EPA Document #: 815-R-05-007
METHOD 331.0 DETERMINATION OF PERCHLORATE IN DRINKING WATER BY
LIQUID CHROMATOGRAPHY ELECTROSPRAY
IONIZATION MASS SPECTROMETRY
Revision 1.0
January 2005
S.C. Wendelken and D. J. Munch (U.S. EPA, Office of Ground Water and Drinking Water)
B.V. Pepich (Shaw Environmental, Inc.)
D. W. Later and C. A. Pohl (Dionex, Inc. Sunnyvale, Ca)
TECHNICAL SUPPORT CENTER
OFFICE OF GROUND WATER AND DRINKING WATER
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 331.0
DETERMINATION OF PERCHLORATE IN DRINKING WATER BY LIQUID
CHROMATOGRAPHY ELECTROSPRAY IONIZATION MASS SPECTROMETRY
(LC/ESI/MS)
1. SCOPE AND APPLICATION
1.1 This is a liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS)
method for the determination of perchlorate in raw and finished drinking waters. This method
can be used to acquire data using either Selected Ion Monitoring (SIM) or Multiple Reaction
Monitoring (MRM) detection. Based on known interferences, MRM detection is
recommended, however, SIM detection may be used if all of the criteria outlined in Section
9.1 are met. Precision and accuracy data have been generated for both SIM and MRM
detection of perchl orate in reagent water, finished groundwater, finished surface water and a
synthetic high ionic strength matrix. The single laboratory Lowest Concentration Minimum
Reporting Level (LCMRL) has also been determined for both detection modes in reagent
water.1
Chemical Abstract Services
Analvte Registry Number (CASRN)
Perchl orate 14797-73-0
1.2 The SIM and MRM mass spectrometry conditions described in this method were developed
using a conventional LC system. Analysts interested in using an ion chromatography system
with MS detection should consult EPA Method 332.0.
1.3 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets the Data
Quality Objectives (DQOs) which are based on the intended use of this method. The single
laboratory LCMRL is the lowest true concentration for which the future recovery is predicted
to fall between 50-150% recovery with 99% confidence. Single laboratory LCMRLs for
perchl orate were 0.022 ng/L for MRM using m/z 83 and 0.056 |J,g/L for SIM using m/z 101.
These values are also provided in Table 2. The procedure used to determine the LCMRL is
described elsewhere.1
1.4 Laboratories using this method are not required to determine an LCMRL, but they will need
to demonstrate that their laboratory MRL for this method meets the requirements described in
Section 9.2.4.
1.5 Detection limit (DL) is defined as the statistically calculated minimum concentration that can
be measured with 99% confidence that the reported value is greater than zero2. The DL is
dependent on sample matrix, fortification concentration, and instrument performance.
Determining the DL for analytes in this method is optional (Sect. 9.2.5). The DLs for
perchlorate were 0.005 ng/L in LC/MRM mode and 0.008 ng/L in the LC/SIM Mode. These
values are also provided in Table 2.
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1.6 This method is intended for use by analysts skilled in the operation of LC/MS instrumentation
and the interpretation of the associated data.
2. SUMMARY OF METHOD
2.1 Water samples are collected in the field using a sterile filtration technique. Prior to analysis,
isotopically enriched perchlorate (C118O4) is added to the sample as an internal standard.
The sample is injected without cleanup or concentration onto a chromatographic column
(Dionex lonPak® AS-21 or equivalent), which separates perchl orate from other anions and
background interferences. Perchlorate is subsequently detected by negative electrospray
ionization mass spectrometry. A remote controlled valve is used to divert early eluting
cations and anions to waste. Prior to the elution of perchl orate, the valve is switched sending
the chromatographic eluent to the mass spectrometer. This diversion helps prevent
unnecessary fouling of the electrospray source. Perchlorate is quantified using the internal
standard technique.
3 DEFINITIONS
3.1 ANALYSIS BATCH - A sequence of samples, which are analyzed within a 30-hour period
and include no more than 20 field samples. Each Analysis Batch must also include all
required QC samples, which do not contribute to the maximum field sample total of 20. The
required QC samples include:
Laboratory Reagent Blank (LRB)
Continuing Calibration Check Standards (CCCs)
Laboratory Fortified Blank (LFB)
Laboratory Fortified Sample Matrix (LFSM)
Laboratory Fortified Sample Matrix Duplicate or Laboratory Duplicate (LFSMD or LD)
3.2 CALIBRATION STANDARD (C AL) - A solution of the target analyte prepared from the
primary dilution standard solution or stock standard solution and the internal standard. The
CAL solutions are used to calibrate the instrument response with respect to analyte
concentration.
3.3 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing the
method analyte, which is analyzed periodically to verify the accuracy of the existing
calibration.
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. 9.2.5), and accurate quantitation is not expected
at this level.2
3.5 INTERNAL STANDARD (IS) - A pure compound added to all standard solutions and field
samples in a known amount. It is used to measure the relative response of the method analyte.
The internal standard must be a compound that is not a sample component.
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3.6 LABORATORY DUPLICATES (LDs) - Two sample aliquots (LDi and LD2), taken in the
laboratory from a single sample bottle, and analyzed separately with identical procedures.
Analyses of LDi and LD2 indicate precision associated specifically with the laboratory
procedures by removing variation contributed from sample collection, preservation and
storage procedures.
3.7 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other blank
matrix to which a known quantity of the method analyte is added. The LFB is analyzed
exactly like a sample including the preservation procedures in Section 8. Its purpose is to
determine whether the methodology is in control, and whether the laboratory is capable of
making accurate measurements.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - An aliquot of a field sample to
which a known quantity of the method analyte is added. The LFSM is processed and
analyzed exactly like a sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concentration of the analyte in the
sample matrix must be determined in a separate aliquot and the measured value in the LFSM
corrected for background concentrations.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A second
aliquot of the field sample used to prepare the LFSM which is fortified and analyzed
identically to the LFSM. The LFSMD is used instead of the Laboratory Duplicate to assess
method precision and accuracy when the occurrence of the target analyte is infrequent.
3.10 LABORATORY FORTIFIED SYNTHETIC SAMPLE MATRIX (LFSSM) - An aliquot of
the Laboratory Synthetic Sample Matrix (Sect. 7.2.4) which is fortified with perchlorate. This
QC sample is processed like a field sample (Sect. 8.1) and is used to confirm that the analyst
has adequate chromatographic resolution between sulfate and perchlorate.
3.11 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other blank
matrix that is treated exactly as a sample including exposure to all filtration equipment,
storage containers and internal standards. The LRB is used to determine if the method analyte
or other interferences are present in the laboratory environment, the reagents, or the apparatus.
3.12 LABORATORY SYNTHETIC SAMPLE MATRIX BLANK (LSSMB) - An aliquot of the
Laboratory Synthetic Sample Matrix (Sect. 7.2.4) that is not fortified with perchlorate. This
QC sample is processed like a field sample (Sect. 8.1) and is used to determine if the method
analyte or other interferences are present in the LSSM solution.
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 50-150% recovery with 99% confidence.1
3.14 MATERIAL SAFETY DATA SHEETS (MSDS) - These sheets contain written information
provided by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire, and reactivity data including storage, spill, and handling precautions.
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3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be reported
by a laboratory as a quantified value for the target 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 the target analyte.
3.16 MULTIPLE REACTION MONITORING (MRM) - A mass spectrometric technique where a
parent ion is first isolated then subsequently fragmented into a product ion(s). Quantitation is
accomplished by monitoring a specific product ion.
3.17 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution containing the method
analyte prepared in the laboratory from stock standard solutions and diluted as needed to
prepare Calibration Standards and other analyte solutions.
3.18 QUALITY CONTROL SAMPLE (QCS) - A solution containing the method analyte at a
known concentration which is obtained from a source external to the laboratory and different
from the source of calibration standards. The QCS is used to verify the calibration
standards/curve integrity.
3.19 REAGENT WATER (RW) - Purified water which does not contain any measurable quantity
of the target analyte or interfering compounds at or above 1/3 the MRL for the target analyte.
3.20 SELECTED ION MONITORING (SIM) - A mass spectrometric technique where only select
ions are monitored. This technique helps to increase sensitivity.
3.21 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing the method
analyte that is prepared in the laboratory using assayed reference materials or purchased from
a reputable commercial source.
4. INTERFERENCES
4.1 All glassware must be meticulously cleaned. Wash glassware thoroughly and rinse with
reagent water. As an alternative to glassware, disposable high-density polyethylene
equipment may be used.
NOTE: Perchl orate has been detected in some common laboratory detergents. If background
contamination is a problem, detergents used during glassware cleaning should be investigated
as a potential source of contamination.
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 l/j the MRL for the target analyte) under the conditions of the analysis. This may be
accomplished by analyzing LRBs as described in Section 3.11. Subtracting blank values
from sample results is not permitted.
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4.3 Matrix interferences may be caused by contaminants that are present in the sample. The
extent of matrix interferences will vary considerably from source to source, depending upon
the nature of the water. Water samples high in organic carbon or dissolved solids may lead to
elevated chromatographic baselines or interfering peaks.
4.3.1 Hydrogen sulfate that is formed from the S isotope of sulfur (H SO4, m/z 99) is a
common interference. It can interfere with the qualitative identification of perchlorate if
there is poor chromatographic resolution between sulfate and perchlorate. This
interference is particularly troublesome when using the SIM mode of detection, as the m/z
of perchlorate (which is required for confirmation) is m/z 99. When using MRM
detection, this ion, at modest concentrations, does not interfere with the identification or
quantitation of perchlorate.
4.4 Equipment used for sample collection and storage has the potential to introduce interferences.
The potential for interferences from these devices must be investigated during the Initial
Demonstration of Capability (Sect. 9.2) by preparing and analyzing a Laboratory Reagent
Blank (LRB). This procedure should be repeated each time that a new brand or lot of
equipment is used to ensure that background contamination does not interfere with the
identification and quantitation of perchlorate.
4.5 The percent of 18O enrichment of the internal standard may vary between manufacturers.
Poor isotopic enrichment of the internal standard may lead to sample contamination by
C116O4 . Consequently, it must be demonstrated that solutions containing a working level of
the internal standard do not contain unlabeled perchlorate at concentrations greater than 1/3 of
the MRL. This is initially confirmed during the analysis of the Laboratory Reagent Blank
(LRB) during the IDC and is monitored in each Analysis Batch.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined. Each chemical should be treated as a potential health hazard, and exposure to these
chemicals should be minimized. Each laboratory is responsible for maintaining an awareness
of OSHA regulations regarding safe handling of chemicals used in this method. A reference
file of MSDSs should be made available to all personnel involved in the chemical analysis.
Additional references to laboratory safety are available.3"5
6. EQUIPMENT AND SUPPLIES (References to specific brands or catalog numbers are included as
examples only and do not imply endorsement of the product.)
6.1 SAMPLE CONTAINERS - 125-mL sterile high-density polyethylene (HOPE) bottles (I-
Chem 125-mL sterile HOPE bottle, Fisher Cat. No. N411-0125 or equivalent).
6.2 SAMPLE FILTERS - Sterile sample filters (Corning 26-mm surfactant free cellulose acetate
0.2-um filter, Fisher Cat. No. 09-754-13 or equivalent). If alternate filters are used they
should be certified as having passed a bacterial challenge test.6 Additionally, if alternate
filters or different lots of the recommended filters are used, they must be tested using a LRB
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and a LFB fortified at the MRL as outlined in Section 9.2 to insure that they do not introduce
interferences or retain perchlorate.
6.3 SYRINGES - Sterile, silicone free disposable syringes (Henke Sass Wolf 20-mL Luer lock,
Fisher Cat. No. 14-817-33 or equivalent).
6.4 VOLUMETRIC FLASKS - Class A, suggested sizes include 5, 100, 250, 500 and 1000 mL
for preparation of standards and mobile phase.
6.5 GRADUATED CYLINDER - Suggested size 25-mL.
6.6 AUTO PIPETTES - Capable of delivering variable volumes from 25 |iL to 2000 |iL.
6.7 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.8 NITROGEN - High purity compressed gas used for desolvation in the mass spectrometer.
The gas purity and pressure requirements will depend on the instrument manufacturers'
specifications.
6.9 ARGON - High purity compressed gas used in the collision cell of the mass spectrometer.
The gas purity and pressure requirements will depend on the instrument
manufactures' specifications.
6.10 SAMPLE PRETREATMENT CARTRIDGES - Cartridges may be needed to clean up
samples that can not be analyzed due to high levels of background interferences. Single-use,
disposable cartridges (OnGuard-II Ba2+ Dionex, Cat. No. 57093 or equivalent) may be used to
remove sulfate from the sample.
6.11 LIQUID CHROMATOGRAPHY ELECTROSPRAY MASS SPECTROMETRY SYSTEM
(LC/ESI/MS)
6.11.1 LC COLUMN - LC column, 2.1 x 250 mm (Dionex IonPak®AS-21 or equivalent). Any
column that provides adequate resolution, peak shape, capacity, accuracy, and precision
(Sect. 9.1) may be used.
NOTE: The IonPak®AS-21 columns are shipped with a sodium hydroxide mobile phase.
During method development new columns were flushed with 200 mM methylamine
mobile phase for approximately 30 minutes before being connected to the mass
spectrometer. Additionally, the retention time of perchlorate on a new lonPak® AS-21
would decrease over several days as the column equilibrated. This shift in retention time
was inconsequential to the analysis due to the presence of the internal standard.
6.11.2 LC SYSTEM - (Waters 2690 or equivalent) The LC system must be compatible with a
basic mobile phase (approximately pH 12). If the LC system contains Vespel®
components, such as injection valve rotor seals, these components must be replaced with
a material suitable for high use at high pH. Suitable materials for high pH include
Tefzel®, PEEK™, Teflon®, UHMWPE and KEL-F®. Consult the instrument
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manufacturer if there are questions regarding the use of a high pH mobile phase in the
system. Additionally, the system must be capable of consistently injecting up tolOO-|jL
volumes and delivering mobile phase at a constant flow rate of 0.350 mL/min.
6.11.3 MASS SPECTROMETER - (Micromass QuattroMicro or equivalent) The MS must be
capable of providing electrospray ionization with negative ion detection. Due to the
potential for H34SO4 interference, MRM detection is recommended.
6.11.4 DATA SYSTEM - An interfaced data system is required to acquire, store, and output MS
data. The computer software should have the capability of processing stored LC/MS data
by recognizing a chromatographic peak within a given retention time window. The
software must allow integration of the ion 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 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be used in all
analyses. Unless otherwise indicated, it is intended that 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, as long as the
reagent is of sufficiently high purity to permit its use without lessening the quality of the
determination.
7.1.1 METHYLAMINE - (CASRN 74-89-5) - (40 wt.%) in water (Aldrich Cat. No. 426466 or
equivalent).
7.1.2 METHYLAMINE MOBILE PHASE (200 mM) - Add 20 mL of the 40 wt.%
methylamine solution in water to a 1L volumetric flask. Dilute to volume with reagent
water. During method development, the mobile phase was found to be stable for at least
48 hours. Laboratories are encouraged to institute their own quality control (QC)
procedures to determine when solutions need to be replaced.
7.1.3 REAGENT WATER - Purified water that does not contain any measurable quantity of
the target analyte or interfering compounds at or above V3 the MRL for the target analyte
(Sect. 3.19). The reagent water used during method development was generated from tap
water using a Millipore ELIX-3 followed by a Millipore Gradient A10 system.
7.1.4 SODIUM CHLORIDE - (NaCl, CASRN 7647-14-5) - Fisher Cat. No. S-271 or
equivalent.
7.1.5 SODIUM BICARBONATE - (NaHCO3, CASRN 497-19-8) - Fluka Cat. No. 71627 or
equivalent.
7.1.6 SODIUM SULFATE - (Na2SO4, CASRN 7757-82-6) - Fluka Cat. No. 71959 or
equivalent.
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7.2 STANDARD SOLUTIONS - When a compounds' purity is assayed to be 96 percent or
greater, the weight can be used without correction to calculate the concentration of the stock
standard. Solution concentrations listed in this section were used to develop this method and
are included as an example. Stock standard solutions are estimated to be stable for one year
and any fortified or dilute solutions made from the stock standards are stable for at least 30
days Although estimated stability times for standard solutions are given, laboratories
should use standard QC practices to determine when their standards need to be
replaced.
7.2.1 INTERNAL STANDARD SOLUTION - This method uses the internal standard
NaCl18O4.
7.2.1.1 INTERNAL STANDARD STOCK STANDARD (ISSS) (100 ng/mL C118O4) -
Prepare, or purchase commercially, an ISSS at a concentration of 100 |j,g /mL. To
prepare this solution from a solid standard, weigh out 12.1 mg of NaCl18O4into a
100-mL volumetric flask and dilute to 100 mL with reagent water. The internal
standard used during method development was custom synthesized by Isotech
(Miamisburg, OH). Subsequently, several manufacturers offer this product as a
standard item. Because the degree of 18O enrichment could vary between
manufacturers, it must be confirmed during the IDC (Sect. 9.2) and in subsequent
LRBs that this standard does not contribute unenriched perchlorate to the sample.
Any background contribution from the IS must be < 1/3 of the perchlorate MRL
when added at working levels (the concentration of the internal standard used
during method development was 1 ng/mL).
7.2.1.2 INTERNAL STANDARD FORTIFICATION SOLUTION (77.0 ng/mL C118O4) -
Place 77.0 |jL of the internal standard ISSS into a 100-mL volumetric flask and
dilute to volume with reagent water. The addition of 25 |jL to 1.9 mL of sample
will produce a final concentration of 1.0 ng/mL. All field samples and CAL
standards must have the same amount of internal standard added.
7.2.2 PERCHLORATE STANDARD SOLUTIONS - Obtain the analyte as a solid standard of
NaClO4 or as a commercially prepared solution from a reputable standard manufacturer.
Prepare the perchlorate stock, primary and secondary standards as described below.
7.2.2.1 PERCHLORATE STOCK STANDARD SOLUTION (PSSS) (100 ng/mL C1O4 ) -
To prepare this solution from a solid NaClO4 standard, weigh out 12.3 mg of
NaClO/tinto a 100-mL volumetric flask and dilute to volume with reagent water.
7.2.2.2 PERCHLORATE PRIMARY DILUTION SOLUTION (PPDS) (1.0 |ig/mL C1O4 )
- Prepare the perchlorate PDS by adding 1.0 mL of the Stock Standard Solution to a
100-mL volumetric flask and dilute to volume with reagent water. This solution is
used to prepare the Perchlorate Secondary Dilution Standard and the Calibration
Standards below.
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7.2.2.3 PERCHLORATE SECONDARY DILUTION SOLUTION (PSDS) (5.0 ng/mL
C1O4 ) - Prepare the 5 ng/mL Analyte SDS by adding 500 |iL of the Analyte PDS
to a 100-mL volumetric flask and dilute to volume with reagent water. This
solution is used to prepare the Calibration Standards below.
7.2.2.4 PERCHLORATE FORTIFICATION SOLUTIONS (PFS) (100, 200 and 1000
|ig/mL) - The Analyte Fortification Solutions are prepared by dilution of the
Perchlorate Secondary Dilution Solution and are used to fortify the LFB, LFSSM,
the LFSMs and the LFSMDs with method analytes. It is recommended that
multiple concentrations be prepared so that the fortification levels can be rotated or
adjusted to the concentration of the target analyte in the native samples.
7.2.3 CALIBRATION (CAL) STANDARDS - Prepare a calibration curve from dilutions of
the perchlorate PDS and the perchlorate SDS using a minimum of five Calibration
Standards that span the concentration range of interest. The lowest concentration
calibration standard must be at or below the MRL. An example of the dilutions used to
prepare the Calibration Standards used to collect the data in Section 17 are shown in the
table below.
PREPARATION OF CALIBRATION CURVE STANDARDS
CAL
Level
1
2
3
4
5
6
Vol. of
Analyte PDS
(HL)
50
100
500
Vol. of
Analyte SDS
(HL)
400
1000
2000
Final Vol. of CAL
Std. (mL)
100
100
100
100
100
100
Final Cone, of CAL
Std. (ng/mL)
0.020
0.050
0.10
0.50
1.0
5.0
A constant amount of the internal standard is added to each Calibration Standard (1.0
ng/mL in the final volume). This is accomplished for each standard by taking 1.9 mL of
the calibration standard and placing it in a 2.0-mL autosampler vial and adding 25 |jL of
the 77 ng/mL Internal Standard Fortification Solution.
NOTE: CAL standards are not processed with the sample collection equipment. This
step must be omitted for the CALs in order to identify any potential losses associated
with the sample filtration or collection protocols.
7.2.4 LABORATORY SYNTHETIC SAMPLE MATRIX (LSSM) - Prepare a LSSM that
contains the common anions chloride, sulfate and bicarbonate at 1000 mg/L follows.
7.2.4.1 Weigh out 1.40 g of NaHCO3, 1.48 g of Na2SO4, and 1.54 g of NaCl (Fluka 71627,
Fluka 71959, Fisher S-271, respectively or equivalent). Add these to a 1-L
volumetric flask using a powder funnel and dilute to volume using reagent water.
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7.2.5 LABORATORY FORTIFIED SYNTHETIC SAMPLE MATRIX (LFSSM) - An aliquot
of the Laboratory Synthetic Sample Matrix fortified using the Perchlorate Fortification
Solutions prepared above (Sect. 7.2.2.4).
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE COLLECTION
8.1.1 Grab samples must be collected in accordance with conventional sampling practices.7
8.1.2 When sampling from a cold water tap, open the tap and allow the system to flush until the
water temperature has stabilized (usually approximately 3 to 5 minutes). Collect a
representative sample from the flowing system using a beaker of appropriate size. Use
this bulk sample to generate individual samples as needed. A volume of at least 20-mL is
required for each individual sample.
8.1.3 When sampling from an open body of water, fill a beaker with water sampled from a
representative area. Use this bulk sample to generate individual samples as needed. A
volume of at least 20-mL is required for each individual field sample.
8.1.4 Once representative samples are obtained, they must be filtered to remove any native
microorganisms. Perchlorate is known to be susceptible to microbiological degradation
by anaerobic bacteria8. Samples are filtered to remove microbes and stored with
headspace to minimize the possibility that anaerobic conditions develop during storage.
At a minimum, leave the top one third of the bottle empty to reduce the potential for
degradation by any remaining anaerobic organisms.
8.1.4.1 Remove a sterile syringe (Sect. 6.3) from its package and draw up 20 mL of the bulk
sample. Remove a sterile syringe filter (Sect 6.2) from its package without touching
the exit Luer connection. Connect the filter to the syringe making sure that no water
from the syringe drops on the exterior of the filter. Open a sterile sample container
(Sect. 6.1) without touching the interior. Using gentle pressure, pass the sample
through the filter into the sample container. During this process do not let the syringe
or filter make contact with the sample container. Following filtration, seal the sample
container tightly, label and prepare the container for shipment. Syringes and filters
are single use items and must be discarded after each sample.
8.2 SAMPLE SHIPMENT AND STORAGE - Samples must be chilled during shipment and
must not exceed 10 °C during the first 48 hours after collection. Samples should be
confirmed to be at or below 10 °C when they are received at the laboratory. Samples stored
in the lab must be held at or below 6 °C until analysis. Samples should not be frozen.
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8.3 SAMPLE HOLDING TIMES - Samples should be analyzed as soon as possible. Samples
that are collected and stored as described in Sections 8.1 and 8.2 may be held for a maximum
of 28 days.
9. QUALITY CONTROL
9.1 Quality Control (QC) requirements include the Initial Demonstration of Capability and
ongoing QC requirements that must be met when preparing and analyzing field samples. This
section describes each QC parameter, its required frequency, and the performance criteria that
must be met in order to meet EPA quality objectives. The QC criteria discussed in the
following sections are summarized in Section 17, Tables 5 and 6. 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 the separation
technique, LC columns, mobile phase composition, LC conditions and MS conditions.
However, each time such method modifications are made, the analyst must repeat the
procedures of the IDC (Sect. 9.2).
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
meet the calibration requirements outlined in Section 10. Requirements for the initial
demonstration of laboratory capability are described in the following sections and are
summarized in Table 5.
9.2.1 DEMONSTRATION OF LOW SYSTEM BACKGROUND - Analyze a LRB and a
LSSMB after processing both through all sample collection steps outlined in Section 8.1.
Confirm that the blanks are reasonably free of contamination and that the criteria in
Section 9.3.1 are met. The LRB and LSSMB must be filtered using the same sample
collection devices that are used for field samples.
9.2.1.1 Concentration dependent carry-over is manifest by signals in subsequent samples
that increase proportionally to the concentration of the previously injected standard.
Analysis of reagent water blank (non-filtered) must be performed after the highest
CAL standard during the IDC to determine if carry-over is present. The results for
this sample must meet the criteria outlined in section 9.3.1. If the reagent water
blank fails to meet the criteria there is likely system carry-over. The source can
often be traced to injection valve problems or an inadequate autosampler rinse
protocol. System carry-over should be eliminated by determining the source of the
problem and taking corrective action.
9.2.2 DEMONSTRATION OF PRECISION - Prepare and analyze 7 replicate LFBs and
LFSSMs. These samples should be fortified near the midrange of the initial calibration
curve. All samples must be processed using the sample collection devices described in
Section 8.1. The percent relative standard deviation (%RSD) of the concentrations of the
replicate analyses must be < 20%.
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n / ^ o^ Standard Deviation of Measured Concentrations , nn
%RSD = xlOO
Average Concentration
9.2.3 DEMONSTRATION OF ACCURACY - Using the same sets of replicate data generated
for section 9.2.2, calculate the average percent recovery. The average percent recovery
of the replicate analyses must be within 80-120% of the true value.
.. _. Average Measured Concentration , ^
% Recovery = x 100
Fortified Concentration
9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Establish a target
concentration for the MRL based on the intended use of the method. The lowest
calibration standard used to establish the initial calibration (as well as the low-level
Continuing Calibration Check) 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 or validate the MRL following the procedure outlined below.
9.2.4.1 Fortify and analyze seven replicate Laboratory Fortified Blanks at the target MRL
concentration. All samples must be processed using the sample collection devices
described in Section 8.1. Calculate the mean (Mean) and standard deviation (S) for
these replicates. Determine the Half Range for the prediction interval of results
(HRpiR) using the equation below
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 Result (PIR =
Mean + HRPiR) meet the upper and lower recovery limits as shown below
The Upper PIR Limit must be <150 percent recovery.
Mean + HRPR xlOO<150%
Fortified Concentration
The Lower PIR Limit must be > 50 percent recovery.
Mean - HR
"PIR
Fortified Concentration
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 determined again at a higher concentration.
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9.2.5 DETECTION LIMIT DETERMINATION (optional) - While DL determination is not a
specific requirement of this method, it may be required by various regulatory bodies
associated with compliance monitoring. It is the responsibility of the laboratory to
determine ifDL determination is required based upon the intended use of the data.
Analyses for this procedure should be done over at least 3 days. Prepare at least 7
replicate LFBs processing the reagent water through all sample collection steps outlined
in Section 8.1. Use the solutions described in Section 7.2.2.4 to fortify at a concentration
estimated to be near the DL. This concentration may be estimated by selecting a
concentration at 2-5 times the noise level. Analyze the seven replicates through all steps
of Section 11.
NOTE: If an MRL confirmation data set meets these requirements, a DL may be
calculated from the MRL confirmation data, and no additional analyses are necessary.
Calculate the DL using the following equation:
DL= St(n_iji_a = o.99)
where:
t(n-i,i-a = 0.99)= Student's t value for the 99% confidence level with n-1 degrees of
freedom
n = number of replicates
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing DL calculations.
9.3 ONGOING QC REQUIREMENTS - This section describes the ongoing QC criteria that must
be followed when processing and analyzing field samples. Table 6 summarizes these
requirements.
9.3.1 LABORATORY REAGENT BLANK (LRB) - A LRB is analyzed during the IDC and is
required with each Analysis Batch (Sect. 3.1) to confirm that background contaminants
are not interfering with the identification or quantitation of perchlorate. If the LRB
produces a peak within the perchlorate retention time window, identify the source of
contamination and eliminate the interference before processing samples. The LRB must
contain the IS at the same concentration used to fortify all field samples and CAL
standards. LRBs must be processed in the same manner as field samples including
exposure to all sample collection devices (i.e., sterile filtration into sample containers). If
samples are collected using devices that have not been previously evaluated by the
laboratory, duplicates of the sample collection devices must be sent with the samples so a
LRB (and a LFB) may be processed in the laboratory. If perchlorate or other
interferences are detected in the LRB at concentrations > 1/3 of the MRL, then all data
for the analyte must be considered invalid for all samples in the Analysis Batch.
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NOTE: Although quantitative data below the MRL may not be accurate enough for data
reporting, such data are useful in determining the magnitude of a background
interference. Therefore, blank contamination levels may be estimated by extrapolation,
when the concentration is below the lowest calibration standard.
9.3.2 CONTINUING CALIBRATION CHECK (CCC) - CCC standards are analyzed at the
beginning of each Analysis Batch, after every ten field samples and at the end of the
Analysis Batch. See Section 10.4 for concentration requirements and acceptance criteria.
9.3.3 LABORATORY FORTIFIED BLANK (LFB) - A LFB is required with each Analysis
Batch. The LFB fortification level must be rotated between low, medium, and high from
batch to batch. The low concentration LFB must be as near as practical to the MRL.
Results of LFBs fortified at < MRL must be within 50-150% of the true value. Results of
LFB analysis from all other concentrations must be 80-120% of the true value. If the LFB
results do not meet these criteria, then all data for perchlorate must be considered invalid
for all samples in the Analysis Batch.
NOTE: LFBs must be processed in the same manner as field samples including exposure
to all sample collection devices (i.e., sterile filtration into sample containers). If samples
are collected using devices that have not been previously evaluated by the laboratory,
duplicates of the sample collection devices must be sent with the samples so a LFB (and a
LRB) may be processed in the laboratory.
9.3.4 INTERNAL STANDARD (IS) - The analyst must monitor the peak area of the internal
standard in all injections of the Analysis Batch. The IS response (as indicated by peak
area) for any chromatographic run must not deviate by more than + 30% of the IS area
measured during the first CCC of the Analysis Batch. If the IS area counts for a sample
do not meet this criterion, inject a second aliquot of the suspect sample as part of the
same or a new Analysis Batch.
9.3.4.1 If the reinjected aliquot produces an acceptable internal standard response, report
results for that aliquot.
9.3.4.2 If the IS area counts of the reinjected aliquot still do not meet the IS criterion, check
the IS area of the most recent CCC. If the IS criterion is met in the CCC but not the
sample, report the sample results as suspect/matrix.
9.3.4.3 If the IS area criterion is not met in both the sample and the CCC, instrument
maintenance such as sample cone cleaning may be necessary. Perform the
appropriate instrument maintenance and then reinject the sample in a subsequent
analytical batch.
9.3.5 ISOTOPE AREA COUNT RATIO ACCEPTANCE CRITERIA - All CAL standards,
QC samples and field samples must meet the area count ratio requirement for
confirmation of perchlorate. Perchlorate has a strong M+2 ion due to the presence of the
37C1 isotope. The analyst must confirm that the ratio of the molecular ion to its M+2 ion
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(99/101) for the SIM technique, or the ratio of their product ions (83/85) for the MRM
technique, are within ± 25% of the theoretical value of 3.08 (2.31 to 3.85)9. If a CAL
standard or QC sample fails this requirement, remedial action is required. Necessary
actions may include mass calibration, column replacement/regeneration or instrument
cleaning. If the area count ratio for a field sample falls outside this range, the cause is
frequently interference from sulfate in the sample matrix. In this case the sample may
require dilution or pretreatment with a barium cartridge to remove the sulfate (see Sect
11.3.4.2 regarding required remedial action).
9.3.6 RELATIVE RETENTION TIME ACCEPTANCE CRITERIA - All CAL standards, QC
samples and field samples must meet the Relative Retention Time (RRT) requirement for
confirmation of perchlorate. The RRT of perchlorate can be calculated using the
equation
Retention Time of Perchl orate Ion
RRI
Retention Time of Internal Standard Ion
The RRT must be 1.0 + 2% (0.98-1.02) for a peak to be identified as perchlorate.
9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of a LFSM (Sect.
3.8) is required in each Analysis Batch. The LFSM is used to assess whether the sample
matrix affects method accuracy. If a variety of different sample matrices are analyzed
regularly, for example drinking water from groundwater and surface water sources,
performance data should be collected for each source.
9.3.7.1 Within each Analysis Batch, a minimum of one field sample is fortified as an LFSM
for every 20 samples analyzed. The LFSM is prepared by fortifying a sample with
an appropriate amount of the Perchlorate Fortification Solution (Sect. 7.2.2.4). The
fortification should be delivered in the smallest volume possible to minimize
dilution of the sample. Select a spiking concentration that is greater than or equal to
the native background concentration, if known. 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 and rotate through low, medium and
high calibration concentrations when selecting a fortifying concentration.
9.3.7.2 Calculate the perchlorate recovery (%REC) using the equation
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.7.3 Recoveries for samples fortified at concentrations < MRL should be 50-150%.
Recoveries for samples fortified at all other concentrations should be 80-120%. If
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the accuracy for a LFSM falls outside the designated range, and the laboratory
performance for perchlorate is shown to be in control in the CCCs, the recovery is
judged to be matrix biased. The result for perchlorate in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect due to
matrix effects.
9.3.7.3.1 Field Samples that have native perchlorate concentrations below the MRL and
are fortified at concentrations at or near the MRL should be corrected for the
native levels in order to obtain meaningful %REC values. This is the only
case where background subtraction of results below the MRL is
permitted.
9.3.8 LABORATORY DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX
DUPLICATE (LD or LFSMD) - Within each Analysis Batch, a minimum of one
Laboratory Duplicate (LD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD)
must be analyzed. Laboratory Duplicates check the precision associated with laboratory
procedures. If perchlorate is not routinely observed in field samples, a LFSMD should be
analyzed rather than a LD.
9.3.8.1 Calculate the relative percent difference (RPD) for duplicate measurements (LDi
and LD2) using the equation
ILD, -LD,
RPD l
(LDj+LDj/2
9.3.8.2 RPDs for Laboratory Duplicates should be < 20%. Greater variability may be
observed when Laboratory Duplicates have perchlorate concentrations that are
within a factor of < 2 x MRL. At these concentrations, Laboratory Duplicates
should have RPDs that are < 50%. If the RPD of the analyte falls outside the
designated range, and the laboratory performance for the analyte is shown to be in
control in the CCC, the precision is judged to be matrix influenced. The result from
the unfortified sample is labeled suspect/matrix to inform the data user that the
results are suspect due to matrix effects.
9.3.8.3 If a LFSMD is analyzed instead of a Laboratory Duplicate, calculate the relative
percent difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the
equation
LFSM - LFSMD
RPD=-, r—xlOO
(LFSM
9.3.8.4 RPDs for duplicate LFSMs should be < 20%. Greater variability may be observed
when fortified LFSMs have analyte concentrations that are within < 2 x MRL.
LFSMs at these concentrations should have RPDs that are < 50%. If the RPD of the
analyte falls outside the designated range, and the laboratory performance for the
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analyte is shown to be in control in the CCC, the precision is judged to be matrix
influenced. The result from the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix effects.
9.4 QUARTERLY INSTRUMENT PERFORMANCE CHECK USING THE LSSMB AND LFSSM
- Analysis of a LFSSM (Sect. 3.10) must be performed at least quarterly to assess instrumental
performance with respect to samples high in common anions. An aliquot of LFSSM (fortified at
the mid-range of the calibration curve) must be processed and analyzed as a sample along with a
LSSMB. Both solutions must be from the same stock of LSSM. Results for the LSSMB and
LFSSM should meet the criteria set forth in Sections 9.3.1 and 9.3.3, respectively. If the LSSMB
contains perchlorate at a concentration > 1/3 the MRL, then the source of the contamination
should be identified and eliminated. If the LFSSM does not meet the QC acceptance criteria for
LFB recovery, instrument maintenance or column replacement may be required.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable MS mass calibration and initial analyte
calibration are required before any samples are analyzed. If the initial calibration is
successful, continuing calibration checks are required at the beginning and end of each period
in which analyses are performed, and after every tenth sample. Verification of mass
spectrometer mass calibration should be repeated each time a major instrument modification
is made or maintenance is performed.
Note: CAL solutions and CCC standards are not processed with the sample collection
devices. This step must be omitted for the CALs and CCCs in order to identify any potential
losses associated with the sample filtration or collection devices._
10.2 MASS SPECTROMETER CALIBRATION AND OPTIMIZATION
10.2.1 MASS CALIBRATION - Calibrate the mass spectrometer according to the
manufacturer's recommendations. The user should be aware that many current LC/MS
instruments are designed to analyze macromolecules having large m/z ratios.
Subsequently, many of the LC/MS calibration procedures are designed to cover the full
scanning range of the instrument. Since this method uses the lower portion of the mass
range, it may be necessary to use alternate calibration compounds that provide ions of a
lower m/z ratio to properly define the mass calibration over the range needed for the
analysis of perchl orate. For our studies, a sodium iodide/rubidium iodide mixture was
used as a calibration compound. It is recommended that the analyst contact the
instrument manufacturer regarding appropriate mass calibration standards. Mass
calibration must be verified prior to the analysis of field samples by injecting a high-level
calibration standard and acquiring a full scan continuum mass spectrum that covers the
m/z range of the target analyte. The perchl orate ion (35 C1O4) should have a m/z of 99 ±
0.3, the (37C1O4) isotope should have a m/z of 101 ± 0.3 and the internal standard
(35 Cl 18O4) should have a m/z of 107 ± 0.3.
10.2.2 OPTIMIZING MS PARAMETERS - LC/MS instruments have a large number of
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instrumental parameters that must be optimized. Each LC/MS system will have different
optimal conditions which are influenced by the source geometry and system design. Due
to the differences in design, the recommendations of the instrument manufacturer should
be followed when tuning the instrument. MS conditions should be established by
infusing a solution of the target compound into the mobile phase while the analyst tunes
(or varies) the MS parameters using the same mobile phase flow rate and composition as
in the final analysis. The response for the parent ions must be optimized for MS analysis
and three MS/MS transitions must be optimized for the MRM analysis of perchlorate.
These transitions are listed in the following table.
Precursor Ion (m/z)
35C1O4(99)
37C1O4(101)
35C118O4(107)
Fragment Lost (m/z )
16O (16)
16O (16)
18O(18)
Product Ion (m/z )
35C103(83)
37C1O3 (85)
35C118O3(89)
10.2.3 INSTRUMENT CONDITIONS - Operating conditions are described in Section 17
Table 1. Conditions different from those described may be used if the QC criteria in
Section 9.2 are met. Different conditions include alternate LC columns, mobile phases
and MS conditions.
10.3 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 is not permitted.
10.3.1 CALIBRATION STANDARDS - Prepare a set of at least five CAL standards as
described in Section 7.2.3. The lowest concentration of the calibration standards must be
at or below the MRL. The MRL must be confirmed using the procedure outlined in
Section 9.2.4 after establishing the initial calibration. Additionally, field samples must be
quantified using a calibration curve that spans the same concentration range used to
collect the IDC data (Sect. 9.2).
10.3.2 CALIBRATION - The LC/MS system is calibrated using peak areas and the internal
standard technique. Concentrations may be calculated through the use of a linear or
quadratic calibration curve. Quantitation ions are listed in Table 1 for both the MRM and
SIM detection techniques.
10.3.3 CALIBRATION ACCEPTANCE CRITERIA - The validation of the calibration is
determined by calculating the concentration of the analyte from each of the analyses used
to generate the calibration curve. Calibration points that are < MRL should calculate to be
50-150% of their true value. All other calibration points should calculate to be 80 to
120% 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, restrict the range of calibration, or select an alternate method of
calibration.
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10.3.4 INITIAL CALIBRATION VERIFICATION - Analyze a QCS sample fortified near the
midpoint of the calibration range. The QCS sample should be from a source different
than the source of the calibration standards. If a second vendor is not available, then a
different lot of the standard should be used. The QCS should be prepared and analyzed
just like a CCC. The acceptance criterion for the QCS is that the calculated amount of
perchlorate must be 80-120% of the true value. If the measured analyte concentration
does not meet this criterion, check the entire analytical procedure to locate and correct the
problem before analyzing any field samples.
10.4 CONTINUING CALIBRATION CHECKS (CCCs) - The CCC verifies the calibration at the
beginning, after every tenth field sample and at the end of each Analysis Batch. CCCs are
not counted as samples. The beginning CCCs for each Analysis Batch must include a
CCC at or below the MRL and a CCC fortified at a level near the midpoint of the
curve. These CCCs verify instrument sensitivity and the accuracy of the curve prior to the
analyses of samples. Subsequent CCCs should alternate between a low, medium and high
concentration.
10.4.1 Inject an aliquot of the appropriate concentration calibration solution and analyze with
the same conditions used during the initial calibration.
10.4.2 Determine that the absolute area of the quantitation ion of the internal standard has not
changed by more than + 30% from the first CCC of the Analysis Batch.
NOTE: The IS area counts of the first CCC of the Analysis Batch should be > 50% of
the average of the IS area counts of the CAL standards from the initial calibration. If the
IS response drifts below 50% of the average determined during the initial calibration,
instrument maintenance or ESI/MS detector inlet cleaning is generally required.
10.4.3 Calculate the concentration of the analyte in the CCC. CCCs fortified at < MRL must
calculate to be 50-150% of the true value. CCCs fortified at all other levels must
calculate to be 80-120%. If these conditions do not exist, then all data for the analyte
must be considered invalid, and remedial action (Sect. 10.4.4) should be taken. The
remedial action may require recalibration. Any field samples that have been analyzed
since the last acceptable calibration verification and are still within holding time should
be reanalyzed after adequate calibration has been restored.
10.4.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria requires remedial
action. Maintenance such as cleaning an ion source probe, sample cone, ion lenses
and/or regenerating or replacing LC columns may be required. Following major
maintenance, the analyst must return to the initial calibration step (Sect. 10.3).
11. PROCEDURE
11.1 Important aspects of this analytical procedure include proper sample collection and storage
(Section 8), ensuring that the instrument is properly calibrated (Section 10) and that all
required QC are met (Section 9). This section describes the procedures for sample
preparation and analysis.
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11.2 SAMPLE PREPARATION
11.2.1 Collect and store field samples as described in Section 8.1. Allow field samples to reach
room temperature prior to analysis.
11.2.2 Process all LRBs, LFBs, LFSSMs and LFSSMBs using the sample collection devices
described in Section 8.1.
11.2.3 Transfer a 1.9-mL aliqout of each Field or QC Sample to an autosampler vial. Add 25
|jL of the IS Fortification Solution (Sect. 7.2.1.2), cap and mix well. Larger amounts of
sample may be used, but the amount of internal standard must be increased to give a final
concentration that is equal to the CAL IS concentration.
11.3 SAMPLE ANALYSIS
11.3.1 Establish operating conditions as described in Table 1 of Section 17.
11.3.2 Establish a valid initial calibration following the procedures outlined in Section 10.3 or
confirm that the calibration is still valid by running a CCC as described in Section 10.4.
If establishing an initial calibration for the first time, complete the IDC as described in
Section 9.2.
11.3.3 Analyze aliquots of Field and QC Samples at appropriate frequencies (Section 9) with the
LC/MS conditions used to acquire the initial calibration. At the conclusion of data
acquisition, use the same software settings that were used in the calibration procedure to
identify peaks in the predetermined retention time windows.
11.3.4 COMPOUND IDENTIFICATION - Establish an appropriate retention time window for
the target analyte and internal standard so they may be identified in QC and field sample
chromatograms. Because the retention time will vary with the ionic strength of the
sample the absolute retention time windows may need to be wider than usual.
11.3.4.1 High ionic strength samples will cause retention times to decrease and introduce
band broadening. This is unavoidable due to the high elutropic strength of the
matrix in these samples. Because of these effects, the relative retention time of
perchlorate is used as one component for qualitative identification (Section 9.3.6).
The relative retention time must be 1.0 + 2% (0.98-1.02) for a peak to be
identified as perchlorate.
11.3.4.2 Perchlorate has a strong M+2 ion due to the presence of 37C1. The analyst must
confirm that the ratio of the molecular ion to its M+2 ion (99/101) for the SIM
technique or the ratio of their daughter ions (83/85) for the MRM technique are
within ± 25% of the theoretical value of 3.08 (2.31 to 3.85). If this ion ratio
requirement is not met in any QC sample, then all samples in the Analysis Batch
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are considered invalid and must be reanalyzed after reestablishing acceptable
instrument performance.
11.3.4.2.1 If the isotope area ratio criteria are not met when using MRM detection the
analyst should report the associated values as "suspect due to lack of
confirmation."
11.3.4.2.2 When using SIM detection, remedial action is required if the following
conditions exist. If a peak is detected at m/z 101, its concentration is > MRL,
the relative retention time criterion is met and the isotope area ratio
requirement is not met. In this case, sample pretreatment must be performed.
The most frequent cause of ratio failure is poor chromatographic resolution
between sulfate and perchlorate. If the ratio failure is due to a high sulfate
level in the sample, dilution or sulfate removal using a commercial barium
pretreatment cartridge (Sect. 6.10) may be successful. Consult the
manufacturer's instructions for preparation of the barium pretreatment
cartridge prior to use with samples. Generally, the procedure requires rinsing
the cartridge with a minimum volume of reagent water. It has been found that
rinsing with approximately twice the recommended volume of water yields
better results. Add IS to the sample prior to sample pretreatment using the
cartridges. Additionally, chromatographic conditions may be changed to
eliminate the coelution, however the IDC must be repeated using the new
conditions.
NOTE: If cartridges are used, a reagent water blank with IS should be
processed to insure that the cartridges don't contribute interferences or retain
perchlorate.
11.3.4.2.3 Following pretreatment, reanalyze the sample as part of the same or a
subsequent Analysis Batch. If the ratio criteria of the reinjected sample pass,
report the results of the reinjected sample. If the ratio criteria are still not met
the analyst should report the associated values as "suspect due to lack of
confirmation."
11.3.5 EXCEEDING 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 sample may be diluted with reagent water, with the appropriate
amount of internal standard added to match the original level, and the diluted sample
reinjected. Incorporate the dilution factor into final concentration calculations. The
dilution will also affect analyte MRLs.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify the analyte in the Field and QC Samples as described in Section 11.3.4.
12.1.1 Sulfate has an ion at m/z 99 (H34SO4) that can interfere with confirmation if the
chromatography is not optimized or if the sulfate concentration in the native samples is
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very high. The SIM version of this method is significantly more susceptible to
interferences from hydrogen sulfate than the MRM version of this method. The M + 2
ion (m/z 101) must be used for quantitation of perchlorate when using SIM.
12.2 Calculate analyte concentrations using the multipoint calibration established in Section 10.3.
Report only those values that fall between the MRL and the highest calibration standard.
Samples with target analyte responses that exceed the highest standard require dilution and
reanalysis (Sect. 11.3.5).
12.3 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.
12.4 Prior to reporting data, the laboratory is responsible for assuring that QC requirements have
been met or that any appropriate qualifier is documented.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY AND DETECTION LIMITS - Tables for these data are
presented in Section 17. Instrumental conditions and monitored ions are presented in Table 1.
The LCMRL for perchlorate is presented in Table 2 and was calculated using a procedure
described elsewhere1. Single laboratory precision and accuracy data are presented in Tables 3
and 4. Table 5 summarizes the requirements for the initial demonstration of capability (IDC)
and Table 6 summarizes the requirements for the required ongoing quality control. Figure 1
shows a representative chromatogram from a MRM analysis. Figure 2 shows a representative
chromatogram from a SIM analysis and Figure 3 shows a total ion chromatogram (m/z 99, 101
and 107) of a LSSM that contains the common anions chloride, sulfate and bicarbonate at
1000 mg/L.
13.2 SECOND LABORATORY DEMONSTRATION -- The performance of this method was
demonstrated by a second laboratory, with results similar to those reported in Section 17. The
authors wish to acknowledge the work of the U.S. EPA National Risk Management Research
Laboratory, Ada, OK and the U.S. EPA New England Laboratory, North Chelmsford, MA for
their participation in the second laboratory demonstration.
14. POLLUTION PREVENTION
14.1 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, or on-line at
http://www.ups.edu/community/storeroom/Chemical Wastes/wastearticles.htm
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15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small amounts of waste
since only small amounts of reagents and solvents are used. The matrices of concern are
finished drinking water or source 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. Also, compliance is required with any sewage
discharge permits and regulations, particularly the hazardous waste identification rules and
land disposal restrictions. For further information on waste management, see the publications
of the American Chemical Society's Laboratory Environment, Health & Safety Task Force on
the Internet at http://membership.acs.Org/c/ccs/labehs/publications.htm. Or see "Laboratory
Waste Minimization and Pollution Prevention," Copyright © 1996 Battelle Seattle Research
Center, which can be found at http://www.p2pays.org/ref/01/text/00779/index2.htm.
16. REFERENCES
1. Statistical Protocol for the Determination of the Single-Laboratory Lowest Concentration Minimum
Reporting Level (LCMRL) and Validation of the Minimum Reporting Level (MRL), available at
www.epa.gov/OGWDW/methods/sourcalt.html
2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
Wastewaters", Environ. Sci. Technol.. 15 (1981) 1426_1435.
3. "Carcinogens - Working With Carcinogens," Department of Health, Education, and Welfare, Public
Health Service, Center for Disease Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety, 3rd Edition, 1979.
6. Blosse, P.T., Boulter, E.M., Sundaram, S., "Diminutive Bacteria Implications for Sterile Filtration",
Pall Corporation, East Hills, NY.
7. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
8. Xu, J., Y. Song, B. Min, L. Steinberg, and B.E. Logan. 2003. Microbial degradation of perchlorate:
principles and applications. Environ. Engin. Sci, 20(5): 405-422.
9. Lange's Handbook of Chemistry (13th Edition); Dean, J. A., Ed; McGraw-Hill: New York, NY, 1985.
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17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. INSTRUMENTAL CONDITIONS, RETENTION TIMES,
QUANTITATION IONS
Micromass QuattroMicro Mass Spectrometer Settings:
Parameter
Ion Source
Polarity
Capillary Voltage
Cone Voltage
Extractor
RFLens
Source Temperature
Desolvation Temperature
Cone Gas Flow
Desolvation Gas Flow
LM 1 Resolution
HM 1 Resolution
Ion Energy
Entrance
Collision
Exit
LM 2 Resolution
HM 2 Resolution
Ion Energy
Multiplier
Gas Cell Pressure
Function dwell time
Data Smoothing (mean)
LC/MRM
Electrospray
Negative Ion
0.58 kV
40V
3V
0.3V
120 °C
320 °C
60 L/hr
N2 800 L/hr
15
15
0.6V
1
24
1
14
14
IV
650V
Ar 3.54e-3 mbar
300ms
Window/Smoothes, 3/2
LC/SIM
Electrospray
Negative Ion
0.58 kV
40V
3V
0.3V
120 °C
320 °C
60 L/hr
N2 800 L/hr
15
15
0.6V
50
2
50
15
15
IV
650V
Off
300ms
Window/Smoothes, 3/2
Chromatographic conditions: LC; Waters 2690, column; Dionex IonPak®AS-21 2.1 x 150 mm
7 |j,m particle diameter, injection volume; 100 |jL, mobile phase; 200 mM methylamine isocratic
at 350 |jL/min.
Monitored Ions and Retention Times
Analyte
J5C103
37C103
35C118O3 (IS)
*Precursor Ion
(m/z)
99
101
107
fProduct Ion
(m/z)
83
85
89
RT
(min)
8.68
8.68
8.65
*In SIM analysis only the precursor ions are monitored and m/z 101 must be used for quantitation.
In MRM analysis either perchlorate product ion may be used for quantitation.
331.0-25
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TABLE 2. LOWEST CONCENTRATION MRL AND DLs FOR PERCHLORATE
Analysis Method
LC/MRM
LC/SIM
Analyte
C104
CIO"
LCMRL (ng/L)
0.022
0.056
*DL (ng/L)
a0.005
b0.008
*The DL was calculated from data acquired on a single day using m/z 83 (MRM) or m/z 101 (SIM)
"Fortification 0.010
fortification 0.075
TABLE 3. LC/MRM PRECISION AND RECOVERY DATA FOR PERCHLORATE IN
VARIOUS MATRICES (n=7 REPLICATES)
Matrix
Reagent Water
Surface Water A
Surface Water B
Ground Water
* Synthetic High Ionic Strength
Unfortified
Concentration
(Hg/L)
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TABLE 5. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL
REQUIREMENTS
Method
Reference
Requirement
Specification and
Frequency
Acceptance Criteria
Section
9.2.1
Demonstration of
Low System
Background
Analyze a LRB and
LSSMB prior to any other
IDC steps.
Demonstrate that the target
analyte is < 1/3 of the MRL
and that possible interferences
from sampling devices do not
prevent the identification and
quantitation of perchlorate.
Section
9.2.1.1
Test For System
Carryover
Analyze a reagent water
blank after the high CAL
standard during the IDC
calibration.
Demonstrate that the target
analyte is < 1/3 of the MRL
and that carry-over from
previous samples does not
prevent the identification and
quantitation of perchl orate.
Section
9.2.2
Demonstration of
Precision
Analyze 7 replicate LFBs
and 7 replicate LFSSMs
fortified near the midrange
concentration.
%RSD must be < 20%
Section
9.2.3
Demonstration of
Accuracy
Calculate average recovery
for replicates used in
Section 9.2.2.
Mean recovery 80-120% of the
true value
Section
9.2.4
Minimum
Reporting Limit
(MRL)
Confirmation
Fortify and analyze 7
replicate LFBs at the
proposed MRL
concentration. Calculate
the mean and the Half
Range (HR). Confirm that
the Upper PIR and Lower
PIR (Sect. 9.2.4.2) meet
the recovery criteria.
Upper PIR < 150%
Lower PIR > 50%
331.0-27
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TABLE 6. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and
Frequency
Acceptance Criteria
Section
8.3
Sample Holding
Time
28 days when processed and
stored according to Sections
8.1 and 8.2.
Sample results are valid only if
samples are analyzed within the
sample holding time.
Section
10.3
Initial Calibration
Use the internal standard
calibration technique to
generate a linear or quadratic
calibration curve. Use at
least 5 standard
concentrations. Check the
calibration curve as
described in Section 10.3.3.
When each calibration standard
is calculated as an unknown
using the calibration curve, the
lowest level standard should be
within 50-150% of the true
value. All other points should
be within 80-120% of the true
value.
Section
9.3.1
Laboratory Reagent
Blank (LRB)
Daily, or with each Analysis
Batch of up to 20 field
samples, whichever is more
frequent.
Results must be < 1/3 the MRL.
If the background exceeds 1/3
the MRL, the results for
perchlorate in the Analysis
Batch are invalid.
Section
10.4
Continuing
Calibration Check
(CCC)
Verify initial calibration by
analyzing a Low CCC and a
Mid CCC at the beginning of
each Analysis Batch.
Subsequent CCCs are
required after every 10 field
samples, and after the last
field sample in a batch.
The lowest level CCC must be
within 50-150% of the true
value. All other points must be
within 80-120% of the true
value.
Results for field samples that
are not bracketed by acceptable
CCCs are invalid.
331.0-28
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TABLE 6. (CONTINUED)
Method
Reference
Requirement
Specification and
Frequency
Acceptance Criteria
Section
9.3.3
Laboratory
Fortified Blank
(LFB)
Daily, or with each Analysis
Batch of up to 20 field
samples, whichever is more
frequent.
For LFBs fortified at
concentrations < MRL, the
result must be within 50-150%
of the true value. All other
LFBs must be within 80-120%
of the true value.
Section
9.3.4
Internal Standard
(IS)
Compare IS area to the IS
area of the first CCC in the
Analysis Batch for all QC
and field samples.
Peak area counts for the IS must
be within + 30% of the IS area
of the first CCC of the Analysis
Batch. If the IS area does not
meet this criterion, the
corresponding perchlorate
results are invalid.
Section
9.3.5
Isotope Area Count
Ratio
Monitor the isotope ratio for
all QC and field samples in
the Analysis Batch.
The calculated area count ratio
(m/z 99/101 for SIM, or m/z
85/83 MRM) must be within
±25% (2.31-3.85) of the
theoretical value of 3.086.
Section
9.3.6
Relative Retention
Time
Monitor the relative retention
time for all QC and field
samples in the Analysis
Batch.
The relative retention time for
perchlorate vs. the IS must be
between 0.98-1.02.
Section
9.3.7
Laboratory
Fortified Sample
Matrix (LFSM)
Analyze one LFSM per
Analysis Batch. Fortify the
LFSM with perchlorate at a
concentration close to but
greater than the native
concentration (if known).
Calculate LFSM recoveries.
For LFSMs fortified at
concentrations < MRL, the
result must be within 50-150%
of the true value. All other
LFSMs must be within 80-
120% of the true value.
331.0-29
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TABLE 6. (CONTINUED)
Method
Reference
Requirement
Specification and
Frequency
Acceptance Criteria
Section
9.3.8
Laboratory
Fortified Sample
Matrix Duplicate
(LFSMD) or
Laboratory
Duplicate(LD)
Analyze at least one LFSMD
or LD daily, or with each
Analysis Batch of up to 20
field samples, whichever is
more frequent.
For LFSMDs or LDs having
perchlorate concentrations < 2 x
MRL, the RPD must be within
50-150%. All other LFSMDs
and LDs must have RPDs
within 80-120%.
Section
10.3.4
Quality Control
Sample (QCS)
Analyzed with every new
calibration curve.
Results must be 80-120% of the
true value.
331.0-30
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TABLE 7. EXAMPLE SEQUENCE FOR AN ANALYTICAL BATCH
Sample
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Sample Description
CCC < MRL
Mid Level CCC
LRB
LFB
Field Sample- 1
Field Sample-2
Field Sample-3
Field Sample-4
Field Sample-5
Field Sample-6
Field Sample-7
Field Sample-8
Field Sample-9
Field Sample- 10
CCC (rotate level)
LFSM
LD or LFSMD
Field Sample- 11
Field Sample-12
Field Sample- 13
Field Sample-14
Field Sample- 15
Field Sample- 16
Field Sample- 17
Field Sample- 18
Field Sample- 19
Field Sample-20
CCC (rotate level)
*Acceptance Criteria
5 0-1 50% of true value
80- 120% true value
< 1/3 MRL
< MRL 50-150% of true value
> MRL 80-120% of true value
< MRL 50-150% of true value
> MRL 80-120% of true value
< MRL 50-150% of true value
> MRL 80-120% of true value
<2xMRL50-150%RPD
> 2 x MRL 80-120% RPD
< MRL 50-150% of true value
> MRL 80-120% of true value
* Additionally, all samples must meet the isotope area count ratio
criteria (Section 9.3.5) and the relative retention time criteria (Section
9.3.6).
331.0-31
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FIGURE 1
LC/MRM CHROMATOGRAM OF A 0.5 ^g/L PERCHLORATE STANDARD
04-25-04-69
100-,
Internal Standard m/z 107>89
MRM of 3 Channels ES-
107 > 89
6.74e3
04-25-04-69
100-1
Perchlorate m/z 101>85
MRM of 3 Channels ES-
101 >85
1.58e3
04-25-04-69
100-,
Perchlorate m/z 99>83
MRM of 3 Channels ES-
99>83
4.10e3
Time
0.00
1.00
2.00
3.00
4.00
5.00 33ao&-32 7.00
8.00
9.00
10.00
11.00
12.00
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FIGURE 2
LC/SIM CHROMATOGRAM OF A 0.5 ^g/L PERCHLORATE STANDARD
07-13-04-07
100-1
0-^^
Internal Standard m/z 107
SIR of 3 Channels ES-
107
1.13e5
07-13-04-07
100-1
Perchlorate m/z 101
SIR of 3 Channels ES-
101
3.78e4
07-13-04-07
100-,
Perchlorate m/z 99
SIR of 3 Channels ES-
99
1.01e5
Time
4.00 4.50 5.00 5.50
6.00
6.50 7.00
331.0-33
7.50
8.00 8.50 9.00 9.50
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07-13-04-01
100n
FIGURE 3
LC/SIM TIC CHROMATOGRAM (m/z 99,101,107) OF A LFSSM (1000 mg/L)
FORTIFIED WITH 0.5 (ig/L PERCHLORATE
SIR of 3 Channels ES-
TIC
9.89e5
331.0-34
/ \
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
Time
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