&EFA
*g"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)
Office of Water (MLK 140) EPA Document No. 815-B-10-001 November 2010 http://water.epa.gov/drink/
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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
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METHOD 539
DETERMINATION OF HORMONES IN DRINKING WATER BY SOLID PHASE
EXTRACTION (SPE) AND LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION
TANDEM MASS SPECTROMETRY (LC/E SI-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 drink-
ing 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:
Chemical Abstracts Services
Analvte Registry Number (CASRN)
16a-Hydroxyestradiol (Estriol) 50-27-1
17p-Estradiol 50-28-2
17a-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 ana-
lytes 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).
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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 (CIS)
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 CIS 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 Calibra-
tion 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
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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 production
(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.
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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 °C for two hours or solvent rinsed. Volumetric glassware should be
solvent rinsed and never heated in an oven above 120 °C.
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).
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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 >l/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 (|iL).
6.4 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 gram (g).
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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 jim, 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 |im 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 (CIS) 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 ELECTROSPRAYIONIZATION 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
CIS 2.1 x 150 mm, 3.5 |im dp column (Waters Part No. 186000408). Any column
539-8
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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 ELECTROSPRAYIONIZATION-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).
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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-l-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
16a-Hydroxyestradiol-<5?2
(Estriol-d2)
13C6-Estradiol
1 3C2-Ethynylestradiol
Testosterone-c/3
CASRNa
53866-32-3
None
None
77546-39-5
Neat Materials Catalog
No.
C/D/N Isotopes, Cat. No.
D-5279
None
None
None
Solution Standards , Cat. No.
N/A
Cambridge Isotope Labs, 100 ug/mL in
Methanol, Cat. No. CLM-7936-1.2
Cambridge Isotope Labs, 100 ug/mL in
Acetonitrile, Cat. No. CLM-3375-1.2
Sigma Drag Std., 100 ug/mL in
dimethoxyethane, Cat. No. T5536
CASRN = Chemical Abstract Registry Number.
7.2.1.1 INTERNAL STANDARD STOCK STANDARDS (ISSS) (500 |ig/mL, except as
noted) - Prepare a-hydroxyestradiol-t/2 (estriol-^) 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
|ig/mL solutions.
7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (IS PDS) (1.0-
4.0 |ig/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 °C. Use 5 jiL of the 1.0 - 4.0 |ig/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
539-10
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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
16a-Hydroxyestradiol-
-------
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 |ig/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
16a-Hydroxyestradiol
(Estriol)
Estrone
17(3-Estradiol
17a-Ethynylestradiol
Equilin
4-Androstene-3,17-dione
Testosterone
Stock
Concentration
(HS/mL)
1000
1000
1000
1000
1000
1000
1000
Stock Volume QiL)
20
20
25
35
20
10
10
Final Volume
(mL 50%
MeOH)
10
Analyte PDS
Concentration
(|ag/mL)
2.0
2.0
2.5
3.5
2.0
1.0
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 |im syringe filters. During method development, the CAL standards
were shown to be stable for at least two weeks when stored at a temperature < 6 °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.
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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
Sodium thiosulfate
2-mercaptopyridine- 1 -oxide,
sodium salt
Amount
80mg/L
65mg/L
Purpose
Removes free chlorine
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.
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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%.
n / ™ r.^ Standard Deviation of Measured Concentrations , ^
% RSD = x 100
Average Concentration
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.
_. _, Average Measured C oncentrati on
% Recovery = x 100
Fortified Concentration
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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 = 3.9638
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 + HRPTa
„ ... ,„ . x 100< 150%
FortijiedConcentration
The Lower PIR Limit must be > 50 percent recovery.
Mean- HRPTP
PIR -xlOO>50%
Fortified Concentration
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.
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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:
= S X ?(n-l,l-a = 0.99)
where
*(n-i,i-a = 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
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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:
(i±\
%R= — xlOO
IfiJ
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
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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:
c
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
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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 (FDi and
FD2) using the equation:
FD, -FD2
(FD, +FD2)/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:
LFSM-LFSMD
(LFSM + LFSMD)/2
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.
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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.
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10.1.2.1 Optimize the response of the precursor ion (Sect. 3.17) for each analyte by infusing
approximately 0.5 - 1.0 |ig/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 |ig/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
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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.
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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. 1 1.5). If using
weight to determine volume, weigh the full sample bottle before extraction.
1 1 . 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-|iL aliquot of SUR PDS was added to 1-L
samples for final concentrations of 25 ng/L bisphenol A-d]6 and 70 ng/L ethynylestradiol-
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.
1 1 .2 DISK SPE PROCEDURE
1 1.2.1 Assemble the extraction glassware onto the vacuum manifold, placing disks on a support
screen between the funnel and base.
1 1.2.2 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.
1 1 .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.
1 1.2.3.1 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.
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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 °C). Rinse the collection tube with 500
jiL 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-|jL ali-
quot 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
l.Og/mL.
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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.
539-25
-------
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.
539-26
-------
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. I; Hallberg, G.R.; Munch D.J.; Frebis, C.P.; Hedrick, E.J.;
Krop, RA. 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.
539-27
-------
17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. HPLC CONDITIONS
HPLC
Column: Waters Xterra® MS CIS, 2.1 x 150 mm, 3.5 jim dD
Column Temperature: 35 °C
Column Flow Rate: 0.200 mL/min.
Injection Volume: 50 |iL
Gradient:
Time
(min.)
0
16.5
17.5
30
31
35
35.1
50
%RW
40
40
25
25
5
5
40
40
%MeOH
50
50
65
65
85
85
50
50
%NH4OHa
(0.2% v/v)
10
10
10
10
10
10
10
10
"Preparation of 0.2% (v/v) ammonium hydroxide: Filter 1000 mL reagent water using 0.22 (im PVDF disk
filter (Sect. 6.7). Add 8 mL 29% ammonium hydroxide (Sect. 7.1.2) and mix well (pH 10 - 10.5). The set-
up 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
Polarity
Capillary Voltage, kV
Source Temperature, °C
N2 Desolvation Temperature, °C
N2 Desolvation Gas Flow, L/hr
Cone Gas Flow, L/hr
Extractor Lens, V
RF Lens, V
Collision Cell Pressure, mbar
HPLC/MS/MS
ESI+ & ESI-
3.0
120
350
900
50
2
0.1
3.4e-3
539-28
-------
TABLE 3. LC-ESI-MS/MS ANALYTE RETENTION TIMES, PRECURSOR AND
PRODUCT IONS, CONE VOLTAGE, AND COLLISION ENERGY
Analyte
Estriol
Bisphenol K-d\6
Equilin
Estrone
Androstenedione
l?p-Estradiol
17a-
Ethynylestradiol
Ethynylestradiol-
d4
Testosterone
Ret.
Time
(min.)
4.99
9.31
16.92
19.49
19.97
20.84
22.74
22.57
24.24
ESI
Mode
ESI-
ESI-
ESI-
ESI-
ESI+
ESI-
ESI-
ESI-
ESI+
Precursor
Ion
287
241. T
267.1
268.9
287.1
271.2
295.1
299
289.1
Product
Ion
144.7
223
142.7
144.7
96.6
144.7
144.7
144.7
96.8
Cone
Voltage,
V
55
40
35
55
30
55
50
55
35
Collision
Energy,
eV
40
18
32
40
20
40
35
40
25
Internal Standard
Estriol-t/2
13C6-Estradiol
13C6-Estradiol
13C6-Estradiol
Testosterone-t/3
13C6-Estradiol
13C2-Ethynylestradiol
13C2-Ethynylestradiol
Testosterone-t/3
aThe bisphenol A-Ji6 readily exchanges two deuterium atoms in solution. As such, the m/z observed in
MS analysis is based on bisphenol A-du
TABLE 4. LC-ESI-MS/MS INTERNAL STANDARD RETENTION TIMES, PRECURSOR
AND PRODUCT IONS, CONE VOLTAGE, AND COLLISION ENERGY
Internal Standard
Estriol-t/2
13C6-Estradiol
13C2-Ethynylestradiol
Testosterone-t/3
Ret. Time
(min.)
4.94
20.98
22.70
24.15
Precursor
Ion
289
277.1
297
292.1
Product
Ion
146.7
144.7
144.7
96.7
Cone
Voltage,
V
55
55
55
35
Collision
Energy,
eV
40
40
38
26
539-29
-------
TABLE 5. LC-ESI-MS/MS LOWEST CONCENTRATION MINIMUM REPORTING
LEVELS (LCMRLs) and DETECTION LIMITS (DLs)
Analyte
Estriol
Estrone
17p-Estradiol
1 7a-Ethynylestradiol
Androstenedione
Testosterone
Equilin
DL Fortified
Concentration (ng/L)a
1.10
1.05
1.30
1.75
0.50
0.50
1.25
DL (ng/L)b
0.24
0.19
0.39
0.33
0.20
0.04
2.94
LCMRL (ng/L)c
0.28
4.0
0.32
1.3
0.37
0.062
3.0
fortification level used to determine DLs
bDetection limits were obtained as described in Section 9.2.6
°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
Estriol
Estrone
17p-Estradiol
1 7a-Ethynylestradiol
Androstenedione
Testosterone
Equilin
Ethynylestradiol-t/4
Bisphenol A-t/i6
Fortified
Concentration (ng/L)
11
10.5
13
17.5
10
10
12.5
67
24
Avg. %Recovery
83.5
86.5
90.6
90.1
87.2
93.5
80.4
98.1
84.5
%RSD
6.3
1.2
6.0
3.1
18
3.3
5.1
2.2
8.5
539-30
-------
TABLE 7. LC-ESI-MS/MS PRECISION AND ACCURACY IN FORTIFIED
CHLORINATED GROUND WATER" (n=5)
Analyte
Estriol
Estrone
17p-Estradiol
1 7a-Ethynylestradiol
Androstenedione
Testosterone
Equilin
Ethynylestradiol-t/4
Bisphenol A-di6
Fortified
Concentration (ng/L)
11
10.5
13
17.5
10
10
12.5
67
24
Avg. %Recovery
89.5
96.5
93.6
105
94.6
88.3
99.9
93.0
93.2
%RSD
5.8
3.0
2.9
3.5
12
2.4
3.1
3.4
1.9
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 WATER" (n=5)
Analyte
Estriol
Estrone
17p-Estradiol
1 7a-Ethynylestradiol
Androstenedione
Testosterone
Equilin
Ethynylestradiol-t/4
Bisphenol A-t/i6
Fortified
Concentration (ng/L)
11
10.5
13
17.5
10
10
12.5
67
24
Avg. %Recovery
89.3
86.8
88.1
99.6
102
86.2
92.4
94.5
93.2
%RSD
3.3
4.8
5.9
6.1
3.7
3.1
11
3.6
13
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.
539-31
-------
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
Estriol
Estrone
l?P-Estradiol
17a-
Ethynylestradiol
Androstenedione
Testosterone
Equilin
Ethynylestradiol-
ck
Bisphenol A-di6
Fortified
Cone.
(ng/L)
11
10.5
13
17.5
10
10
12.5
67
24
DayO
Avg.
%Rec
85.5
84.1
91.8
97.6
104
93.0
103
85.9
85.8
%RSD
4.3
3.6
2.9
3.9
3.8
6.0
13
5.7
6.3
Day?
Avg.
%Rec
83.4
82.8
85.3
90.4
103
88.9
92.6
86.2
87.7
%RSD
4.0
5.3
5.3
4.0
3.4
2.5
4.1
4.5
4.7
Day 14
Avg.
%Rec
86.7
89.1
92.3
97.4
93.5
85.9
85.7
90.6
85.6
%RSD
3.5
5.8
4.8
4.9
5.9
3.2
14
5.5
8.7
Day 21
Avg.
%Rec
86.9
91.9
88.1
90.4
115
91.7
91.7
88.8
127
%RSD
3.5
6.4
4.0
3.2
7.1
1.5
11
3.6
7.3
Day 28
Avg.
%Rec
86.1
84.3
87.3
85.1
112
95.9
86.3
85.0
94.7
%RSD
3.8
3.1
2.4
3.3
4.6
2.3
5.0
7.1
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
Estriol
Estrone
17p-Estradiol
17a-
Ethyny le stradiol
Androstenedione
Testosterone
Equilin
Ethynylestradiol-
d4
Bisphenol A-J16
Fortified
Cone.
(ng/L)
11
10.5
13
17.5
10
10
12.5
67
24
DayO
Avg.
%Rec
85.5
84.1
91.8
97.6
104
93.0
103
85.9
85.8
%RSD
4.3
3.6
2.9
3.9
3.8
6.0
13
5.7
6.3
Day?
Avg.
%Rec
82.3
85.6
86.0
91.1
104
89.8
90.1
89.4
89.5
%RSD
5.6
4.9
8.9
6.8
6.0
4.2
4.8
6.7
3.7
Day 14
Avg.
%Rec
84.0
83.4
89.3
86.6
96.0
90.4
79.5
88.6
87.6
%RSD
6.9
7.9
5.5
6.7
8.9
2.9
9.3
5.9
15
Day 21
Avg.
%Rec
82.3
88.8
88.6
90.4
119
96.2
91.0
86.4
131
%RSD
4.4
3.8
5.6
3.0
8.2
4.9
4.1
2.1
3.9
Day 28
Avg.
%Rec
86.1
84.2
89.6
88.4
111
96.6
88.7
87.8
99.8
%RSD
5.2
3.5
4.0
4.8
3.4
4.1
4.4
4.6
3.9
539-32
-------
TABLE 11. INITIAL DEMONSTRATION OF CAPABILITY (IDC) QUALITY CONTROL REQUIREMENTS
Method
Reference
Section 9.2.1
Section 9.2.2
Section 9.2.3
Section 9.2.4
Section 9.2.5
Requirement
Demonstration of low
system background
Demonstration of
precision
Demonstration of
accuracy
MRL confirmation
Quality Control Sample
(QCS)
Specification and Frequency
Analyze a Laboratory Reagent Blank (LRB)
prior to any other IDC steps.
Prepare, extract, and analyze 4-7 replicate
Laboratory Fortified Blanks (LFBs) fortified
near the midrange concentration.
Calculate average percent recovery for
replicates used in Section 9.2.2.
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.
Analyze mid-level QCS.
Acceptance Criteria
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.
Percent relative standard deviation must be < 20%.
Average percent recovery within + 30% of the true
value.
Upper PIR < 150%
Lower PIR > 50%
Results must be within + 30% of the true value.
539-33
-------
TABLE 12. ONGOING QUALITY CONTROL REQUIREMENTS
Method
Reference
Section
10.2
Section
9.3.1
Section
9.3.3
Section
10.3
Section
9.3.4
Section
9.3.5
Section
9.3.6
Section
9.3.7
Section
9.3.8
Requirement
Initial calibration
Laboratory Reagent Blank
(LRB)
Laboratory Fortified Blank
(LFB)
Continuing Calibration
Check (CCC)
Internal standard (IS)
Surrogate analyte
Laboratory Fortified
Sample Matrix (LFSM)
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD) or Field
Duplicate (FD)
Quality Control Sample
(QCS)
Specification and Frequency
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.
Analyze one LRB with each extraction batch.
Extract and analyze one LFB with each extraction
batch.
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.
Isotopically labeled internal standards are added to
all standards and samples.
Fortify the surrogate analytes into all samples prior
to extraction.
Analyze one LFSM per extraction batch. Fortify
the LFSM with method analytes at a concentration
greater than the native concentrations.
Analyze at least one LFSMD or FD with each
extraction batch.
Analyze mid-level QCS at least quarterly.
Acceptance Criteria
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.
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.
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.
The lowest level CCC must be within + 50% of the true
value. All other points must be within j^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.
Peak area counts for each IS must be within + 50% of the
average peak areas in the initial calibration.
Surrogate recovery must be in the range of 70 to 130%.
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.
For LFSMDs or FDs, the calculated relative percent
difference must be < 30%. (< 50% if concentration <_2 x
MRL.)
Results must be + 30% of the true value.
539-34
-------
FIGURE 1. EXAMPLE CHROMATOGRAM OF LC-ESI-MS/MS TRANSITIONS FOR
METHOD 539 ANALYTES.
CAL6
100^
0.00
041410-8
100^
0.00
041410-8
100^
24.24
8: (.
><>
"'".annels
•
1.87e5
0.00 10.00 20.00 30.00 40.00 50.00 60.00
041410-8 7: MRM of 3 Channels ES-
22.74 295.1 > 144.7 (Ethynylestradiol)
1 UU
0.
| 4.24e3
DO 10.00 20.00 30.00 40.00 50.00
60.00
041410-8 6: MRM of 2 Channels ES-
100^
0.
20.84 271.2 > 144.7 (Estradiol)
h 3.10e3
_J\
DO 10.00 20.00 30.00 40.00 50.00
041410-8 5: MRM
100^
0.
19.49 268.9
j \_22.61
i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i
DO 10.00 20.00 30.00 40.00 50.00
60.00
of 1 Channel ES-
> 144.7(Estrone)
9.67e3
60.00
4: of 1 ES+
100^
19.97 287 1 > 966
ft
II
5,10e4
10.00 20.00
16.90
30.00 40.00 50.00 60.00
3: MRM of 2 Channels ES-
267.1 > 142.7 (Equilin)
5.41e3
4.98
10.00 20.00 30.00 40.00 50.00 60.00
1: MRM of 2 Channels ES-
287 > 144.7(Estriol)
3.38e3
0.00
10.00
20.00
30.00
40.00
50.00
60.00
539-35
-------
FIGURE 2. EXAMPLE CHROMATOGRAM OF LC-ESI-MS/MS TRANSITIONS FOR
METHOD 539 SURROGATES AND INTERNAL STANDARDS.
CAL6
0.00
041410-8
100n
0
0.00
041410-8
100n
0
0.00
041410-8
100n
0
0.00
041410-8
100n
0.00
10.00
5.18
24.15
8: i' , ES+
> 96,7
1 UU
0"
0.
100n
n
DO 10.00 20.00
22
I
3,05e4
29.44
30.00 40.00 50.00 60.00
7:
57
1
10.00 20.00 30.00
22.66
10.00
4.92
5.23
10.00
40.00
50.00 60.00
7: MRM of 3 Channels ES-
297 > 144.7 (13C2-Ethynylestradiol)
3.17e3
20.00
20.98
30.00 40.00 50.00 60.00
6: MRM of 2 Channels ES-
277.1 > 144.7 (13C6-Estradiol)
2.21e3
20.00 30.00 40.00 50.00 60.00
2: MRM of 1 Channel ES-
241.1 >223(BisphenolA-d16)
5.12e3
20.00
30.00
40.00
50.00 60.00
1: MRM of 2 Channels ES-
289 > 146.7(Estriol-d2)
1.45e3
20.00
30.00
40.00
50.00
Time
60.00
539-36
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