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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 

332.0-1

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 (ClO 4- ) 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 ClO 4- , 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 ClO 4- in reagent 

water was 0.02 µg/L (Table 5). 

1.6 The two predominant ClO 4 

- 

ions that occur naturally at a ratio of 3.086:1 are 35 Cl 16 O 4- , 

m/z 99, and 37 Cl 16 O 4- , m/z 101, respectively. 3 Due to fewer mass spectral interferences, 

the concentration of ClO 4 

- 

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


atio and relative retention time are used for confirmation of ClO 4 

- 

in samples. An 

oxygen-18 ( 18 O) enriched ClO 4 

- 

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 ClO 4 

- 

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 ClO 4- 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, ClO 4 

- 

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 18 O-enriched 35 Cl 18 O 4- 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 ClO 4- ions with 

respect to the internal standard. Qualitative confirmation of ClO 4- 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) 


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 ClO 4 

- 


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

chromatographic resolution between sulfate and ClO 4- . 

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 - , SO 4 

2- 

and CO 3 

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. 


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 ClO 4- 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, ClO 4- elutes later than the common 

inorganic anions (Cl - , SO 4 

2- 

, CO 3 

2- 

, and HCO 3- ). Separation of ClO 4 

- 

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 ClO 4- . 

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

concentration, direct chromatographic co-elution of a contaminant with ClO 4 

- 

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 ClO 4- , 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 ClO 4- . Any of these conditions could lead to a positive or negative bias of 

ClO 4 

- 

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 


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 (P 2 O 7 

4- 

), 

tripolyphosphate (P 3 O 10 

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 ClO 4 

- 

without 

compromising data quality. 

4.2.2 Concentration-Dependent Interference by Sulfate (SO 4 

2- 

): Of the common 

anions found in drinking waters (Cl - , SO 4 

2- 

, CO 3 

2- 

, HCO 3- ), sulfate can be the 

most problematic. Sulfate elutes before ClO 4 

- 

on most of the anion 

chromatography columns currently being used for ClO 4 

- 

analysis; however, it has 

a tendency to elute broadly, tailing into the retention time of ClO 4- . Formation of 

H 32 SO 4- (m/z 97) and H 34 SO 4- (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 ClO 4 

- 

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 ClO 4- (Sect. 9.3.5). As the column ages and the 

retention time of ClO 4 

- 

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 ClO 4- 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 ClO 4 

- 

(see Figures 1 and 2). This is 


ecause 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 ClO 4 

- 

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 

ClO 4 

- 

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 ClO 4 

- 

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 18 O enrichment of the internal standard may vary between standard 

manufacturers. Poor isotopic enrichment may lead to sample contamination by native 

Cl 16 O 4- (m/z 99) in the internal standard. Therefore, it must be demonstrated that the IS 

does not contain unlabeled ClO 4 

- 

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. 


5.2 Pure ClO 4 

- 

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 O C 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 


[(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 HSO 4 

- 

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 


ate (>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 ClO 4 

- 

(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 ClO 4 

- 

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 


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 Ba 2+ 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 Ba 2+ 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). 


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 - Na 2 SO 4 , 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 - Na 2 CO 3 , 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 O C or less when not in use. Even though stability times for standard 

solutions are suggested in the following sections, laboratories should use standard QC 

practices to determine when their standards need to be replaced. 

7.2.1 INTERNAL STANDARD STOCK STANDARD SOLUTION (IS-SSS) - 

1,000 mg Cl 18 O 4- /L. (NaCl 18 O 4 , F.W.=130.4, CASRN 7601-89-0, 90% enriched 

on 18 O, 98% pure NaCl 18 O 4 , or better, Isotec, Inc., Miamisburg, OH or 


equivalent,). A 1,000 mg/L solution of Cl 18 O 4 

- 

is prepared by dissolving 

0.0123 g NaCl 18 O 4 in 10 mL of RW. The solution may be stored in an HDPE 

screw-cap bottle (Sect. 6.3). The anhydrous NaCl 18 O 4 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 Cl 18 O 4- /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 Cl 18 O 4- /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 ClO 4- /L. 

(NaClO 4 , 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 ClO 4 

- 

is prepared by dissolving 0.1231 g of NaClO 4 in 

100 mL of RW. The solution may be stored in a HDPE screw-cap bottle 

(Sect. 6.3). The anhydrous NaClO 4 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 ClO 4- /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 ClO 4- /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. 


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 - , SO 4 

2- 

, CO 3 

2- 

. Add 1.48 g of Na 2 SO 4 (Sect. 7.1.5), 1.65 g of NaCl 

(Sect. 7.1.6) and 1.77 g of Na 2 CO 3 (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. 


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. 


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 

ClO 4- 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 


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 (HR PIR ) 

using the equation below: 

HR PIR = 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 + HR PIR ) meet the upper and lower recovery limits 

as shown below: 

The Upper PIR Limit must be < 150% recovery. 

Mean + HR PIR X 100 < 150% 

Fortified Concentration 

The Lower PIR Limit must be > 50% recovery. 

Mean - HR PIR X 100 > 50% 

Fortified Concentration 


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 


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 


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 ClO 4- . 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 

ClO 4 

- 

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 

Cl 18 O 4 

- 

IS has the same retention time as naturally occurring ClO 4- , 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 ClO 4 

- 

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 


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 ClO 4- concentrations < the 

MRL. At these concentrations, LFSM sample recovery should be 

50-150%. If the accuracy of ClO 4 

- 

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 ClO 4 

- 

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 ClO 4- 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. 


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 ClO 4 

- 

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 2 X MRL to highest calibration 

standard 

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 


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 ClO 4- . 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 ClO 4- by performing a simple experiment. 

Prepare a high CAL standard containing equal amounts of ClO 4- and the IS. While the 

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

ClO 4- 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 ClO 4 

- 

ions that have varying numbers 

of 18 O 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 ClO 4- , 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 


counts for ClO 4 

- 

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

dissociation with subsequent formation of ClO 3 

- 

(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 ClO 4- . 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 


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 ClO 4- 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. 


11. PROCEDURE 

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 ClO 4 

- 

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). 

11.1 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 (

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 Cl 18 O 4 

- 

/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 ClO 4 

- 

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). 


11.5 COMPOUND IDENTIFICATION - Identification/confirmation of ClO 4 

- 

in a sample is 

made by detecting ClO 4 

- 

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 

Cl 18 O 4 

- 

IS has the same retention time as naturally occurring ClO 4- , 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 ClO 4 

- 

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 ClO 4 

- 

(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 ClO 4 

- 

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 ClO 4- 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 

ClO 4- . 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. 


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 Ba 2+ 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 ClO 4- 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. 


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 ClO 4- . 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 ClO 4- . 

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 0.1 µg/L ClO 4- . 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 ClO 4- . 

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 ClO 4- 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 HSO 4 

- 

. 

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 ClO 4 

- 

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 


16. REFERENCES 

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. 

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,” 3 rd 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. 


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 O C 

Auxiliary Pump Flow Rate 2 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 o C, or 500 o C 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. 

2 

Auxiliary 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 ClO 4 

- 

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. 


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 O C 

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 o C 

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. 


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 


3 LRB 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) 

16 LFSM of a field sample 

previously analyzed 

17 Laboratory Duplicate or a 

LFSMD of field sample 

previously analyzed. 

Choose LFSMD if 

samples are low in 

perchlorate. 

80 - 120% recovery using 

Initial Calibration for 

concentrations > MRL 

50-150% recovery for 

concentrations < MRL 

At fortification concentrations 

> MRL concentration, 80- 

120% recovery. 

At fortification concentrations 

< MRL, 50-150% of true 

value. 

RPD 2 X MRL 

RPD MRL 

Instrument maintenance and 



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) 


TABLE 5. DETECTION LIMIT AND LCMRL FOR PERCHLORATE IN REAGENT 

WATER 

Detection Limit* 0.02 

LCMRL 0.10 

Concentration 

(µg/L) - m/z 101 

*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 ND 1 0.50 3.05 102 3.6 

0.05 2.77 105 14 

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 Water 2 

ND 

0.20 2.93 104 8.6 

1.0 3.04 95 1.5 

Ground Water

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. 

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


Reference 


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 o C to be received within 48 hours. 

Once received at the lab, samples should be 

analyzed as soon as possible. Sterile filtered 

samples must be stored with head space. Leave 

1/3 of bottle empty. Store at 6 o C or less. 

Section 

10.3 

Initial Calibration 

Use internal standardization calibration 

and a minimum of 5 calibration standards. 

Use peak area for calibration and 

quantitation. 

80 - 120% recovery of all reprocessed standards 

at > the MRL. 

50 - 150% recovery of reprocessed standards < 

the MRL. 

Section 

9.3.1 

Laboratory Reagent Blank (LRB) 

Analyze one LFB per Analysis Batch 

(every 20 field samples). 

Demonstrate that the target analyte is

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


Reference 


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. 

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

FIGURE 2. Alternative IC-ESI/MS Configuration 

* static mixing tee 


FIGURE 3. MASS CHROMATOGRAM OF A STANDARD CONTAINING 0.1 µg/L ClO 4 

- 

AND 1.0 µg/L INTERNAL STANDARD 


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

- 

AND 

1.0 µg/L INTERNAL STANDARD 


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 ClO 4- . 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 ClO 4- ), 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. 


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 ClO 4- . 

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. 


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 / DF LOF 

SSPE / DF PE 

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. 

DF LOF = degrees of freedom for SSLF. Equals c - 2 for 1 st order polynomial. 

Equals c - 3 for second order polynomial (quadratic). 

DF PE = degrees of freedom for SSPE. Equals n - c. 

c 

n 

= number of concentration levels. 

= 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 1 st 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. 


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. 


APPENDIX A 

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

1 1 X 

A B C D E F G 

2 1/Xij Yij=(m/z 101/107)*(1/X) 

2 Y 

4 Pred Y 

3 Mean 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 1 st 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.

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