332.0-1

EPA Document #: EPA/600/R-05/049

METHOD 332.0 DETERMINATION OF PERCHLORATE IN DRINKING WATER BY

ION CHROMATOGRAPHY 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
332.0-2

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 (ClO4

-

) 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 IC operational description (Sect. 10) and quality control requirements

(Sect. 9) of Method 332.0 may be 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 ClO4

-

, as defined

in Section 3.13, was 0.10 μg/L using the quantitation ion at m/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 ClO4

-

 in reagent

water was 0.02 μg/L (Table 5).

1.6 The two predominant ClO4

-

 ions that occur naturally at a ratio of 3.086:1 are 35Cl16O4

-

,

 m/z 99, and 37Cl16O4

-

, m/z 101, respectively.3 Due to fewer mass spectral interferences,

the concentration of ClO4

-

 using the m/z 101 ion is reported. The m/z 99/101 area count
332.0-3

ratio and relative retention time are used for confirmation of ClO4

-

 in samples. An

oxygen-18 (18O) enriched ClO4

-

 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 ClO4

-

 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 ClO4

-

 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, ClO4

-

 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 35Cl18O4

-

 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 ClO4

-

 ions with

respect to the internal standard. Qualitative confirmation of ClO4

-

 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)
332.0-4

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 ClO4

-
332.0-5

and processed like a field sample (Sect. 8). It is used to confirm that there is adequate

chromatographic resolution between sulfate and ClO4

-

.

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-, SO4

2-and CO3

2-) 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. 
332.0-6

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 ClO4

-

 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, ClO4

-

 elutes later than the common

inorganic anions (Cl-

, SO4

2-, CO3

2-, and HCO3

-

). Separation of ClO4

-

 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 ClO4

-

. 

4.2.1 Direct Chromatographic Co-elution of Contaminants: At sufficiently high

concentration, direct chromatographic co-elution of a contaminant with ClO4

-

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 ClO4

-

, 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 ClO4

-

. Any of these conditions could lead to a positive or negative bias of

ClO4

-

 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 coeluting 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
332.0-7

eluent strength or modifying the eluent with organic solvents (if compatible with

the IC 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 (P2O7

4-),

tripolyphosphate (P3O10

5-) 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 ClO4

-

 without

compromising data quality.

4.2.2 Concentration-Dependent Interference by Sulfate (SO4

2-): Of the common

anions found in drinking waters (Cl-, SO4

2-, CO32-, HCO3-

), sulfate can be the

most problematic. Sulfate elutes before ClO4

-

 on most of the anion

chromatography columns currently being used for ClO4

-

 analysis; however, it has

a tendency to elute broadly, tailing into the retention time of ClO4

-

. Formation of

H

32SO4

-

 (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

(m/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 ClO4

-

 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 ClO4

-

 (Sect. 9.3.5). As the column ages and the

retention time of ClO4

-

 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 ClO4

-

 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 ClO4

-

 (see Figures 1 and 2). This is
332.0-8

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 ClO4

-

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

ClO4

-

 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 ClO4

-

 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

Cl16O4

-

 (m/z 99) in the internal standard. Therefore, it must be demonstrated that the IS

does not contain unlabeled ClO4

-

 at a concentration > 1/3 of the MRL when added at the

appropriate concentration to samples (a concentration of 1 μg/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.
332.0-9

5.2 Pure ClO4

-

 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-ESI/MS 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 IC-ESI/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 μL), a conductivity suppressor, a conductivity detector and a data

acquisition and management system that has been interfaced with the ESI/MS.

6.1.1 CHROMATOGRAPHIC PUMP - 2-mm isocratic IC 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, IC 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 IonPac 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 OC is recommended but not required for this method [(Dionex

Model No. LC30) or (Metrohm Advanced IC 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
332.0-10

[(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 IC 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 IC Conductivity Detector,

Metrohm, Model 819, Part No. 2.819.0010) or equivalent].

6.1.9 SAMPLE LOOPS - 50 to 200 μL size. A 200 μL 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 IC or MS. A system that allows

control of both the IC 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,

v10.02, 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
332.0-11

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 ClO4

-

 (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 water:acetonitrile 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 ClO4

- 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 (HDPE), 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 (HDPE) bottles

(IChem 125-mL sterile HDPE bottle, Fisher Scientific, Suwanee, GA, Cat. No. N411-

0125 or equivalent) or disposable single-use, sterile polystyrene, 150 mL, with screwcap for sterile filtered samples (Fisher Scientific, Suwanee, GA, Part No. 09-761-140 or
332.0-12

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 μm 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 μL, 1000 μL 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).
332.0-13

7.1.2 ACETONITRILE - ACN, CASRN 75-05-8 (Fisher Scientific, Suwanee, GA,

Cat. No. A998-1 or equivalent). ACN is only required if using the IC-ESI/MS

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.= 56.11, 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 IC

system equipped with an eluent generator (Sect. 6.1.3), KOH eluent preparation

is not necessary. 

If using a Metrohm IC 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 - Na2SO4, F.W.=142.04, CASRN 7757-82-6 (Fisher

Scientific, Suwanee, GA., Cat. No. S421-500 or equivalent).

7.1.6 SODIUM CHLORIDE - NaCl, F.W.=58.44, CASRN 7647-14-5 (Fisher

Scientific, Suwanee, GA., Cat. No. S271-500 or equivalent).

7.1.7 SODIUM CARBONATE - Na2CO3, 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 OC 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 Cl18O4

-

/L. (NaCl18O4, F.W.=130.4, CASRN 7601-89-0, 90% enriched

on 18O, 98% pure NaCl18O4

, or better, Isotec, Inc., Miamisburg, OH or
332.0-14

equivalent,). A 1,000 mg/L solution of Cl18O4

-

 is prepared by dissolving

0.0123 g NaCl18O4

 in 10 mL of RW. The solution may be 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 Cl18O4

-

/L. Prepared gravimetrically, using an

analytical balance having +0.1 mg accuracy, by adding 0.1 g (100 μL) 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 μg Cl18O4

-

/L. 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 may be

used. (Dionex Corporation, Part No. 062923 or equivalent). 

7.2.2 PERCHLORATE STOCK STANDARD SOLUTION (SSS) - 1,000 mg ClO4

-

/L. 

(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 ClO4

-

 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 ClO4

-

/L. Prepared gravimetrically using an analytical balance

having +0.1 mg accuracy, by adding 0.1 g (100 μL) 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 μg ClO4

-

/L. 

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.
332.0-15

7.2.3 CALIBRATION STANDARDS (CAL) - The following guide may be used for

preparing 100-mL CAL solutions containing 1.0 μg/L of the IS. The holding

time for CAL solutions is one month.

 Final CAL Conc. (μg/L) Volume (mL) FS to add Volume (mL) IS-FS to add

(LRB) 0 0 1.0

0.1 0.1 1.0

0.2 0.2 1.0

0.5 0.5 1.0

1 1 1.0

5 5 1.0

7 7 1.0

10 10 1.0

7.2.4 LABORATORY SYNTHETIC SAMPLE MATRIX (LSSM) - 1,000 mg/L each

of Cl-, SO4

2-, CO3

2-. Add 1.48 g of Na2SO4

 (Sect. 7.1.5), 1.65 g of NaCl

(Sect. 7.1.6) and 1.77 g of Na2CO3 (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.
332.0-16

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 μm) may help to prevent clogging or rupture of

the 0.2 μm 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.
332.0-17

9.1.1 METHOD MODIFICATIONS - The analyst is permitted to modify IC columns,

mobile phases, chromatographic and ESI/MS 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

ClO4

-

 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 IC 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
332.0-18

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 + HRPIR X 100 < 150%

Fortified Concentration

The Lower PIR Limit must be > 50% recovery.

Mean - HRPIR X 100 > 50%

 Fortified Concentration
332.0-19

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 if DL 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:

DL = S * t( n - 1, 1 - alpha = 0.99)

where,

t

(n-1,1-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 a peak 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
332.0-20

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) may be 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 Fortified Concentration Range LFB Recovery Limits

< MRL 50 - 150%

>MRL to highest calibration standard 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
332.0-21

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 ClO4

-

. 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

ClO4

-

 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

Cl18O4

-

 IS has the same retention time as naturally occurring ClO4

-

, 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 ClO4

-

 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
332.0-22

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 ClO4

-

 concentrations < the

MRL. At these concentrations, LFSM sample recovery should be 

50-150%. If the accuracy of ClO4

-

 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 ClO4

-

 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 ClO4

-

 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.
332.0-23

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 ClO4

-

 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:

9.3.8.2 If an LFSMD is analyzed instead of a LD, calculate the relative percent

difference (RPD) for duplicate concentrations of the LFSMs (LFSM and

LFSMD) using the equation:

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.

Concentration Range RPD Acceptance Criteria

< 2 X MRL <50%

> 2 X MRL to highest calibration

standard 

<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 ClO4

-

 at a concentration

>1/3 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
332.0-24

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 ClO4

-

. 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 ClO4

-

 by performing a simple experiment. 

Prepare a high CAL standard containing equal amounts of ClO4

-

 and the IS. While the

CAL standard is being infused, scan over the range of 95 - 115 amu and verify that the

ClO4

-

 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 ClO4

-

 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 ClO4

-

, 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
332.0-25

counts for ClO4

-

 at m/z 99 while minimizing in-source collisionally induced

dissociation with subsequent formation of ClO3

-

 (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 IC

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 μL 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 ClO4

-

. 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
332.0-26

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 ClO4

-

 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 (midlevel 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 may be 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.
332.0-27

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 ClO4

-

 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 IC should be allowed to operate until the conductivity of

the eluate from the conductivity suppressor stabilizes (<1 μS for the data presented in

this method), at which time it may be connected to the ESI/MS. It is recommended that

the IC-ESI/MS operate for approximately 30 minutes prior to the analysis of samples.

11.2.1 For some IC-ESI/MS instrumentation it may be necessary to use a second sixport 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.
332.0-28

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 μL of the 100 μg/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 μg Cl18O4

-

 /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 ClO4

-

 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 μL 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 (m/z 99, 101, and 107). 
332.0-29

11.5 COMPOUND IDENTIFICATION - Identification/confirmation of ClO4

-

 in a sample is

made by detecting ClO4

-

 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

Cl18O4

-

 IS has the same retention time as naturally occurring ClO4

-

, 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 ClO4

-

 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 ClO4

-

 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 ClO4

-

 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

ClO4

-

. 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.
332.0-30

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 μm 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 ClO4

-

 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.
332.0-31

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 μg/L ClO4

-

. Accuracy, as measured by percent recoveries of

fortified drinking water samples and external Quality Control samples, was 90 - 110%

for concentrations > 0.1 μg/L ClO4

-

.

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 μg/L ClO4

-

. 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 μg/L ClO4

-

.

13.2 Figure 3 shows chromatograms of a 0.1 μg/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 μg/L ClO4

-

 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 ClO4

-

 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/community/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
332.0-32

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.org/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.p2pays.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, D3370-82, “Standard Practice for

Sampling Water,” American Society for Testing and Materials, Philadelphia, PA, 1986.

8. Xu, J., Y. Song, B. Min, L. Steinberg, and B.E. Logan, “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.
332.0-33

17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA

TABLE 1. DIONEX IC-MS OPERATING CONDITIONS*

Ion Chromatograph Dionex Corporation, Sunnyvale, CA

Mobile Phase 75 mM KOH, or 65 mM KOH 1 

Guard and Separator Columns Dionex AG16 + AS16, 250 mm X 2 mm

Flow Rate 0.3 mL/min

Conductivity Suppressor and Current ASRS-MS, 75 mA, or 70 mA 1

Column Temperature 30 OC

Auxiliary Pump Flow Rate2

0.3 mL/min RW, or 50/50 v/v acetonitrile/water 1

Injection Volume 200 μL

Mass Spectrometer MSQ with Enhanced Low Mass Option (ELMO)

ThermoFinnigan, San Jose, CA

Ion Energy (V) 0.3

Low Mass Resolution 12.7

High Mass Resolution 12.5

Capillary Voltage -3 kV

Sampling Cone Voltage -70 V

Probe Temperature 400 oC, or 500 oC 1

Nitrogen pressure 80 psi

Selected Ion Monitoring m/z 99, 101, 107

Mass Scan Range 0.3 amu

Dwell Time per mass 0.75 sec, or 0.3 sec 1

Smoothing/Points/Range 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 ClO4

-

 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.
332.0-34

TABLE 2. METROHM-PEAK IC-MS OPERATING CONDITIONS*

Ion Chromatograph Metrohm-Peak, Houston, TX

Mobile Phase 30 mM NaOH/30% Methanol 

Guard and Separator Columns Metrohm ASUPP4/5 + ASUPP5-100, 100 mm X 4 mm

Flow Rate 0.7 mL/min

Suppressor Regenerant 60 mM nitric acid/10% Methanol, Rinse 10% Methanol

Column Temperature 30 OC

Injection Volume 100 μL

Mass Spectrometer 1100 Series MSD Quad SL, Agilent Technologies, Wilmington, DE

Low Mass Resolution 0.65

Capillary Voltage -2 kV

Nitrogen Pressure 80 psi

Fragmentor Voltage 150 V

Drying Gas Temperature 320 oC

Drying Gas Flow Rate 9 - 10 L/min

Selected Ion Monitoring m/z 99, 101, 107

Mass Scan Range 0.1 amu

Dwell Time Per Mass 0.25 sec

*Instrumentation, when specified, does not constitute endorsement. Brand names are included for

illustration only.
332.0-35

TABLE 3. SAMPLE ANALYSIS BATCH

Injection # Sample Description Acceptance Criteria Remedial Action

1 Mid-Level CCC 80 - 120 % recovery using

Initial Calibration

Instrument maintenance and

recalibration.

2 MRL CCC 50 - 150% recovery Instrument maintenance to

recover sensitivity and

recalibration.

3 LRB <1/3 MRL concentration Find and correct source of

contamination.

4 LFB

< MRL concentration

>MRL to highest CAL std

50 - 150% recovery

80 - 120% recovery

Identify and correct source of

problem.

5

.

.

14

Field Samples 1 - 10 Pass RT, m/z 99/101 area

count ratio, and IS area count

QC criteria at concentrations

>MRL concentration.

If problem is due to sulfate,

clean up sample using Ba form

cartridge, otherwise report.

15 CCC (rotating

concentrations)

80 - 120% recovery using

Initial Calibration for

concentrations > MRL

50-150% recovery for

concentrations < MRL

Instrument maintenance and

recalibration.

16 LFSM of a field sample

previously analyzed 

At fortification concentrations

> MRL concentration, 80-

120% recovery.

At fortification concentrations

< MRL, 50-150% of true

value.

If problem is due to sulfate,

clean up sample using Ba form

cartridge, otherwise report.

17 Laboratory Duplicate or a

LFSMD of field sample

previously analyzed. 

Choose LFSMD if

samples are low in

perchlorate.

RPD <20% for concentrations

> 2 X MRL

RPD <50% for samples < 2 X

MRL

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”.

18

.

.

27

Field Samples 11 - 20 Pass RT, m/z 99/101 area

count ratio, and IS area count

QC criteria.

If problem is due to sulfate,

clean up sample using Ba form

cartridge, otherwise report.

28 Final CCC (rotating

concentrations)

80 - 120 % recovery using

Initial Calibration for

concentration > MRL

Instrument maintenance and

recalibration.
332.0-36

TABLE 4. EXAMPLE TEMPLATE FOR TABULATION OF SAMPLE DATA FOR QC

REQUIREMENTS

Sample

ID

Ion

(m/z)

Area

Counts

Retention

Time (min)

Relative Retention

Time Ratio 

(m/z 99/107,

m/z 101/107)*

Area Count Ratio

(m/z 99/101)**

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)
332.0-37

TABLE 5. DETECTION LIMIT AND LCMRL FOR PERCHLORATE IN REAGENT

WATER

Concentration

(μg/L) - m/z 101

Detection Limit* 0.02

LCMRL 0.10

*Fortification concentration - 0.05 μg/L. 

 Seven replicates over three days.

TABLE 6. PRECISION AND RECOVERY DATA FOR PERCHLORATE IN VARIOUS

MATRICES (N=7)

Matrix Background

Conc. (μg/L)

Fortification

Conc. (μg/L)

m/z 99/101

Area Ratio

Avg. %

Recovery %RSD

Reagent Water ND1

0.05 2.77 105 14

0.50 3.05 102 3.6

LSSM ND

0.20 2.64 90 11

1.0 2.70 90 3.0

Surface Source Tap

Water

0.27 1.0 2.98 99 1.6

High TOC Surface

Source Tap Water2 ND

0.20 2.93 104 8.6

1.0 3.04 95 1.5

Ground Water <LCMRL

3

0.20 2.83 99 7.4

1.0 2.99 93 2.4

1 - Not detected.

2 - 15 mg/L total organic carbon.

3 - LCMRL = 0.10 μg/L
332.0-38

TABLE 7. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS 

Method

Reference

Requirement Specification and Frequency Acceptance Criteria

Section

9.2.1

Demonstration of Low System

Background

Analyze an LRB and LSSMB prior to any

other IDC steps and when modifications

are made. 

<1/3 MRL (Sect. 9.3.1)

Section

9.2.1.1

Concentration Dependent Carry-Over During IDC and when modifications are

made. Analyze a RW blank after the high

CAL during Initial Calibration.

Demonstrate that carry-over from one

injection to the next is <1/3 the MRL. 

Section 9.2.2 Demonstration of Precision in LFBs

and LFSSMs

Analyze 7 replicates fortified near midrange of calibration.

<20% RSD

Section 9.2.3 Demonstration of 

Accuracy in LFBs and LFSSMs 

Calculate the average recovery for

replicates in Section 9.2.2.

80 - 120% recovery at the mid-level

concentration

Section 9.2.4 Minimum Reporting Level (MRL)

Confirmation

Analyze 7 replicate LFBs at the target

MRL. Use the equation provided to

verify the MRL. Repeat after major

instrument or operational changes.

MRL acceptance based on project DQOs

or regulatory requirements.

Upper PIR < 150%

Lower PIR > 50% 
332.0-39

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 μm filter with the filtrate

collected in a sterile bottle.

Ship at < 10 oC 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 oC 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 <1/3 the

MRL (at both m/z 99 and m/z 101 at the RT for

ClO4

- ), and that possible interferences do not

prevent the identification and quantification of

ClO4

- 

.

Section 10.4 Continuing Calibration Check (CCC) With each Analysis Batch (20 field

samples), verify Initial Calibration by

analyzing a mid-level and an MRL CCC. 

Analyze a CCC after every 10 field

samples and after the last sample in the

Analysis Batch. Alternate between low

(<MRL), medium and high CAL

standards.

Mid-level CCC must be 

80 - 120% recovered.

MRL CCC must be 50 - 150% recovered.

The peak area of the IS must be within +30% of

the peak area of the first CCC of the Analysis

Batch (Sect. 10.4.1). 

Section

9.3.3

Laboratory Fortified Blank (LFB) With each Analysis Batch (20 field

samples).

50 - 150% rec. at conc.< MRL

80 - 120% rec. at conc. > 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. 
332.0-40

TABLE 8 (Continued). ON-GOING QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Reference

Requirement Specification and Frequency Acceptance Criteria

Section

9.3.5

Area Count Ratio (m/z 99/101)

Acceptance Criteria

In all standards, field samples, LFBs,

CCCs, etc., analyzed during each

Analysis Batch.

The calculated m/z 99/101 area count ratio

must be within + 25% (2.31 - 3.85).

Section

9.3.6

Relative Retention Time Acceptance

Criteria

In all standards, field samples, CCCs,

LFBs, LFSMs, etc.

Relative retention times of m/z 99/107 and

m/z 101/107 must be within 0.98 - 1.02.

Section

9.3.7

Laboratory Fortified Sample Matrix

(LFSM)

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.

Recoveries not within 80 - 120% of the

fortified amount at > MRL may indicate a

matrix effect. Conc. < MRL may be

recovered 50 - 150%.

Section

9.3.8

Laboratory Duplicate (LD) or

Laboratory Fortified Sample Matrix

Duplicate (LFSMD)

Analyze at least one LFSMD or LD with

each Analysis Batch of up to 20 field

samples.

<50% RPD at conc. <2 X MRL

<20% RPD at conc. > 2 X MRL

Section

10.3.4

Initial Calibration Verification Each time the Initial Calibration is

repeated or new standards are prepared,

analyze a QCS.

80 - 120% recovery of the certified

concentration at the mid-range of the

calibration. 
332.0-41

FIGURE 1. IC-ESI/MS Configuration Used to Generate Data in Method

FIGURE 2. Alternative IC-ESI/MS Configuration

* static mixing tee
332.0-42

FIGURE 3. MASS CHROMATOGRAM OF A STANDARD CONTAINING 0.1 μg/L ClO4

-

AND 1.0 μg/L INTERNAL STANDARD
332.0-43

FIGURE 4. MASS CHROMATOGRAM OF AN LFSSM CONTAINING 1.0 μg/L ClO4

-

 AND

1.0 μg/L INTERNAL STANDARD 
332.0-44

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 may be 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-ESI/MS 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 μg/L ClO4

-

. If, however, the upper range was extended to 5.0 or 10.0 μg/L, a weighted

linear regression was required (weight factor = 1/x) to achieve the same results. Using a nonweighted linear regression across the range 0.1-5.0 or 10.0 μg/L resulted in consistently high

recoveries (115-131%) for the 0.1 μg/L CAL standards. Choosing a short range over which

the variance is constant (e.g. 0.1-1.0 μg/L ClO4

-

), 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.
332.0-45

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 (μg/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 (μg/L) of ClO4

-

. 

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 A1). 

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 A1). 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. 
332.0-46

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(1-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, Inc,

Boston, MA.
332.0-47

APPENDIX A

TABLE A1. SAMPLE DATA COMPILATION AND DETERMINATION OF ACCURACY OF

CALIBRATION MODEL

A B C

1 X = Conc. 1

μg/L

Pred Xij 2

μg/L

%Recovery

2 0.1 0.1094 109

3 0.1 0.0926 92.6

4 0.5 0.4828 96.6

5 0.5 0.4609 92.2

6 1 1.0196 102

7 1 0.9882 98.8

8 5 5.0781 102

9 5 5.0889 102

10 10 10.155 102

12 10 10.349 104

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.
332.0-48

APPENDIX A

TABLE A2. SPREADSHEET TABULATION OF DATA TO DETERMINE F-TEST FOR LACK OF FIT

A B C D E F G

1

1X

2Y

4Pred Y

2 1/Xij Yij=(m/z 101/107)*(1/X) 3Mean Yj 4(Pred Yij)*(1/X) (MeanYj - PredYij)^2 (Yij-MEAN Yj)^2

3 10 0.409412140738561 0.3790897717 0.3753877 1.37053349457528E-005 0.000919446063506114

4 10 0.348767402681377 0.3790897717 0.3753877 1.37053349457528E-005 0.000919446063506114

5 2 0.35171017276265 0.3437878062 0.3640693 0.000411338989721146 6.27638915474167E-005

6 2 0.335865439688544 0.3437878062 0.3640693 0.000411338989721146 6.27638915474176E-005

7 1 0.369735548824696 0.3640767720 0.3626545 2.02285782420544E-006 3.20217544267144E-005

8 1 0.358417995303424 0.3640767720 0.3626545 2.02285782420544E-006 3.20217544267138E-005

9 0.2 0.367171010012092 0.3675596088 0.36152266 3.64447517258711E-005 1.51009076672299E-007

10 0.2 0.367948207738986 0.3675596088 0.36152266 3.64447517258711E-005 1.51009076672299E-007

11 0.1 0.366984493885163 0.3705014438 0.36138118 8.31792126127104E-005 1.23689370239679E-005

12 0.1 0.374018393805966 0.3705014438 0.36138118 8.31792126127104E-005 1.23689370239683E-005

13 5

SSLF = 0.00109338229365937 0.00205350331116177 =

6

SSPE

14 SSLF/c-2 = 0.000364460764553124 0.000410700662232354 =SSPE/n-c

15 7

F* = (SSLF/3)/(SSPE/5)= 0.887

1 X = concentration of CAL standard with weighting factor applied. Levels of X = 1 - j, replicates = 1 - i. NOTE: If not using a weighted regression, then X = concentration. 

2 Y = (Yij m/z 101/107area count ratio) times (the weighting factor, 1/X). NOTE: If not using a weighted regression, then Y = m/z 101/107 area count ratio.

3 Mean Yj = mean of Y for a given replicate level, i.

4 Pred Y = predicted Yij using the chosen regression model for a given X with weighting factor applied. NOTE: If not using a weighted regression, then Pred Y would not

 have the weighting factor applied. The weighted linear regression equation was y = 0.0014148 + 0.3612397 X.

5

SSLF = lack of fit sum of squares. Obtained by summing cells E3..E12. Degrees of freedom = c - 2 for 1st order polynomial. For this example, degrees of freedom for SSLF = 3. 

 NOTE: Degrees of freedom for a quadratic fit would be c - 3.

6

 SSPE = sum of squares pure error. Obtained by summing cells F3..F12. For this example, degrees of freedom for SSPE = 5.

7

F* = calculated F for the given regression model.