry Number.

7.2.1.1 INTERNAL STANDARD STOCK STANDARDS (ISSS) (500 μg/mL, except as 

noted) – Prepare α-hydroxyestradiol-d2 (estriol-d2) stock standard solution by 

weighing 5 mg of the solid material into a tared 10-mL volumetric flask and diluting 

to volume with methanol. The remaining internal standards can be purchased as 100 

μg/mL solutions. 

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (IS PDS) (1.0 – 

4.0 μg/mL) – The table below can be used as a guide for preparing the IS PDS. The 

IS PDS can be prepared in acetonitrile. The IS PDS is stable for about six months 

provided it is stored at a temperature < 6 o

C. Use 5 μL of the 1.0 – 4.0 μg/mL IS PDS 

to fortify the final 1-mL extracts. This will yield a final concentration of 5.0 – 20 

ng/mL of each IS in the 1-mL extracts. Analysts are permitted to use other IS PDS 
539-11 

concentrations and volumes provided all extracts and calibration standards contain the 

same final concentration of the internal standards and adequate signal is obtained to 

maintain precision.

Internal Standard Conc. of IS Stock, 

μg/mL

Volume of IS 

Stock, μL

Final Volume of 

IS PDS, mL

Final Conc. of IS 

PDS (μg/mL)

16α-Hydroxyestradiol-d2

(Estriol-d2) 500 40 10 2.0

13C6-Estradiol 100 400 10 4.0

13C2-Ethynylestradiol 100 400 10 4.0

Testosterone-d3 100 100 10 1.0

7.2.2 SURROGATE ANALYTES – Two isotopically labeled surrogates were evaluated during 

method development and are listed in the following table. Only one surrogate, however, 

is required to be evaluated with each extraction batch. The analyst is permitted to select 

the surrogate that performs best under the LC-MS/MS conditions employed for the 

analysis. Other alternate surrogate standards may be used provided they meet the QC 

requirements described in Section 9.3.5. 

Surrogate Analyte CASRNa Neat Materials Catalog No.

Ethynylestradiol-d4 350820-06-3 C/D/N Isotopes, Cat. No. D-4319

Bisphenol A-d16 96210-87-6 Sigma, Cat. No. 451835 a CASRN = Chemical Abstract Registry Number.

7.2.2.1 SURROGATE STOCK STANDARDS (1000 μg/mL) – Prepare individual solutions 

of the surrogate standards by weighing 10 mg of the solid material into tared 10-mL 

volumetric flasks and diluting to volume with methanol. 

7.2.2.2 SURROGATE ANALYTE PRIMARY DILUTION STANDARD (SUR PDS) (2.5 – 

7.0 μg/mL) – The table below can be used as a guide for preparing the SUR PDS. 

The SUR PDS can be prepared in methanol and is stable for about six months 

provided it is stored at a temperature < 6 o

C. Use 10 μL of SUR PDS to fortify 1-L 

samples. This will yield a final concentration of 70 ng/mL ethynylestradiol-d4 or 25 

ng/mL bisphenol A-d16 in the 1-mL extracts.

Surrogate Analyte Conc. of SUR Stock, 

μg/mL

Volume of SUR 

Stock, μL

Final Volume of 

SUR PDS, mL

Final Conc. of 

SUR PDS (μg/mL)

Ethynylestradiol-d4 1000 70 10 7.0

Bisphenol A-d16 1000 25 10 2.5

7.2.3 METHOD ANALYTE STANDARD SOLUTIONS

7.2.3.1 ANALYTE STOCK STANDARD SOLUTION (1000 μg/mL) – Obtain the analytes 

listed in the table in Section 1.1 as ampoulized solutions or as neat materials. Prepare 

stock standards individually by weighing 10 mg of the solid standards into tared 10-

mL volumetric flasks and diluting to volume with methanol. 
539-12 

NOTE: The androgens, testosterone and androstenedione, can be purchased as 1 

mg/mL drug standards. 

7.2.3.2 ANALYTE PRIMARY DILUTION SOLUTION (Analyte PDS) (1.0 – 3.5 μg/mL) – 

Prepare the Analyte PDS by diluting the Analyte Stock Standard solutions into 50% 

methanol in reagent water. An example preparation of the Analyte PDS that was 

used to collect data presented in Section 17 is provided in the table below. The 

concentrations vary based on the instrumental sensitivity. The Analyte PDS is used 

to prepare calibration standards, and to fortify LFBs, LFSMs, and LFSMDs with the 

method analytes. 

Analyte Stock

Stock

Concentration 

(μg/mL) Stock Volume (μL)

Final Volume 

(mL 50% 

MeOH)

Analyte PDS 

Concentration 

(μg/mL)

16α-Hydroxyestradiol 

(Estriol) 1000 20

10

2.0

Estrone 1000 20 2.0

17β-Estradiol 1000 25 2.5

17α-Ethynylestradiol 1000 35 3.5

Equilin 1000 20 2.0

4-Androstene-3,17-dione 1000 10 1.0

Testosterone 1000 10 1.0

7.2.4 CALIBRATION (CAL) STANDARDS – Prepare at least five calibration standards over 

the concentration range of interest by diluting aliquots of Analyte PDS into 50% 

methanol in reagent water. The lowest calibration standard must be at or below the 

MRL. Using the IS PDS and SUR PDS, add a constant amount of each internal standard 

and surrogate to each calibration standard. The concentration of the surrogate should 

match the concentration in the sample extracts, assuming 100% recovery through the 

extraction process. The CAL standards may also be used as CCCs (Sect. 9.3.2). If the 

extracts are filtered (Sect. 11.4), it is recommended that the CAL standards also be 

filtered using 0.2 μm syringe filters. During method development, the CAL standards 

were shown to be stable for at least two weeks when stored at a temperature < 6 o

C.

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE BOTTLE PREPARATION

8.1.1 SAMPLE CONTAINERS – One-liter amber glass bottles with PTFE-lined screw caps 

and sufficient capacity to allow subsequent preparation of all required sample and QC 

aliquots.

NOTE: Smaller sample volumes (e.g., 500-mL) can be collected if the laboratory 

demonstrates acceptable performance in meeting the required MRLs (Sect. 9.2.4) using 

the smaller sample volume. The amount of added preservatives and surrogate/analyte 

fortification levels should be adjusted accordingly. 
539-13 

8.1.2 ADDITION OF PRESERVATIVES – Preservation reagents, listed in the table below, are 

added to each sample bottle prior to shipment to the field (or prior to sample collection).

Compound Amount Purpose

Sodium thiosulfate 80 mg/L Removes free chlorine

2-mercaptopyridine-1-oxide, 

sodium salt

65 mg/L Microbial inhibitor

8.2 SAMPLE COLLECTION – Grab samples must be collected in accordance with conventional 

sampling practices.5 Fill sample bottles taking care not to flush out the preservatives. 

Because the method analytes are not volatile, it is not necessary to ensure that the sample 

bottles are completely headspace-free.

 

8.2.1 SAMPLING FROM A TAP – When sampling from a cold water tap, remove the aerator, 

open the tap, and allow the system to flush until the water temperature has stabilized 

(approximately five minutes). Invert the bottles several times to mix the sample with the 

preservation reagents.

8.3 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment and 

must not exceed 10 °C during the first 48 hours after collection. Samples must be confirmed 

to be at or below 10 °C when they are received at the laboratory. In the laboratory, samples 

must be stored at or below 6 °C and protected from light until analysis. Samples must not be 

frozen.

8.4 SAMPLE HOLDING TIMES – Results of the sample storage stability study (Table 9) 

indicated that all compounds listed in the method have adequate stability for 28 days when 

collected, preserved, shipped and stored as described in Sections 8.1 – 8.3. Therefore, 

samples should be extracted as soon as possible, but must be extracted within 28 days. 

Extracts must be stored at 0 °C or less and analyzed within 28 days after extraction. The 

extract storage stability study data are presented in Table 10. 

9. QUALITY CONTROL

9.1 QC requirements include the IDC (Sect. 9.2) and ongoing QC requirements (Sect. 9.3). This 

section describes each QC parameter, its required frequency, and the performance criteria 

that must be met in order to satisfy EPA quality objectives. The QC criteria discussed in the 

following sections are summarized in Section 17, Tables 11 and 12. 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, internal standards and surrogate standards, MS and MS/MS conditions. Each 

time such method modifications are made, the analyst must repeat the procedures of the 

IDC. Modifications to LC conditions should minimize co-elution of method analytes to 

reduce the probability of suppression/enhancement effects.
539-14 

NOTE: Ultraperformance liquid chromatography (UPLC) was evaluated concurrently 

with high performance liquid chromatography (HPLC) during development of this 

method. The UPLC columns were found to be less stable under the basic mobile phase 

conditions that were used in the HPLC analyses. Basic conditions will improve the 

negative electrospray ionization response of the estrogens. The UPLC analyses also 

exhibited a greater degree of matrix suppression when chlorinated (finished) groundwater 

and surface water samples were evaluated. This does not preclude the use of UPLC for 

this method. However, analysts must verify that UPLC analyses can meet the established 

MRLs (Sect. 9.2.4) and that acceptable recoveries are obtained in real drinking water 

matrices (Sect. 9.4.2). 

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – The IDC must be successfully 

performed prior to analyzing any field samples. Prior to conducting the IDC, the analyst 

must generate an acceptable initial calibration following the procedure outlined in Section 

10.2. 

9.2.1 DEMONSTRATION OF LOW SYSTEM BACKGROUND – Analyze an LRB. 

Confirm that the blank is free of contamination as defined in Section 9.3.1. 

9.2.1.1 Depending on the source and purity, labeled internal standards may contain a small 

percentage of the corresponding native analyte. Therefore, the analyst must 

demonstrate that the internal standards do not contain the unlabeled analytes at a 

concentration > 1/3 of the MRL when added at the appropriate concentration to 

samples.

9.2.1.2 The system should also be checked for carry-over by analyzing a RW blank 

immediately following the highest CAL standard. If this RW sample does not meet 

the criteria outline in Section 9.3.1, then carry-over is present and should be identified 

and eliminated. 

9.2.2 DEMONSTRATION OF PRECISION – Prepare, extract and analyze four to seven 

replicate LFBs. Fortify these samples near the midrange of the initial calibration curve. 

The method preservatives must be added to the LFBs as described in Section 8.1.2. The 

percent relative standard deviation (%RSD) of the results of the replicate analyses must 

be ≤ 20%. 

100

AverageConcentrat ion

 Standard Deviation of Measured Concentrat ions = RSD % ×

9.2.3 DEMONSTRATION OF ACCURACY – Using the same set of replicate data generated 

for Section 9.2.2, calculate the average percent recovery. The average percent recovery 

for each analyte must be within + 30% of the true value. 

100

Fortified Concentrat ion

AverageMeasured Concentrat ion Recovery % = ×
539-15 

9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target 

concentration for the MRL based on the intended use of the method. Analyze an initial 

calibration following the procedures in Section 10. The lowest calibration standard used 

to establish the initial calibration (as well as the low-level CCC) 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. 

9.2.4.1 Fortify, extract and analyze seven replicate LFBs at or below the proposed MRL 

concentration. The LFBs must contain the method preservatives as specified in 

Section 8.1.2. Calculate the mean (Mean) and standard deviation for these replicates. 

Determine the Half Range for the Prediction Interval of Results (HRPIR) using the 

equation 

HRPIR = 3.963S

where S is the standard deviation and 3.963 is a constant value for seven replicates.1

9.2.4.2 Confirm that the Upper and Lower limits for the Prediction Interval of Results (PIR = 

Mean + HRPIR) meet the upper and lower recovery limits as shown below. 

The Upper PIR Limit must be ≤ 150 percent recovery.

Mean HR

FortifiedConcentration

PIR +

× ≤ 100 150%

The Lower PIR Limit must be ≥ 50 percent recovery.

 Mean HR

FortifiedConcentration

PIR − × ≥ 100 50%

9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the criteria 

described above. If these criteria are not met, the MRL has been set too low and must 

be confirmed again at a higher concentration. 

NOTE: These equations are only valid for seven replicate samples.

9.2.5 QUALITY CONTROL SAMPLE (QCS) – Analyze a mid-level Quality Control Sample 

(Sect. 9.3.8) to confirm the accuracy of the primary calibration standards. 

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 

ascertain whether DL determination is required based upon the intended use of the data.
539-16 

Analyses for this procedure 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 two to five times the noise level. The 

method preservatives must be added to the samples as described in Section 8.1.2. 

Process the seven replicates through all steps of Section 11. Do not subtract blank values 

when performing DL calculations. 

 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 = s × t(n-1,1-α = 0.99)

where

 t(n-1,1-α = 0.99) = Student's t value for the 99% confidence level with n-1 degrees of 

freedom (for seven replicate determinations, the Student’s t value 

is 3.143 at a 99% confidence level), 

n = number of replicates, and 

s = standard deviation of replicate analyses.

9.3 ONGOING QC REQUIREMENTS – This section describes the ongoing QC elements that 

must be included when processing and analyzing field samples. 

9.3.1 LABORATORY REAGENT BLANK (LRB) – Analyze an LRB (Sect. 3.12) with each 

extraction batch (Sect. 3.5). The LRB must contain the method preservatives and the 

surrogate analytes at the same concentration used to fortify field samples. Background 

from method analytes or contaminants that interfere with the measurement of method 

analytes must be < 1/3 of the MRL. If method analytes are detected in the LRB at 

concentrations greater than this level, then all data for the problem analyte(s) must be 

considered invalid for all samples in the extraction batch. Subtracting blank values from 

sample results is not permitted.

NOTE: Although quantitative data below the MRL may not be accurate enough for data 

reporting, such data are useful in determining the magnitude of background interference. 

Therefore, blank contamination levels may be estimated by extrapolation when the 

concentration is below the MRL.

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – Analyze CCC standards at the 

beginning of each analysis batch (Sect. 3.1), after every ten field samples, and at the end 

of the analysis batch. See Section 10.3 for concentration requirements and acceptance 

criteria for CCCs.

 

9.3.3 LABORATORY FORTIFIED BLANK – An LFB (Sect. 3.9) is required with each 

extraction batch (Sect. 3.5). The concentration of the LFB must be rotated between low, 

medium, and high concentrations from batch to batch. The low concentration LFB must 
539-17 

be as near as practical to, but no more than two 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 must be within 

+ 50% of the true value for each analyte. Results of the medium and high-level LFB 

analyses must be within + 30% of the true value for each analyte. If the LFB results do 

not meet these criteria, 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 

internal standards in all injections of the analysis batch (Sect. 3.1). The internal standard 

responses (as indicated by peak areas) for any chromatographic run must not deviate by 

more than ± 50% from the average areas measured during the initial calibration for the 

internal standards. If the IS areas in a chromatographic run do not meet these criteria, 

inject a second aliquot from the same autosampler vial. 

9.3.4.1 If the re-injected aliquot produces an acceptable IS response, report results for that 

aliquot.

9.3.4.2 If the re-injected aliquot fails the IS criterion, the analyst should check the calibration 

by injecting the most recent CAL standard that passed. If the CAL standard fails the 

IS criterion, recalibration is in order as per Section 10.2. If the CAL standard is 

acceptable, report results from the re-injected aliquot, but annotate as “suspect/IS 

recovery”. Alternatively, collect a new sample and reanalyze.

9.3.5 SURROGATE RECOVERY – The surrogate standard is fortified into all field samples, 

LRB, LFB, LFSMs, LFSMDs, and FDs prior to extraction. It is also added to the CAL 

standards. Calculate the percent recovery (%R) for each surrogate using the following 

equation:

100

B

A %R ×



 



 =

where 

A = calculated surrogate concentration for the QC or field sample, and 

B = fortified concentration of the surrogate. 

9.3.5.1 Surrogate recovery must be in the range of 70 to 130%. When a surrogate fails to 

meet this criterion, check 1) calculations to locate possible errors, 2) standard 

solutions for degradation, 3) contamination, and 4) instrument performance. Correct 

the problem and reanalyze the extract. 

9.3.5.2 If the repeat analysis meets the surrogate recovery criterion, report only data for the 

reanalyzed extract.

9.3.5.3 If the extract reanalysis fails the 70 to 130% recovery criterion after corrective action, 

check the calibration by injecting the last CAL standard that passed. If the CAL 
539-18 

standard fails the criteria of Section 10.3, recalibrate as described in Section 10.2. If 

the CAL standard is acceptable, extraction of the sample should be repeated provided 

a sample is available and still within the holding time. If the re-extracted sample also 

fails the recovery criterion, report all data for that sample as “suspect/surrogate 

recovery.” Alternatively, collect a new sample and re-analyze.

9.3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Within each extraction 

batch (Sect. 3.5), analyze a minimum of one LFSM (Sect. 3.10) for every 20 samples. 

The background concentrations of the analytes in the sample matrix must be determined 

in a separate aliquot and subtracted from the measured values in the LFSM. If various 

sample matrices are analyzed regularly, for example, drinking water processed from 

ground water and surface water sources, performance data should be collected for each 

source.

9.3.6.1 Prepare the LFSM by fortifying a Field Duplicate with an appropriate amount of the 

Analyte PDS (Sect. 7.2.3.2). Generally, select a spiking concentration that is greater 

than or equal to the native concentration for most analytes. Selecting a duplicate 

aliquot of a sample that has already been analyzed aids in the selection of an 

appropriate spiking level. If this is not possible, use historical data when selecting a 

fortifying concentration. 

9.3.6.2 Calculate the percent recovery (%R) using the equation:

( ) 100

C

B-A %R ×=

where

A = measured concentration in the fortified sample,

B = measured concentration in the unfortified sample, and 

C = fortification concentration. 

9.3.6.3 Recoveries for samples fortified at concentrations at or near the MRL (within a factor 

of two times the MRL concentration) must be within + 50% of the true value. 

Recoveries for samples fortified at all other concentrations must be within + 30% of 

the true value. If the accuracy for any analyte falls outside the designated range, and 

the laboratory performance for that analyte is shown to be in control in the CCCs and 

in the LFB, the recovery is judged matrix biased. Report the result for the 

corresponding analyte in the unfortified sample as “suspect/matrix.” 

NOTE: In order to obtain meaningful percent recovery results, correct the 

measured values in the LFSM and LFSMD for the native levels in the unfortified 

samples, even if the native values are less than the MRL. This situation and the 

LRB are the only permitted uses of analyte results below the MRL.

9.3.7 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX 

DUPLICATE (FD or LFSMD) – Within each extraction batch, analyze a minimum of 
539-19 

one Field Duplicate or one Laboratory Fortified Sample Matrix Duplicate. If the method 

analytes are not routinely observed in field samples, analyze an LFSMD rather than an 

FD.

9.3.7.1 Calculate the relative percent difference (RPD) for duplicate measurements (FD1 and 

FD2) using the equation: 

( ) 100

FDFD 2/

FDFD

RPD

1 2

1 2

×

+

− =

9.3.7.2 RPDs for Field Duplicates shoud be ≤ 30% for each analyte. Greater variability may 

be observed when Field Duplicates have analyte concentrations that are at or near the 

MRL (within a factor of two times the MRL concentration). At these concentrations, 

Field Duplicates must have RPDs that are ≤ 50%. If the RPD of an analyte falls 

outside the designated range, and the laboratory performance for the analyte is shown 

to be in control in the CCC and in the LFB, the precision is judged matrix influenced. 

Report the result for the corresponding analyte in the unfortified sample as 

“suspect/matrix.”

9.3.7.3 If an LFSMD is analyzed instead of a Field Duplicate, calculate the RPD for the 

LFSM and LFSMD using the equation: 

9.3.7.4 RPDs for duplicate LFSMs should be ≤ 30% for each analyte. Greater variability 

may be observed when the matrix is fortified at analyte concentrations at or near the 

MRL (within a factor of two times the MRL concentration). LFSMs at these 

concentrations must have RPDs that are ≤ 50%. If the RPD of an analyte falls outside 

the designated range, and the laboratory performance for the analyte is shown to be in 

control in the CCC and in the LFB, the precision is judged matrix influenced. Report 

the result for the corresponding analyte in the unfortified sample as “suspect/matrix.”

9.3.8 QUALITY CONTROL SAMPLE (QCS) – As part of the IDC (Sect. 9.2), 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. Fortify the QCS near 

the midpoint of the calibration range. The acceptance criteria for the QCS are the same 

as the mid- and high-level CCCs (Sect. 10.3). If the accuracy for any analyte fails the 

recovery criterion, prepare fresh standard dilutions and repeat the QCS evaluation. 

9.4 METHOD MODIFICATION QC REQUIREMENTS – The analyst is permitted to modify 

the separation technique, LC column, mobile phase composition, LC conditions and MS 

conditions. 

( ) 100

LFSM /2LFSMD

LFSM LFSMD

RPD ×

+

− =
539-20 

9.4.1 Each time method modifications are made, the analyst must repeat the procedures of the 

IDC (Sect. 9.2) and verify that all QC criteria can be met in ongoing QC samples (Sect. 

9.3). 

9.4.2 The analyst is also required to evaluate and document method performance for the 

proposed modifications in real matrices that span the range of waters that the laboratory 

analyzes. This additional step is required because modifications that perform acceptably 

in the IDC, which is conducted in reagent water, could fail ongoing method QC 

requirements in real matrices. This is particularly important for methods subject to 

matrix effects, such as LC/MS-based methods. For example, a laboratory may routinely 

analyze drinking water from municipal treatment plants that process ground water, 

surface water, or a blend of surface and ground water. In this case, the method 

modification requirement could be accomplished by assessing precision and accuracy 

(Sects. 9.2.2 and 9.2.3) in a surface water with moderate to high total organic carbon 

(e.g., 2 mg/L or greater) and a hard ground water (e.g., 250 mg/L as calcium carbonate 

(CaCO3) equivalent, or greater).

9.4.3 The results of Sections 9.4.1 and 9.4.2 must be appropriately documented by the analyst 

and independently assessed by the laboratory’s QA officer prior to analyzing field 

samples. When implementing method modifications, it is the responsibility of the 

laboratory to closely review the results of ongoing QC, and in particular, the results 

associated with the LFSM (Sect. 9.3.6), FD (Sect. 9.3.7), CCCs (Sect. 10.3), and the 

internal standard area counts (Sect. 9.3.4). If repeated failures are noted, the modification 

must be abandoned. 

10. CALIBRATION AND STANDARDIZATION

Demonstration and documentation of acceptable MS calibration and initial analyte calibration are 

required before performing the IDC (Sect. 9.2) and prior to analyzing field samples. Verification 

of the initial calibration should be repeated each time a major instrument modification or 

maintenance is performed.

10.1 LC-ESI-MS/MS CALIBRATION AND OPTIMIZATION

10.1.1 MASS CALIBRATION – Calibrate the mass spectrometer with the calibration 

compounds and procedures specified by the manufacturer. 

10.1.2 OPTIMIZING MS PARAMETERS – Each LC-ESI-MS/MS system will have different 

optimal conditions, which are influenced by the source geometry and system design. Due 

to the differences in design, follow the recommendations of the instrument manufacturer 

when tuning the instrument. During the development of this method, instrumental 

parameters were optimized for the precursor and product ions listed in Section 17, Table 

3. Product ions other than those listed may be selected; however, the analyst is cautioned 

to avoid using ions with lower mass and/or common ions that may not provide sufficient 

discrimination between the analytes of interest and co-eluting interferences.
539-21 

10.1.2.1 Optimize the response of the precursor ion (Sect. 3.17) for each analyte by infusing 

approximately 0.5 – 1.0 μg/mL of each analyte directly into the mass spectrometer as 

recommended by the instrument manufacturer. Vary the MS parameters (source 

voltages, source and desolvation temperatures, gas flows, etc.) until optimal analyte 

responses are obtained. The target analytes may have different optimal instrument 

parameters, thus requiring some compromise on the final operating conditions. See 

Section 17, Table 2 for the ESI-MS/MS conditions used in method development. 

10.1.2.2 Optimize the response of the product ion (Sect. 3.19) for each analyte by infusing 

approximately 0.5 – 1.0 μg/mL of each analyte directly into the mass spectrometer as 

recommended by the instrument manufacturer. Vary the MS/MS parameters 

(collision gas pressure, collision energy, etc.) until optimal product ion responses are 

determined. 

10.1.3 LIQUID CHROMATOGRAPHY INSTRUMENT CONDITIONS – Establish LC 

operating parameters that optimize resolution. Suggested LC operating conditions are 

described in Section 17, Table 1. Conditions different from those listed (e.g., LC 

columns and mobile phase compositions) may be used if the QC criteria in Sections 9.2 

and 9.3 are met and chromatographic separation of the method analytes is achieved. 

NOTE: Chromatographic separation as defined does not include the isotopically 

enriched internal standards and surrogates, which are mass separated. Co-elution of the 

internal standards with their analogous method analytes helps mitigate matrix suppression 

and/or enhancement effects.

10.1.4 ESTABLISH LC-ESI-MS/MS RETENTION TIMES AND MRM SEGMENTS – Inject a 

mid- to high-level calibration standard under optimized LC-ESI-MS/MS conditions to 

obtain the retention times of each method analyte. Divide the chromatogram into 

segments that contain one or more chromatographic peaks. For maximum sensitivity in 

subsequent MS/MS analyses, minimize the number of MRM (Sect. 3.16) transitions that 

are simultaneously monitored within each segment. 

10.2 INITIAL CALIBRATION - During method development, daily calibrations were performed; 

however, it is permissible to verify the calibration with daily CCCs. Calibration must be 

performed using peak areas and the internal standard technique. Calibration using peak 

heights or external standard calibration is not permitted.

10.2.1 CALIBRATION STANDARDS – Prepare a set of at least five calibration standards as 

described in Section 7.2.4. The analyte concentrations in the lowest calibration standard 

must be at or below the MRL. Field samples must be quantified using a calibration curve 

that spans the same concentration range used to collect the IDC data (Sect. 9.2), i.e., 

analysts are not permitted to use a restricted calibration range to meet the IDC criteria 

and then use a larger dynamic range during analysis of field samples.

10.2.2 CALIBRATION – Calibrate the LC-ESI-MS/MS and fit the calibration points with either 

a linear regression or quadratic regression (response vs. concentration). Weighting may 
539-22 

be used. Forcing the calibration curve through the origin is not recommended. The 

MS/MS instruments used during method development were calibrated using weighted 

(1/x) quadratic curves. Internal standard assignments appropriate for each method 

analyte and surrogate analyte are presented in Table 3. The MRM transitions for the 

internal standards are provided in Table 4. 

10.2.3 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 should 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. In this case, corrective action should be taken to reanalyze 

the calibration standards or restrict the range of calibration.

10.3 CONTINUING CALIBRATION CHECKS (CCCs) – Analyze a CCC to verify the initial 

calibration at the beginning of each analysis batch, after every tenth field sample, and at the 

end of each analysis batch. The beginning CCC for each analysis batch must be at or below 

the MRL. This CCC verifies instrument sensitivity prior to the analysis of samples. 

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 Verify that the absolute areas of the quantitation ions of each of the internal standards 

have not changed by more than ± 50% from the average areas measured during the initial 

calibration. If this limit is exceeded, corrective action is necessary (Sect. 10.4).

10.3.3 The calculated concentration of the surrogate analytes must be within + 30% of the true 

value. If the surrogate analytes fail this criterion, corrective action is necessary (Sect. 

10.4). 

10.3.4 Calculate the concentration of each method analyte in the CCC. Each analyte fortified at 

a level ≤ MRL must calculate to be within + 50% of the true value. The calculated 

concentration of the method analytes in CCCs fortified at all other levels must be within 

+ 30%. If these limits are exceeded, then all data for the failed analytes must be 

considered invalid. Any field samples analyzed since the last acceptable CCC that are 

still within holding time must be reanalyzed after an acceptable calibration has been 

restored.

10.4 CORRECTIVE ACTION – Failure to meet CCC QC performance criteria requires corrective 

action. Acceptable method performance may be restored simply by flushing the column at 

the highest eluent concentration in the gradient. Following this and other minor remedial 

action, check the calibration with a mid-level CCC and a CCC at the MRL, or recalibrate 

according to Section 10.2. If internal standard and calibration failures persist, maintenance 

may be required, such as servicing the ESI-MS/MS system or replacing the LC column. 
539-23 

These later measures constitute major maintenance, and the analyst must return to the initial 

calibration step (Sect. 10.2). 

11. PROCEDURE

This section describes the procedures for sample preparation and analysis. Important aspects of 

this analytical procedure include proper sample collection and storage (Sect. 8), ensuring that the 

instrument is properly calibrated (Sect. 10), and that all required QC elements are included (Sect. 

9). 

11.1 SAMPLE PREPARATION 

11.1.1 Samples are dechlorinated, preserved, collected and stored as described in Section 8. All 

field and QC samples must contain the dechlorinating and preservation agents listed in 

Section 8.1.2, including the LRB. Before extraction, mark the level of the sample on the 

outside of the sample bottle for later sample volume determination (Sect. 11.5). If using 

weight to determine volume, weigh the full sample bottle before extraction. 

11.1.2 Add an aliquot of the SUR PDS (Sect. 7.2.2.2) to each sample, cap the sample, and invert 

to mix. During method devlopment, a 10-μL aliquot of SUR PDS was added to 1-L 

samples for final concentrations of 25 ng/L bisphenol A-d16 and 70 ng/L ethynylestradiold4. 

11.1.3 Fortify LFBs, LFSMs, or LFSMDs, with an appropriate volume of Analyte PDS (Sect. 

7.2.3.2). Cap and invert each sample several times to mix.

11.2 DISK SPE PROCEDURE

11.2.1 Assemble the extraction glassware onto the vacuum manifold, placing disks on a support 

screen between the funnel and base.

11.2.2 DISK CLEANING – Add a 10-mL aliquot of methanol and draw through the disk until 

dry. Add another 5-mL aliquot of methanol and draw through the disk until dry. 

11.2.3 DISK CONDITIONING – The conditioning step is critical for recovery of analytes and 

can have a marked effect on method precision and accuracy. Once the conditioning has 

begun, do not allow the disk to dry until the last portion of the sample is drawn 

through the disk. If the disk goes dry during the conditioning phase, the conditioning 

must be repeated.

11.2.3.1 CONDITIONING WITH METHANOL – Add approximately 10 mL of methanol to 

each disk. Pull about 1 mL of solvent through the disk and turn off the vacuum 

temporarily; let the disk soak for about one minute. Draw most of the remaining 

solvent through the disk, but leave a thin layer of methanol on the surface of the disk. 

The disk must not be allowed to go dry from this point until the end of the sample 

extraction.
539-24 

11.2.3.2 CONDITIONING WITH WATER – Add 10 mL of reagent water to each disk and 

draw through, leaving a thin layer of liquid on the surface of the disk. Follow this 

with another 10-mL aliquot of reagent water. Draw the water through each disk, 

again being careful to keep the water level above the disk surface. Turn off the 

vacuum. 

11.2.4 SAMPLE EXTRACTION – Add the sample to the extraction reservoir containing the 

conditioned disk and turn on the vacuum (approximately 10 to 15 in. Hg). Do not let the 

disk go dry before the entire sample volume is extracted. When the sample has been 

drawn through the disk, add a 10-mL aliquot of 15% methanol to the sample container 

and wash the disk with the rinsate from the container. Pull air through the disk by 

maintaining full vacuum for 10 – 15 minutes. After drying, turn off and release the 

vacuum. 

11.2.5 DISK ELUTION – Detach the glassware base from the manifold without disassembling 

the funnel from the base. Insert collection tubes into the manifold to catch the extracts as 

they are eluted from the disk. The collection tube must fit around the drip tip of the base 

to ensure collection of all the eluent. Reattach the base to the manifold. Add 5 mL of 

methanol to the disk and, with vacuum, pull enough methanol into the disk to soak the 

sorbent. Allow the disk to soak for about one minute. Using vacuum, pull the remaining 

methanol slowly through the disk into the collection tube. Elute with additional 2 x 5-mL 

aliquots of methanol. Detach glassware from manifold and remove collection tube.

11.3 EXTRACT CONCENTRATION – Concentrate the extract to approximately dryness under a 

gentle stream of nitrogen in a warm water bath (~45 o

C). Rinse the collection tube with 500 

μL of 50% methanol, and transfer the rinsate to a 1-mL volumetric. Add IS PDS solution, 

and adjust to the 1-mL volume with 50% methanol. During method development, a 5-μL aliquot of the IS PDS (Sect. 7.2.1.2) was added to each extract. 

11.4 FILTERING EXTRACTS – It is highly recommended that all samples be filtered prior to 

analysis. Finished drinking water matrices will yield extracts that may contain particulates. 

If filtering is incorporated as part of the sample preparation, the first lot of syringe filters 

must be included in the procedure when conducting the IDC (Sect. 9.2) to ensure that they do 

not introduce interferences or retain any of the method analytes. Verification of subsequent 

lots of syringe filters can be accomplished by examining CAL standards. Filter aliquots of at 

least two prepared CAL standards. Compare the filtered samples to the unfiltered CAL 

standards. The filtered and unfiltered area counts should agree within 15% of each other. If 

the difference is greater than 15%, another lot of syringe filters should be obtained. 

11.5 SAMPLE VOLUME OR WEIGHT DETERMINATION – 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 the volume of each sample to the nearest 10 mL for use in the final 

calculations of analyte concentration. If using weight to determine volume, reweigh the 

empty sample bottle to the nearest 10 g. From the weight of the original sample bottle 

measured in Section 11.1.1, subtract the empty bottle weight. Assume a sample density of 

1.0 g/mL. 
539-25 

11.6 SAMPLE ANALYSIS

11.6.1 Establish LC-ESI-MS/MS operating conditions equivalent to those summarized in Tables 

1-4 of Section 17 as per the guidance in Section 10.1. Column choice and instrument 

parameters should be optimized prior to initiation of the IDC (Sect. 9.2). 

11.6.2 Establish a valid initial calibration following the procedures in Section 10.2 or confirm 

that the calibration is still valid by analyzing a CCC (Sect. 10.3). If establishing an initial 

calibration for the first time, complete the IDC as described in Section 9.2 prior to 

analyzing field samples.

11.6.3 Analyze field and QC samples at appropriate frequencies in the analysis batch as 

described in Section 11.7. 

11.7 THE ANALYSIS BATCH – An analysis batch is a sequence of samples, analyzed within a 

24-hour period, of no more than 20 field samples and includes all required QC samples 

(LRB, CCCs, LFB, LFSM and LFSMD (or FD)). The required QC samples are not included 

in counting the maximum field sample total of 20. LC-MS/MS conditions for the analysis 

batch must be the same as those used during calibration.

11.7.1 After a valid calibration is established, begin every analysis batch by analyzing an initial 

low-level CCC at or below the MRL. This initial CCC must be within ± 50% of the true 

value for each method and surrogate analyte and must pass the IS area criterion (Sect. 

10.3.2). The initial CCC confirms that the calibration is still valid. Failure to meet the 

QC criteria may indicate that recalibration is required prior to analyzing samples. After 

the initial CCC, continue the analysis batch by analyzing an LRB, followed by field and 

QC samples at appropriate frequencies (Section 9.3). Analyze a mid- or high-level CCC 

after every ten field samples and at the end of each analysis batch. Do not count QC 

samples (LRBs, LFBs, FDs, LFSMs, LFSMDs) when calculating the required frequency 

of CCCs. 

11.7.2 A final CCC completes the analysis batch. The acquisition start time of the final CCC

must be within 24 hours of the acquisition start time of the initial low-level CCC at the 

beginning of the analysis batch. More than one analysis batch within a 24-hour period is 

permitted.

12. DATA ANALYSIS AND CALCULATIONS

12.1 Establish an appropriate retention time window for each analyte to identify them in QC and 

field sample chromatograms. Base this assignment on measurements of actual retention time 

variation for each compound in standard solutions over the course of time. The suggested 

variation is plus or minus three times the standard deviation of the retention time for each 

compound for a series of injections. The injections from the initial calibration and from the 

IDC (Sect. 9.2) may be used to calculate the retention time window. However, the 

experience of the analyst should weigh heavily on the determination of an appropriate range.
539-26 

12.2 At the conclusion of data acquisition, use the same software settings established during the 

calibration procedure to identify peaks of interest in the predetermined retention time 

windows. Confirm the identify of each analyte by comparison of its retention time with that 

of the corresponding analyte peak in an initial calibration standard or CCC. 

12.3 Calculate analyte concentrations using the multipoint calibration established in Section 10.2. 

Report only those values that fall between the MRL and the highest calibration standard. 

12.4 Calculations must use 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. 

12.5 Prior to reporting the data, the chromatograms must be reviewed for any incorrect peak 

identifications or improper integration. The laboratory is responsible for ensuring that QC 

requirements have been met and that any appropriate qualifier is assigned. 

12.6 EXCEEDING THE CALIBRATION RANGE – The analyst must not extrapolate beyond the 

established calibration range. If an analyte result exceeds the range of the initial calibration 

curve, the extract may be diluted using 50% methanol containing the appropriate amount of 

internal standard added to match the original level. Re-inject the diluted sample. Incorporate 

the dilution factor into final concentration calculations. The resulting data must be annotated 

as a dilution, and the reported MRLs must reflect the dilution factor. Acceptable surrogate 

performance must be determined from the undiluted sample extract. 

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND DETECTION LIMITS – 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 four water matrices: reagent water (Table 6); 

chlorinated (finished) groundwater (Table 7); moderate TOC chlorinated surface water 

(Table 8); and, the aqueous holding time study in chlorinated surface water (a separate source 

from the moderate TOC chlorinated surface water) (Table 9).

13.2 SECOND LABORATORY DEMONSTRATION – The performance of this method was 

demonstrated in multi-lab studies using triple quadrupole, ion trap, and “hybrid” triple 

quadrupole/ion trap mass spectrometers. The authors wish to acknowledge the work of 

MWH Laboratories (Monrovia, CA), Southern Nevada Water Authority (Henderson, NV), 

and Suffolk County Water Authority (Hauppauge, NY) for assisting in the review of this 

method and participation in the second laboratory demonstration. 

13.3 ANALYTE STABILITY STUDY - Chlorinated surface water samples, fortified with method 

analytes at 5 – 10 ng/L, were preserved as required in Section 8 and stored over a 28-day 

period. The accuracy and precision of six replicate analyses, conducted on days 0, 7, 14, 21 

and 28, are presented in Section 17, Table 9. 
539-27 

13.4 EXTRACT STORAGE STABILITY – Extract storage stability studies were conducted on 

extracts obtained from a chlorinated surface water fortified with the method analytes. The 

precision and accuracy of six replicates conducted on days 0, 7, 14, 21, and 28, are presented 

in Section 17, Table 10. 

14. POLLUTION PREVENTION

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 environment as compared to the use of large volumes of organic 

solvents in conventional liquid-liquid extractions. For information about pollution prevention that 

may be applicable to laboratory operations, consult “Less is Better: Laboratory Chemical 

Management for Waste Reduction” available from the American Chemical Society’s Department 

of Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C., 20036.

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 matrix of concern is finished 

drinking water. However, the Agency requires that laboratory waste management practices 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. 

In addition, compliance is required with any sewage discharge permits and regulations, particularly 

the hazardous waste identification rules and land disposal restrictions. 

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.; Foerst, D.L.; McKee, G.D.; Quave, S.A.; Budde, W.L. Trace Analyses for 

Wastewaters. Environ. Sci. Technol. 1981; 15, 1426-1435. 

3. Occupational Exposures to Hazardous Chemicals in Laboratories; 29 CFR 1910.1450, Occupational 

Safety and Health Administration, 1990. 

4. Safety in Academic Chemistry Laboratories; American Chemical Society Publication, Committee on 

Chemical Safety, 7th Edition: Washington, D.C., 2003. 

5. Standard Practice for Sampling Water from Closed Conduits; ASTM Annual Book of Standards, 

Section 11, Volume 11.01, D3370-08; American Society for Testing and Materials: Philadelphia, 

PA, 2008. 
539-28 

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. HPLC CONDITIONS

HPLC

Column: Waters Xterra® MS C18, 2.1 x 150 mm, 3.5 μm dp

Column Temperature: 35 oC

Column Flow Rate: 0.200 mL/min.

Injection Volume: 50 μL

Gradient:

Time

(min.)

%RW %MeOH %NH4OHa

(0.2% v/v)

0 40 50 10

16.5 40 50 10

17.5 25 65 10

30 25 65 10

31 5 85 10

35 5 85 10

35.1 40 50 10

50 40 50 10 a

Preparation of 0.2% (v/v) ammonium hydroxide: Filter 1000 mL reagent water using 0.22 μm PVDF disk 

filter (Sect. 6.7). Add 8 mL 29% ammonium hydroxide (Sect. 7.1.2) and mix well (pH 10 – 10.5). The setup described in the table entails quarternary mixing; if this option is not available, an alternative approach 

is to incorporate 0.02% ammonium hydroxide in the RW and MeOH eluents.

As the ammonia solutions age, analyte responses may drop – this is an indication that fresh mobile phase 

needs to be prepared.

TABLE 2. ESI-MS/MS METHOD CONDITIONS

MS Parameter HPLC/MS/MS

Polarity ESI+ & ESICapillary Voltage, kV 3.0

Source Temperature, oC 120

N2 Desolvation Temperature, oC 350

N2 Desolvation Gas Flow, L/hr 900

Cone Gas Flow, L/hr 50

Extractor Lens, V 2

RF Lens, V 0.1

Collision Cell Pressure, mbar 3.4 e-3
539-29 

TABLE 3. LC-ESI-MS/MS ANALYTE RETENTION TIMES, PRECURSOR AND 

PRODUCT IONS, CONE VOLTAGE, AND COLLISION ENERGY

Analyte Ret. 

Time 

(min.)

ESI 

Mode

Precursor 

Ion 

Product 

Ion 

Cone 

Voltage, 

V

Collision 

Energy, 

eV

Internal Standard

Estriol 4.99 ESI- 287 144.7 55 40 Estriol-d2

Bisphenol A-d16 9.31 ESI- 241.1a 223 40 18 13C6-Estradiol

Equilin 16.92 ESI- 267.1 142.7 35 32 13C6-Estradiol

Estrone 19.49 ESI- 268.9 144.7 55 40 13C6-Estradiol

Androstenedione 19.97 ESI+ 287.1 96.6 30 20 Testosterone-d3

17β-Estradiol 20.84 ESI- 271.2 144.7 55 40 13C6-Estradiol

17αEthynylestradiol 22.74 ESI- 295.1 144.7 50 35 13C2-Ethynylestradiol

Ethynylestradiold4 22.57 ESI- 299 144.7 55 40 13C2-Ethynylestradiol

Testosterone 24.24 ESI+ 289.1 96.8 35 25 Testosterone-d3

a

The bisphenol A-d16 readily exchanges two deuterium atoms in solution. As such, the m/z observed in 

MS analysis is based on bisphenol A-d14

TABLE 4. LC-ESI-MS/MS INTERNAL STANDARD RETENTION TIMES, PRECURSOR 

AND PRODUCT IONS, CONE VOLTAGE, AND COLLISION ENERGY

Internal Standard 

Ret. Time 

(min.)

Precursor 

Ion 

Product 

Ion 

Cone 

Voltage, 

V 

Collision 

Energy, 

eV

Estriol-d2 4.94 289 146.7 55 40

13C6-Estradiol 20.98 277.1 144.7 55 40

13C2-Ethynylestradiol 22.70 297 144.7 55 38 

Testosterone-d3 24.15 292.1 96.7 35 26
539-30 

TABLE 5. LC-ESI-MS/MS LOWEST CONCENTRATION MINIMUM REPORTING 

LEVELS (LCMRLs) and DETECTION LIMITS (DLs)

Analyte

DL Fortified 

Concentration (ng/L)a DL (ng/L)b LCMRL (ng/L)c 

Estriol 1.10 0.24 0.28

Estrone 1.05 0.19 4.0

17β-Estradiol 1.30 0.39 0.32

17α-Ethynylestradiol 1.75 0.33 1.3

Androstenedione 0.50 0.20 0.37

Testosterone 0.50 0.04 0.062

Equilin 1.25 2.94 3.0

a

Fortification level used to determine DLs

b

Detection limits were obtained as described in Section 9.2.6

c

LCMRLs were calculated according to the procedure in reference 1 with the following modification: 

Instead of evaluating seven replicates at four concentration levels, LCMRLs are now obtained by 

analyzing four replicates at seven concentration levels.

TABLE 6. LC-ESI-MS/MS PRECISION AND ACCURACY IN FORTIFIED REAGENT 

 WATER (n=5)

Analyte

Fortified 

Concentration (ng/L)

Avg. %Recovery %RSD

Estriol 11 83.5 6.3

Estrone 10.5 86.5 1.2

17β-Estradiol 13 90.6 6.0

17α-Ethynylestradiol 17.5 90.1 3.1

Androstenedione 10 87.2 18

Testosterone 10 93.5 3.3

Equilin 12.5 80.4 5.1

Ethynylestradiol-d4 67 98.1 2.2

Bisphenol A-d16 24 84.5 8.5
539-31 

TABLE 7. LC-ESI-MS/MS PRECISION AND ACCURACY IN FORTIFIED 

CHLORINATED GROUND WATERa (n=5) 

Analyte

Fortified 

Concentration (ng/L)

Avg. %Recovery %RSD

Estriol 11 89.5 5.8

Estrone 10.5 96.5 3.0

17β-Estradiol 13 93.6 2.9

17α-Ethynylestradiol 17.5 105 3.5

Androstenedione 10 94.6 12

Testosterone 10 88.3 2.4

Equilin 12.5 99.9 3.1

Ethynylestradiol-d4 67 93.0 3.4

Bisphenol A-d16 24 93.2 1.9 a Ground water physical parameters: total hardness = 334 milligrams/liter (mg/L) (as CaCO3); free chlorine = 

0.60 mg/L; total chlorine = 0.79 mg/L. 

TABLE 8. LC-ESI-MS/MS PRECISION AND ACCURACY IN FORTIFIED MODERATE 

TOC CHLORINATED SURFACE WATERa

 (n=5) 

Analyte

Fortified 

Concentration (ng/L)

Avg. %Recovery %RSD

Estriol 11 89.3 3.3

Estrone 10.5 86.8 4.8

17β-Estradiol 13 88.1 5.9

17α-Ethynylestradiol 17.5 99.6 6.1

Androstenedione 10 102 3.7

Testosterone 10 86.2 3.1

Equilin 12.5 92.4 11

Ethynylestradiol-d4 67 94.5 3.6

Bisphenol A-d16 24 93.2 13 a Surface water physical parameters: total hardness = 113 milligrams/liter (mg/L) (as CaCO3); free chlorine = 

1.35 mg/L; total chlorine = 1.56 mg/L.
539-32 

TABLE 9. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFACE 

WATERa, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED ACCORDING TO 

METHOD SECTION 8 (n = 6)

Analyte Fortified 

Conc. 

(ng/L)

Day 0 Day 7 Day 14 Day 21 Day 28

Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD

Estriol 11 85.5 4.3 83.4 4.0 86.7 3.5 86.9 3.5 86.1 3.8

Estrone 10.5 84.1 3.6 82.8 5.3 89.1 5.8 91.9 6.4 84.3 3.1

17β-Estradiol 13 91.8 2.9 85.3 5.3 92.3 4.8 88.1 4.0 87.3 2.4

17αEthynylestradiol 17.5 97.6 3.9 90.4 4.0 97.4 4.9 90.4 3.2 85.1 3.3

Androstenedione 10 104 3.8 103 3.4 93.5 5.9 115 7.1 112 4.6

Testosterone 10 93.0 6.0 88.9 2.5 85.9 3.2 91.7 1.5 95.9 2.3

Equilin 12.5 103 13 92.6 4.1 85.7 14 91.7 11 86.3 5.0

Ethynylestradiold4 67 85.9 5.7 86.2 4.5 90.6 5.5 88.8 3.6 85.0 7.1

Bisphenol A-d16 24 85.8 6.3 87.7 4.7 85.6 8.7 127 7.3 94.7 4.9 a Surface water physical parameters: total hardness = 134 milligrams/liter (mg/L) (as CaCO3); free chlorine = 1.05 mg/L; total chlorine = 1.25 

mg/L.

TABLE 10. EXTRACT HOLDING TIME DATA FOR SAMPLES FROM CHLORINATED SURFACE WATER FORTIFIED WITH 

METHOD ANALYTES AND PRESERVED AND STORED ACCORDING TO METHOD SECTION 8 (n = 6)

Analyte Fortified 

Conc. 

(ng/L)

Day 0 Day 7 Day 14 Day 21 Day 28

Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD Avg. 

%Rec

%RSD

Estriol 11 85.5 4.3 82.3 5.6 84.0 6.9 82.3 4.4 86.1 5.2

Estrone 10.5 84.1 3.6 85.6 4.9 83.4 7.9 88.8 3.8 84.2 3.5

17β-Estradiol 13 91.8 2.9 86.0 8.9 89.3 5.5 88.6 5.6 89.6 4.0

17αEthynylestradiol 17.5 97.6 3.9 91.1 6.8 86.6 6.7 90.4 3.0 88.4 4.8

Androstenedione 10 104 3.8 104 6.0 96.0 8.9 119 8.2 111 3.4

Testosterone 10 93.0 6.0 89.8 4.2 90.4 2.9 96.2 4.9 96.6 4.1

Equilin 12.5 103 13 90.1 4.8 79.5 9.3 91.0 4.1 88.7 4.4

Ethynylestradiold4

67 85.9 5.7 89.4 6.7 88.6 5.9 86.4 2.1 87.8 4.6

Bisphenol A-d16 24 85.8 6.3 89.5 3.7 87.6 15 131 3.9 99.8 3.9
539-33 

TABLE 11. INITIAL DEMONSTRATION OF CAPABILITY (IDC) QUALITY CONTROL REQUIREMENTS

Method 

Reference Requirement Specification and Frequency Acceptance Criteria

Section 9.2.1 Demonstration of low 

system background

Analyze a Laboratory Reagent Blank (LRB) 

prior to any other IDC steps.

Demonstrate that all method analytes are < 1/3 of 

the Minimum Reporting Level (MRL) and that 

possible interferences from reagents and glassware 

do not prevent the identification and quantitation 

of method analytes.

Section 9.2.2 Demonstration of 

precision

Prepare, extract, and analyze 4 - 7 replicate 

Laboratory Fortified Blanks (LFBs) fortified 

near the midrange concentration.

Percent relative standard deviation must be ≤ 20%.

Section 9.2.3 Demonstration of 

accuracy

Calculate average percent recovery for 

replicates used in Section 9.2.2.

Average percent recovery within + 30% of the true 

value.

Section 9.2.4 MRL confirmation

Fortify, extract, and analyze 7 replicate LFBs at 

the proposed MRL concentration. Confirm that 

the Upper Prediction Interval of Results (PIR) 

and Lower PIR (Sect. 9.2.4.1 and Sect. 9.2.4.2) 

meet the recovery criteria.

Upper PIR ≤ 150%

Lower PIR ≥ 50%

Section 9.2.5 Quality Control Sample 

(QCS) Analyze mid-level QCS. Results must be within + 30% of the true value.
539-34

TABLE 12. ONGOING QUALITY CONTROL REQUIREMENTS

Method 

Reference Requirement Specification and Frequency Acceptance Criteria

Section 

10.2 Initial calibration

Use the internal standard calibration technique to 

generate a linear or quadratic calibration curve. 

Use at least five calibration concentrations. 

Validate the calibration curve as described in 

Section 10.2.3.

When each calibration standard is calculated as an unknown 

using the regression equation, the lowest level standard must 

be within + 50% of the true value. All other points must be 

within + 30% of the true value.

Section 

9.3.1

Laboratory Reagent Blank 

(LRB) Analyze one LRB with each extraction batch.

Demonstrate that all method analytes are < 1/3 the Minimum 

Reporting Level (MRL), and that possible interference from 

reagents and glassware do not prevent identification and 

quantitation of method analytes.

Section 

9.3.3

Laboratory Fortified Blank 

(LFB)

Extract and analyze one LFB with each extraction 

batch.

For LFBs fortified at concentrations < 2 x MRL, the result 

must be within + 50% of the true value. At concentrations > 

2 x MRL, the result must be + 30% of the true value.

Section 

10.3

Continuing Calibration 

Check (CCC)

Verify initial calibration by analyzing a low-level 

CCC at the beginning of each analysis batch. 

Subsequent CCCs are required after every 10 field 

samples, and at the end of the analysis batch.

The lowest level CCC must be within + 50% of the true 

value. All other points must be within + 30% of the true 

value. Surrogate analytes must be + 30% of the true value. 

Internal standards must be + 50% of the average peak areas in 

the initial calibration.

Results for field samples that are not bracketed by acceptable 

CCCs are invalid.

Section 

9.3.4 Internal standard (IS) Isotopically labeled internal standards are added to all standards and samples.

Peak area counts for each IS must be within ± 50% of the 

average peak areas in the initial calibration.

Section 

9.3.5 Surrogate analyte Fortify the surrogate analytes into all samples prior to extraction. Surrogate recovery must be in the range of 70 to 130%.

Section 

9.3.6

Laboratory Fortified 

Sample Matrix (LFSM)

Analyze one LFSM per extraction batch. Fortify 

the LFSM with method analytes at a concentration 

greater than the native concentrations. 

For LFSMs fortified at concentrations < 2 x MRL, the 

calculated recovery must be within + 50% of the true value. 

At concentrations greater than the 2 x MRL, the recovery 

must be + 30% of the true value.

Section 

9.3.7

Laboratory Fortified 

Sample Matrix Duplicate 

(LFSMD) or Field 

Duplicate (FD)

Analyze at least one LFSMD or FD with each 

extraction batch.

For LFSMDs or FDs, the calculated relative percent 

difference must be ≤ 30%. (≤ 50% if concentration < 2 x 

MRL.)

Section 

9.3.8

Quality Control Sample 

(QCS) Analyze mid-level QCS at least quarterly. Results must be + 30% of the true value.
539-35

FIGURE 1. EXAMPLE CHROMATOGRAM OF LC-ESI-MS/MS TRANSITIONS FOR 

METHOD 539 ANALYTES.

CAL 6

Time

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

041410-8 8: MRM of 2 Channels ES+ 

289.1 > 96.8 (Testosterone)

1.87e5

24.24

041410-8 7: MRM of 3 Channels ES295.1 > 144.7 (Ethynylestradiol)

4.24e3

22.74

041410-8 6: MRM of 2 Channels ES271.2 > 144.7 (Estradiol)

3.10e3

20.84

041410-8 5: MRM of 1 Channel ES268.9 > 144.7 (Estrone)

9.67e3

19.49

22.61

041410-8 4: MRM of 1 Channel ES+ 

287.1 > 96.6 (Androstenedion)

5.10e4

19.97

041410-8 3: MRM of 2 Channels ES267.1 > 142.7 (Equilin)

5.41e3

16.90

041410-8 1: MRM of 2 Channels ES287 > 144.7 (Estriol)

3.38e3

4.98
539-36

FIGURE 2. EXAMPLE CHROMATOGRAM OF LC-ESI-MS/MS TRANSITIONS FOR 

METHOD 539 SURROGATES AND INTERNAL STANDARDS.

CAL 6

Time

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

0.00 10.00 20.00 30.00 40.00 50.00 60.00

%

0

100

041410-8 8: MRM of 2 Channels ES+ 

292.1 > 96.7 (Testosterone-d3)

3.05e4

24.15

29.44

041410-8 7: MRM of 3 Channels ES299 > 144.7 (Surrogate)

2.17e3

22.57

041410-8 7: MRM of 3 Channels ES297 > 144.7 (13C2-Ethynylestradiol)

3.17e3

22.66

041410-8 6: MRM of 2 Channels ES277.1 > 144.7 (13C6-Estradiol)

2.21e3

20.98

041410-8 2: MRM of 1 Channel ES241.1 > 223 (Bisphenol A-d16)

5.12e3

9.26

9.11

5.18

9.49

9.66

041410-8 1: MRM of 2 Channels ES289 > 146.7 (Estriol-d2)

1.45e3

4.92

5.23