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EPA/600/R-08/101 

METHOD 522 DETERMINATION OF 1,4-DIOXANE IN DRINKING WATER BY 

SOLID PHASE EXTRACTION (SPE) AND GAS CHROMATOGRAPHY/ 

MASS SPECTROMETRY (GC/MS) WITH SELECTED ION 

MONITORING (SIM)

Version 1.0 

September, 2008 

Jean W. Munch and Paul E. Grimmett 

 

NATIONAL EXPOSURE RESEARCH LABORATORY 

OFFICE OF RESEARCH AND DEVELOPMENT 

U. S. ENVIRONMENTAL PROTECTION AGENCY 

CINCINNATI, OHIO 45268 
 

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METHOD 522 

DETERMINATION OF 1,4-DIOXANE IN DRINKING WATER BY SOLID PHASE 

EXTRACTION (SPE) AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY 

(GC/MS) WITH SELECTED ION MONITORING (SIM)

1. SCOPE AND APPLICATION

1.1 This is a gas chromatography/mass spectrometry (GC/MS) method for the 

determination of 1,4-dioxane (CASRN 123-91-1) in drinking water. Accuracy and 

precision data have been generated in reagent water, finished ground and surface 

waters. Although this method was developed and demonstrated using selected ion 

monitoring (SIM) GC/MS for maximum sensitivity, it can also be used with full scan 

GC/MS if the sensitivity achieved meets the user’s data requirements. 

1.2 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets 

Data Quality Objectives (DQOs) that are developed based on the intended use of this 

method. Two single laboratory Lowest Concentration Minimum Reporting Levels 

(LCMRLs) of 0.036 and 0.047 μg/L have been determined in reagent water. The 

single laboratory LCMRL is the lowest true concentration for which the future 

recovery is predicted to fall, with high confidence (99%), between 50 and 150% 

recovery. The procedure used to determine the LCMRLs is described elsewhere.1

1.3 Laboratories using this method will not be required to determine the LCMRL for this 

method, but will need to demonstrate that their laboratory MRL for this method meets 

the requirements described in Section 9.2.4. 

1.4 Determining the Detection Limit (DL) for analytes in this method is optional (Sect. 

9.2.6). Detection limit 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 extraction efficiency, sample 

matrix, fortification concentration, and instrument performance. 

1.5 This method is intended for use only by analysts skilled in solid phase extraction 

(SPE), the operation of GC/MS instruments, and the interpretation of the associated 

data. 

1.6 METHOD FLEXIBILITY – In recognition of technological advances in analytical 

systems and techniques, the laboratory is permitted to modify the GC column, GC 

conditions and MS conditions (Sect. 9.4). Changes may not be made to sample 

collection and preservation (Sect. 8), the quality control (QC) requirements (Sect. 9), 

or to the sample extraction steps (Sect. 11). Method modifications should be 

considered only to improve method performance. Modifications that are introduced in 

the interest of reducing cost or sample processing time, but result in poorer method 

performance, should not be used. For example, modifications should not sacrifice 
 

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chromatographic separations in the interest of method turnaround time. Because the 

method analyte, surrogate analyte (SUR) and internal standard (IS) have only very low 

mass ions in their mass spectra, and SIM is being used, it is important to strive for 

chromatographic separation from potential interferences. In all cases where method 

modifications are proposed, the analyst must perform the procedures outlined in the 

initial demonstration of capability (IDC; Sect. 9.2), verify that all QC acceptance 

criteria in this method (Sect. 9) are met, and demonstrate that method performance can 

be verified in a real sample matrix (Sect. 9.3.7 and 9.4). 

NOTE: The above method flexibility section is intended as an abbreviated summation 

of method flexibility. Method Sections 4-12 provide detailed information of specific 

portions of the method that may be modified. If there is any perceived conflict 

between the general method flexibility statement in this section and specific 

information in Sections 4-12, Sections 4-12 supersede Section 1.6. 

2. SUMMARY OF METHOD

2.1 A water sample that has been dechlorinated and preserved with a microbial inhibitor is 

fortified with the isotopically labeled SUR, 1,4-dioxane-d8. The sample is extracted 

by one of two SPE options. In option 1, a 500-mL sample is passed through an SPE 

cartridge containing 2 g of coconut charcoal to extract the method analyte and SUR. 

In option 2, a 100-mL sample is extracted on a Waters AC-2 Sep-Pak or Supelco 

Supelclean ENVI-Carb Plus cartridge. In either option, the compounds are eluted 

from the solid phase with a small amount of dichloromethane (DCM), approximately 

9 mL or 1.5 mL, respectively. The extract volume is adjusted, and the IS, 

tetrahydrofuran-d8 (THF-d8), is added. Finally, the extract is dried with anhydrous 

sodium sulfate. Analysis of the extract is performed by GC/MS. The data provided in 

this method were collected using splitless injection with a high-resolution fused silica 

capillary GC column that was interfaced to an MS operated in the SIM mode. The 

analyte, SUR and IS are separated and identified by comparing the acquired mass 

spectra and retention times to reference spectra and retention times for calibration 

standards acquired under identical GC/MS conditions. The concentration of the 

analyte is determined by comparison to its response in calibration standards relative to 

the IS. Although the performance data presented in Section 17 of this method were 

obtained in the SIM mode for maximum method sensitivity, the sample extract 

analysis may also be performed in full scan mode if the sensitivity achieved meets the 

data user’s requirements. 

3. DEFINITIONS

3.1 ANALYSIS BATCH – A set of samples that is analyzed on the same instrument 

during a 24-hour period that begins and ends with the analysis of the appropriate 

Continuing Calibration Check (CCC) Standards. Additional CCCs may be required 

depending on the length of the analysis batch and/or the number of Field Samples. 
 

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3.2 CALIBRATION STANDARD (CAL) – A solution prepared from stock standard 

solution(s) and the ISs and SURs. The CAL solutions are used to calibrate the 

instrument response with respect to analyte concentration. 

3.3 CONTINUING CALIBRATION CHECK (CCC) STANDARD – A calibration 

standard containing the method analyte, IS and SUR which is analyzed periodically to 

verify the accuracy of the existing calibration for those analytes. 

3.4 DETECTION LIMIT (DL) – The minimum concentration of an analyte that can be 

identified, measured and reported with 99% confidence that the analyte concentration 

is greater than zero. This is a statistical determination (Sect. 9.2.6), and accurate 

quantitation is not expected at this level.2

3.5 EXTRACTION BATCH – A set of up to 20 Field Samples (not including QC 

samples) extracted together by the same person(s) during a work day using the same 

lot of SPE devices, solvents, SUR solution, and fortifying solutions. Required QC 

samples include Laboratory Reagent Blank (LRB), Laboratory Fortified Blank (LFB), 

Laboratory Fortified Sample Matrix (LFSM), and either a Field Duplicate (FD1 and 

FD2) or Laboratory Fortified Sample Matrix Duplicate (LFSMD). 

3.6 FIELD DUPLICATES (FD1 and FD2) – Two separate samples collected at the same 

time and place under identical circumstances, and treated exactly the same throughout 

field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the 

precision associated with sample collection, preservation, and storage, as well as with 

laboratory procedures. 

3.7 INTERNAL STANDARD (IS) – A pure analyte, which is extremely unlikely to be 

found in any sample, which is added to an extract or standard solution in a known 

amount and used to measure the relative responses of the method analyte and SUR. 

The IS must be an analyte that is not a sample component. 

3.8 LABORATORY FORTIFIED BLANK (LFB) – An aliquot of reagent water or other 

blank matrix to which known quantities of the method analyte and all the preservation 

compounds are added. The LFB is processed and analyzed exactly like a sample, and 

its purpose is to determine whether the methodology is in control, and whether the 

laboratory is capable of making accurate and precise measurements. 

3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – An aliquot of a Field 

Sample to which known quantities of the method analyte and all the preservation 

compounds 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 concentrations of the analyte in the sample matrix 

must be determined in a separate aliquot or duplicate sample and the measured values 

in the LFSM corrected for background concentrations. 
 

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3.10 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) – A Field 

Sample Duplicate of the Field Sample used to prepare the LFSM, which is fortified, 

extracted and analyzed identically to the LFSM. The LFSMD is used instead of the 

Field Duplicate to access method precision and accuracy when the occurrence of the 

method analyte at a concentration greater than the MRL is infrequent. 

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 glassware, 

equipment, solvents, reagents, sample preservatives, ISs, and SURs that are used in the 

extraction batch. The LRB is used to determine if the method analyte or other 

interferences are present in the laboratory environment, the reagents, or the apparatus. 

3.12 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) – The 

single-laboratory LCMRL is the lowest true concentration for which the future 

recovery is predicted to fall, with high confidence (99%), between 50 and 150% 

recovery.1

3.13 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. 

3.14 MINIMUM REPORTING LEVEL (MRL) – The minimum concentration that can be 

reported by a laboratory as a quantitated value for a target analyte in a sample 

following analysis. This defined concentration must meet the criteria defined in 

Section 9.2.4 and must not be any lower than the concentration of the lowest 

calibration standard for that analyte. A laboratory may be required to demonstrate a 

specific MRL by a regulatory body if this method is being performed for compliance 

purposes. 

3.15 QUALITY CONTROL SAMPLE (QCS) – A sample or standard prepared using a 

Stock Standard Solution (SSS) of the method analyte that is obtained from a source 

external to the laboratory and different from the source of calibration standards. The 

second source SSS is used to fortify the QCS at a known concentration. The QCS is 

used to check calibration standard integrity. 

3.16 SELECTED ION MONITORING (SIM) – A GC/MS technique where only one or a 

few ions are monitored. When used with gas chromatography, the set of ions 

monitored is usually changed periodically throughout the chromatographic run, to 

correlate with the characteristic ions of the analyte, SUR and IS as they elute from the 

chromatographic column. 

3.17 STOCK STANDARD SOLUTION (SSS) – A concentrated solution containing the 

method analyte prepared in the laboratory using assayed reference materials or 

purchased from a reputable commercial source. 
 

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3.18 SURROGATE ANALYTE (SUR) – A pure analyte, which is extremely unlikely to be 

found in any sample, and which is added to a sample aliquot in a known amount 

before extraction or other processing, and is measured with the same procedures used 

to measure other sample components. The purpose of the SUR is to monitor method 

performance with each sample. 

4. INTERFERENCES

4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap 

water, rinse with tap water, followed by reagent water. Non-volumetric glassware 

should be heated in a muffle furnace at 400 °C for 2 hours. Volumetric glassware 

should be solvent rinsed with DCM or purge and trap grade methanol after washing, 

and dried in a low temperature oven (<120 °C) or air dried. 

4.2 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 method analyte) under the conditions of the analysis by analyzing 

LRBs as described in Sections 9.2.1 and 9.3.1. Subtracting blank values from 

sample results is not permitted.

4.3 Purge and trap grade methanol must be used for all steps where methanol is used 

in this method. Other grades of methanol contain numerous low molecular 

weight compounds that contain interfering ions which may prohibit accurate 

identification and quantitation of the analyte, SUR and IS. 

4.4 Matrix interferences may be caused by contaminants that are co-extracted from the 

sample. The extent of matrix interferences will vary considerably from source to 

source, depending upon the nature of the water. 

4.5 Preservatives (Sect. 8.1) are added to samples to ensure sample stability during 

shipping and storage prior to analysis. The potential exists for trace-level organic 

contaminants in these reagents. Interferences from these sources should be monitored 

by analysis of LRBs, particularly when new lots of reagents are acquired. 

4.6 Solid phase extraction cartridges may be a source of interferences. The analysis of 

field and laboratory reagent blanks can provide important information regarding the 

presence or absence of such interferences. Brands and lots of SPE devices should be 

tested to ensure that contamination does not preclude analyte identification and 

quantitation. 

4.7 Analyte carry-over may occur when a relatively “clean” sample is analyzed 

immediately after a sample containing relatively high concentrations of compounds. 

Syringes and splitless injection port liners must be cleaned carefully or replaced as 

needed. After analysis of a sample containing high concentrations of compounds, a 
 

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LRB should be analyzed to ensure that accurate values are obtained for the next 

sample. During automated GC/MS analyses, extracts with positive results that were 

analyzed immediately following a sample with high concentrations of the analyte, 

should be reanalyzed after analyzing an acceptable LRB. If the analyte is not detected 

in extracts analyzed immediately after a high concentration extract, no reanalysis is 

necessary. 

4.8 Silicone compounds may be leached from punctured autosampler vial septa, 

particularly when particles of the septa sit in the vial. This can occur after repeated 

injections from the same autosampler vial. If this method is performed in full scan 

mode, these silicone compounds may appear as regularly spaced chromatographic 

peaks with similar fragmentation patterns. They can unnecessarily complicate the 

total ion chromatogram and may cause interferences at high levels. 

4.9 High laboratory background levels of 1,4-dioxane have been reported to be associated 

with air contamination. If 1,4-dioxane is detected in LRBs, room air should be 

considered as a possible source.3,4

5. SAFETY

5.1 The toxicity or carcinogenicity of each reagent used in this method has not been 

precisely defined. Each chemical should be treated as a potential health hazard, and 

exposure to these chemicals should be minimized. 1,4-Dioxane is classified as a class 

B2 or probable human carcinogen.5 Each laboratory is responsible for maintaining an 

awareness of OSHA regulations regarding safe handling of chemicals used in this 

method. A reference file of MSDSs should be made available to all personnel 

involved in the chemical analysis. Additional references on laboratory safety are 

available.6-8

5.2 Pure standard materials and stock standard solutions of these compounds should be 

handled with suitable protection to skin and eyes, and care should be taken not to 

breathe the vapors or ingest the materials. 

5.3 Sodium bisulfate is used as a sample preservative to inhibit microbial growth and 

potential decay of 1,4-dioxane. Sodium bisulfate is highly acidic and should be used 

with appropriate caution. 

6. EQUIPMENT AND SUPPLIES (References to specific brands or catalog numbers are 

included for illustration only, and do not imply endorsement of the product.) 

6.1 SAMPLE CONTAINERS – Glass bottles fitted with polytetrafluoroethylene (PTFE) 

lined screw caps. Although samples do not need to be collected headspace free, the 

size of the sample bottle should be selected based upon the sample volume to be 

extracted. 
 

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6.2 VIALS – Various sizes of glass vials with PTFE-lined screw caps for storing standard 

solutions and extracts, including glass 2-mL autosampler vials with PTFE-faced septa. 

6.3 VOLUMETRIC FLASKS – Class A, various sizes, including 1, 5, and 10 mL for 

preparation of standards. A 2-mL volumetric collection tube may also be used for the 

collection of the extract when SPE Option 2 is used (Sect. 11.5.3). 

6.4 GRADUATED CYLINDERS – Glass, various sizes, including 100 and 500 mL for 

measurement of sample volumes. 

6.5 MICRO SYRINGES – Suggested sizes include 10, 25, 50, 100, 250, 500, and 

1000 μL. 

6.6 CONICAL CENTRIFUGE TUBES – 15 mL, glass, with graduations at 0.1 mL and 

PTFE-lined screw caps (KIMAX #45166 or equivalent), suitable for collection of the 

eluent from the 2-g solid phase cartridge after extraction. 

6.7 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g. 

6.8 SOLID PHASE EXTRACTION (SPE) APPARATUS 

6.8.1 SPE CARTRIDGES – SPE carbon cartridges acceptable for use in this method 

are made of either coconut charcoal or synthetic carbon as described in Sections 

6.8.1.1 and 6.8.1.2 below. Graphitized carbon does not retain 1,4-dioxane and 

may not be used in this method. 

6.8.1.1 Option 1 (for use with sample volumes of ≤ 500 mL) – 6-mL polypropylene 

tubes packed with 2 g coconut charcoal (80-120 mesh, approximately 

150 μm). At the time of method development, pre-packed cartridges were 

obtained from Restek Corporation (cat. # 26032) and United Chemical 

Technologies (UCT; cat. # EU52112M6). Equivalent brands of coconut 

charcoal SPE cartridges with the specified particle size and whose 

performance meets all QC criteria in Section 9 may be used. 

6.8.1.2 Option 2 (for use with samples volumes of ≤ 100 mL) – Waters AC-2 SepPak 400-mg activated carbon SPE cartridge (cat. # JJAN20229; 85 μm 

particle size) or Supelco Supelclean ENVI-Carb Plus 400-mg synthetic 

carbon SPE cartridge (cat. # 54812-U). 

6.8.2 VACUUM EXTRACTION MANIFOLD – Equipped with flow/vacuum control 

(Supelco cat. # 57250-U or equivalent). 

6.8.3 SAMPLE DELIVERY SYSTEM – Use of a transfer tube system (Supelco 

“Visiprep”, cat. # 57275 or equivalent), which transfers the sample directly from 

the sample container to the SPE cartridge, is recommended. Sample reservoirs 
 

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(Varian, cat. #12131012 or equivalent), which attach to the cartridge, may be 

used, although they hold only a limited volume of sample. 

6.8.4 An automatic or robotic system designed for use with SPE cartridges may be used 

if all QC requirements discussed in Section 9 are met. Automated systems may 

use either vacuum or positive pressure to process samples and solvents through 

the cartridge. All extraction and elution steps must be the same as in the 

manual procedure. Extraction and/or elution steps may not be changed or 

omitted to accommodate the use of an automated system. 

6.9 LABORATORY OR ASPIRATOR VACUUM SYSTEM – Sufficient capacity to 

maintain a vacuum of approximately 15 to 25 inches of mercury. 

6.10 GAS CHROMATOGRAPH/MASS SPECTROMETER (GC/MS) SYSTEM 

6.10.1 FUSED SILICA CAPILLARY GC COLUMN – Data presented in this method 

were obtained with a column typically used for volatiles analysis: Varian CP 

Select 624, 30 m x 0.25-mm i.d. with a 1.4 μm film thickness, or equivalent. 

Other types of columns may be used. However, use of shorter or thinner film 

columns is not recommended because this will result in loss of resolution and 

very short retention times. Because the method analyte, SUR and IS have only 

very low mass ions in their spectra, and SIM is being used, it is important to strive 

for chromatographic separation from any potential interferences. See Section 9.4 

regarding the use of alternate GC columns. 

CAUTION: If a GC column and/or temperature program different than that 

described in this method is used, the analyst must verify that the temperature 

program and subsequent column bake-out is sufficient to remove all injected 

material from the column. Although the retention times of the method analyte, 

IS and SUR are short, many chemicals may be co-extracted by carbon SPE and 

these need to be eluted from the GC column to prevent them from appearing as 

interferences in subsequent analyses. 

6.10.2 GC INJECTOR AND OVEN – The performance data in Section 17 were obtained 

using hot, splitless injection. Other injection techniques such as temperature 

programmed injections, cold on-column injections and large volume injections 

may be used if the QC criteria in Section 9 are met. Equipment designed 

appropriately for these alternate types of injections must be used if these options 

are selected. 

6.10.3 GC/MS INTERFACE – The interface should allow the capillary column or 

transfer line exit to be placed within a few millimeters of the ion source. Other 

interfaces, such as jet separators, are acceptable as long as the system has 

adequate sensitivity and QC performance criteria in Section 9 are met. 
 

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6.10.4 MASS SPECTROMETER (MS) – Any type of mass spectrometer may be used 

(i.e., quadrupole, ion trap, time of flight, etc.), although the SIM option may not 

provide enhanced sensitivity, or be an available option on all instruments. The 

spectrometer must produce a mass spectrum that meets all criteria in Table 1 

when a solution containing approximately one to two nanograms of bromofluorobenzene (BFB) is injected into the GC/MS. This test must be performed in 

the full scan mode. Use a single spectrum at the apex of the BFB peak, an 

average spectrum of the three highest points of the peak, or an average spectrum 

across the entire peak to evaluate the performance of the system. Appropriate 

background subtraction is permitted. The scan time should be set so that there is a 

minimum of five scans across the chromatographic peak. Ten scans across 

chromatographic peaks are recommended. 

6.10.5 DATA SYSTEM – An interfaced data system is required to acquire, store, and 

output MS data. The computer software should have the capability of processing 

stored GC/MS data by recognizing a GC peak within a given retention time 

window. The software must allow integration of the ion abundance of any 

specific ion between specified times or scan number limits. The software must be 

able to construct linear regressions and quadratic calibration curves, and calculate 

analyte concentrations. 

6.11 N, N-DIETHYL-P-PHENYLENEDIAMINE (DPD) CHLORINE TEST KIT – Used 

to verify sample dechlorination when samples are received at the analytical laboratory 

(Hach model CN-66; cat. # 2231-01 or equivalent). 

7. REAGENTS AND STANDARDS

7.1 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used in 

all steps. Unless otherwise indicated, it is intended that all reagents will conform to 

the specifications of the Committee on Analytical Reagents of the American Chemical 

Society (ACS), where such specifications are available. Other grades may be used, 

provided it is first determined that the reagent is of sufficiently high purity to permit its 

use without lessening the quality of the determination. During method development, 

only purge and trap grade methanol was found to be sufficiently free from low 

molecular weight interferences (as described in Sect. 4.3). Therefore that grade must

be used. 

7.1.1 HELIUM – 99.999 % or better, GC carrier gas. 

7.1.2 REAGENT WATER – Purified water which does not contain any measurable 

quantities of the method analyte or interfering compounds at or above 1

/3 the 

MRL for 1,4-dioxane. In addition, there must be no interferences with the SUR 

or IS. 

7.1.3 METHANOL (CASRN 67-56-1) – Purge and trap grade only (see Section 4.3). 
 

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7.1.4 DICHLOROMETHANE (DCM) (CASRN 75-09-02) – High purity, demonstrated 

to be free of analytes and interferences (B&J Brand GC2, Capillary GC or GC/MS 

Grade or equivalent). 

7.1.5 SODIUM SULFATE, ANHYDROUS (CASRN 7757-82-6) – Soxhlet extracted 

with DCM for a minimum of four hours or heated to 400 °C for two hours in a 

muffle furnace. An “ACS grade, suitable for pesticide residue analysis”, or 

equivalent, of anhydrous sodium sulfate is recommended. 

7.1.6 SAMPLE PRESERVATION REAGENTS 

7.1.6.1 SODIUM SULFITE (CASRN 7757-83-7) – Reduces free and combined 

chlorine in samples that have been disinfected with chlorine and/or 

chloramine. 

7.1.6.2 SODIUM BISULFATE (CASRN 7681-38-1) – Anhydrous, technical grade. 

It is added to acidify the samples to pH < 4 to act as a microbial inhibitor 

during sample shipping and storage. 

7.2 STANDARD SOLUTIONS – 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 to develop this 

method and are included as examples. Alternate concentrations may be used as 

necessary depending on instrument sensitivity and the calibration range used. 

Standards for sample fortification generally should be prepared in the smallest volume 

that can be accurately measured to minimize the addition of excess organic solvent to 

aqueous samples. During method development, standard solutions were stored in a 

freezer at about -15 to -20 °C for several months. Experience indicates that the most 

likely cause of standard deterioration for this method is solvent evaporation. 

Therefore, it is recommended that standard solutions be stored at 0 °C or less, with 

minimal headspace. Laboratories should use standard QC practices to determine when 

standards need to be replaced. Standard QC practices, such as monitoring area counts 

and recovery percentages of CCCs, would help to determine if a standard requires 

replacement. As mentioned above, the major form of “standard deterioration” is 

solvent evaporation, which would result in a continuous increase in area counts, and in 

some cases, target and SUR recoveries. During method development, SSSs and 

dilutions of SSSs of 1,4-dioxane, THF-d8, 1,4-dioxane-d8, and BFB were valid for at 

least 6 months after opening/preparation. Expiration dates provided by the vendor 

should be used as an indicator of SSS shelf life prior to opening. 

NOTE: Only purge and trap grade methanol may be used in the preparation of 

standards and sample fortification solutions.

7.2.1 STOCK STANDARD SOLUTIONS (SSS) – Stock standard solutions of the 

method analyte, SUR and IS are commercially available from multiple 

commercial sources. It is recommended that these solutions be purchased from 
 

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commercial sources. However, they may be prepared from neat materials. 

Utilizing stock standards prepared in methanol allows the same stock standard to 

be used to prepare both calibration standards and sample fortification solutions. 

The concentrations of the stock standard solutions used during method 

development were 1000-2000 μg/mL. 

7.2.1.1 PREPARATION OF STOCK STANDARD SOLUTIONS (SSS) FROM 

NEAT MATERIALS 

7.2.1.1.1 Place about 9.8 mL of methanol in a 10-mL volumetric flask. Allow 

the flask to stand, unstoppered, for about 10 min or until all alcohol 

wetted surfaces have dried. Weigh to the nearest 0.1 mg. 

7.2.1.1.2 Using a 100 μL-syringe, quickly add two or more drops of the neat 

standard material to the flask. Be sure that the standard falls directly 

into the alcohol, without contacting the neck of the flask. 

7.2.1.1.3 Quickly reweigh, dilute to volume and mix by inverting the flask 

several times. Calculate the concentration in μg/μL from the net gain 

in weight. 

7.2.1.1.4 Store SSS in 12-15 mL glass vials with PTFE lined screw caps. 

7.2.2 CALIBRATION STANDARD SOLUTIONS (CAL) – Prepare a series of 

calibration standards to encompass the desired calibration range. Calibration 

standards must contain varying amounts of 1,4-dioxane, and a fixed amount of 

both the SUR and the IS, and be prepared in DCM. The number of standards 

required is determined by the calibration range. Three standards are required for 

one order of magnitude, six standards for two orders of magnitude and nine 

standards for three orders of magnitude. The calibration curve associated with the 

demonstration data in Section 17 contained nine standards with concentrations of 

1,4-dioxane ranging from 0.002 μg/mL to 1.0 μg/mL. (This corresponds to a 

concentration range of 0.04-20 μg/L in the drinking water samples.) The 

concentrations of both 1,4-dioxane-d8 (SUR) and the THF-d8 (IS) were 

0.500 μg/mL in each standard. 

7.2.3 SAMPLE FORTIFICATION SOLUTIONS 

 

7.2.3.1 ANALYTE FORTIFICATON SOLUTION – Prepare one or more solutions 

in methanol for use in preparing LFBs and LFSMs (Sect. 11.3.5). The 

number of solutions needed depends upon the calibration range and/or 

sample volume. During method development, two solutions at 

concentrations of 20 μg/mL and 200 μg/mL were used to fortify 100-mL 

and 500-mL samples, respectively. 
 

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7.2.3.2 SURROGATE ANALYTE (SUR) FORTIFICATION SOLUTION – 

Prepare one or more solutions of 1,4-dioxane-d8 in methanol to be used for 

fortification of the SUR into samples, LFBs, LRBs and LFSMs 

(Sect.11.3.4). The concentrations used during method development were 

200 μg/mL and 2000 μg/mL (STOCK) to fortify 100-mL and 500-mL 

samples, respectively. 

7.2.3.3 INTERNAL STANDARD (IS) FORTIFICATION SOLUTION – Prepare 

one or more solutions of THF-d8 in DCM to be used to add the IS to all 

extracts (Sect. 11.4.4 and 11.5.4). During method development, the 

concentrations of these solutions were 100 μg/mL and 1000 μg/mL 

(STOCK), used to fortify 2-mL and 10-mL extracts, respectively. 

7.2.4 GC/MS TUNE CHECK SOLUTION – Stock standard solutions of BFB (CASRN 

460-00-4) are available commercially. Prepare a BFB solution at a concentration 

of 1-2 μg/mL in DCM by dilution of the stock standard. Store this solution in an 

amber glass screw cap vial at 0 °C or less. To prepare a SSS of BFB from neat 

material, see Section 7.2.1.1. 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE PRESERVATIVES – Preservation reagents, listed in the table below, are 

added to each sample at the time of sample collection. Sodium sulfite must be added 

first and may be placed as a dry material in the sample bottles prior to shipment to the 

field. Aqueous solutions of sodium sulfite may not be added to sample bottles prior to 

shipment to the collection site because these solutions are unstable and cannot be 

relied upon to completely dechlorinate the samples. Sodium bisulfate is added only 

after the sodium sulfite has been dissolved in the aqueous sample. See Section 8.2.2 

and 8.2.3 for complete instructions. 

 

Compound Amount Purpose

Sodium sulfite 50 mg/L Reduce chlorine/chloramine residual 

Sodium bisulfate 1 g/L (approx) Microbial inhibitor

 

8.2 SAMPLE COLLECTION 

8.2.1 Open the tap and allow the system to flush until the water temperature has 

stabilized (approximately three to five min). Collect samples from the flowing 

system. 

8.2.2 Fill sample bottles, taking care not to flush out the sample dechlorination reagent. 

Samples do not need to be collected headspace free.

8.2.3 After collecting the sample, cap the bottle and agitate by hand until the sodium 

sulfite is dissolved. Add enough sodium bisulfate such that the final 
 

522-14 

concentration will be 1 g/L. Cap the bottle and mix until dissolved. Unless field 

verification of pH is to be performed, keep the sample sealed until just prior to 

extraction. 

8.2.4 Field verification of pH 4 (optional). It is anticipated that 1 g/L of sodium 

bisulfate will be sufficient to acidify most samples to < pH 4. If there is reason to 

suspect that more may be needed, the pH can be verified with narrow range pH 

paper at the time of sample collection. After acidification and mixing, pour a 

small amount of sample over a strip of the pH paper (do not dip the strip in the 

sample). Read the result as instructed on the pH paper package. If the pH is ≥ 4, 

add additional sodium bisulfate until pH < 4 is obtained. Seal the bottle, and keep 

the sample sealed until extraction. 

8.3 SAMPLE SHIPMENT AND STORAGE – Samples must be chilled during shipment 

and must not exceed 10 °C during the first 48 hours after collection. Sample 

temperature must be confirmed to be at or below 10 °C when they are received at the 

laboratory. Depending upon the water temperature at the time of collection, samples 

may need to be refrigerated to reduce their temperature before being packed on ice for 

shipment. Samples stored in the lab must be held at or below 6 °C until extraction, but 

should not be frozen. Freezing samples may compromise the sealed cap or result in 

sample bottle breakage. 

8.3.1 Verification of sample dechlorination – Upon the receipt of samples at the 

laboratory, verify that Field Samples were dechlorinated at the time of collection. 

The absence of total chlorine can be verified with a DPD chlorine test kit (Sect. 

6.11). 

8.4 SAMPLE AND EXTRACT HOLDING TIMES – Aqueous samples may be stored as 

described above for up to 28 days from collection. Sample extracts, as prepared in 

Section 11, may be stored at -5 °C and protected from light for an additional 28 days. 

9. QUALITY CONTROL

9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing 

QC requirements that must be met when preparing and analyzing Field Samples. This 

section describes each QC parameter, its required frequency, and the performance 

criteria that must be met in order to meet USEPA quality objectives. The QC criteria 

discussed in the following sections are summarized in Section 17, Tables 6 and 7. 

These QC requirements are considered the minimum acceptable QC criteria. 

Laboratories are encouraged to institute additional QC practices to meet their specific 

needs. If method modifications are under consideration by the laboratory, refer to 

Sect. 9.4 for detailed requirements. 

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) – The IDC must be 

successfully performed prior to analyzing any Field Samples. Prior to conducting the 
 

522-15 

IDC, the analyst must first generate an acceptable Initial Calibration following the 

procedure outlined in Section 10.2. 

9.2.1 INITIAL DEMONSTRATION OF LOW BACKGROUND – Before any samples 

are analyzed, or any time a new lot or brand of SPE materials or solvents are 

received from a supplier, it must be demonstrated that a LRB is reasonably free of 

any contamination that would prevent the identification or quantitation of 

1,4-dioxane, the IS or SUR. 

9.2.1.1 A source of potential contamination is the SPE materials which may contain 

phthalate esters, silicon compounds, and other contaminants that could 

interfere with the determination of the method analyte, SUR or IS. 

Although extraction media are generally made of inert materials, they may 

still contain extractable organic material. If the background contamination 

is sufficient to prevent accurate and precise measurements, the condition 

must be corrected before proceeding with the IDP (Sect. 9.2.2). 

9.2.1.2 Other sources of background contamination are solvents, reagents (including 

reagent water), room air and glassware. Background contamination must be 

reduced to an acceptable level before proceeding with the IDP (Sect. 9.2.2). 

Background from 1,4-dioxane and interferences must be ≤ 1/3 the MRL. If 

background contamination is an on-going or intermittent issue, the analyst 

must not attempt to verify an MRL less than the mean LRB concentration 

+ 3σ, or three times the mean LRB concentration, whichever is greater. 

 NOTE: Although quantitative data below the MRL may not be reliably 

accurate enough for data reporting, such data are useful in determining an 

MRL cut off for the method analyte if it is found in the LRB. Therefore, 

blank contamination levels may be estimated by extrapolation, when the 

concentration is below the lowest calibration standard. This also applies to 

estimating LRB concentrations for daily low background verification. 

9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) – Prepare, extract, and 

analyze four to seven replicate LFBs fortified near the midrange of the initial 

calibration curve according to the procedure described in Section 11. Sample 

preservatives as described in Section 8.1 must be added to these samples. The 

relative standard deviation (RSD) of the results of the replicate analyses must be 

less than 20%. 

9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) – Using the same set of 

replicate data generated for Section 9.2.2, calculate average recovery. The 

average recovery of the replicate values must be within ± 20% of the true value. 

9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION – Establish a target 

concentration for the MRL for 1,4-dioxane based on the intended use of the 

method. In some cases, the MRL may be dictated by USEPA or another 
 

522-16 

regulatory body. Establish an Initial Calibration following the procedure outlined 

in Section 10.2. The lowest calibration standard used to establish the Initial 

Calibration (as well as the low-level CCC) must be at or below the concentration 

of the MRL. Establishing the MRL concentration too low may cause repeated 

failure of ongoing QC requirements. Validate the MRL following the procedure 

outlined below. 

9.2.4.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL 

concentration. These LFBs must contain all method preservatives described 

in Section 8.1. Calculate the mean (Mean) and standard deviation for these 

replicates. Determine the Half Range for the prediction interval of results 

(HRPIR) using the equation below 

HR S PIR = 3963 .

where S is the standard deviation, and 3.963 is the constant value for seven 

replicates.1 

NOTE: The mass spectrum (either SIM or full scan) for the method analyte 

in the LFBs must meet all the analyte identification criteria in Sections 12.1 

and 12.2, i.e., the MRL verification may not be performed on LFBs where 

only the base peak is observed. If during MRL confirmation all 

identification ions are not observed, the MRL selected is too low. 

9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result 

(PIR = Mean + HRPIR) meet the upper and lower recovery limits as shown

below. 

 The Upper PIR Limit must be ≤ 150% recovery. 

×100% ≤ 150% +

FortifiedConcentration

Mean HRPIR

 The Lower PIR Limit must be ≥ 50% recovery. 

×100% ≥ 50% −

FortifiedConcentration

Mean HRPIR

9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the 

criteria described above (Sects. 9.2.4.2). If these criteria are not met, the 

MRL for 1,4-dioxane has been set too low and must be re-evaluated at a 

higher concentration. 

9.2.5 CALIBRATION CONFIRMATION – Analyze a QCS as described in Section 

9.3.9 to confirm the accuracy of the standards and calibration curve. 
 

522-17 

9.2.6 DETECTION LIMIT (DL) 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 upon the intended use of the data. 

Replicate analyses for this procedure should be done over at least three days (i.e., 

both the sample extraction and the GC analyses should be done over at least three 

days). Prepare at least seven replicate LFBs containing 1,4-dioxane at a 

concentration estimated to be near the DL. This concentration may be estimated 

by selecting a concentration at two to five times the noise level. The appropriate 

fortification concentrations will be dependent upon the sensitivity of the GC/MS 

system used. All preservation reagents listed in Section 8.1 must also be added to 

these samples. Analyze the seven replicates through all steps of Section 11. 

NOTE: If an MRL confirmation data set meets these requirements, a DL may be 

calculated from the MRL confirmation data, and no additional analyses are 

necessary. 

 Calculate the DL using the equation 

 

 ( − 1 ,1 − = 99.0 )= ×

n α

DL s t

 

 where: 

s = standard deviation of replicate analyses 

t (n-1, 1-α=0.99) = Student's t value for the 99% confidence level with n-1 

degrees of freedom 

n = number of replicates. 

NOTE: There are no precision and accuracy requirements for the DL replicates. 

Do not subtract blank values when performing DL calculations. 

9.3 ONGOING QC REQUIREMENTS – This section summarizes the ongoing QC 

criteria that must be followed when processing and analyzing Field Samples. 

9.3.1 LABORATORY REAGENT BLANK (LRB) – An LRB is required with each 

extraction batch to confirm that potential background contaminants are not 

interfering with the identification or quantitation of the method analyte, SUR or 

IS. If the LRB produces a peak within the retention time window of any analyte 

that would prevent the identification or quantitation of that analyte, determine the 

source of contamination and eliminate the interference before processing samples. 

Background contamination must be reduced to an acceptable level before 

proceeding. 
 

522-18 

NOTE: Absence of interferences must be verified for 1,4-dioxane at both the 

quantitation ion (QI) (m/z 88) and the confirmation ion (m/z 58). Background 

from contaminants that interfere with the measurement of 1,4-dioxane must be 

below 1/3 of the MRL. Blank contamination may be estimated by extrapolation, if 

the concentration is below the lowest calibration standard. This procedure is not 

allowed for sample results as it may not meet data quality objectives. If the 

method analyte is detected in the LRB at concentrations equal to or greater than 

this level, then all data for the analyte must be considered invalid for all samples 

in the extraction batch. 

9.3.2 CONTINUING CALIBRATION CHECK (CCC) – CCC Standards are analyzed 

at the beginning of each analysis batch, after every ten Field Samples, and at the 

end of the analysis batch. See Section 10.3 for concentration requirements and 

acceptance criteria. 

9.3.3 LABORATORY FORTIFIED BLANK (LFB) – An LFB is required with each 

extraction 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, but no more than two times the 

MRL. Similarly, the high concentration LFB should be near the high end of the 

calibration range established during the initial calibration (Sect. 10.2). Results of 

the low-level LFB analyses must be 50-150% of the true value. Results of the 

medium and high-level LFB analyses must be 70-130% of the true value. If the 

LFB results do not meet these criteria for 1,4-dioxane, then all data must be 

considered invalid for all samples in the extraction batch. 

9.3.4 MS TUNE CHECK – A complete description of the MS Tune Check is found in 

Section 10.2.1. Acceptance criteria for the MS Tune Check are summarized in 

Section 17, Table 1. The MS Tune Check must be performed each time a major 

change is made to the mass spectrometer, and prior to establishing and/or reestablishing an initial calibration (Sect.10.2). In this method, daily BFB analysis 

is not required. 

9.3.5 INTERNAL STANDARDS (IS) – The analyst must monitor the peak area of the 

IS in all injections during each analysis day. The IS response (peak area) in any 

chromatographic run must not deviate from the response in the most recent CCC 

by more than 30%, and must not deviate by more than 50% from the mean area 

measured during initial analyte calibration. If the IS area in a chromatographic 

run does not meet these criteria, inject a second aliquot of that extract. 

9.3.5.1 If the reinjected aliquot produces an acceptable IS response, report results 

for that aliquot. 

9.3.5.2 If the reinjected extract fails again, the analyst should check the calibration 

by reanalyzing the most recently acceptable calibration standard. If the 

calibration standard fails the criteria of Section 10.3, recalibration is in order 
 

522-19 

per Section 10.2. If the calibration standard is acceptable, extraction of the 

sample may need to be repeated provided the sample is still within the 

holding time. Otherwise, report results obtained from the reinjected extract, 

but annotate as “suspect/IS recovery.” Alternatively, collect a new sample 

and re-analyze. 

9.3.6 SURROGATE (SUR) RECOVERY – The SUR standard is fortified into the 

aqueous portion of all samples, LRBs, LFBs, CCCs, LFSMs and LFSMDs prior 

to extraction. It is also added to the calibration standards. The SUR is a means of 

assessing method performance from extraction to final chromatographic 

measurement. Calculate the percent recovery (%R) for the SUR using the 

equation 

%  ×100 









=

B

A

R

where: 

A = calculated SUR concentration for the QC or Field Sample 

 B = fortified concentration of the SUR. 

9.3.6.1 SUR recovery must be in the range of 70-130% of the true value. When 

SUR recovery is less than 70% or greater than 130%, check the 1) 

calculations to locate possible errors, 2) standard solutions for degradation, 

3) contamination, and 4) instrument performance. Correct the problem and 

reanalyze the extract. 

9.3.6.2 If the extract reanalysis meets the SUR recovery criterion, report only data 

for the reanalyzed extract. 

9.3.6.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst 

should check the calibration by injecting the last CCC that passed. If the 

CCC fails the criteria of Section 9.3.6.1, recalibration is in order per Section 

10.2. If the calibration standard is acceptable, extraction of the sample 

should be repeated provided the sample is still within the holding time. If 

the re-extracted sample also fails the recovery criterion, report all data for 

that sample as “suspect/SUR recovery” to inform the data user that the 

results are suspect due to SUR recovery. Alternatively, collect a new 

sample and re-analyze. 

9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) – Analysis of an 

LFSM is required in each extraction batch and is used to determine that the 

sample matrix does not adversely affect method accuracy. If the occurrence of 

1,4-dioxane in the samples is infrequent, or if historical trends are unavailable, a 

LFSMD must be prepared, extracted, and analyzed from a duplicate Field Sample 

to assess method precision (Sect. 9.3.8). Extraction batches that contain LFSMDs 

do not require the analysis of a Field Duplicate. If a variety of different sample 
 

522-20 

matrices are analyzed regularly, for example, drinking water from groundwater 

and surface water sources, method performance should be established for each. 

Over time, LFSM data should be documented by the laboratory for all routine 

sample sources analyzed. 

9.3.7.1 Within each extraction batch, a minimum of one Field Sample is fortified as 

an LFSM for every 20 samples extracted. The LFSM is prepared by spiking 

a sample with an appropriate amount of the Analyte Fortification Solution 

(Sect. 7.2.3.1). Select a spiking concentration that is greater than or equal to 

the matrix background concentration, if known. Use historical data and 

rotate through low, medium and high concentrations when selecting a 

fortifying concentration. 

9.3.7.2 Calculate the percent recovery (%R) for the analyte using the equation 

( ) % ×100 −

=

C

A B

R

where: 

A = measured concentration in the fortified sample 

 B = measured concentration in the unfortified sample

 C = fortification concentration. 

NOTE: Field Samples that have native analyte concentrations above the 

DL but below the MRL, and are fortified at concentrations at or near the 

MRL, should be corrected for the native levels in order to obtain 

meaningful %R values. This example and the LRB (Sect. 9.3.1) are the only 

permitted use of analyte results below the MRL. 

9.3.7.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or 

above their native concentration, recoveries should range between 70-130%, 

except for low-level fortification near or at the MRL (within a factor of two 

times the MRL concentration) where 50-150% recoveries are acceptable. If 

the accuracy of any analyte falls outside the designated range, and the 

laboratory performance for that analyte is shown to be in control in the 

CCCs, the recovery is judged to be matrix biased. The result for that analyte 

in the unfortified sample is labeled “suspect/matrix” to inform the data user 

that the results are suspect due to matrix effects.

9.3.8 FIELD DUPLICATE (FD) OR LABORATORY FORTIFIED SAMPLE 

MATRIX DUPLICATE (LFSMD) – Within each extraction batch, a minimum of 

one Field Duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate 

(LFSMD) must be analyzed. Duplicates check the precision associated with 

sample collection, preservation, storage, and laboratory procedures. If method 

analytes are not routinely observed in Field Samples, an LFSMD should be 

analyzed rather than an FD. 
 

522-21 

9.3.8.1 Calculate the relative percent difference (RPD) for duplicate FD 

measurements (FD1 and FD2) using the equation 

( ) 1001 2 2/

1 2

×

+

−

=

FD FD

FD FD

RPD

9.3.8.2 RPDs for FDs should be ≤ 30%. Greater variability may be observed when 

FDs have analyte concentrations that are within a factor of two of the MRL. 

At these concentrations, FDs should have RPDs that are ≤ 50%. If the RPD 

of the analyte falls outside the designated range, and the laboratory 

performance for the analyte is shown to be in control in the CCC, the 

recovery is judged to be matrix biased. The result for that analyte in the 

unfortified sample is labeled “suspect/matrix” to inform the data user that 

the results are suspect due to matrix effects. Failure to meet the FD criteria 

is a reflection of the performance of the method for that individual sample, 

and does not constitute analysis or extraction batch failure. 

9.3.8.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent 

difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the 

equation 

( ) 1002/

×

+

−

=

LFSM LFSMD

LFSM LFSMD

RPD

9.3.8.4 RPDs for duplicate LFSMs should be ≤ 30% for samples fortified at or 

above their native concentration. Greater variability may be observed when 

LFSMs are fortified at analyte concentrations that are within a factor of two 

of the MRL. LFSMs fortified at these concentrations should have RPDs 

that are ≤ 50% for samples fortified at or above their native concentration. 

If the RPD of the analyte falls outside the designated range, and the 

laboratory performance for the analyte is shown to be in control in the CCC, 

the recovery is judged to be matrix biased. The result for that analyte in the 

unfortified sample is labeled “suspect/matrix” to inform the data user that 

the results are suspect due to matrix effects. Failure to meet the 

LFSM/LFSMD criteria is a reflection of the performance of the method for 

that individual sample, and does not constitute analysis or extraction batch 

failure. 

9.3.9 QUALITY CONTROL SAMPLES (QCS) – As part of the IDC (Sect. 9.2), each 

time new calibration standards are prepared, and at least quarterly, analyze a QCS 

sample from a source different from the source of the calibration standards. The 

QCS should be prepared and analyzed just like a CCC (Sect. 10.3). The 

calculated amount for the analyte in the QCS must be ± 20% of the expected 

value. If measured analyte concentrations are not of acceptable accuracy, check 
 

522-22 

the entire analytical procedure to locate and correct the problem, or obtain another 

QCS. 

9.4 METHOD MODIFICATION QC REQUIREMENTS – The analyst is permitted to 

modify the GC injection technique, GC column and conditions, and MS conditions. 

9.4.1 Each time method modifications are made, the analyst must repeat the procedures 

of the IDC (Sect. 9.2) and verify that all QC criteria can be met in ongoing QC 

samples (Sect. 9.3). 

9.4.2 The analyst is also required to evaluate and document method performance for the 

proposed method modifications in real matrices that span the range of waters that 

the laboratory analyzes. This additional step is required because modifications 

that perform acceptably in the IDC, which is conducted in reagent water, can fail 

ongoing method QC requirements in real matrices. This is particularly important 

for methods subject to matrix effects. If, for example, the laboratory analyzes 

finished waters from both surface and groundwater municipalities, this 

requirement can be accomplished by assessing precision and accuracy (Sects. 

9.2.2 and 9.2.3) in a surface water with moderate to high Total Organic Carbon 

(TOC) (e.g., 2 mg/L or greater) and a hard groundwater (e.g., 250 mg/L or greater 

as calcium carbonate). 

9.4.3 The results of Sections 9.4.1 and 9.4.2 must be appropriately documented by the 

analyst and should be independently assessed by the laboratory’s Quality 

Assurance (QA) officer prior to analyzing Field Samples. 

9.4.3.1 When implementing method modifications, it is the responsibility of the 

laboratory to closely review the results of ongoing QC, and in particular, the 

results associated with the LFBs (Sect. 9.3.3), LFSMs (Sect. 9.3.7), FDs or 

LFSMDs (Sect. 9.3.8), CCCs (Sect. 9.3.2), and the IS area counts (Sect. 

9.3.5). If repeated failures are noted, the modification must be abandoned. 

10. CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of acceptable mass spectrometer (MS) tune and 

initial calibration is required before any samples are analyzed. After the initial 

calibration is successful, a CCC is required at the beginning and end of each period in 

which analyses are performed, and after every tenth Field Sample. Verification of 

mass spectrometer tune must be repeated each time a major instrument modification is 

made or maintenance is performed, and prior to establishing or re-establishing an 

initial calibration. 

10.2 INITIAL CALIBRATION 

10.2.1 MS TUNE/MS TUNE CHECK – Operate the MS in the electron ionization (EI) 

mode. Calibrate the mass and abundance scales of the MS with calibration 
 

522-23 

compounds and procedures prescribed by the manufacturer with any 

modifications necessary to meet tuning requirements. Inject 2 ng or less of the 

BFB solution (Sect. 7.2.4) into the GC/MS system. Acquire a mass spectrum that 

includes data for m/z 45 to 180. The GC and MS parameters must be set such that 

a minimum of five scans (10 scans are recommended) are obtained during the 

elution of the BFB chromatographic peak Use a single spectrum of the BFB 

peak, an average spectrum of the three highest points of the peak, or an average 

spectrum across the entire peak to evaluate the performance of the system. 

Appropriate background subtraction is permitted. If the BFB mass spectrum does 

not meet all criteria in Table 1, the MS must be re-tuned and adjusted to meet all 

criteria before proceeding with the initial calibration. 

10.2.2 ANALYTE CALIBRATION – Operating conditions used during method 

development are described below. Data were obtained using SIM on a 

quadrupole MS system using the GC column described in Section 6.10.1. 

Conditions different from those described may be used if QC criteria in Section 9 

are met. Different conditions include alternate GC columns, temperature 

programs, MS conditions, and injection techniques and volume, such as cold oncolumn and direct injection port liners and/or large volume injection techniques. 

Equipment specifically designed for alternate types of injections must be used if 

these options are selected. Full scan MS data may be used if the sensitivity 

achieved meets the data user’s needs. 

10.2.2.1 GAS CHROMATOGRAPH (GC) CONDITIONS – Inject a 1-μL aliquot of 

each CAL prepared as in Section 7.2.2 into a hot, splitless injection port 

held at 200 EC with a split delay of 0.5 min. Temperature program the GC 

as follows: initial oven temperature of 30 EC, hold for 1.0 min, ramp at 

7 EC/min to 90 EC, ramp at 20 EC/min to a final temperature of 200 EC and 

hold for 3.0 min, for a total run time of 18.07 min. Begin data acquisition at 

6.0 min. During method development, the GC was operated in a constant 

flow rate mode at a rate of 1.0 mL per min. The MS ion source and the GC 

injector were both maintained at 200 EC. 

 

NOTE: Using these conditions, all compounds of interest eluted before 

9 min. However, it is important to complete the GC temperature program to 

ensure that co-extracted materials have eluted from the GC column prior to 

initiating the next analysis. 

10.2.2.2 MASS SPECTROMETER (MS) CONDITIONS – Any type of MS may be 

used as detailed in Sect. 6.10.4. Data shown in Section 17 (Tables 2-5) were 

obtained in the SIM mode. Operation of the MS in this mode enhances 

sensitivity. However, less mass spectral data are obtained for all peaks 

including the method analyte, SUR, IS and any potential interferences. 

Because of this, and because the selected ions for the compounds of interest 

are very low masses that are likely to occur more frequently in interferences 

than most higher mass ions, the analyst should also rely on chromatography 
 

522-24 

to reduce the possibility of false positives. It is highly recommended that a 

GC column at least 30 m in length with a film thickness of at least 1.4 μm 

be used to provide adequate separation of the compounds of interest from 

possible interferences. During method development, the MS was scanned in 

SIM mode for m/z 46, 78, and 80 (IS) at a rate of 0.36 s/scan in Segment 1 

(6 to 8 min). Segment 2 (8 to 18 min) was set to scan for m/z 58 and 88 

(TARGET), as well as 62, 64, and 96 (SUR) at 0.60 s/scan, resulting in at 

least nine scans across each chromatographic peak. A minimum of five 

scans during the elution of each GC peak are required. Ten scans across 

each GC peak are recommended. This requirement is applicable to both full 

scan and SIM analysis. Timing of the SIM segments must be set such that 

no chromatographic peak of interest begins or ends within 5 s of the 

beginning or end of the segment. An example chromatogram obtained 

during method development using the instrumental conditions described in 

this section is shown in Figure 1. 

10.2.3 CALIBRATION CURVE – Use the GC/MS software to create a calibration curve 

for 1,4-dioxane using the IS technique. Concentrations may be calculated through 

the use of a linear or quadratic calibration curve. A weighted curve is permitted at 

the discretion of the analyst. Because the SUR is added to all samples and 

standards at a single concentration, calibrate for the SUR using the average 

response factor. 

10.2.4 CALIBRATION ACCEPTANCE CRITERIA – When quantitated using the 

calibration curve, each calibration point, except the lowest point, should calculate 

to be within 80-120% of its true value. The lowest point should calculate to be 

within 60-140% of its true value. If these criteria cannot be met, the analyst will 

have difficulty meeting ongoing QC criteria. It is recommended that corrective 

action be taken to re-analyze the CALs, restrict the range of calibration, or select 

an alternate method of calibration. 

10.3 CONTINUING CALIBRATION CHECK (CCC) – The CCC verifies the initial 

calibration at the beginning and end of each group of analyses, and after every tenth 

sample during analyses. In this context, a “sample” is considered to be a Field 

Sample. The LRBs, LFBs, LFSMs, LFSMDs, FDs and CCCs are not counted as 

samples. The beginning CCC for each analysis batch must be at or below the MRL in 

order to verify instrument sensitivity prior to any analyses. Subsequent CCCs must 

alternate between medium and high concentration CALs. 

10.3.1 Inject an aliquot of the appropriate concentration CAL solution and analyze with 

the same conditions used during the initial calibration. 

10.3.2 Determine that the absolute area of the QI of the IS has not changed by more than 

± 50% from the average area measured during initial calibration, or more than 

± 30% from the most recent CCC. If the IS area has changed by more than this 
 

522-25 

amount, remedial action must be taken (Sect. 10.3.4). Control charts are useful 

aids in documenting system sensitivity changes. 

10.3.3 Calculate the concentration of 1,4-dioxane and the SUR in the check standard. 

The calculated amount for 1,4-dioxane for medium and high level CCCs must be 

± 30% of the true value. The calculated amount for the lowest calibration level 

for 1,4-dioxane, which must be at a concentration less than or equal to the MRL, 

must be within ± 50% of the true value. If these criteria are not met, then all data 

for the problem analyte must be considered invalid, and remedial action 

(Sect. 10.3.4) should be taken. This may require recalibration. Any Field Sample 

extracts that have been analyzed since the last acceptable calibration verification 

should be re-analyzed after adequate calibration has been restored, with the 

following exception: if the CCC fails at the end of an analytical sequence because 

the calculated concentration of the target compound or SUR is greater than 130% 

(150% for the low-level CCC), and Field Sample extracts show no detection for 

the target compound, non-detects may be reported without re-analysis. 

10.3.4 REMEDIAL ACTION – Failure to meet CCC QC performance criteria may 

require remedial action. Major maintenance such as cleaning the ion source, 

cleaning the mass analyzer, replacing filament assemblies, replacing the GC 

column, etc., require returning to the initial calibration step (Sect. 10.2). 

11. PROCEDURE

11.1 This procedure may be performed manually or in an automated mode (Sect. 6.8.4) 

using a robotic or automatic sample preparation device. If an automatic system is used 

to prepare samples, follow the manufacturer's operating instructions, but all extraction 

and elution steps must be the same as in the manual procedure. Extraction and/or 

elution steps may not be changed or omitted to accommodate the use of an 

automated system.

 

11.2 Important aspects of this analytical procedure include proper preparation of laboratory 

glassware and sample containers (Sect. 4.1), as well as sample collection and storage 

(Sect. 8). This section details the procedures for sample preparation, SPE using 2-g 

coconut charcoal or 0.4-g carbon cartridges as described in Sect. 6.8.1, and extract 

analysis. 

 NOTE: The SPE cartridges and sodium sulfate drying materials described in this 

section are designed as single use items and must be discarded after use. They may 

not be refurbished for re-use in subsequent analyses. 

 NOTE: Only purge and trap grade methanol may be used in this extraction (see 

Sect. 4.3).

 
 

522-26 

11.3 SAMPLE PREPARATION 

11.3.1 All Field and QC samples (LRBs and LFBs) must contain the dechlorinating 

agent, sodium sulfite. Verify that Field Samples were checked for total chlorine 

upon sample receipt at the laboratory, and that no chlorine was present (Sect. 

8.3.1). Add sodium sulfite to all LRBs and LFBs according to the instructions in 

Section 8, prior to fortification with the SUR and analyte. 

 11.3.2 All Field and QC samples (LRBs and LFBs), must contain the anti-microbial 

agent sodium bisulfate. Verify that Field Samples were acidified to < pH 4 at the 

time of collection by checking with narrow range pH paper (this verification may 

take place at sample receipt if preferred by the laboratory). Add sodium bisulfate 

to all LRBs and LFBs according to the instructions in Section 8, prior to 

fortification with the SUR and analyte. 

 11.3.3 If the sample bottles used contain a sample volume close to the sample volume to 

be extracted (± 10%), mark the sample level on the outside of the sample 

container for subsequent measurement of the sample volume. After the sample 

has been extracted, determine the exact sample volume by filling the bottle to the 

mark with water and transferring to an appropriately sized graduated cylinder. 

Measure to within ± 5 mL for 100 mL samples, and to within ± 25 mL for 

500-mL samples. If a much larger sample has been collected than will be 

extracted, transfer either 100 mL or 500 mL of the sample to a clean glass 

container with a clean graduated cylinder. The sample volume will depend upon 

the SPE being used (Sects. 11.4, 11.5). 

 Alternatively, the sample volume may be calculated by weighing the sample 

bottle before and after extraction (or sample transfer) and using a density of 

1.0 g/mL. 

11.3.4 ADDITION OF SURROGATE (SUR) ANALYTE – Add an aliquot of the 

Surrogate Analyte Fortification Solution (Sect. 7.2.3.2) to all samples and mix by 

swirling the sample. Addition of 2.5 μL of a 2000-μg/mL solution to a 500-mL 

sample will result in a concentration of 10 μg/L. Addition of 5 μL of a 

200-μg/mL solution to a 100-mL sample will also result in a concentration of 

10 μg/L. 

11.3.5 FORTIFICATION WITH METHOD ANALYTE – If the sample is an LFB, 

LFSM, or LFSMD, add the necessary amount of Analyte Fortification Solution 

(Sect. 7.2.3.1). Swirl each sample to ensure all components are properly mixed. 

Addition of 2.5 μL of a 200-μg/mL solution to a 500-mL sample will result in a 

concentration of 1 μg/L. Addition of 5 μL of a 20-μg/mL solution to a 100-mL 

sample will also result in a concentration of 1 μg/L. 

11.3.6 Proceed with sample extraction Option 1 (Sect. 11.4) or Option 2 (Sect. 11.5). 
 

522-27 

11.4 SPE PROCEDURE OPTION 1; EXTRACTION OF 500-ML SAMPLES – Proper 

conditioning of the solid phase sorbent can have a marked effect on method precision 

and accuracy. This section describes the SPE procedure using 2-g coconut charcoal 

cartridges and the transfer tube system and SPE manifold as described in Section 6.8. 

 

11.4.1 CARTRIDGE CONDITIONING 

11.4.1.1 Fill the cartridge with approximately 3 mL of DCM, turn on the vacuum, 

and pull the solvent through, aspirating completely. 

11.4.1.2 Fill the cartridge with approximately 3 mL of methanol, turn on the vacuum, 

and pull the solvent through, aspirating completely. 

11.4.1.3 Fill the cartridge with approximately 3 mL of methanol and elute with 

vacuum to just above the top frit - not allowing the cartridge to go dry at the 

end. From this point forward, do not allow the cartridge to go dry. 

11.4.1.4 Fill the cartridge with approximately 3 mL of reagent water, turn on the 

vacuum, and pull the water through, repeat five times, without allowing the 

cartridge to go dry in between washes or at the end. 

11.4.2 SAMPLE EXTRACTION – Attach a transfer tube from each sample bottle to 

each cartridge and then turn on the vacuum. Adjust the vacuum so that the 

approximate flow rate is 10 mL/min. After all the sample has passed through 

each SPE cartridge, detach the transfer tube and draw air through the cartridge for 

10 min at full vacuum. Turn off and release the vacuum. Proceed immediately 

with cartridge elution. 

11.4.3 CARTRIDGE ELUTION – Lift the extraction manifold top and insert a rack with 

collection tubes into the vacuum manifold tank to collect the extracts as they are 

eluted from the cartridges. Fill each cartridge with DCM. Pull enough of the 

solvent into the cartridge at low vacuum to soak the sorbent. Turn off the vacuum 

and vent the system. Allow the sorbent to soak in DCM for approximately 1 min. 

Apply a low vacuum and pull the DCM through the cartridge in a dropwise 

fashion into the collection tube. Continue to add DCM to the cartridge as it is 

being drawn through until the volume of extract is about 9 mL, determined by the 

markings on the side of the collection tube. 

11.4.4 Remove collection tubes containing the extract from the vacuum manifold. 

Adjust the final volume as closely as possible to 10 mL with DCM. Small 

amounts of residual water from the sample container and the SPE cartridge may 

form a small immiscible layer with the extract. Read the volume on the collection 

tube at the level of the DCM layer. Add the IS. Addition of 5 μL of a 

1000-μg/mL IS solution to a 10-mL extract will result in an IS concentration of 

500 ng/mL. Mix well. A vortex mixer is recommended. To eliminate residual 

water, add approximately 2 g of anhydrous sodium sulfate. Mix well. Transfer 
 

522-28 

aliquots of the extract to autosampler vials, or other glass vials, for storage until 

analysis. 

 NOTE: Experiments during method development indicated that this extract 

cannot be reliably concentrated by nitrogen evaporation because of the volatility 

of the method analyte. Therefore, extract concentration to enhance sensitivity is 

not permitted. 

 

11.5 SPE PROCEDURE OPTION 2; EXTRACTION OF 100-ML SAMPLES – Proper 

conditioning of the solid phase sorbent can have a marked effect on method precision 

and accuracy. This section describes the SPE procedure using Waters AC-2 Sep-Pak 

or Supelco Supelclean ENVI-Carb Plus cartridges with 60-mL reservoirs and the SPE 

manifold as described in Sect. 6.8. 

11.5.1 CARTRIDGE CONDITIONING 

11.5.1.1 Fill the cartridge with approximately 1 mL of DCM, turn on the vacuum, 

and pull the solvent through, aspirating completely. 

11.5.1.2 Place a reservoir over each cartridge and fill the cartridge with 

approximately 2 mL of methanol, turn on the vacuum, and pull the solvent 

through, aspirating completely. 

11.5.1.3 Fill the cartridge with approximately 2 mL of methanol and elute with 

vacuum to just above the top of the cartridge - not allowing the cartridge to 

go dry at the end. From this point forward, do not allow the cartridge to go 

dry. 

11.5.1.4 Fill the cartridge with approximately 3 mL of reagent water, turn on the 

vacuum, and pull the water through, without allowing the cartridge to go 

dry. 

11.5.2 SAMPLE EXTRACTION – Fill each reservoir with sample. Adjust the vacuum 

so that the approximate flow rate is 10 mL/min. A 60-mL reservoir will require a 

second filling to complete sample loading. After all the sample has passed 

through each SPE cartridge, detach the reservoir and draw air through the 

cartridge for 10 min at full vacuum. Turn off and release the vacuum. Proceed 

immediately with cartridge elution. 

11.5.3 CARTRIDGE ELUTION – Lift the extraction manifold top and insert a rack with 

2-mL volumetric collection tubes into the extraction tank to collect the extracts as 

they are eluted from the cartridges. (It is not necessary to reverse the ENVI-Carb 

Plus cartridge as described in the manufacturer’s literature.) Fill each cartridge 

with DCM. Pull enough of the solvent into the cartridge at low vacuum to soak 

the sorbent. Turn off the vacuum and vent the system. Allow the sorbent to soak 

in DCM for approximately 1 min. Apply a low vacuum and pull the DCM 
 

522-29 

through the cartridge in a dropwise fashion into the collection tube. Continue to 

add DCM to the cartridge as it is being drawn through until the volume of extract 

is about 1.5 mL, estimated by the volumetric mark on the side of the collection 

tube. 

11.5.4 Remove collection tubes containing the extracts from the vacuum manifold. 

Adjust the final volume as closely as possible to 2 mL with DCM. Small amounts 

of residual water from the sample container and the SPE cartridge may form a 

small immiscible layer with the extract. Read the volume on the collection tube at 

the level of the DCM layer. Add the IS. Addition of 10 μL of a 100-μg/mL IS 

solution to a 2-mL extract will result in an IS concentration of 500 ng/mL. Mix 

well. A vortex mixer is recommended. To eliminate residual water, pass the 

2-mL extracts through a small column containing a minimum of 0.4 g of dry 

anhydrous sodium sulfate. A fritted 3-mL polypropylene column or a 5-8 mm 

diameter disposable pipette with a small piece of glass wool may be used. Collect 

the extracts from the drying column directly in 2-mL autosampler vials for storage 

until analysis. 

NOTE: Experiments during method development indicated that this extract 

cannot be reliably concentrated by nitrogen evaporation because of the volatility 

of the method analyte. Therefore, extract concentration to enhance sensitivity is 

not permitted. 

11.6 Analyze an aliquot of the sample extract prepared using Option 1 or 2, with the 

GC/MS system under the same conditions used for the initial and continuing 

calibrations (Sect. 10). 

12. DATA ANALYSIS AND CALCULATIONS

12.1 IDENTIFICATION OF ANALYTES – At the conclusion of data acquisition, use the 

same software that was used in the calibration procedure to identify peaks in 

predetermined retention time windows of interest. Use the data system software to 

examine the ion abundances of components of the chromatogram. Identify a sample 

component by comparison of its SIM (or full scan) mass spectrum to a SIM (or full 

scan) reference spectrum in the user-created database. The GC retention time of a 

method analyte should be within one to two sec of the retention time observed for that 

same compound in the most recently analyzed CCC standard. Ideally, the width of the 

retention time window should be based upon measurements of actual retention time 

variations of standards over the course of a day. Three times the standard deviation of 

a retention time can be used to calculate a suggested window size for an analyte. 

However, the experience of the analyst should weigh heavily in the interpretation of 

the chromatogram. When this method is performed in the SIM mode, verification of 

retention times is particularly important because less mass spectral information is 

being collected. 
 

522-30 

12.2 IDENTIFICATION VERIFICATION USING ION RATIOS – When the QI of 

1,4-dioxane (m/z 88) is observed at the correct retention time, verify that it is truly 

present by verifying that the ratio of the confirmation ion (m/z 58) to the QI (m/z 88) is 

within an absolute ±20% of the ratio observed in a mid-range calibration standard. For 

example, if the ratio in the standard is 70%, then the ratio in a Field Sample must be 

within 50-90%. Ratios must be determined using the integrated areas for the QI 

and confirmation ion from their respective mass chromatograms. Using relative 

abundances obtained directly from the mass spectrum, whether or not it has undergone 

background subtraction, is not acceptable. Variations in system software and analyst 

subjectivity in selecting background spectra for subtraction can affect the validity of 

the ratio calculation when the relative abundance from the spectrum is used. Using a 

raw spectrum (not corrected for background) can also affect the validity of the ratio 

calculation. 

 Use similar procedures for identification of the IS and SUR. Suggested QIs and 

confirmation ions for these compounds may be found in Table 2 (Sect. 17). 

12.3 Calculate analyte and SUR concentrations using the multi-point calibration established 

in Section 10.2. Do not use daily CCC data to quantitate 1,4-dioxane or the SUR in 

samples. The integrated abundances of the QIs of the analyte, SUR and IS should be 

used for all calculations. Adjust the final analyte concentrations to reflect the actual 

sample volume as determined in Sect.11.3.3. Field Sample extracts that require 

dilution should be treated as described in Section 12.4. 

12.4 EXCEEDING CALIBRATION RANGE – An analyst must not extrapolate beyond the 

established calibration range. If an analyte result exceeds the range of the initial 

calibration curve, the extract may be diluted with DCM, with the appropriate amount 

of IS added to match the original concentration, and the diluted extract injected. 

Acceptable SUR performance (Sect. 9.3.6.1) should be determined from the undiluted 

sample extract. Incorporate the dilution factor into final concentration calculations. 

The resulting sample should be documented as a dilution, and the MRL adjusted 

accordingly. 

12.5 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), typically two, and not more than three significant figures. 

NOTE: Some data in Section 17 of this method are reported with more than two 

significant figures. This is done to better illustrate the method performance data. 

13. METHOD PERFORMANCE

13.1 Method performance data presented in Section 17 were obtained with a Thermo 

Finnigan Trace DSQ GC/MS system. Any GC/MS system that meets all of the QC 

requirements of the method may be used. 
 

522-31 

13.2 PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS – Single 

laboratory DLs and LCMRLs are presented in Table 3. Single laboratory accuracy 

and precision data from both fortified reagent water and fortified drinking water 

matrices are presented in Tables 4 and 5, respectively. Two of the matrices tested 

were selected specifically as potentially difficult matrices because of either their 

relatively high TOC or high mineral content (hardness). There was no apparent 

difference in method performance when results from reagent water studies were 

compared to these drinking water matrices. 

13.3 METHOD PERFORMANCE IN SAMPLES WITH HIGH CONCENTRATIONS OF 

1,1,1-TRICHLORETHANE – Because of its widespread use as a chlorinated solvent 

stabilizer, 1,4-dioxane may be found in the presence of high concentrations of 

 1,1,1-trichloroethane. The method was tested in a high TOC water matrix with up to 

500 μg/L of 1,1,1-trichloroethane added as a co-contaminant. No adverse affect was 

observed, and the method performed well within QC limits (data not shown). 

13.4 SAMPLE STORAGE STABILITY STUDIES 

13.4.1 AQUEOUS SAMPLES – Chlorinated drinking water samples from a surface 

water source were fortified with 1,4-dioxane and preserved and stored as required 

in Section 8. Samples were extracted and analyzed in replicate (n=7) on day 0, 

and at five additional times points up to and beyond 28 days. Data from these 

analyses validate the 28 day holding time, and are presented in Figure 2. 

13.4.2 EXTRACTS – Sample extracts stored at -5 °C and protected from light were 

analyzed in replicate (n=7) on day 0, and at three additional time points up to and 

beyond 28 days. Data from these analyses validate the 28 day holding time, and 

are presented in Figure 3. 

13.5 MULTIPLE LABORATORY DEMONSTRATION – The performance of this method 

was demonstrated in two additional laboratories, with results similar to those reported 

in Section 17. The authors wish to acknowledge the assistance of the analysts and 

laboratories listed below for their participation in the multi-lab demonstration. 

13.5.1 Mr. Alan Zaffiro of Shaw Environmental and Infrastructure, Inc. under contract to 

the USEPA Office of Ground Water and Drinking Water Technical Support 

Center, Cincinnati, OH. 

13.5.2 Dr. Peggy Knight and Ms. Megan Pickett of the USEPA Region 10 Laboratory, 

Port Orchard, WA. 

14. POLLUTION PREVENTION

14.1 This method utilizes SPE to extract the analyte from water. It requires the use of very 

small volumes of organic solvent and very small quantities of pure analyte, thereby 

minimizing the potential hazards to both the analyst and the environment as compared 
 

522-32 

to the use of large volumes of organic solvents in conventional liquid-liquid 

extractions. 

14.2 For information about pollution prevention that may be applicable to laboratory 

operations, consult “Less is Better: Laboratory Chemical Management for Waste 

Reduction” available from the American Chemical Society’s Department of 

Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C., 

20036 or on-line at http://membership.acs.org/c/ccs/pub_9.htm. (accessed September 

2008). 

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts 

of waste since only small amounts of reagents and solvents are used. The matrices of 

concern are finished drinking water or source water. Waste management practices 

must be conducted consistent with all applicable rules and regulations, and 

laboratories must protect the air, water, and land by minimizing and controlling all 

releases from fume hoods and bench operations. Compliance is required with any 

sewage discharge permits and regulations, particularly the hazardous waste 

identification rules and land disposal restrictions. 

16. REFERENCES

1. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P., 

Hedrick, E.J. and R.A. Krop, Statistical Procedures for Determination and Verification of 

Minimum Reporting Levels for Drinking Water Methods. Environ. Sci. Technol., 40, (2006) 

281-288. 

2. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A. and W.L. Budde, Trace Analyses for 

Wastewaters. Environ. Sci. Technol., 15, (1981) 1426-1435. 

3. Kadokami, K., Koga, M. and Otsuki, Gas Chromatography/Mass Spectrometric 

Determination of Traces of Hydrophilic and Volatile Organic Compounds in Water after 

Preconcentration with Activated Carbon. Analytical Sciences, 6, (1990) 843-849. 

4. Isaacson, C., Mohr, T.K.G. and J.A. Field, Quantitative Determination of 1,4-Dioxane and 

Tetrahydrofuran in Groundwater by Solid Phase Extraction GC/MS/MS. Environ. Sci. 

Technol., 40, (2006) 7305-7311. 

5. USEPA, 2006 Edition of the Drinking Water Standards and Health Advisories. APA #822-R06-013. 

6. Carcinogens - Working With Carcinogens. Department of Health, Education, and Welfare, 

Public Health Service, Center for Disease Control, National Institute for Occupational Safety 

and Health, Publication No. 77-206, Aug. 1977. 
 

522-33 

7. OSHA Safety and Health Standards, General Industry. (29CFR1910), Occupational Safety 

and Health Administration, OSHA 2206, (Revised, July 1, 2001). 

8. Safety in Academic Chemistry Laboratories. American Chemical Society Publication, 

Committee on Chemical Safety, 7th Edition. Information on obtaining a copy is available at 

http://membership.acs.org/C/CCS/pub_3.htm. (accessed September 2008). Also available by 

request at OSS@acs.org 
 

522-34 

17. TABLES, DIAGRAMS, FLOWCHARTS AND DEMONSTRATION DATA 

Table 1. Bromofluorobenzene (BFB) Tune Verification Criteria 

Mass (m/z) Relative Abundance Criteria 

50 15-40% of mass 95 

75 30-80% of mass 95 

95 Base peak, 100% relative abundance 

96 5-9% of mass 95 

173 <2% of mass 174 

174 >50% of mass 95 

175 5-9% of mass 174 

176 >95% but <101% of mass 174 

177 5-9% of mass 176 

Table 2. Retention Times and Quantitation Ions (QIs) 

Compound Retention Time (min) SIM Ions (m/z) 

1,4-dioxane 8.85 58a, 88

1,4-dioxane-d8 (SUR) 8.77 62, 64, 96

THF-d8 (IS) 6.68 46, 78, 80 

Note: Suggested quantitation ion in bold. 

 a. Ion traps may give a confirmation ion of m/z 57 for 1,4-dioxane instead of the typical m/z 58 

in quadrupole spectra and in many mass spectral libraries. If the instrument has been 

calibrated and passes bromofluorobenzene (BFB) criteria, then m/z 57 may be used as the 

confirmation ion on ion trap instruments. 

 

Table 3. Lowest Concentration Minimum Reporting Level (LCMRL) and Detection 

Limit (DL) Calculated for Each Extraction Option 

Compound/Extraction Option LCMRL 

(μg/L) 

DL (μg/L) 

1,4-dioxane (500 ml w/2-g Coconut charcoal) 0.047 0.026a 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 0.036 0.020b 

a. Calculated from replicates fortified at 0.040 μg/L, n=7. 

b. Calculated from replicates fortified at 0.030 μg/L, n=8. 
 

522-35 

Table 4. Demonstration of Method Performance in Reagent Water Fortified with 

 1,4-Dioxane at Three Concentrations 

Reagent Water Fortified at 

0.030 or 0.040 μg/L

(n=7 or 8) a

Compound/Extraction Option 

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 110 19 

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 92 3.5 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 110 20 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

102 3.8 

Reagent Water Fortified at 

1.0 μg/L (n=6)

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 98.0 6.2 

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 97.1 5.2 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 101 4.2 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

105 4.8 

Reagent Water Fortified at 

10.0 μg/L (n=7) 

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 93.9 2.8 

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 90.1 2.8 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 95.2 2.9 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

99.3 3.0 

a. Samples (500 mL) extracted on 2-g Coconut charcoal cartridges were fortified at 0.040 μg/L, 

n=7. Samples (100 mL) extracted on Waters AC-2 Sep-Pak cartridges were fortified at 0.030 

μg/L, n=8. 
 

522-36 

Table 5. Demonstration of Method Performance in Drinking Water Matrices Using Each 

of the Solid Phase Extraction (SPE) Options; Matrix Samples Fortified at 1.0 

μg/L (n=7 for each matrix) 

Compound/Extraction Option Surface Water 

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 99.0a 4.9 

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 100 2.5 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 97.0b 4.6 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

98.5 2.5 

Surface Water (high in 

TOC)c 

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 102 3.5

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 99.8 2.4 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 98.5 5.6 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

101 4.2 

Groundwater (high in 

mineral content) d

Mean % 

recovery 

RSD (%)

1,4-dioxane (500 ml w/2-g Coconut charcoal) 95.9e 2.1 

1,4-dioxane-d8 (SUR) (500 ml w/2-g Coconut charcoal) 98.2 2.5 

1,4-dioxane (100 ml w/Waters AC-2 Sep-Pak cartridge) 101f 3.3 

1,4-dioxane-d8 (SUR) (100 ml w/Waters AC-2 Sep-Pak 

cartridge) 

104 5.9 

a. Percent recovery after correction for matrix background of 0.66 μg/L. 

b. Percent recovery after correction for matrix background of 0.42 μg/L. 

Note: Groundwater used for these Laboratory Fortified Blanks (LFBs) was collected from the 

same source as the 500 mL LFBs using coconut charcoal, but was collected on different days, 

thus the difference in matrix background concentration. 

c. TOC measured at 4.950 mg/L. 

d. Hardness measured at 289 mg/L as calcium carbonate. 

e. Percent recovery after correction for matrix background of 0.070 μg/L. 

f. Percent recovery after correction for matrix background of 0.080 μg/L. 
522-37 

Table 6. Initial Demonstration of Capability (IDC) and Quality Control (QC) Requirements (Summary)

Method Reference 

Requirement Specification and Frequency Acceptance Criteria 

Sect. 9.2.1 Initial Demonstration of 

Low Background 

Analyze LRB prior to any other IDC steps. When a new lot of SPE media is obtained, verify that background is 

at acceptable limits. 

Demonstrate that the method 

MRL, and that possible interextraction media do not prevand/or quantification of the or IS. Note: This includes the absboth the QIs and confirmatiointerest. 

Sect. 9.2.2 Initial Demonstration of 

Precision (IDP) 

Analyze 4-7 replicate LFBs fortified near the midrange 

calibration concentration. 

%RSD must be ≤ 20% 

Sect. 9.2.3 Initial Demonstration of 

Accuracy (IDA) 

Calculate average recovery for replicates used in IDP. Mean recovery ± 20% of tru

Sect. 9.2.4 Minimum Reporting 

Limit (MRL) Confirmation 

Fortify, extract and analyze 7 replicates at the proposed MRL concentration. Calculate the mean, standard deviation and HRPIR. Confirm that the upper and lower 

limits for the Prediction Interval of Result (Upper PIR, and Lower PIR, Sect. 9.2.4.2) meet the recovery criteria. 

Upper PIR ≤ 150% 

Lower PIR ≥ 50% 

Sect. 9.2.5 & 9.3.9 

Calibration Confirmation, Quality Control Sample (QCS) 

Analyze a standard from a second source (QCS) to verify the initial calibration curve. 

± 20% of the expected value

NOTE: Table 6 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because t

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional informatis needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table. 
522-38 

Table 7. Ongoing Quality Control (QC) Requirements (Summary)

Method Reference 

Requirement Specification and Frequency Acceptance Criteria 

Sect. 8.3 Sample Holding Time 28 days with appropriate preservation and storage as 

described in Sections 8.1-8.3. 

Sample results are valid only extracted within sample hold 

Sect. 8.4 Extract Holding Time 28 days stored at -5 °C and protected from light Sample results are valid only 

analyzed within extract hold 

Sect. 9.3.1 Laboratory Reagent Blank 

(LRB) 

One LRB with each extraction batch of up to 20 Field Samples. 

Demonstrate that the method is ≤ 1/3 the MRL, and confirm 

interferences do not prevent background concentration ex

results for the extraction bat

Sect. 9.3.3 Laboratory Fortified 

Blank (LFB) 

One LFB is required for each extraction batch of up to 

20 Field Samples. Rotate the fortified concentrations between low, medium, and high amounts. 

Results of LFB analyses at mfortifications must be 70-13the analyte and SUR. Resulmust be 50-150% of the true 

Sect. 9.3.5 Internal Standard (IS) Compare IS area to the average IS area in the initial 

calibration and the most recent CCC. 

Peak area counts for all inje± 50% of the average peak athe initial calibration and ± 3

recent CCC. If the IS does n

target analyte results are inv9.3.5 for further information

Sect. 9.3.6 Surrogate(SUR) 

Standards 

The SUR standard added to all calibration standards and samples, including QC samples. Calculate SUR recoveries. 

SUR recovery must be 70-1If a SUR fails this criterion, 

sample as suspect/SUR reco

Sect. 9.3.7 Laboratory Fortified 

Sample Matrix (LFSM) 

Analyze one LFSM per extraction batch (of up to 20 Field Samples) fortified with the method analyte at a 

concentration close to but greater than the native concentration. Calculate LFSM recoveries. 

See Sect. 9.3.7.3 for instructinterpretation of LFSM resu
522-39 

Table 7.Ongoing Quality Control (QC) Requirements (Summary) (Continued) 

Sect. 9.3.8 Laboratory Fortified 

Sample Matrix Duplicate (LFSMD) or Field Duplicates (FD) 

Extract and analyze at least one FD or LFSMD with each extraction batch of up to 20 Field Samples. A

LFSMD may be substituted for a FD when the frequency of detects for 1,4-dioxane are low. Calculate RPDs. 

Method analyte RPDs for thbe ≤ 30% at mid and high le

≤ 50% near the MRL. Failumay indicate a matrix effect

Sect. 9.3.9 Quality Control Sample 

(QCS) 

Analyze QCS during the IDC, each time CAL solutions

are prepared. A QCS must be analyzed at least quarterly. 

Results must be 80-120% of 

Sect. 10.2 Initial Calibration Use IS calibration technique to generate a linear or 

quadratic calibration curve. The number of standards required is determined by the calibration range (Sect. 7.2.2). Check the calibration curve as described in Section 10.2.4. 

When each calibration standunknown using the calibratioshould be 80-120% of the trthe lowest standard, which sthe true value. If this criteri

CALs, select a different metrecalibrate over a shorter ran

Sects. 10.1 and 10.2.1 

MS Tune Check Analyze BFB to verify MS tune each time the 

instrument is calibrated. 

Criteria are given in Table 1

Sect. 10.3 Continuing Calibration 

Check (CCC) 

Verify initial calibration by analyzing a calibration standard at the beginning of each analysis batch prior to analyzing samples, after every 10 samples, and after the last sample. The first CCC daily must be at or below the MRL. Subsequent CCCs alternate between medium and high concentrations. Low CCC – at or below the MRL concentration Mid CCC – near midpoint in initial calibration curve High CCC – near the highest calibration standard. 

Low: ± 50% of true value 

Mid: ± 30% of true value 

High: ± 30% of true value 

Note: Table 7 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the 

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional informatis needed. In all cases, the full text of Sections 8-10 in the method supersedes any missing or conflicting information in this table.
 

522-40 

Figure 1. Reconstructed total ion current chromatogram and mass chromatograms for THF-d8 (IS), 

1,4-dioxane-d8 (SUR), and 1,4-dioxane at 0.5 μg/mL each (the standard is equivalent to an extract of 

a 10 μg/L aqueous sample). * Peak at 6.845 min is chloroform, a chemical present in DCM. 
 

522-41 

Stability of 1,4-Dioxane in Drinking Water

99.2% 95.3%

103.2% 99.4% 98.9%

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

0 7 14 21 28

days

% Recovery 

Figure 2. Stability of 1,4-dioxane in preserved drinking water stored at 4°C in the dark, over a 35-

day time period. Replicate samples (n = 7) were fortified to a concentration of 1 μg/L 1,4-dioxane. 

Matrix blank data were used to correct for native analyte concentrations. 

Stability of 1,4-Dioxane in Extracts

93.9% 92.0% 91.2%

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

0 14 28

days

% Recovery

Figure 3. Stability of 1,4-dioxane in sample extracts stored at -5°C, in the dark, over a 42-day time

period. Replicate samples (n = 7) were fortified to a concentration of 10 μg/L 1,4-dioxane.