EPA Document #: EPA/600/R-05/049
METHOD 332.0 DETERMINATION OF PERCHLORATE IN DRINKING WATER BY
ION CHROMATO GRAPH Y WITH SUPPRESSED CONDUCTIVITY
AND ELECTROSPRAY IONIZATION MASS SPECTROMETRY
Revision 1.0
March 2005
Elizabeth Hedrick and Thomas Behymer, U.S. EPA, Office of Research and Development
Rosanne Slingsby, Dionex Corporation
David Munch, U.S. EPA, Office of Ground Water and Drinking Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 332.0
DETERMINATION OF PERCHLORATE IN DRINKING WATER BY ION
CHROMATOGRAPHY WITH SUPPRESSED CONDUCTIVITY AND ELECTROSPRAY
IONIZATION MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This method is applicable to the identification and quantitation of perchlorate (C1O4~) in
raw and finished drinking waters. The approach used is ion chromatography with
suppressed conductivity and electrospray ionization mass spectrometry (IC-ESI/MS).
Chemical Abstract Services
Analyte Registry Number (CASRN)
Perchlorate 14797-73-0
1.2 The ion chromatographic conditions described in this method may be used with a
tandem mass spectrometer (MS/MS) detector as described in EPA Method 331.0.
Specifically, the 1C operational description (Sect. 10) and quality control requirements
(Sect. 9) of Method 332.0 maybe used in combination with the MS/MS operational
description and quality control requirements in Method 331.0.
1.3 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets
the Data Quality Objectives (DQOs) that are developed based upon the intended use of
the method. The Lowest Concentration MRL (LCMRL) is the lowest true concentration
for which a future recovery is predicted to fall, with 99 percent confidence, between 50
and 150 percent. The method development laboratory s LCMRL for C1O4", as defined
in Section 3.13, was 0.10 (ig/L using the quantitation ion atm/z 101 (Table 5). The
procedure used to determine LCMRLs is described elsewhere.1
1.4 Laboratories using this method are not required to determine an LCMRL for this
method, but must determine a single laboratory MRL using the procedure 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 zero.2
The DL is compound dependent and is dependent on sample matrix, fortification
concentration, and instrument performance. Determining the DL in this method is
optional (Sect. 9.2.5). The method development laboratory's DL for C1O4" in reagent
water was 0.02 (ig/L (Table 5).
1.6 The two predominant C1O4" ions that occur naturally at a ratio of 3.086:1 are 35C116O4",
m/z 99, and 37C116O4", m/z 101, respectively.3 Due to fewer mass spectral interferences,
the concentration of C1O4" using the m/z 101 ion is reported. The m/z 99/101 area count
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ratio and relative retention time are used for confirmation of C1O4" in samples. An
oxygen-18 (18O) enriched C1O4" internal standard is used to improve accuracy and
ruggedness of the method.
1.7 This method is intended for use by or under the supervision of analysts with prior
experience using ion chromatography and mass spectrometry with electrospray
ionization and interpretation of associated data. This method has been developed for
raw and finished drinking waters; however, with further method development the basic
approach may be suitable for measuring C1O4" in other matrices. For example, sample
preparation, sample clean-up and the identification of possible interferences would
require further study. In addition, the IC-ESI/MS conditions may require optimization.
Finally, precision, accuracy and minimum reporting limits would need to be determined
for the matrices of interest.
2.0 SUMMARY OF METHOD
2.1 This method describes the instrumentation and procedures necessary to identify and
quantify low levels of C1O4" in drinking waters using IC-ESI/MS. Drinking water
samples are collected using a sterile filtration technique. A small volume of sample is
injected into an ion chromatograph. Using an anion exchange column, C1O4" is
separated from constituent cations and anions in the sample using a potassium
hydroxide mobile phase. Due to the use of a non-volatile mobile phase, the eluate from
the column is passed through a conductivity suppressor to remove the potassium (K+)
ions of the mobile phase and to remove the analyte counter cations prior to the eluate
entering the mass spectrometer. An 18O-enriched 35C118O4" internal standard (m/z 107) is
used for quantitation to improve accuracy and ruggedness of the method. Identification
is made by verifying the relative retention time of the two predominant C1O4" ions with
respect to the internal standard. Qualitative confirmation of C1O4" is made by
confirming that the m/z 99/101 area count ratio is within a specified range. If these
conditions are met, along with passing all other QC requirements defined in Section 9,
then the concentration obtained using the m/z 101 quantitation ion is reported.
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. An Analysis Batch must 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 Checks (CCCs)
Laboratory Fortified Blank (LFB)
Laboratory Fortified Sample Matrix (LFSM)
Either a Laboratory Duplicate (LD) or a Laboratory Fortified Sample Matrix
Duplicate (LFSMD)
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3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the secondary dilution
standard and 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 and internal standard, which is analyzed periodically to verify the
accuracy of the existing calibration for the method analyte.
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 a statistical determination (Sect. 9.2.5), and accurate quantitation
is not expected at this concentration.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.
3.6 LABORATORY DUPLICATES (LDs) - Two sample aliquots (LD1 and LD2), taken in
the laboratory from a single sample bottle, and analyzed separately with identical
procedures. Analyses of LD1 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 known quantities of the method analyte and internal standard are
added in the laboratory. The LFB is analyzed exactly like a sample, including
preservation procedures, and its purpose is to determine whether the method, inclusive
of sample processing, is in control, and whether the laboratory is capable of making
accurate and precise measurements.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - An aliquot of a field
sample to which a known quantity of the method analyte and internal standard are
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 values 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.
3.10 LABORATORY FORTIFIED SYNTHETIC SAMPLE MATRIX (LFSSM) - An aliquot
of the Laboratory Synthetic Sample Matrix Blank (Sect. 3.12) that is fortified with C1O4"
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and processed like a field sample (Sect. 8). It is used to confirm that there is adequate
chromatographic resolution between sulfate and C1O4".
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 apparatus.
3.12 LABORATORY SYNTHETIC SAMPLE MATRIX BLANK (LSSMB) - A solution of
1,000 mg/L each of chloride, sulfate and carbonate (Cl~, SO42"and CO32") anions that is
processed like a field sample. The LSSMB is a reagent blank that must be analyzed
with each LFSSM.
3.13 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) - The
single laboratory LCMRL is the lowest true concentration for which a future recovery is
predicted to fall, with 99 percent confidence, between 50 and 150 percent recovery.1
3.14 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a target analyte in a sample following analysis. This
defined concentration can be no lower than the concentration of the lowest calibration
standard for that analyte, and can only be used if acceptable quality control criteria for
the analyte at this concentration are met.
3.16 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 solutions and other needed analyte solutions.
3.17 QUALITY CONTROL SAMPLE (QCS) - A solution containing the method analyte at a
known concentration that is obtained from a source external to the laboratory and
different from the source of calibration standards. It is used to verify that the standard
solution has been properly prepared, and stored to maintain its integrity.
3.18 REAGENT WATER (RW) - Purified water which does not contain any measurable
quantity of the method analyte at or above 1/3 the MRL, or interfering compounds that
would affect the determination of the method analyte.
3.19 SECONDARY DILUTION STANDARD (SDS) - A dilution made from the primary
dilution standard (PDS) that is used to prepare the calibration standards.
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3.20 SELECTED ION MONITORING (SIM) - A mass spectrometric technique where only
one or a few ions are monitored to improve 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 Method interferences may be caused by contaminants in solvents, reagents (including
reagent water), sample bottles and caps, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the chromatograms. All items such as
these must be routinely demonstrated to be free from interferences (less than 1/3 the
MRL for the target analyte) under the conditions of the analysis by analyzing LRBs as
described in Section 9.3.1. Subtracting blank values from sample results is not
permitted.
NOTE: The use of low or high density polyethylene plastic is recommended in place of
glass when possible. If glassware is used, it should be washed with detergent and tap
water and rinsed thoroughly with reagent water since C1O4" was found in common lab
detergent during method development.
4.2 In anion chromatography, cations are not retained on the analytical column and, in
theory, pass through in the void volume. The anions are separated by charge, size and
polarizability. As a large polarizable molecule, C1O4" elutes later than the common
inorganic anions (Cl~, SO42", CO32", and HCO3"). Separation of C1O4" from the matrix
ions combined with the specificity of mass spectrometry has resulted in a method that
minimizes interferences for drinking water matrices. There are, however, the following
known conditions or contaminants that, if present, could result in positive or negative
bias in the reporting of C1O4".
4.2.1 Direct Chromatographic Co-elution of Contaminants: At sufficiently high
concentration, direct chromatographic co-elution of a contaminant with C1O4"
could result in ionization suppression of one or more of the ions of interest
(m/z 99, 101, and/or 107). Alternatively, the contaminant could have the same
m/z as C1O4", or in-source collisionally induced dissociation of a co-eluting
contaminant in the ESI interface could produce a fragment ion with the same m/z
as C1O4". Any of these conditions could lead to a positive or negative bias of
C1O4" depending on the affected ion.
If a contaminant is present at a concentration detectable by conductivity, a full
mass scan on a replicate analysis may reveal the presence and m/z of the co-
eluting contaminant. Direct chromatographic coelution problems or
concentration dependent coelution problems may be solved by achieving
adequate chromatographic separation. This may be done by modifying the
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eluent strength or modifying the eluent with organic solvents (if compatible with
the 1C column and suppressor), changing the detection systems (e.g., MS/MS),
or selective removal of the interference with sample pretreatment. Sample
dilution will only be beneficial if the coelution is a result of column overloading.
High concentrations of polar anions such as pyrophosphate (P2O74~),
tripolyphosphate (P3O105~) and thio compounds, including aromatic sulfonates,
are potential chromatographic interferants. A 75 mM hydroxide mobile phase
concentration was found to elute the polyphosphates well before C1O4" without
compromising data quality.
4.2.2 Concentration-Dependent Interference by Sulfate (SO42~): Of the common
anions found in drinking waters (Cl~, SO42", CO32", HCO3"), sulfate can be the
most problematic. Sulfate elutes before C1O4" on most of the anion
chromatography columns currently being used for C1O4" analysis; however, it has
a tendency to elute broadly, tailing into the retention time of C1O4". Formation of
H32SO4" (m/z 97) and H34SO4" (m/z 99) are favored in the conductivity suppressor
as the pH of the eluate leaving the suppressor becomes strongly acidic. They are
also formed in the electrospray ionization interface. In general, the result of high
sulfate concentrations was observed to be either (1) an inability to detect the
m/z 99 ion, whereas the m/z 101 ion was still detected, or (2) an area count ratio
(rn/z 99/101) that did not meet the QC requirement (Sect. 9.3.5). If either of
these effects are observed, the analyst must evaluate the background counts at
m/z 99 in the half minute before C1O4" elutes. If the background counts are high
(approximately 10-20 times higher than the background counts at m/z 99 in the
first CCC of the Analysis Batch, Sect. 10.4.1), sample dilution or pretreatment to
reduce/remove the sulfate is required to meet the m/z 99/101 area count ratio
requirement for confirmation of C1O4" (Sect. 9.3.5). As the column ages and the
retention time of C1O4" becomes shorter, the analyst might note that the
m/z 99/101 area count ratio is more severely affected by the presence of high
concentrations of sulfate. Column cleaning or replacement is recommended if
this occurs.
4.2.3 ESI/MS Detector Inlet Fouling: The effect of ESI/MS detector inlet fouling is
deterioration of signal intensity for the three ions monitored in this method
(m/z 99, 101 and 107). The deterioration can be rapid (after the analysis of one
problematic matrix) or it can be gradual. To a large extent, the IS will correct
for gradual and minor loss of signal intensity due to ESI/MS inlet fouling.
However, continued loss of signal intensity may eventually affect sensitivity to
the point that it is no longer possible to detect C1O4" at the MRL, and/or the QC
criteria for IS area counts will fail (Sect. 9.3.4). Not all mass spectrometers
exhibit this problem to the same extent; however, if the problem is observed to
be gradual and significant over the course of a week, it may be greatly reduced
by using an instrument configuration that bypasses the mass spectrometer until
1.5 to 2 minutes prior to the elution of C1O4" (see Figures 1 and 2). This is
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because the ions that have the greatest potential for ESI/MS detector inlet
fouling elute in the first few minutes after sample injection. For the
instrumentation used to collect the data that is presented in this method,
bypassing the mass spectrometer until just prior to the elution of C1O4"
dramatically improved system ruggedness and reduced the need for ESI/MS
detector inlet cleaning.
4.2.4 System Carry-over: Carry-over from one analysis may affect the detection of
C1O4" in a second or subsequent analysis. It can occur when the analysis of a low
concentration sample immediately follows the analysis of a high concentration
sample. Carry-over from one analysis to a subsequent analysis may occur if
using an autosampler or if the injection valve is switched back to the load
position too soon after injection of a sample. If C1O4" carry-over is discovered in
blanks proportional to the concentration of the previously injected standard, the
problem must be corrected prior to further analyses.
4.3 Every effort has been made to address known interferences in this method and to inform
the analyst regarding interpretation of chromatographic and mass spectrometric data to
determine if an interferant is present. There are also mandatory QC requirements that, if
failed, should alert the analyst to the possibility of an interferant. Modifications in
sample pretreatment, chromatography and instrumentation are allowed to overcome
interferences.
NOTE: Although modifications are acceptable, the analyst must demonstrate that the
modifications do not introduce any adverse affects on method performance by repeating
and passing all the QC criteria described in Section 9.2, in addition to meeting all the
ongoing QC requirements. Changes are not permitted in sample collection or
preservation (Sect. 8.1).
4.4 The percent of 18O enrichment of the internal standard may vary between standard
manufacturers. Poor isotopic enrichment may lead to sample contamination by native
C116O4" (m/z 99) in the internal standard. Therefore, it must be demonstrated that the IS
does not contain unlabeled C1O4" at a concentration > 1/3 of the MRL when added at the
appropriate concentration to samples (a concentration of 1 ng/L was used during method
development). This is initially confirmed during the IDC and is monitored in each
Analysis Batch by analysis of the Laboratory Reagent Blank (LRB, Sect. 9.3.1).
5. SAFETY
5.1 The toxicity or carcinogenicity of many of the chemicals used in this method have 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 awareness of OSHA regulations regarding safe handling of chemicals used
in this method.4"6 Each laboratory should maintain a file of applicable MSDSs.
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5.2 Pure C1O4" salts are classified as oxidizers and the potassium hydroxide used in the
mobile phase is caustic. Pure standard materials and stock standards of these
compounds should be handled with suitable protection to skin and eyes.
6. EQUIPMENT AND SUPPLIES (References to specific brands or catalog numbers are
included for illustration only, and do not imply endorsement of the product).
The analytical equipment consists of an ion chromatograph and a mass spectrometer. Figures 1
and 2 show two configurations of the Dionex IC-ESFMS system that yielded acceptable results
during method development. Figure 1 and Table 1 show the configuration and operating
conditions used to generate the data presented in this method. Table 2 shows the recommended
operating conditions for Metrohm-Peak/Agilent instrumentation. Other instrumentation and
configurations are acceptable provided the QC requirements of the method are met.
6.1 1C-ES1/MS SYSTEM - An analytical system consisting of a microbore chromatographic
pump, a guard and anion separator column, a six-port injection valve, varying sample
loop sizes (50-200 |jL), a conductivity suppressor, a conductivity detector and a data
acquisition and management system that has been interfaced with the ESFMS.
6.1.1 CHROMATOGRAPHIC PUMP - 2-mm isocratic 1C pump capable of precisely
delivering flow rates from 0.01-1.0 mL/min [(Dionex Corporation, Sunnyvale,
CA, Model IP25) or an isocratic, metal free, 1C pump capable of precisely
delivering flow rates from 0.01-5.0 mL/min, (Metrohm-Peak Inc., Houston, TX,
Model 818) or equivalent].
6.1.2 ANION TRAP COLUMN - A continuously re-generated, high capacity anion
exchange resin column placed before the eluent generator used to remove anions
in the RW (Dionex lonPac CR-ATC-2 mm, Part No. 060477 or equivalent).
6.1.3 ELUENT GENERATOR - An eluent generator is optional (Dionex Model EG40
with EGC-KOH or equivalent). Preparation of mobile phase from high purity
potassium hydroxide (KOH) is permissible. Frequent preparation from KOH
salt may be necessary to maintain a carbonate-free solution.
6.1.4 CHROMATOGRAPHY OVEN - Temperature controlled chromatography oven.
The chromatography oven contains the 6-port injection valve, the guard and
separator columns, the conductivity suppressor and detector. Temperature
maintained at 30 °C is recommended but not required for this method [(Dionex
Model No. LC30) or (Metrohm Advanced 1C Separation Center, Metrohm
Model No. 820, Part Nos. 2.820.0220 and 2.833.0010) or equivalent].
6.1.5 ANION GUARD COLUMN - A guard column packed with the same material as
the separator column. It protects the separator column from particulate matter
and compounds that could foul the exchange sites of the separator column
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[(Dionex AG16, 2-mm internal diameter (I.D.), Part No. 55379) or (Metrohm
ASUPP-4/5 guard, 4-mm I.D., Metrohm Part No. 6.1006.500) or equivalent].
6.1.6 ANION SEPARATOR COLUMN - A 100-250 mm column packed with a solid
phase specially engineered to achieve separation of the anions of interest
[(Dionex AS16, 2-mm I.D. X 250-mm length, Part No. 55378) or (Metrohm
ASUPP5-100, 4-mm I.D. X 100-mm length, Part No. 6.1006.510) or
equivalent].
6.1.7 CONDUCTIVITY SUPPRESSOR - An electrolytic suppressor operated with an
external source of RW. A chemical conductivity suppressor is acceptable,
although sulfuric acid should not be used as the chemical regenerant due to mass
spectrometric interferences caused by HSO4" at m/z 99 [(Dionex Anion Self
Regenerating Suppressor ASRS-MS, 2-mm, Part No. 63008) or (Metrohm
Advanced 1C Separation Center, Metrohm Model No. 820, Part No. 2.820.0220
and 2.833.0010) or equivalent].
6.1.8 CONDUCTIVITY DETECTOR - A flow-through detector with an internal
volume that does not introduce analyte band broadening [(Dionex Conductivity
Detector, Model CD25A) or (Metrohm Advanced 1C Conductivity Detector,
Metrohm, Model 819, Part No. 2.819.0010) or equivalent].
6.1.9 SAMPLE LOOPS - 50 to 200 \\L size. A 200 |iL size was used to generate the
data presented in this method. Smaller or larger injection volumes may be used
as long as the Initial Demonstration of Capability (Sect. 9.2), and all calibrations
and sample analyses are performed using the same injection volume.
6.1.10 DATA SYSTEM - Data management software differs from vendor to vendor
and may be recommended by the supplier of the 1C or MS. A system that allows
control of both the 1C and MS is recommended [(Dionex Chromeleon
Chromatography Management Software, Version 6.4 MSQ) or (Metrohm ICNet
2.3 data management software and Agilent LCMS Chemstation, Metrohm,
vlO.02, Part No. G2710AA) or equivalent].
6.1.11 HELIUM - High purity, compressed gas with a pressure of at least 80 psi to
activate valves, sparge eluent and deliver water to the suppressor.
6.1.12 MASS SPECTROMETER - MS equipped with an ESI interface. Operated in
SIM mode [(Dionex Model MSQ-ELMO, manufactured by Thermo Electron,
San Jose, CA) or (Agilent 1100 Series MSD Quad SL, Part No. G1956B,
manufactured by Agilent Technologies, Wilmington, DE) or equivalent].
6.1.13 NITROGEN - Compressed gas for ESI operation, 80 psi. The purity should be
consistent with the MS manufacturer's recommendations. Due to the high flow
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rate (>15 L/min), liquid nitrogen or a nitrogen generator is recommended for
long periods of operation.
NOTE: The following instrumentation, used to generate the data presented in this method,
is recommended but not required.
6.1.14 AUXILIARY PUMP - Pump capable of precisely delivering flow rates from
0.01 - 1.0 mL/min. This pump is used to deliver continuous liquid flow to the
mass spectrometer while the eluate flow from the column is diverted to waste
until 1.5-2 minutes prior to the elution C1O4" (Dionex high performance
metering pump, Model No. AXP-MS or equivalent). See Figures 1 and 2 for
placement of the pump.
6.1.15 AUXILIARY SIX-PORT VALVE - Electronic, 6-port, rear-loading valve
(Rheodyne, LLC, Rohnert Park, CA, Part No. 9126-038 or equivalent). This
valve may be placed between the exit of the column and the entrance of the
suppressor, as was done for the data reported in this method (Figure 1), or
alternatively, it may be placed between the conductivity detector and the MS
(Figure 2). In the latter configuration, a 50:50 watenacetonitrile mixture is
mixed with the eluate before it enters the MS using a static mixing tee. The flow
rate to the MS during the time of C1O4" elution in Figure 2 is 0.6 mL/min or
0.3 mL/min in Figure 1. As long as all the QC requirements of the method are
met (Sect. 9.2), either configuration is acceptable.
6.1.16 STATIC MIXING TEE - High pressure, microbore, mixing tee. The static
mixing tee is only used in the Figure 2 configuration (UpChurch Scientific, Oak
Harbor, WA, Part No. U466 or equivalent).
6.1.17 AUTOSAMPLER - Used to automate sample analysis. Minimally, the
autosampler should be capable of delivering a volume of sample 10 times the
chosen sample loop size [(Dionex, Model AS40) or (Metrohm Advanced
Sample Processor, Metrohm, Model 788, Part No. 2.788.0010) or equivalent].
6.2 ANALYTICAL BALANCE - Balance capable of+0.1 mg accuracy (Mettler-Toledo,
Inc., Columbus, OH, Mettler AT200 or equivalent).
6.3 STORAGE BOTTLES - Opaque high density polyethylene (HOPE), 30 mL, 125 mL
and 250 mL sizes for storage of standards (Fisher Scientific, Suwanee, GA, Cat. No.
2911974, 2911958 and 2911961 or equivalent).
6.4 SAMPLE CONTAINERS - 125-mL sterile high-density polyethylene (HOPE) bottles
(IChem 125-mL sterile HOPE bottle, Fisher Scientific, Suwanee, GA, Cat. No. N411-
0125 or equivalent) or disposable single-use, sterile polystyrene, 150 mL, with screw-
cap for sterile filtered samples (Fisher Scientific, Suwanee, GA, Part No. 09-761-140 or
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equivalent). The latter can be used directly with a sterile vacuum filter unit if not using
syringe filtration.
6.5 SAMPLE FILTERS - Sterile, single-use, disposable surfactant-free cellulose acetate
(SFCA) 26 mm, 0.2 |j,m syringe filter (Fisher Scientific, Suwanee, GA, Corning Brand,
Part No. 09-754-13 or equivalent). For samples high in particulates, filters with built-in
prefilters are available. All samples must be filtered at the time of sample collection.
6.6 SYRINGES - Sterile, single-use, disposable, silicone-free, luer-lok, 20 mL (Fisher
Scientific, Suwanee, GA, Target Brand, Part No. 03-377-30 or equivalent).
6.7 SAMPLE PRETREATMENT CARTRIDGES - Single-use, disposable OnGuard-II H
cartridges (Dionex, Part No. 057085 or equivalent) used to remove high concentrations
of carbonate if it is determined to be an interferant. OnGuard-II Ba2+ cartridges
(Dionex, Part No. 57093 or equivalent) used to remove high concentrations of sulfate if
it is determined to be an interferant. OnGuard-II Ag cartridges (Dionex, Part No. 57089
or equivalent) used to remove high concentrations of chloride if it is determined to be an
interferant. The Ba2+ pretreatment cartridge is the only one that may be required to meet
the QC requirements of this method (Sect. 11.6.2)
6.8 MICRO-PIPETTES - 250 |jL, 1000 |jL and 10 mL sizes with single-use disposable tips
(Rainin, Oakland, CA, Part Nos. EP-250, EP-1000, and EP-10 mL or equivalent).
6.9 VIALS - Single use, disposable autosampler vials with filter caps, or other disposable,
single use vials with caps having a 10 mL or less capacity to be used for sample
preparation.
7. REAGENTS AND STANDARDS
7.1 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be used.
Unless otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society (ACS), where such specifications are available. Solvents should be HPLC
grade or better. Other grades may be used provided it is first determined that the reagent
is of sufficiently high purity to permit its use without lessening the quality of the
determination.
7.1.1 HIGH PURITY REAGENT WATER (RW) - Purified water which does not
contain any measurable quantity of the target analyte or interfering compounds
at concentrations > 1/3 the MRL for the target analyte. The purity of the water
required for this method cannot be overly emphasized. For this work, deionized
water was further purified using a bench model Millipore water purification
system (Millipore Corp, Billerica, MA, Model No. MilliQ Gradient A10 or
equivalent).
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7.1.2 ACETON1TR1LE - ACN, CASRN 75-05-8 (Fisher Scientific, Suwanee, GA,
Cat. No. A998-1 or equivalent). ACN is only required if using the IC-ESFMS
configuration presented in Figure 2.
7.1.3 METHANOL - MeOH, CASRN 67-56- 1 (Fisher Scientific, Suwanee, GA, Cat.
No. A452-1 or equivalent). MeOH is only required if using the Metrohm-Peak
instrumentation.
7.1.4 POTASSIUM HYDROXIDE ELUENT - 75 mM (KOH, F.W.= 56.1 1, CASRN
1310-58-3, 45% (w/w), Certified ACS Grade , or better). 75 mM KOH is
prepared by diluting 9.35 g of a 45% (w/w) solution to 1 L with RW. Filter,
degas by sonication, or sparge with helium, and pressurize with helium to
minimize absorption of carbon dioxide from the atmosphere. If using an 1C
system equipped with an eluent generator (Sect. 6.1.3), KOH eluent preparation
is not necessary.
If using a Metrohm 1C system, the recommended eluent is 30 mM NaOH
(NaOH, F.W.= 40.0, CASRN 1310-73-2, 50% (w/w), Certified ACS Grade, or
better) prepared by diluting 2.4 g of the 50% (w/w) solution to 700 mL of RW.
Add 300 mL of MeOH to bring final volume to 1 L. Degas by sonication, or
sparge with helium, to minimize absorption of carbon dioxide from the
atmosphere.
7.1.5 SODIUM SULFATE - Na^O^ F.W.=1 42.04, CASRN 7757-82-6 (Fisher
Scientific, Suwanee, GA., Cat. No. S42 1-500 or equivalent).
7.1.6 SODIUM CHLORIDE - NaCl, F.W.=58.44, CASRN 7647-14-5 (Fisher
Scientific, Suwanee, GA., Cat. No. S27 1-500 or equivalent).
7.1.7 SODIUM CARBONATE - Naj^, F.W.=106, CASRN 497-19-8 (Sigma
Aldrich Chemical, St Louis, MO, Cat. No. S6139 or equivalent).
7.2 STANDARD SOLUTIONS - Standard solutions may be prepared from certified,
commercially available solutions or from neat compounds. When a compound purity is
assayed to be 96% 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 during the development of this method and are included as an example. Unless
otherwise noted, all standards should be stored in 125-mL HDPE screw-cap bottles
(Sect. 6.3) at 6 °C or less when not in use. Even though stability times for standard
solutions are suggested in the following sections, laboratories should use standard QC
practices to determine when their standards need to be replaced.
7.2. 1 INTERNAL STANDARD STOCK STANDARD SOLUTION (IS-SSS) -
1,000 mg Cl18O4yL. (NaCl18O4, F.W.=130.4, CASRN 7601-89-0, 90% enriched
on 18O, 98% pure NaCl18O4, or better, Isotec, Inc., Miamisburg, OH or
332.0-13
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equivalent,). A 1,000 mg/L solution of C118O4" is prepared by dissolving
0.0123 g NaCl18O4 in 10 mL of RW. The solution maybe stored in an HDPE
screw-cap bottle (Sect. 6.3). The anhydrous NaCl18O4 salt should be stored in a
desiccator to minimize absorption of water from the atmosphere. The
recommended holding time is one year.
7.2.1.1 INTERNAL STANDARD PRIMARY DILUTION STANDARD -
(IS-PDS) - 1.0 mg C118O47L. Prepared gravimetrically, using an
analytical balance having +0.1 mg accuracy, by adding 0.1 g (100 |j,L) of
the IS-SSS to 99.9 g of RW in a 125-mL HDPE storage bottle.
Alternatively, this dilution may be done volumetrically. The
recommended holding time is one year.
7.2.1.2 INTERNAL STANDARD FORTIFICATION SOLUTION - (IS-FS) -
100 ng C118O47L. Prepared by adding 10 mL of the IS-PDS to 90 mL of
RW in a 125-mL HDPE storage bottle. Alternatively, this dilution may
be done by weight using an analytical balance having +0.1 mg accuracy.
The recommended holding time is one year.
NOTE: A commercially prepared internal standard solution maybe
used. (Dionex Corporation, Part No. 062923 or equivalent).
7.2.2 PERCHLORATE STOCK STANDARD SOLUTION (SSS) - 1,000 mg C1O47L.
(NaClO4, anhydrous, 99% pure grade, or better, F.W.= 122.4, CASRN
7601-89-0, Sigma Aldrich Co., St. Louis, MO, Cat. No. S-1513, or equivalent).
A 1,000 mg/L solution of C1O4" is prepared by dissolving 0.1231 g of NaClO4 in
100 mL of RW. The solution may be stored in a HDPE screw-cap bottle
(Sect. 6.3). The anhydrous NaClO4 salt should be stored in a desiccator to
minimize absorption of water from the atmosphere. The recommended holding
time is one year.
7.2.2.1 PERCHLORATE PRIMARY DILUTION STANDARD - (PDS) -
1.0 mg C1O4~/L. Prepared gravimetrically using an analytical balance
having +0.1 mg accuracy, by adding 0.1 g (100 |jL) of the SSS to 99.9 g
of RW in a 125-mL HDPE storage bottle. Alternatively, this dilution
may be done volumetrically. The recommended holding time is one
year.
7.2.2.2 PERCHLORATE FORTIFICATION SOLUTION - (FS) - 100 ng C1O4VL.
Prepared by adding 10 mL of the PDS to 90 mL of RW. The solution may
be stored in a 125-mL HDPE storage bottle. Alternatively, this dilution may
be done by weight using an analytical balance having +0.1 mg accuracy.
The recommended holding time is one year.
332.0-14
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7.2.3 CALIBRATION STANDARDS (CAL) - The following guide may be used for
preparing 100-mL CAL solutions containing 1.0 (jg/L of the IS. The holding
time for CAL solutions is one month.
(LRB) 0
1.0
0.1
0.1
1.0
0.2
0.2
1.0
0.5
0.5
1.0
1.0
1.0
1.0
10
10
1.0
7.2.4 LABORATORY SYNTHETIC SAMPLE MATRIX (LSSM) - 1,000 mg/L each
of Cr, SO42-, CO32\ Add 1.48 g of Na^ (Sect. 7.1.5), 1.65 g of NaCl
(Sect. 7.1.6) and 1.77 g of Na^Oj (Sect. 7.1.7) to 1-L volumetric flask and
dilute to volume with RW. The recommended holding time is one year.
7.2.5 LABORATORY FORTIFIED SYNTHETIC SAMPLE MATRIX (LFSSM) -
Prepare an LFSSM at the mid-level concentration of the calibration curve using
the LSSM (Sect 7.2.4). The LFSSM must contain the IS at the same
concentration as the CAL standards. The holding time is one month.
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.
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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 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 microbial
degradation by anaerobic bacteria.8 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 sample
bottle empty.
8.1.4.1 Remove a sample syringe (Sect. 6.6) from its package and draw up 20
mL of the bulk sample. Remove a sterile sample filter (Sect. 6.5) 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. For samples high in particulates, pre-filtration using
a sterile filter (0.45 - 10 |jm) may help to prevent clogging or rupture of
the 0.2 |j,m filter. Open a sterile sample container (Sect. 6.4) without
touching the interior. Using gentle pressure, pass the sample through the
filter into the sample container, directing the first milliliter of sample to
waste. 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, but should not be frozen.
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 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, their 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 7 and 8.
These QC requirements are considered the minimum acceptable QC criteria.
Laboratories are encouraged to institute additional QC practices to meet their specific
needs.
332.0-16
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9.1.1 METHOD MODIFICATIONS - The analyst is permitted to modify 1C columns,
mobile phases, chromatographic and ESFMS conditions. Each time such
method modifications are made, the analyst must repeat the IDC procedures in
Section 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 first meet the calibration requirements of Section 10.
Requirements for the initial demonstration of laboratory capability are described in the
following sections and are summarized in Table 7.
9.2.1 DEMONSTRATION OF LOW SYSTEM BACKGROUND - Before any
samples are analyzed, or at any time that new reagents, labware or
instrumentation are used, it must be demonstrated that laboratory reagent blanks
are reasonably free of any contaminants that would prevent the determination of
C1O4" and that the criteria of 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 (Sect. 8.1.4.1).
9.2.1.1 Concentration dependent carry-over is manifest by signals in samples
that increase proportionally to the concentration of the previously
injected sample. Analysis of a blank RW sample must be performed
after the highest CAL standard to assess if carry-over has occurred. This
type of blank is not the same as an LRB in that it is not filtered or
processed as a sample. If there is system carry-over, the source can often
be traced to the use of an autosampler, injection valve problems or an
excess of tubing between the 1C and/or MS components. The results for
this sample must meet the criteria outlined in Section 9.3.1. System
carry-over should be eliminated, to the extent possible, by determining
the source of the problem and taking corrective action.
9.2.2 DEMONSTRATION OF PRECISION - Prepare and analyze 7 replicate LFBs
and 7 replicate LFSSMs fortified near the midrange of the Initial Calibration
curve. All samples must be fortified and processed using the sample collection
devices described in Section 8.1.4.1. The relative standard deviation (RSD) of
the measured concentrations at m/z 101 of the replicate analyses must be < 20
percent for both LFB and LFSSM. Calculate %RSD using the equation below:
%RSD = standard deviation of measured concentrations X 100
average measured concentration
332.0-17
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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
recovery must be within 80-120% for both the LFB and the LFSSM data.
Calculate percent recovery (%R) using the following equation:
%R = average measured concentration X 100
fortification concentration
9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Select a target
concentration for the MRL based on the intended use of the method. Establish
an Initial Calibration following the procedure outlined in Section 10.3. The
lowest calibration standard used to establish the Initial Calibration in
Section 10.3 (as well as the low-level CCC) must be at or below the
concentration of the target MRL. Establishing the MRL concentration too low
may cause repeated failure of on-going QC requirements. Confirm the targeted
MRL following the procedure outlined below.
9.2.4.1 Prepare and analyze seven replicate LFBs at the target MRL
concentration. All samples must be processed using the sample
collection devices described in Section 8.1.4.1. Calculate the mean
(Mean) and standard deviation for these replicates using the m/z 101 ion.
Determine the Half Range for the prediction interval of results (HRPIR)
using the equation below:
HRPIR = 3.963 X S
where,
S is the standard deviation, and 3.963 is a constant value for seven
replicates.
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% recovery.
Mean + HRn,n X 100 < 150%
PIR
Fortified Concentration
The Lower PIR Limit must be > 50% recovery.
Mean - HRPIP X 100 >50%
Fortified Concentration
332.0-18
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9.2.4.3 The target 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.
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 on the intended use of the data.
Prepare and analyze at least seven replicate LFBs at a concentration estimated to
be near the Detection Limit over at least 3 days using the procedure described in
Section 11. This fortification level may be estimated by selecting a
concentration with a signal of 2-5 times the noise level.
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 equation:
Q * t
O l( n _ ^ i _ aipha = 0.99)
where,
Vu-alpha = 0.99)= Student's t for the 99% confidence level with n-1 degrees of
freedom. Student's t = 3.143 for n = 7.
n = number of replicates.
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MRL or DL calculations.
9.3 ONGOING REQUIREMENTS - This section summarizes the ongoing QC criteria that
must be followed when processing and analyzing field samples. Table 8 summarizes
ongoing QC requirements.
9.3.1 LABORATORY REAGENT BLANK (LRB) - An 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 the method analyte. If the LRB produces apeak within the
retention time window of the analyte that would prevent the determination of the
method analyte, determine 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 and must
be processed (i.e., sterile filtration) as described in Section 8.1.4.1. Perchlorate
332.0-19
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or other interferences in the LRB must be < 1/3 the MRL. If this criterion is not
met, then all data must be considered invalid for all samples in the Analysis
Batch.
NOTE: 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 an LRB (and an LFB) maybe processed in the
laboratory.
NOTE: Although quantitative data below the MRL may not be reliably accurate
enough for data reporting, such data is 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) - CCCs 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) - An LFB is required with each
Analysis Batch. The fortified concentration of the LFB must be rotated between
low, medium, and high concentrations from batch to batch. The low
concentration LFB must be as near as practical to the MRL. Similarly, the high
concentration LFB should be near the high end of the calibration range
established during the Initial Calibration (Sect. 10.3). Results of LFBs fortified
at concentrations < the MRL must be recovered within 50-150% of the true
value. Results from the analysis at any other concentration must be recovered
within 80-120% of the true value. If the LFB results do not meet these criteria,
then all data must be considered invalid for all field samples in the Analysis
Batch.
NOTE: LFBs must be processed in the same manner as field samples including
all sample preservation and pretreatment requirements (i.e., sterile filtration) as
described in Section 8.1.4.1.
. LFB;Eof tified £oii^»traiflb&R^p:--:i
MRL to highest calibration standard
:i'l3lB\Ile£i>₯er5?; :lMmite f h;>
50- 150%
80- 120%
9.3.4 INTERNAL STANDARD (IS) - The analyst must monitor the peak area of the
internal standard in all injections during each Analysis Batch. The IS response
(as indicated by peak area) for any chromatographic run must not deviate by
332.0-20
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more than ±30 percent from the area counts measured in the first CCC of the
Analysis Batch (Sect. 10.4.1). If the IS area counts do not meet this criterion,
inject a second aliquot of the sample as part of the same or new Analysis Batch
within the holding time of the sample.
9.3.4.1 If the reinjected aliquot produces an acceptable IS 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 cleaning of the MS sample cone, may
be necessary. Once the analyst has re-established proper operating
conditions, the sample, or affected samples, must be reanalyzed provided
that they are still within their holding times.
9.3.5 AREA COUNT RATIO (m/z 99/101) ACCEPTANCE CRITERIA - All CAL
standards, QC samples and field samples must meet the m/z 99/101 area count
ratio requirement for confirmation of C1O4". The measured ratio must fall within
+25% (2.31-3.85). Area count ratios that fall outside this range due to sulfate
interference must be diluted and/or pretreated with barium form pretreatment
cartridges to remove the sulfate to a level that allows better integration of the
C1O4" peak at m/z 99 (Sect. 11.6.2), and thus, better m/z 99/101 area count ratios
for confirmation. If a CAL standard, CCC or LFB fails the area count ratio
acceptance criteria, there may be column, suppressor or instrumental problems.
The source of the problem must be identified and corrected before further
analysis of samples.
9.3.6 RELATIVE RETENTION TIME ACCEPTANCE CRITERIA - Since the
C118O4" IS has the same retention time as naturally occurring C1O4", the retention
time ratio of m/z 99/107 and m/z 101/107 in samples must be within 0.98 - 1.02
(+2% of the ideal ratio of 1) for confirmation of C1O4" in a sample. Use the
equation below to determine the relative retention time:
Relative Retention Time = retention time of m/z 99 or m/z 101 ion
retention time of m/z 107 IS ion
9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of an
LFSM (Sect. 3.8) is required in each Analysis Batch and is used to determine
that the sample matrix does not adversely affect method accuracy. If a variety of
different sample matrices are analyzed regularly, for example drinking water
332.0-21
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from groundwater and surface water sources, performance data should be
collected for each source. Over time, LFSM data should be documented for all
routine sample sources for the laboratory.
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 FS (Sect. 7.2.2.2).
Select a fortification 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 fortification 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 recovery (%R) for the analyte using the following equation:
%R = (A - B) X 100
C
where,
A = measured concentration in fortified sample
B = measured background concentration in an unfortified aliquot of
the same sample
C = fortification concentration
9.3.7.3 Recoveries for LFSM samples should be 80-120%. Greater variability
may be observed when LFSM samples have C1O4" concentrations < the
MRL. At these concentrations, LFSM sample recovery should be
50-150%. If the accuracy of C1O4" falls outside the designated range, and
the laboratory performance is shown to be in control in the CCCs, the
recovery is judged to be matrix biased. The result for C1O4" in the
unfortified sample should be labeled "suspect matrix" to inform the data
user that the results are suspect due to matrix effects.
NOTE: A high concentration of sulfate is a known interferant that may
cause the sample to fail the m/z 99/101 area count ratio criteria
(Sect. 9.3.5). In that case, the sample must be diluted or pretreated to
reduce/remove the sulfate to an acceptable level. Refer to Section 11.6
for required remedial action.
NOTE: Field samples that have detectable native C1O4" concentrations
below the MRL that are fortified at concentrations at or near the MRL
should be corrected for the native levels to obtain more accurate results.
This is the only case where background subtraction of results below the
MRL is permitted.
332.0-22
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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 C1O4" 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 measured
concentrations (LD1 and LD2) using the equation:
RPD-
(LD1+LD2)I2
9.3.8.2 If an LFSMD is analyzed instead of aLD, calculate the relative percent
difference (RPD) for duplicate concentrations of the LFSMs (LFSM and
LFSMD) using the equation:
RPD- \LFSM-LFSMD X1QQ
(LFSM+ LFSMD)/2
9.3.8.3 The RPD acceptance criteria for LDs and duplicate LFSMs are listed in
the table below. If the RPD is not within the control, but the laboratory
performance is shown to meet the acceptance criteria in the LFB, the
recovery problem is judged to be matrix related. The result for the
unfortified sample is labeled "suspect matrix" to inform the data user that
the results are suspect due to matrix effects.
:^OUBCeiitraM0ja-iitogf ;:;Vl;-;-:V-:\:\:V-
< 2 X MRL
> 2 X MRL to highest calibration
standard
/R^-^^aitsCfife^;::"?:
<50%
<20%
9.4 QUARTERLY INSTRUMENT PERFORMANCE CHECK USING THE LSSMB AND
LFSSM - Analysis of an LFSSM (Sect. 3.10) must be performed at least quarterly to
assess instrumental performance with respect to samples high in common anions. An
LFSSM fortified at the mid-range of the calibration curve must be processed and
analyzed as a sample along with an LSSMB (Sect. 3.12). Both solutions must be from
the same stock of LSSM. Results for the LSSMB should meet the criterion set forth in
Sect. 9.3.1 for LRB contamination. If the LSSMB contains C1O4" at a concentration
>l/3 the MRL, then the source of the contamination should be identified and corrected.
The LFSSM should meet the criteria set forth in Sect. 9.3.3. If the LFSSM does not
332.0-23
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meet the QC acceptance criteria for LFB recovery or if the criteria of Sections 9.3.4 -
9.3.6 fail, instrument maintenance such as column or suppressor cleaning is
recommended.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable MS mass calibration and an Initial
Calibration are required before any samples are analyzed. Once the Initial Calibration is
successful, CCCs are required at the beginning and end of an Analysis Batch and after
every tenth field sample. Although not required, it is recommended that the Initial
Calibration be repeated and the mass calibration verified when instrument modifications
(column or suppressor replacement) or maintenance (ESI/MS detector inlet cleaning)
are performed.
NOTE: CAL solutions and CCCs are not processed with the sample collection or
pretreatment devices. This step must be omitted for the CALs and CCCs to identify
potential losses associated with the sample filtration, collection or pretreatment devices.
10.2 MASS CALIBRATION AND INSTRUMENT OPTIMIZATION - MS resolution must
be 1 amu or better. It is recommended that the analyst contact the instrument
manufacturer regarding appropriate mass calibration standards. The user should be
aware that many ESI/MS instruments are designed to analyze macromolecules having
large m/z ratios. As a result, many ESI/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 mass calibration compounds of lower m/z
ratios to achieve a better mass calibration for low m/z ions like C1O4". For the
instrumentation used during this method development, a sodium iodide solution was
used as a calibration compound. After the mass calibration has been performed, the
analyst must check mass accuracy for C1O4" by performing a simple experiment.
Prepare a high CAL standard containing equal amounts of C1O4" and the IS. While the
CAL standard is being infused, scan over the range of 95 - 115 amu and verify that the
C1O4" peaks are symmetric about m/z 99, 101 and 107. (There will also be peaks at
m/z 103, 105 and 109 from the internal standard C1O4" ions that have varying numbers
of 18O atoms.) If the peaks are not symmetric about the mass assignments (i.e., 99 ± 0.3,
101 ± 0.3 and 107 ± 0.3), then a new mass calibration of the MS, or other instrument
maintenance according to the manufacturer's recommendations, should be performed.
10.2.1 OPTIMIZING MS PARAMETERS - MS instruments have a large number of
parameters that may be varied to achieve optimal signal to noise. Due to
differences in MS design, the recommendations of the instrument manufacturer
should be followed when tuning the instrument. MS conditions may be
established by infusing a solution of C1O4", at the same flow rate to be used for
sample analysis, while the analyst optimizes the MS parameters. The cone
voltage determined to be optimal for the instrumentation used in this method
may be adjusted for different MS systems, if necessary, to yield the highest
332.0-24
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counts for C1O4" at m/z 99 while minimizing in-source collisionally induced
dissociation with subsequent formation of C1O3" (m/z 83).
10.2.2 INSTRUMENT CONDITIONS - Suggested operating conditions are listed in
Table 1 for Dionex instrumentation and in Table 2 for Metrohm-Peak
instrumentation. Conditions different from those described may be used if the
QC criteria in Section 9.2 are met. Different conditions include alternate 1C
columns, mobile phases and MS conditions.
10.3 INITIAL CALIBRATION - For the data presented in this method, daily calibrations
were performed using the internal standardization calibration technique; however, it is
permissible to perform an Initial Calibration with daily calibration verification using
CCCs as described in Sections 10.4.1 and 10.4.2. Calibrations must be performed using
peak area (dependent variable) versus concentration (independent variable). Peak
height versus concentration is not permitted.
10.3.1 CALIBRATION SOLUTIONS - Prepare a set of at least five CAL standards as
described in Section 7.2.3. The lowest concentration of the calibration standard
must be at or below the MRL, which will depend on system sensitivity and
intended use of the method. The target MRL must be confirmed using the
procedure outlined in Section 9.2.4 after establishing the Initial Calibration.
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 Inject 200 [iL of each standard into the IC-ESI/MS. Inject a RW blank after the
highest CAL standard to check for carry-over (Sect. 9.2.1.1). Table 4 is
provided to assist in tabulating data for standards and samples. Tabulate the area
counts of m/z 101 and m/z 107, relative retention time ratios of m/z 99/107 and
m/z 101/107, and the m/z 99/101 area count ratio. Evaluate if the m/z 99/101
area count ratio for all the standards are within the acceptance limits of 2.31 -
3.85 (Sect. 9.3.5) and verify that the relative retention time ratios for m/z 99/107
and m/z 101/107 are between 0.98 - 1.02 (Sect. 9.3.6).
NOTE: A different injection volume may be used as long as the data quality
objectives and QC requirements of the method are met and that the same volume
is used for the analysis of samples.
10.3.3 CALIBRATION ACCEPTANCE CRITERIA - Using the data obtained in
Section 10.3.2, perform a regression (e.g., linear, weighted linear, quadratic) of
the m/z 101/107 area count ratio vs. concentration of C1O4". To evaluate if the
chosen regression model yields accurate results across the range, reprocess (do
not re-analyze) CAL standards as unknowns and determine the calculated
concentrations. Determine the percent recoveries of the reprocessed CAL
standards based on the known concentrations. Recoveries at ALL the tested
concentrations must be within 80 - 120% for concentrations > the MRL. For
332.0-25
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concentrations < the MRL, the minimum acceptance criterion is 50 - 150%
recovery. If the recoveries are not within the acceptable ranges, a different
regression model such as a weighted linear, quadratic or weighted quadratic
should be tested. An acceptable calibration has been obtained when recoveries
of reprocessed standards are within the acceptance criteria stated above.
NOTE: For additional verification of the chosen regression model, or if
experiencing problems in meeting QC criteria contained in this method, refer to
Appendix A for instructions on how to statistically verify regression models for
instrument calibration.
10.3.4 INITIAL CALIBRATION VERIFICATION - Analyze a QCS sample
(Sect. 3.17) 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 calculated amount of C1O4" must be 80-120% of the certified 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. Calculate percent recovery (%R) using the following equation:
%R = measured concentration X 100
certified concentration
10.4 CONTINUING CALIBRATION CHECKS (CCCs) - At the beginning of the Analysis
Batch, the Initial Calibration must be verified by analyzing a mid-level and MRL level
CCC. Throughout an Analysis Batch the calibration is verified after every ten field
samples by the analysis of a CCC that is rotated between low (< MRL), medium (mid-
level calibration concentration) and high concentration (upper calibration
concentration). CCCs are not counted as samples. Analyze CCCs under the same
conditions used during the Initial Calibration.
10.4.1. MID-LEVEL CCC - The first CCC of an Analysis Batch must be at or near the
mid-point of the calibration to verify the Initial Calibration. Acceptance criteria
for the mid-level CCC is 80-120% recovery. The IS area count acceptance
criterion (Sect 9.3.4) for subsequent samples must be relative to this first
CCC.
NOTE: If the IS response drifts below 50% of the average IS response of the
CAL standards from the Initial Calibration, instrument maintenance or ESI/MS
detector inlet cleaning maybe required (Sect. 4.2.3).
10.4.2 MRL CONCENTRATION CCC- A CCC at a concentration that is < the MRL
concentration is performed, following the mid-level CCC, to verify instrument
sensitivity prior to any analyses. The acceptance criteria is 50-150% recovery.
332.0-26
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10.4.3 After every tenth field sample and at the end of an Analysis Batch, CCCs must
alternate between low (< MRL), medium (mid-level calibration concentration)
and high concentration (upper calibration concentration). Calculate the
concentration of C1O4" in the CCCs. A CCC 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 the criteria are not met, then all data from the last successful
CCC to the failed CCC must be considered invalid, and remedial action
(Sect. 10.4.4) should be taken. The remedial action may require re-calibration.
Any field samples that have been analyzed since the last acceptable CCC, that
are still within their holding times, should be reanalyzed after calibration has
been restored.
10.4.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria may
require remedial action. Major maintenance such as cleaning the ion source or
mass analyzer, requires returning to the Initial Calibration (Sect. 10.3).
11. PROCEDURE
11.1 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 are met (Sect. 9.2). This section describes the procedures for sample
preparation and analysis.
11.2 IC-ESI/MS START-UP - The 1C should be allowed to operate until the conductivity of
the eluate from the conductivity suppressor stabilizes (<1 |J,S for the data presented in
this method), at which time it may be connected to the ESI/MS. It is recommended that
the IC-ESFMS operate for approximately 30 minutes prior to the analysis of samples.
11.2.1 For some IC-ESFMS instrumentation it may be necessary to use a second six-
port valve (Valve 2 in Figures 1 and 2) and auxiliary pump to improve system
ruggedness and to maintain sensitivity for extended periods of time. To
determine if the additional instrumentation is necessary, the repeated analysis of
a mid-level concentration LFSSM (Sect. 3.10) for one or two days is
recommended. If the IS area counts drift downward over time (to <50% of the
average from the Initial Calibration), then it may be beneficial to install a second
six-port valve and auxiliary pump.
11.3 SAMPLE PREPARATION
11.3.1 Collect and store field samples as described in Section 8.1. For refrigerated or
field samples arriving at the laboratory cold, ensure the samples have
equilibrated to room temperature prior to analysis by allowing the samples to sit
on the bench for at least 30 minutes.
332.0-27
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11.3.2 Process all LRBs, LFBs, LSSMBs and LFSSMs using the sample collection
devices is Section 8.1.
11.3.3 Prepare the sample for analysis by pipetting 5 mL into an autosampler vial, or
other suitable single use vial. Dilution of the sample may be required if the
sample concentration is suspected to exceed the upper calibration standard. Add
50 |jL of the 100 (jg/L IS-FS (Sect. 7.2.1.2), cap the vial and invert several times
to mix. If using a commercially available IS solution, calculate the volume
necessary to achieve a 1.0 |j,g C118O4" /L final IS concentration in the sample.
NOTE: A 1% dilution error introduced by the addition of the IS is considered
insignificant. It is permissible to use a different IS concentration; however, the
analyst must be aware that ionization suppression of the native C1O4" may occur
if the IS concentration is too high.
11.4 SAMPLE ANALYSIS
11.4.1 Establish optimal operating conditions for the IC-ESI/MS instrumentation to be
used. Operating conditions may vary depending on instrumentation. The
analyst is responsible for determining optimal conditions for their
instrumentation. The configuration of Figure 1 and the operating conditions of
Table 1 were used to generate the data presented in this method.
11.4.2 Establish a valid Initial Calibration following the procedures outlined in
Section 10.3 or confirm that the calibration is still valid by analyzing the
required CCCs as described in Section 10.4.
11.4.3 Inject aliquots of field samples and QC samples under the same instrumental
conditions used for the Initial Calibration (a 200 |jL sample size was used in
collection of data for the method). A sample Analysis Batch is presented in
Table 3.
NOTE: If not using an autosampler, use a syringe to withdraw the sample from
the sample vial. Place the injection valve in the Load position and manually
load the sample loop. The loop size must be the same loop size that was used to
calibrate the instrument. Flush the loop with at least three loop volumes of
sample.
11.4.4 At the conclusion of data acquisition, use the same data acquisition method that
was used for the Initial Calibration to identify peaks in the chromatogram. Use
the data acquisition method to determine the relative retention times and
integrate the peak areas of the monitored ions (rn/z 99, 101, and 107).
332.0-28
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11.5 COMPOUND IDENTIFICATION - Identification/confirmation of CIO/ in a sample is
made by detecting C1O4~ at m/z 101 and m/z 99 at the retention time of the internal
standard and by passing the QC criteria established for the m/z 99/101 area count ratio.
11.5.1 RELATIVE RETENTION TIME ACCEPTANCE CRITERIA - Since the
C118O4~ IS has the same retention time as naturally occurring C1O4", the retention
time ratio of m/z 99/107 and m/z 101/107 in samples must be within 0.98 - 1.02
(+2% of ideal ratio of 1) for confirmation of C1O4" in a sample.
11.5.2 AREA COUNT RATIO (m/z 99/101) ACCEPTANCE CRITERIA - All CAL
standards, QC samples and field samples must meet the m/z 99/101 area count
ratio requirement for confirmation of ClO4"(Sect. 9.3.5). The measured ratio
must fall within +25% (2.31-3.85). If this area count ratio requirement is not
met for a CCC or LFB, then all samples in the Analysis Batch are considered
invalid and must be reanalyzed after reestablishing acceptable instrument
performance. Field samples having m/z 99/101 area count ratios falling outside
this range due to sulfate interference must be diluted and/or pretreated with
barium form pretreatment cartridges to remove/reduce sulfate to a level that
allows better integration of the C1O4" peak at m/z 99. Section 11.6 describes the
required remedial action in the case that (1) a peak is detected at m/z 101 at the
retention time of the IS at concentrations > the MRL but no peak is detected at
m/z 99 due to high sulfate concentration in the sample, or (2) peaks are detected
at both the m/z 101 and m/z 99 ions but the ratio is not within control due to high
sulfate concentration in the sample. In either case, the required remedial action
described in Section 11.6 must be performed.
11.6 REQUIRED REMEDIAL ACTION - If C1O4~ is detected at m/z 101 at concentrations
> the MRL, but the m/z 99/101 area count ratio fails due to background counts at m/z
99, remedial action is required (Sect. 11.6.1 and/or Sect 11.6.2). Sample dilution and/or
pretreatment using the barium form pretreatment cartridge are acceptable means to
reduce the background at m/z 99 due to high concentrations of sulfate. Generally, the
background at m/z 99 is considered high if it is approximately 10-20 times higher than
the background at m/z 99 measured in the first CCC of the Analysis Batch
(Sect. 10.4.1).
11.6.1 SAMPLE DILUTION - If the concentration detected at m/z 101 is at least
2 times the MRL, a 2-fold dilution of a fresh aliquot of sample may be attempted
to lower the background at m/z 99 due to sulfate in the sample. The m/z 99/101
area count ratio must be re-evaluated in the diluted sample for confirmation of
C1O4". If the background at m/z 99 still appears high in the diluted sample,
sample pretreatment using the procedure described in Section 11.6.2 must be
attempted.
NOTE: If a sample is diluted, the analyst must be careful not to dilute the
analyte concentration to below the MRL. Add the IS after dilution.
332.0-29
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11.6.2 SAMPLE PRETREATMENT - If a sample is pretreated using pretreatment
cartridges, an LRB must also be processed in the same manner as the sample. If
all of the cartridges described in Section 6.7 are used in series, the sample flow
path must be arranged as follows: (1) the Ba2+ cartridge (used to remove
sulfate), (2) the Ag cartridge (used to remove chloride), (3) a 0.2 |j,m filter to
remove colloidal silver, and (4) the H+ cartridge (used to remove carbonate).
NOTE: Some sample matrices may result in an IS area count QC criteria failure
(Sect 9.3.4), peak shape distortion, high background conductivity, or high
background(s) at m/z 99, 101 and/or 107. In these cases, it may be helpful to use
all three forms of the pretreatment cartridges described in Section 6.7. Consult
the manufacturer's instructions for preparation of the pretreatment cartridges
prior to use with samples. Generally, the procedure requires rinsing each
cartridge with a minimum volume of RW. It has been found that rinsing with
approximately 2 times the recommended volume of water gives better results.
Insufficiently rinsed cartridges often result in random peaks by conductivity
detection. Add the IS to the sample prior to sample pretreatment using the
cartridges.
11.7 EXCEEDING THE CALIBRATION RANGE - The analyst must not extrapolate
beyond the established calibration range. If the calculated C1O4" concentration in a
sample is greater than the highest CAL standard of the Initial Calibration, a fresh aliquot
of the sample must be diluted, IS added, and the sample re-analyzed. Incorporate the
dilution factor into the final concentration calculation.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Tabulate data using Table 4 as a guide. Compute sample concentration on the m/z 101
quantitation ion using the calibration generated in Section 10.4.
12.2 If the measured concentration of a field sample exceeds the calibration range, a fresh
aliquot of the sample must be diluted and re-analyzed and pass the confirmation criteria.
12.3 When using an autosampler, the analyst may be unaware that samples continued to be
analyzed even after the failure of on-going QC. Therefore, if using an autosampler,
check that all the on-going QC requirements of the method were successful in the
interim of the analyst's absence. If a CCC failed at any point during an Analysis Batch,
it will be necessary to re-analyze all samples after the last successful CCC.
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. Report ONLY those
values that fall between the MRL and the highest calibration standard.
332.0-30
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12.4.1 Calculations must utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures
(one digit of uncertainty), with not more than three significant figures.
13. METHOD PERFORMANCE
13.1 SUMMARY - Single laboratory precision in drinking waters, as measured by percent
relative standard deviation (%RSD) of replicate analyses (n=7), was < 10% at
concentrations > 0.2 ng/L C1O4". Accuracy, as measured by percent recoveries of
fortified drinking water samples and external Quality Control samples, was 90-110%
for concentrations > 0.1 (jg/LClO4~.
Single laboratory precision in fortified synthetic waters containing up to 1,000 mg/L of
each of the common anions (LFSSM), as measured by %RSD of replicate analyses
(n=7), was <20% at concentrations > 0.1 (Jg/L C1O4". Accuracy, as measured by percent
recovery of fortified synthetic high ionic waters containing up to 1,000 mg/L of each of
the common anions (LFSSM), was 80 - 120% for concentrations > 0.1 ng/L C1O4".
13.2 Figure 3 shows chromatograms of a 0.1 (jg/L calibration standard with retention times for
the ions monitored in this method (m/z 99, 101 and 107). Figure 4 shows chromatograms
of a 1.0 |Jg/L C1O4" LFSSM solution containing 1,000 mg/L of chloride, sulfate and
carbonate. Figure 4 also illustrates the effect of a high background at m/z 99 due to HSO4".
13.3 Table 5 contains single laboratory DL and LCMRL data in RW.
13.4 Table 6 contains precision (%RSD) and recovery (%R) data for C1O4" in various drinking
water and synthetic water samples at low and high fortification concentrations.
14. POLLUTION PREVENTION
14.1 For information about pollution prevention that may be applicable to laboratories and
research institutions, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction," available from the American Chemical Society's Department of Government
Regulations and Science Policy, 1155 16th Street N.W., Washington D.C. 20036, or on-line
at http://www.ups.edu/communitv/storeroom/Chemical Wastes/wastearticles.htm. last
verified in March 2005.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small amounts of
waste since only small amounts of reagents are used. The matrices of concern are
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. Also, compliance is required with any
332.0-31
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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 Committee on
Chemical Safety at http://membership.acs.0rg/c/ccs/publications.htm. last verified in
March 2005. Or see "Laboratory Waste Minimization and Pollution Prevention,"
Copyright © 1996 Battelle Seattle Research Center, which can be found on-line at
http://www.p2pavs.org/ref/01/text/00779/index2.htm. last verified in March 2005.
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. last verified in March
2005.
2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
Wastewaters," Environ. Sci. Technol 1981, 15, 1426-1435.
3. Lange's Handbook of Chemistry (15th Edition); Dean, J.A., Ed; McGraw-Hill: New York,
NY, 1999.
4. "Carcinogens-Working with Carcinogens," Publication No. 77-206, Department of Health,
Education, and Welfare, Public Health Service, Center for Disease Control, National
Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977.
5. "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical Society
Publication, Committee on Chemical Safety, Washington, D.C., 1979.
6. "OSHA Safety and Health Standards, General Industry," (29CFR1910). Occupational
Safety and Health Administration, OSHA 2206, (Revised, January 1976).
7. ASTM Annual Book of Standards, Part II, Volume 11.01, D3 370-82, "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, "Microbial degradation of
perchlorate: principles and applications," Environ. Engin. Sci, 2003, 20(5), 405-422.
ACKNOWLEDGMENTS
The authors would like to gratefully acknowledge Dr. Douglas W. Later, Dr. William C. Schnute and
Robert J. Joyce of Dionex Corporation for their valuable contributions throughout the development of
this method and in coordinating the collection of second laboratory demonstration data. The authors
also acknowledge Jay Gandhi of Metrohm-Peak, Inc., for providing second laboratory demonstration
data for the method.
332.0-32
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17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. DIONEX IC-MS OPERATING CONDITIONS*
Ion Chromatograph
Mobile Phase
Guard and Separator Columns
Flow Rate
Conductivity Suppressor and Current
Column Temperature
Auxiliary Pump Flow Rate2
Injection Volume
Mass Spectrometer
Ion Energy (V)
Low Mass Resolution
High Mass Resolution
Capillary Voltage
Sampling Cone Voltage
Probe Temperature
Nitrogen pressure
Selected Ion Monitoring
Mass Scan Range
Dwell Time per mass
Smoothing/Points/Range
Dionex Corporation, Sunnyvale, CA
75 mM KOH, or 65 mM KOH 1
Dionex AG16 + AS16, 250 mm X 2 mm
0.3 mL/min
ASRS-MS, 75 mA, or 70 mA '
30 °C
0.3 mL/min RW, or 50/50 v/v acetonitrile/water '
200 |jL
MSQ with Enhanced Low Mass Option (ELMO)
ThermoFinnigan, San Jose, CA
0.3
12.7
12.5
-3kV
-70V
400 °C, or 500 °C '
SOpsi
m/z99, 101, 107
0.3 amu
0.75 sec, or 0.3 sec '
Boxcar/5/6
1 Condition used in IC-MS Configuration 2.
2Auxiliary pump is used to deliver RW or 50/50 v/v acetonitrile/water to the conductivity suppressor
and the mass spectrometer until 1.5 minutes prior to the elution of C1O4" depending on which
configuration is used (Figures 1 or 2). For the data presented in this method, RW was used in the
auxiliary pump.
*Instrumentation, when specified, does not constitute endorsement. Brand names are included for
illustration only.
332.0-33
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TABLE 2. METROHM-PEAKIC-MS OPERATING CONDITIONS*
Ion Chromatograph
Mobile Phase
Guard and Separator Columns
Flow Rate
Suppressor Regenerant
Column Temperature
Injection Volume
Mass Spectrometer
Low Mass Resolution
Capillary Voltage
Nitrogen Pressure
Fragmentor Voltage
Drying Gas Temperature
Drying Gas Flow Rate
Selected Ion Monitoring
Mass Scan Range
Dwell Time Per Mass
Metrohm-Peak, Houston, TX
30 mM NaOH/30% Methanol
Metrohm ASUPP4/5 + ASUPP5-100, 100 mm X 4 mm
0.7 mL/min
60 mM nitric acid/10% Methanol, Rinse 10% Methanol
30 °C
100 ML
1100 Series MSD Quad SL, Agilent Technologies, Wilmington, DE
0.65
-2kV
80psi
150V
320 °C
9- lOL/min
m/z99, 101, 107
0.1 amu
0.25 sec
*lnstrumentation, when specified, does not constitute endorsement. Brand names are included for
illustration only.
332.0-34
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TABLE 3. SAMPLE ANALYSIS BATCH
Injection #
i
2
3
4
5
14
15
16
17
18
27
28
Sample Description
Mid-Level CCC
MRL CCC
LRB
LFB
< MRL concentration
>MRL to highest CAL std
Field Samples 1 - 10
CCC (rotating
concentrations)
LFSM of a field sample
previously analyzed
Laboratory Duplicate or a
LFSMD of field sample
previously analyzed.
Choose LFSMD if
samples are low in
perchlorate.
Field Samples 11-20
Final CCC (rotating
concentrations)
Acceptance Criteria
80 - 120 % recovery using
Initial Calibration
50 - 150% recovery
MRL concentration.
80 - 120% recovery using
Initial Calibration for
concentrations > MRL
50-150% recovery for
concentrations < MRL
At fortification concentrations
> MRL concentration, 80-
120% recovery.
At fortification concentrations
2 X MRL
RPD <50% for samples < 2 X
MRL
Pass RT, m/z 99/101 area
count ratio, and IS area count
QC criteria.
80 - 120 % recovery using
Initial Calibration for
concentration > MRL
Remedial Action
Instrument maintenance and
recalibration.
Instrument maintenance to
recover sensitivity and
recalibration.
Find and correct source of
contamination.
Identify and correct source of
problem.
If problem is due to sulfate,
clean up sample using Ba form
cartridge, otherwise report.
Instrument maintenance and
recalibration.
If problem is due to sulfate,
clean up sample using Ba form
cartridge, otherwise report.
If RPD out of the designated
range, but the laboratory
performance is acceptable in
LFB, the recovery problem is
judged to be matrix related.
Label sample "suspect matrix".
If problem is due to sulfate,
clean up sample using Ba form
cartridge, otherwise report.
Instrument maintenance and
recalibration.
332.0-35
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TABLE 4. EXAMPLE TEMPLATE FOR TABULATION OF SAMPLE DATA FOR QC
REQUIREMENTS
99
101
107
Are area counts of IS +30% of first CCC?
99
101
107
Are area counts of IS +30% of first CCC?
* - Acceptance Criteria (0.98 - 1.02)
** - Acceptance Criteria (2.31 - 3.85)
332.0-36
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TABLE 5. DETECTION LIMIT AND LCMRL FOR PERCHLORATE IN REAGENT
WATER
0.02
0.10
*Fortification concentration - 0.05 |~ig/L.
Seven replicates over three days.
TABLE 6. PRECISION AND RECOVERY DATA FOR PERCHLORATE IN VARIOUS
MATRICES (N=7)
- '- >' !'"' " ,'.:''..' '. ';.'. -"/" ; "'';" ;"'V"'.;
:: .;: ":.':' .-.. - -" fVT ill"!*!^ " ' ' "' "' "' "' "
Reagent Water
LSSM
Surface Source Tap
Water
High TOC Surface
Source Tap Water2
Ground Water
ND1
ND
0.27
ND
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TABLE 7. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS
Method
Reference
Section
9.2.1
Section
9.2.1.1
Section 9.2. 2
Section 9. 2. 3
Section 9.2.4
Requirement
Demonstration of Low System
Background
Concentration Dependent Carry-Over
Demonstration of Precision in LFBs
and LFSSMs
Demonstration of
Accuracy in LFBs and LFSSMs
Minimum Reporting Level (MRL)
Confirmation
Specification and Frequency
Analyze an LRB and LSSMB prior to any
other IDC steps and when modifications
are made.
During IDC and when modifications are
made. Analyze a RW blank after the high
CAL during Initial Calibration.
Analyze 7 replicates fortified near mid-
range of calibration.
Calculate the average recovery for
replicates in Section 9.2.2.
Analyze 7 replicate LFBs at the target
MRL. Use the equation provided to
verify the MRL. Repeat after major
instrument or operational changes.
Acceptance Criteria
50%
332.0-38
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TABLE 8. ON-GOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section 8
Sample Collection, Preservation, and
Holding Time
28 days, samples must be sterile filtered
through a 0.2 |_im filter with the filtrate
collected in a sterile bottle.
Ship at < 10 °C to be received within 48 hours.
Once received at the lab, samples should be
analyzed as soon as possible. Sterile filtered
samples must be stored with head space. Leave
1/3 of bottle empty. Store at 6 °C or less.
Section
10.3
Initial Calibration
Use internal standardization calibration
and a minimum of 5 calibration standards.
Use peak area for calibration and
quantitation.
80 - 120% recovery of all reprocessed standards
at > the MRL.
50 - 150% recovery of reprocessed standards £
the MRL.
Section
9.3.1
Laboratory Reagent Blank (LRB)
Analyze one LFB per Analysis Batch
(every 20 field samples).
Demonstrate that the target analyte is MRL
Section
9.3.4
Internal Standard Area Counts
In all standards, field samples, LFBs,
CCCs, etc., analyzed during each
Analysis Batch.
Deviation of area counts not to exceed +30% of
area counts of first CCC of the Analysis Batch.
332.0-39
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TABLE 8 (Continued). ON-GOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Section
9.3.5
Section
9.3.6
Section
9.3.7
Section
9.3.8
Section
10.3.4
Requirement
Area Count Ratio (m/z 99/101)
Acceptance Criteria
Relative Retention Time Acceptance
Criteria
Laboratory Fortified Sample Matrix
(LFSM)
Laboratory Duplicate (LD) or
Laboratory Fortified Sample Matrix
Duplicate (LFSMD)
Initial Calibration Verification
Specification and Frequency
In all standards, field samples, LFBs,
CCCs, etc., analyzed during each
Analysis Batch.
In all standards, field samples, CCCs,
LFBs, LFSMs, etc.
Analyze one LFSM per Analysis Batch
(every 20 field samples) fortified with
perchlorate at a concentration that is
greater than or equal to the native
concentration.
Analyze at least one LFSMD or LD with
each Analysis Batch of up to 20 field
samples.
Each time the Initial Calibration is
repeated or new standards are prepared,
analyze a QCS.
Acceptance Criteria
The calculated m/z 99/101 area count ratio
must be within +25% (2.31 - 3.85).
Relative retention times of m/z 99/107 and
m/z 101/107 must be within 0.98 - 1.02.
Recoveries not within 80 - 120% of the
fortified amount at > MRL may indicate a
matrix effect. Cone. < MRL may be
recovered 50 - 150%.
<50% RPD at cone. <2 X MRL
<20% RPD at cone. > 2 X MRL
80 - 120% recovery of the certified
concentration at the mid-range of the
calibration.
332.0-40
-------
FIGURE 1. IC-ESI/MS Configuration Used to Generate Data in Method
Liquid
Pump
Auxiliary
Pump /
co nductmty detector
Mass
Spectrometer
FIGURE 2. Alternative IC-ESI/MS Configuration
static mixing tee
Liquid
Pump
(Conductivity detector)
Mass
Spectrometer
332.0-41
-------
FIGURE 3. MASS CHROMATOGRAM OF A STANDARD CONTAINING 0.1 ng/L C1O4
AND 1.0 ng/L INTERNAL STANDARD
4.00C
COilTtS
1.00C
RT=aos
Aim Counts = 58621
98.84-99.16 amu
1.90C -
cents
90C-
fteicMarate
RT=8.0G
Area Courts = 195.37
100.84-101.16 amu
22.00C
COUt5
-2,000
5.00 6.0C
106. 84- 107. 16 amu
f^-Nm^e Itltmml Sfcandarti
RT=7.m
, Area Oou:n£s= 5392. 03
7.00 8.00 9.0C
Tlmejniiuitesf
10.00 11.OC 12.0C
332.0-42
-------
FIGURE 4. MASS CHROMATOGRAM OF AN LFSSM CONTAINING 1.0 ng/L C1O4 AND
1.0 ng/L INTERNAL STANDARD
22.00C
COLltS
-2,000
ftrcftforafe
Area Gout s= 15E.29
98.84-99.16 amu
3.SDC
COLltS
500
7.0QC
COLltS
_L
ftrctforafe
RT=6.50
Area Carts BG2333
Area. Courts = 175&78
T
1 ' ' ' I ' ' ' ' I ' ' ' ' I ' '
5.03 6.00
1 i * i i i i i
7.03 8.0] 9.03
100.34-101.16amu
106.&4-107.16 amu
10.03 11.00 12.00
Time (minutes)
332.0-43
-------
APPENDIX A (Optional)
Statistical Validation of the Regression Model Used For Instrument Calibration
Introduction
Selection of an appropriate regression model for instrument calibration is critical for obtaining accurate,
non-biased results and for the determination of an LCMRL and MRL. The following guidance is
provided for labs that desire additional validation of their instrument calibration model and for labs that
may be experiencing problems meeting the QC criteria contained in Section 9 of the method.
Background
The Calibration Range (CR) is defined as the concentration range over which the instrument has been
calibrated and results maybe reported. During the Initial Demonstration of Capability (Sect. 9.2), the
regression model used to describe the CR must pass certain minimum criteria for accuracy as determined
by the percent recovery of standards that are reprocessed (not re-analyzed) as samples. In practice, a lab
may find that low concentration standards are consistently biased low (or high). It may be that the
analyst has attempted to calibrate over too large of a concentration range for the chosen model. The
analyst may change the range of interest or select a different model but be unsure if the new range and/or
model is appropriate. The F-test for lack of fit, described below, is a statistical metric for determining if
a selected regression model (e.g., linear, quadratic) gives a non-biased estimate of the expected response,
Pred Y (area count ratio, m/z 101/107), as a function of standard concentration, x.
General Recommendations
The instrument should be operationally stable. This may require a period of approximately
30 minutes of operation with liquid flow through the IC-ESFMS system.
If the desired CR is two orders of magnitude or greater, a weighted regression model will
likely be required. Most newer instrumentation automatically allows for this type of
calibration. For the instrumentation used in this method, it was found that a non-weighted
linear regression model yielded 90-110% recoveries of all standards, for concentrations 0.1-
1.0 ng/L C1O4". If, however, the upper range was extended to 5.0 or 10.0 ng/L, a weighted
linear regression was required (weight factor = 1/x) to achieve the same results. Using a non-
weighted linear regression across the range 0.1-5.0 or 10.0 ng/L resulted in consistently high
recoveries (115-131%) for the 0.1 ng/L CAL standards. Choosing a short range over which
the variance is constant (e.g. 0.1-1.0 ng/L C1O4"), or using a weighted regression model, are
both acceptable means to obtain a regression that yields accurate, non-biased results across the
range.
The F-test for lack of fit assumes constant variance across the concentration range. Statistical
software is highly recommended to test for constant variance and to perform the F-test for lack
of fit; however, if statistical software is not available, the mathematical procedures described
below should result in selection of an appropriate regression model.
332.0-44
-------
Procedure
1. Prepare and inject, in duplicate, five standards that span the range of interest. Concentration is
the independent variable, x, and the dependent variable, y, is the area count ratio m/z 101/107.
Evaluate each standard to make sure the IS area counts are within control and that the m/z
99/101 area count ratio is in control. Tabulate concentration (iig/L), x, and response (area
count ratio m/z 101/107), y.
NOTE: To perform the F-test for lack of fit, there must be replicates on some or all of the
levels of concentration, x.
2. Using the data obtained in Step 1, perform a non-weighted linear regression of the area count
ratio (area count ratio m/z 101/107) vs. concentration (ng/L) of C1O4".
3. Decide what will be deemed acceptable recoveries for the data quality objectives of the work.
For the example presented below, it was decided that 90-110% recoveries across the range
would be the criteria for accepting the model (see Table Al).
4. To evaluate if the chosen regression model yields accurate results (i.e., constant variance
across the range), reprocess (do not re-analyze) standards as unknowns and determine the
calculated concentrations. Determine the percent recoveries of the reprocessed standards
based on the known concentrations (see example in Table Al). Recoveries should meet the
recovery criteria and be consistent across the range, i.e., recoveries at ALL the tested
concentrations must be within the recovery range (in the example presented here that range is
90- 110%).
5. If the recoveries are not consistent across the range, a weighted linear regression model should
be tested. Reprocess the data and re-evaluate the recalculated recoveries. If the results are still
unacceptable, delete the highest standard from the regression model and reprocess the data. If
unacceptable results are still encountered when the range has been reduced to one order of
magnitude, there may be very poor precision between duplicate analyses. This may signal that
instrument maintenance is required.
6. Since the F-test for lack of fit assumes normally distributed data with equal variances for the
Y distribution (i.e., across the range of concentrations), a weighted regression model should be
tried before proceeding to the F-test for lack of fit. Weighting will generally give better
recoveries across a wide calibration range. When an acceptable range and model have been
chosen that yields recoveries of reprocessed standards within the set criterion for recoveries of
reprocessed standards, proceed to Step 7, the F-test for lack of fit.
7. F-TEST FOR LACK OF FIT - The use of statistical software to perform the F-test for lack of
fit is highly recommended. If this option is not available, however, use a spreadsheet software
program and the following directions to perform the test. Prepare a table exactly like Table A2
and enter the data required in each column.
332.0-45
-------
The test statistic involves calculating F* for the chosen model and comparing it to a critical F
value from a standard table of F values.1 The test statistic is as follows:
F* = SSLF / DFLOF
SSPE / DFPE
where,
F* = calculated F for regression model
SSLF = lack of fit sum of squares. See Table A2 for calculation.
SSPE = pure error sum of squares. See Table A2 for calculation.
DFLOF = degrees of freedom for SSLF. Equals c - 2 for 1st order polynomial.
Equals c - 3 for second order polynomial (quadratic).
DFPE = degrees of freedom for SSPE. Equals n - c.
c = number of concentration levels.
n = total number of observations.
Table A2 shows the mathematical calculation of SSLF and SSPE from the data obtained from
the chosen regression model (a weighted 1st order linear regression model). The table was
completed by entering the required data into a software spreadsheet program. In the example
provided in Table A2, n = 10 and c = 5. Critical F(l-alpha, c-2, n-c) = F(0.95, 3, 5) = 9.01.
The Decision rule was:
If F* < 9.01, then conclude that the regression model is appropriate.
If F* > 9.01, then conclude that the regression model is not appropriate.
In this example, the calculated F* using a weighted linear regression model was 0.8874 which
is less than the critical F value of 9.01. It was concluded that the selected model was
appropriate. If the calculated F* had been greater than the critical F value, then a different
model (quadratic or weighted quadratic) would have been evaluated and consistent recoveries
and lack of fit would have been tested again with proper modification of the degrees of
freedom for SSLF. Return to Step 4.
Reference
1. Neter, J., W. Wasserman and M. Kutner, Applied Linear Regression Models. 1989, Irwin, Jnc,
Boston, MA.
332.0-46
-------
APPENDIX A
TABLE Al. SAMPLE DATA COMPILATION AND DETERMINATION OF ACCURACY OF
CALIBRATION MODEL
1' :'''. .«-'" ' -.'.""
:: JX<:-;'--V
;;>X'-'C':;"::':: ;
iiWv>':- -
:: ^|; ;- ...;:::..::..::..::..
^k-
7-''1' '' . '. ': ' -
V:> ;v.i.:^.v
jj'o,- . '"> :'".''.
::,O.;V .,::............:..
ll&i^isSv-
;; - -..:..," .. ..-
.. .-. .;. .-. ,
il& ?--:
isss
::; A r'"- ---:-:-
X = Cone. '
Hg/L
0.1
0.1
0.5
0.5
1
1
5
5
10
10
&&:£????£??£i
PredXij 2
Hg/L
0.1094
0.0926
0.4828
0.4609
1.0196
0.9882
5.0781
5.0889
10.155
10.349
^:r^m
%Recovery
109
92.6
96.6
92.2
102
98.8
102
102
102
104
1 X = concentration of CAL standard. Levels of X = 1 - j, replicates = 1 - i
2 Predicted Xij = concentration calculated from regression model for a given Yij.
NOTE: The weighted linear regression equation was y = 0.0014148 + 0.3612397 X.
332.0-47
-------
APPENDIX A
TABLE A2. SPREADSHEET TABULATION OF DATA TO DETERMINE F-TEST FOR LACK OF FIT
::. .' ..
^,':: £:'..
:: ::-"2:"^<-
:;.-:3';r .i
.;,:4.-:-::-:;;
7. ,-5; ;;?.:'
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.: ..:">>. -..
5:?v7.:r;-:
M;*i:l:
r~.'::.,9-'"^..
;;-'j-ip;-:x
:::-ft:.:.:-::.
:;.12A....-i
. ;. '..-...-. .~.~.~.~f~-.: .~ .~ .~ .~ .~ .~ .~ .~ ;
(Yij-MEAN Yj)A2
0.000919446063506114
0.000919446063506114
6.27638915474167E-005
6.27638915474176E-005
3.20217544267144E-005
3.20217544267138E-005
1.51009076672299E-007
1.51009076672299E-007
1.23689370239679E-005
1.23689370239683E-005
0.00205350331116177
0.000410700662232354
Lj f;; |