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 EPA Document #: EPA/600/R-05/052

METHOD 529 DETERMINATION OF EXPLOSIVES AND RELATED

COMPOUNDS IN DRINKING WATER BY SOLID PHASE

EXTRACTION AND CAPILLARY COLUMN GAS

CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)

Revision 1.0

September, 2002

J.W. Munch 

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
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METHOD 529

DETERMINATION OF EXPLOSIVES AND RELATED COMPOUNDS IN DRINKING

WATER BY SOLID PHASE EXTRACTION AND CAPILLARY COLUMN 

GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)

1. SCOPE AND APPLICATION

1.1 This method provides procedures for the determination of explosives and related

compounds in finished drinking water. The method may be applicable to untreated

source waters and other types of water samples, but it has not been evaluated for

these uses. The method is applicable to a variety of explosives that are efficiently

partitioned from the water sample onto a solid phase extraction (SPE) sorbent, and

sufficiently volatile and thermally stable for gas chromatography. The method

includes the following compounds:

ANALYTE CAS NUMBER

2-amino-4,6-dinitrotoluene 35572-78-2

4-amino-2,6-dinitrotoluene 19406-51-0

3,5-dinitroanaline 618-87-1

1,3-dinitrobenzene 99-65-0

2,4-dinitrotoluene 121-14-2

2,6-dinitrotoluene 606-20-2

hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) 121-82-4

nitrobenzene 98-95-3

2-nitrotoluene 88-72-2

3-nitrotoluene 99-08-1

4-nitrotoluene 99-99-0

1,3,5-trinitrobenzene 99-35-4

2,4,6-trinitrophenylmethylnitramine (Tetryl) 479-45-8

2,4,6-trinitrotoluene (TNT) 118-96-7
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1.2 Detection limit (DL) is defined as the statistically calculated minimum concentration

that can be measured with 99% confidence that the reported value is greater than

zero (1). The DL is compound dependent and is particularly dependent on

extraction efficiency, sample matrix, and instrument performance. DLs for method

analytes are listed in Tables 3, 9, and 15. 

1.3 This method should be performed only by analysts with experience in solid phase

extractions and GC/MS analyses. 

2. SUMMARY OF METHOD

Analytes and surrogates are extracted by passing a 1 L water sample through a solid phase

extraction (SPE) cartridge containing 500 mg of a divinylbenzene/vinylpyrrolidone

copolymer or a 47 mm disk containing a reverse phase sulfonated polystyrene divinylbenzene phase. The organic compounds are eluted from the solid phase with a small

quantity of ethyl acetate. The sample components are separated, identified, and measured

by injecting an aliquot of the concentrated extract into a high resolution fused silica

capillary column of a GC/MS system with a programmed temperature vaporizing (PTV)

injector. Compounds eluting from the GC column are identified by comparing their

measured mass spectra and retention times to reference spectra and retention times in a

data base. Analysis may be performed using either full scan or selected ion monitoring

(SIM) mass spectrometry. Reference spectra and retention times for analytes are obtained

by the measurement of calibration standards under the same conditions used for samples. 

The concentration of each identified component is measured by relating the MS response

of the quantitation ion(s) produced by that compound to the MS response of the

quantitation ion(s) produced by a compound that is used as an internal standard. Surrogate

analytes, whose concentrations are known in every sample, are measured with the same

internal standard calibration procedure.

The method provides instructions and demonstration data for both the SPE disk and

cartridge extraction techniques and for three different GC/MS techniques: PTV splitless

injection with full scan mass spectrometry, cold on-column injection with full scan mass

spectrometry, and PTV splitless injection with SIM mass spectrometry.

3. DEFINITIONS

3.1 ANALYSIS BATCH -- A set of samples 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.

3.2 CALIBRATION STANDARD (CAL) -- A solution prepared from the primary

dilution standard solution or stock standard solutions and the internal standards and
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surrogate analytes. The CAL solutions are used to calibrate the instrument response

with respect to analyte concentration.

3.3 CONTINUING CALIBRATION CHECK (CCC) -- A calibration standard

containing one or more method analytes, 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. 9.2.4),

and accurate quantitation is not expected at this level (1).

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 solid phase extraction devices and solvents, surrogate solution, and fortifying

solutions. Required QC samples for each extraction batch include: Laboratory

Reagent Blank, Laboratory Fortified Blank, Laboratory Fortified Matrix, and either a

Field Duplicate or Laboratory Fortified Matrix Duplicate.

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(s) added to a sample, extract, or

standard solution in known amount(s) and used to measure the relative responses of

other method analytes and surrogates that are components of the same solution. The

internal standard 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 analytes are added in the

laboratory. The LFB is analyzed exactly like a sample, including the use of sample

preservatives, 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 (LFM) -- An aliquot of an

environmental sample to which known quantities of the method analytes are added

in the laboratory. The LFM is 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 analytes in the sample matrix must be determined

in a separate aliquot and the measured values in the LFM corrected for background

concentrations.
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3.10 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) -- A

second aliquot of the Field Sample, or duplicate Field Sample, that is used to prepare

the LFM. The LFMD is fortified, extracted and analyzed identically to the LFM. 

The LFMD is used instead of the Laboratory Duplicate to assess method precision

when the occurrence of target analytes are low.

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, internal standards, surrogates, and sample

preservatives that are used with other samples. The LRB is used to determine if

method analytes or other interferences are present in the laboratory environment, the

reagents, or the apparatus.

3.12 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.

3.13 MINIMUM REPORTING LEVEL (MRL) -- The minimum concentration that can

be reported as a quantitated value for a target analyte in a sample following analysis. 

This defined concentration can be no lower than the concentration of the lowest

calibration standard for that analyte, and can only be used if acceptable quality

control criteria for the analyte at this concentration are met.

3.14 PRIMARY DILUTION STANDARD SOLUTION (PDS) -- A solution of several

analytes prepared in the laboratory from stock standard solutions and diluted as

needed to prepare calibration solutions and other needed analyte solutions.

3.15 PROGRAMMED TEMPERATURE VAPORIZING INJECTOR (PTV) -- A GC

injector capable of rapid heating. Typical use of a PTV injector involves introducing

the sample with the injector cool, then rapidly heating it at 100-200 degrees per

minute to volatilize the analytes onto the GC column. One advantage of this type of

injection is that thermally labile analytes in a mixture can be transferred to the GC

column at a lower temperature than in conventional hot injections.

3.16 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of known

concentrations that is obtained from a source external to the laboratory and different

from the source of calibration standards. It is used to check laboratory performance

with externally prepared test materials.

3.17 SELECTED ION MONITORING (SIM) -- A chromatographic/mass spectrometric

technique where only one or a few ions are monitored. When used with gas

chromatography, the set of ions monitored is usually changed periodically
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throughout the chromatographic run, to correlate with the characteristic ions of each

analyte or group of analytes as they elute from the chromatographic column.

3.18 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing one

or more method analytes prepared in the laboratory using assayed reference materials

or purchased from a reputable commercial source.

3.19 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 During analysis, major contaminant sources are reagents and SPE devices. Analyses

of laboratory reagent blanks provide information about the presence of

contaminants. Solid phase extraction devices described in this method have two

potential sources of contamination, both the solid phase sorbent and for cartridge

extractions, the polypropylene cartridge that it is packed in. Manufacturers’ brands

and lot numbers of these devices should be monitored and tracked to ensure that

contamination will not preclude analyte identification and quantitation.

4.2 Interfering contamination may occur when a sample containing low concentrations

of compounds is analyzed immediately after a sample containing relatively high

concentrations of compounds. Injection port liners must be replaced as needed

(cleaning and deactivation by the analyst is not recommended). After analysis of a

sample containing high concentrations of compounds, a laboratory reagent blank

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

In the case of automated analysis, the analyst may not be aware of high concentration

samples until after an entire batch is analyzed. In this situation, the analyst should

carefully review data from samples analyzed immediately after high concentration

samples, and reanalyze them if necessary.

5. SAFETY

5.1 The toxicity or carcinogenicity of many of the chemicals used in this method has not

been precisely defined; each chemical should be treated as a potential health hazard,

and exposure to these chemicals should be minimized. Each laboratory is

responsible for maintaining awareness of OSHA regulations regarding safe handling

of chemicals used in this method. Each laboratory should maintain a file of

applicable MSDSs. 
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5.2 Some method analytes including 2,4- and 2,6-dinitrotoluene, RDX, and TNT have

been classified as known or suspected human or mammalian carcinogens. Pure

standard materials and stock standard solutions of these compounds should be

handled with suitable protection to skin, eyes, etc. (2-5).

5.3 RDX, Tetryl and TNT are explosives and the neat materials require special handling to

be used safely. It is highly recommended that only dilute solutions of these

materials, available as standards from commercial suppliers be used for this

method (6).

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 GLASSWARE -- All glassware must be meticulously cleaned. This may be

accomplished by washing with detergent and water, rinsing with water, distilled

water, and solvent rinsing or heating (where appropriate) at 400° C for 2 h in a

muffle furnace. Volumetric glassware should never be heated to the temperatures

obtained in a muffle furnace.

6.2 SAMPLE CONTAINERS -- 1 L or 1 qt amber glass bottles fitted with

polytetrafluoroethylene (PTFE) lined polypropylene screw caps. Amber bottles are

highly recommended since some of the method analytes are sensitive to light and

may degrade upon exposure. Clear glass bottles may be used if they are wrapped in

foil, or samples are stored in boxes that prevent exposure to light.

6.3 VOLUMETRIC FLASKS -- various sizes.

6.4 LABORATORY OR ASPIRATOR VACUUM SYSTEM -- Sufficient capacity to

maintain a vacuum of approximately 25 cm (10 in.) of mercury.

6.5 MICRO SYRINGES -- various sizes.

6.6 VIALS -- Various sizes of amber vials with PTFE lined screw caps for storing

standard solutions and extracts.

6.7 DRYING COLUMN -- The drying tube should contain about 5 to 7 grams of

anhydrous sodium sulfate to remove residual water from the extract. Any small tube

may be used, such as a syringe barrel, a glass dropper, etc. as long as no particulate

sodium sulfate passes through the column into the extract.

6.8 ANALYTICAL BALANCE -- Capable of weighing 0.0001 g accurately. 
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6.9 FUSED SILICA CAPILLARY GAS CHROMATOGRAPHY COLUMN -- Any

capillary column that provides adequate resolution, capacity, accuracy, and precision

can be used. Medium polarity, low bleed columns are recommended for use with

this method to provide adequate chromatography and minimize column bleed. 

Deactivated injection port liners are highly recommended. Example retention times

are presented in Table 2.

6.9.1. Column - 15 m × 0.25 mm i.d. fused silica capillary column coated with a

0.25 :m bonded film of polyphenylmethylsilicone, (Agilent DB-5ms or

equivalent).

NOTE: Although other columns may be used, longer or thicker film

columns are not recommended because increased residence time in the

column has been shown to contribute to analyte degradation (7). 

6.10 GAS CHROMATOGRAPH/MASS SPECTROMETER/DATA SYSTEM

(GC/MS/DS)--

6.10.1 The GC must be capable of temperature programming and should be

equipped for PTV splitless or cold on-column injection. The injection

system should not allow the analytes to contact hot stainless steel or other

metal surfaces that promote decomposition. Satisfactory data could not be

demonstrated for this method using hot splitless injections.

A large volume injector may be used to increase method sensitivity, or to

avoid extract evaporation, if all criteria in Sections 9 and 10 are met.

6.10.2 Deactivated injection port liners should be used, and it is highly

recommended that they be replaced when necessary with a new deactivated

liner. Cleaning and deactivation of injection port liners by the analyst is not

recommended. The injection port liner should be replaced as necessary to

meet all QC requirements. The frequency will vary by the type of

instrument used and the type and number of samples analyzed. 

In general, packed injection port liners should not be used with this method. 

The use of liner packings such as glass wool and fused silica wool during

development of this method caused significant breakdown of some method

analytes, particularly RDX. Carbofrits (Restek) did show acceptable

performance and may be used with PTV splitless injection. (The data

shown in this method in Tables 3-8 were generated using a Carbofrit.)

6.10.3 The mass spectrometer must be capable of electron ionization at a nominal

electron energy of 70 eV to produce positive ions. The spectrometer must
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be capable of scanning at a minimum from 45 to 250 amu with a complete

scan cycle time (including scan overhead) of 0.7 sec or less. (Scan cycle

time = total MS data acquisition time in sec divided by number of scans in

the chromatogram). The spectrometer must produce a mass spectrum that

meets all criteria in Table 1 when an injection of approximately 5 ng of

BFB is introduced into the GC. A single spectrum at the apex of the

chromatographic peak, or an average of the three spectra at the apex of the

peak, or an average spectrum across the entire GC peak may be used to

evaluate the performance of the system. Background subtraction is

permitted. The scan time must be set so that all analytes have a minimum

of 5 scans across the chromatographic peak. Seven to ten scans across

chromatographic peaks are strongly recommended.

NOTE: Although BFB is most commonly used as the tune check for

volatile compound analysis, it was selected for use in this method for

semivolatile analytes because the ions used for the BFB tune check are

similar to the analyte quantitation ions in this method.

6.10.4 If the SIM option is selected for use with this method, the mass

spectrometer must be capable of this technique. Some ion trap mass

spectrometers have an option similar to SIM known as Selected Ion Storage

(SIS). Selected ion storage may be used with this method, however a

preliminary assessment during method development showed little if any

increase in method sensitivity using SIS.

6.10.5 An interfaced data system is required to acquire, store, reduce, and output

mass spectral data. The computer software should have the capability of

processing stored GC/MS data by recognizing a GC peak within any given

retention time window. The software must also allow integration of the ion

abundance of any specific ion between specified time or scan number

limits, calculation of response factors as defined in Sect. 10.2.5 or

construction of a linear regression calibration curve, and calculation of

analyte concentrations.

6.11 VACUUM MANIFOLD -- A vacuum manifold (Supelco # 57030 and #57275 for

extraction cartridges or Varian #12146001 for disks, or equivalent) is required for

processing samples through the extraction/elution procedure. An automatic or

robotic sample preparation system designed for use with solid phase extraction

cartridges/disks may be utilized in this method if all quality control requirements

discussed in Sect. 9 are met. Automated systems may use either vacuum or positive

pressure to process samples and solvents through the disk or cartridge. All

extraction and elution steps must be the same as in the manual procedure. 
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Extraction and/or elution steps may not be changed or omitted to accommodate the

use of an automated system. 

7. REAGENTS AND STANDARDS

7.1 HELIUM -- carrier gas, purity as recommended by the GC/MS manufacturer. 

7.2 SOLID PHASE EXTRACTION CARTRIDGES -- Waters Porapak RDX. 

Cartridges are inert non-leaching plastic, for example polypropylene, or glass, and

must not contain plasticizers that leach into the ethyl acetate eluant and prevent the

identification and quantitation of method analytes. The polypropylene cartridges (6

mL volume) are packed with 500 mg of a divinylbenzene/vinylpyrrolidone

copolymer. The packing must have a narrow size distribution and must not leach

interfering organic compounds into the eluting solvent.

7.3 SOLID PHASE EXTRACTION DISKS – 3M Empore SDB-RPS, 47mm.

NOTE: This SPE material is a chemically modified polystyrene divinyl benzene

material. Use of conventional polystyrene divinyl benzene materials will not result

in acceptable data.

7.4. COPPER SULFATE -- ACS grade. Used as a sample preservative for inhibiting

microbial activity in stored samples.

7.5 TRIZMA PRE-SET CRYSTALS (pH 7.0 BUFFER) -- Tris (hydroxymethyl)

aminomethane and Tris hydrochloride (Sigma Chemical Co.). Used as a binding

agent for free chlorine, and to keep the copper in solution. Alternatively, a two

component buffer of tris(hydroxymethyl)aminomethane [CAS# 77-86-1] and

tris(hydroxymethyl)aminomethane hydrochloride [CAS# 1185-53-1] may be used. 

Proportions of each to be used are listed in Sect. 8.2.1.

7.6 SOLVENTS --

7.6.1 ETHYL ACETATE AND METHANOL -- High purity pesticide quality or

equivalent. 

7.6.2 REAGENT WATER -- Water in which an interference is not observed at

>1/3 the MRL of any of the compounds of interest. Prepare reagent water

by passing tap water through a filter bed containing about 0.5 kg of

activated carbon or by using a water purification system. Store in clean,

narrow-mouth bottles with PTFE lined septa and screw caps.

7.7 SODIUM SULFATE, ANHYDROUS -- ACS grade. Material should be Soxhlet

extracted with methylene chloride for a minimum of 4 h or heated to 400° C for 2 h

in a muffle furnace.
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7.8 ANALYTE STOCK STANDARD SOLUTIONS -- It is recommended that

individual solutions of analytes, or mixtures of analytes, be purchased from

commercial suppliers. All work done during development of this method was done

with commercial mixtures. RDX, Tetryl and TNT are explosives and require special

handling to be used safely (6). They are not readily available as neat materials. 

Stock standard solutions of explosives are frequently provided in acetonitrile. This

is acceptable because the acetonitrile will be sufficiently diluted in calibration

standards and spiking solutions. Store the amber vials at 0° C or less, or according

to the supplier’s recommendations.

Stock standards of method analytes that are not explosive may be prepared from neat

materials. Typical concentrations of stock standards that can be used conveniently

are 1-10 mg/mL.

7.9 ANALYTE PRIMARY DILUTION STANDARD SOLUTION(S) -- The stock

standard solutions may be used to prepare a primary dilution standard (PDS)

solution that contains multiple method analytes in ethyl acetate. Aliquots of each of

the stock standard solutions are combined to produce the primary dilution in which

the concentration of the analytes is at least equal to the concentration of the most

concentrated calibration solution, typically, 10 ng/:L. Store the primary dilution

standard solution in an amber vial at 0° C or less, and check regularly for signs of

degradation or evaporation, especially just before preparing calibration solutions. 

Alternatively, mixtures of method analytes to be used as primary dilution standards

may also be purchased from commercial suppliers. Typically these will be in

acetonitrile. This is acceptable because the acetonitrile will be sufficiently diluted in

calibration standards and spiking solutions. Store the primary dilution standard

solution in an amber vial at 0° C or less, or according to the supplier’s

recommendations.

7.10 CALIBRATION SOLUTIONS -- These standards are prepared by combining

appropriate aliquots of a primary dilution standard solution (Sect. 7.9) and the

fortification solution of internal standards and surrogates (Sect. 7.11). All

calibration solutions should contain at least 80% ethyl acetate to avoid gas

chromatographic problems due to mixed solvents. The following calibration ranges

are suggested, but the range may be modified depending on instrument sensitivity. 

For full scan mass spectral analysis, prepare a series of six calibration solutions in

ethyl acetate which contain analytes of interest at suggested concentrations of 10,

5.0, 2.0, 1.0, 0.50, and 0.10 ng/μL. For SIM analysis, prepare a series of seven

calibration solutions in ethyl acetate which contain analytes of interest at suggested

concentrations of 2.0, 1.0, 0.50, 0.25, 0.10, 0.050, and 0.025 ng/μL. Add the

internal standard to each CAL standard at a constant concentration (5-7 ng/μL is

recommended). Surrogate analytes are also added to each CAL solution, and may be
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added at a constant concentration or varied concentrations (similar to those for

method analytes), at the discretion of the analyst. If the surrogate analytes are added

at a constant concentration, 1-5 ng/μL is recommended for full scan analysis and

1ng/μL is recommended for SIM analysis. Store these solutions in amber vials at 0°

C or less. Check these solutions regularly for signs of evaporation and/or

degradation. During method development these solutions were stable for at least 60

days.

7.11 INTERNAL STANDARD SOLUTION AND SURROGATE STOCK SOLUTIONS -- 

7.11.1 INTERNAL STANDARD STOCK - Prepare a stock standard of the internal

standard, 3,4-dinitrotoluene [CAS# 610-39-9], at approximately 2.0 mg/mL in

ethyl acetate. Store at 0° C or less. During method development, this solution

was stable for at least 1 year. 

7.11.2. SURROGATE STANDARD STOCK SOLUTIONS - Prepare or purchase

individual stock standards of the following surrogate analytes: 1,3,5-

trimethyl-2-nitrobenzene commonly known as 2-nitromesitylene [CAS#

603-71-4], 1,2,4-trimethyl-5-nitrobenzene commonly known as 2-

nitropseudocumene [CAS# 610-91-3], and nitrobenzene-d5[CAS# 4165-

60-0]. Standards of 2-nitromesitylene and 2-nitropseudocumene should be

prepared in methanol at concentrations of 1-2 mg/mL. Nitrobenzene-d5

should be prepared in methylene chloride at a concentration of 1-2 mg/mL.

Store at 0° C or less. During method development, these solutions were

stable for at least 1 year.

7.12 SAMPLE FORTIFICATION SOLUTIONS -- 

7.12.1 INTERNAL STANDARD FORTIFICATION SOLUTION - Prepare a

dilution of the stock standard in ethyl acetate such that the final

concentration is 0.20 mg/mL. This solution is used to add the internal

standard to each sample, LRB, LFB, and LFM extract. Add 5-25 μL of this

solution to each 1 mL extract to obtain the recommended final

concentration of 1-5 ng/μL. Check these solutions regularly for signs of

evaporation and/or degradation. During method development these

solutions were stable for at least 60 days.

7.12.2 ANALYTE FORTIFICATION SOLUTION(S) - This solution contains all

method analytes of interest in methanol. These solutions are used to fortify

LFBs and LFMs with method analytes. It is recommended that more than

one concentration of this solution be prepared. During the method

development, two solutions were used. One containing 100 μg/mL of each

analyte, was used for higher concentration fortifications, and the other

containing 10 μg/mL of each analyte in methanol was used for lower level
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fortifications. Check these solutions regularly for signs of evaporation

and/or degradation. During method development these solutions were

stable for at least 60 days.

7.12.3 SURROGATE ANALYTE FORTIFICATION SOLUTION - Prepare a

solution of the three surrogate standards listed in Sect. 7.11.2 in methanol. 

If a single concentration of each was used to prepare calibration standards,

then prepare this solution such that a convenient fortification volume will

correspond to an equivalent concentration in the extracts. If the surrogates

have been calibrated over a concentration range, add surrogate to the

samples, LRBs, LFBs, LFMs at a single concentration in the range of 1-5

μg/L. Check these solutions regularly for signs of evaporation and/or

degradation. During method development these solutions were stable for at

least 60 days.

7.13 GC/MS TUNE CHECK SOLUTION -- Bromofluorobenzene (BFB) [CAS# 460-00-4]

in ethyl acetate; suggested concentration, 5 μg/mL. Store this solution in an amber vial

at 0° C or less. 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE

8.1 SAMPLE COLLECTION -- When sampling from a water tap, open the tap and

allow the system to flush until the water temperature has stabilized (usually about 2

min). Adjust the flow to about 500 mL/min and collect samples from the flowing

stream. The sample should nearly fill the 1 L or 1 qt bottle, but does not need to be

headspace free. If preservatives have been added to the bottle prior to sampling, be

careful not to rinse them out during sample collection. Keep samples sealed from

collection time until analysis. When sampling from an open body of water, fill the

sample container with water from a representative area. Sampling equipment,

including automatic samplers, must be free of plastic tubing, gaskets, and other parts

that may leach interfering analytes into the water sample. 

8.2 SAMPLE DECHLORINATION AND PRESERVATION -- All samples must be

dechlorinated and preserved with a microbial inhibitor at the time of collection. 

Jenkins, et al. (8) have clearly shown the need for microbial preservation of several

of the method analytes. Copper sulfate pentahydrate is added as a microbial

inhibitor at a concentration of 0.5 g/L of water (9). The Trizma Pre-Set Crystals

pH7 buffer (Sect. 7.5) is added at a concentration of 5.0 g/L. The buffer acts to tie

up free chlorine, and also keeps the copper in solution (9). The buffer should be

added to all samples, even those that are unchlorinated. The copper sulfate and

Trizma may be added to the sample bottles as dry salts prior to transporting the

sample bottles to the sampling site. 
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8.2.1 As an alternative to the Trizma Pre-Set Crystals pH7 buffer, the following

may be used : 0.3 g of tris(hydroxymethyl)aminomethane plus 4.7 g of

tris(hydroxymethyl)aminomethane hydrochloride for each 1 L sample.

8.3 SAMPLE TRANSPORT AND STORAGE -- All samples should be iced during

shipment and must not exceed 10o C during the first 48 hours. Samples should be

confirmed to be at or below 10o C when they are received at the laboratory. Samples 

stored in the lab must be held at or below 6o C until extraction, but should not be

frozen.

Samples that are significantly above 10° C at the time of collection, may need to be

iced or refrigerated for a period of time, in order to chill them prior to shipping. This

will allow them to be shipped with sufficient ice to meet the above requirements.

8.4 HOLDING TIME -- Samples must be extracted within 14 days of collection. 

Sample extracts may be stored for up to 30 days after sample extraction, when stored

in amber vials at 0° C or less. 

9. QUALITY CONTROL

9.1 Quality control (QC) requirements include: the initial demonstration of laboratory

capability (summarized in Table 23) followed by regular analyses of continuing

calibration checks, laboratory performance check standards, laboratory reagent

blanks, laboratory fortified blanks, and laboratory fortified matrix samples. In

addition, a DL must be determined for each analyte of interest. These criteria are

considered the minimum acceptable QC criteria, and laboratories are encouraged to

institute additional QC practices to meet their specific needs. The laboratory must

maintain records to document the quality of the data generated. A complete

summary of QC requirements is summarized in Table 24.

 

9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) -- Requirements for the

initial demonstration of laboratory capability are described in the following sections

and summarized in Table 23.

9.2.1 INITIAL DEMONSTRATION OF LOW CARTRIDGE/DISK

EXTRACTION BACKGROUND AND SYSTEM BACKGROUND --

Before any samples are analyzed, or any time a new lot or brand of solid

phase extraction disks or cartridges is received from a supplier, it must be

demonstrated that a laboratory reagent blank (LRB) is reasonably free of

any contamination that would prevent the determination of any analyte of

concern (Sect. 9.2.1.2 and 9.4).

9.2.1.1 A source of potential contamination is the solid phase extraction

media which may contain phthalate esters, silicon compounds,
529-15

and other contaminants that could interfere with the

determination of method analytes. 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 initial

demonstration.

9.2.1.2 Other sources of background contamination are solvents,

reagents, and glassware. Background contamination must be

reduced to an acceptable level before proceeding with the next

section. Background from method analytes and interferences

should be # 1/3 the MRL.

9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) -- Prepare 4-7

replicate LFBs fortified at 2-5 :g/L, or other mid-range concentration, using

solutions described in Sect. 7.12.2 and 7.12.3. This concentration may be

lower if SIM is being used. Sample preservatives described in Sect. 8.2

must be added to these samples. Extract and analyze these replicates

according to the procedure described in Sect. 11. The relative standard

deviation (RSD) of the results of the replicate analyses must be less than or

equal to 20% for all method analytes and surrogates. 

9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) -- Using the same

set of replicate data generated for Sect. 9.2.2, calculate average recovery. 

The average recovery of the replicate values for all analytes and surrogates

must be within 70-130% of the true value. 

9.2.4 DL DETERMINATION -- Replicate analyses for this procedure should be

done over at least 3 days (both the sample extraction and the GC analyses

should be done over at least 3 days). Prepare at least 7 replicate LFBs using

solutions described in Sect. 7.12.2 and 7.12.3, at a concentration estimated

to be near the DL. This concentration may be estimated by selecting a

concentration at 2-5 times the noise level. Concentrations shown in the

example data in Tables 3, 9, and 15 may be used as a guide, however the

appropriate concentration will be dependent upon the injection technique

and the sensitivity of the GC/MS system used. Sample preservatives

described in Sect. 8.2 must be added to these samples. Analyze the seven

replicates through all steps of Sect. 11. Calculate the DL using the

following equation:

DL = St( n - 1, 1 - alpha = 0.99)
529-16

where:

t

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

n-1 degrees of freedom

n = number of replicates

S = standard deviation of replicate analyses.

NOTE: Do not subtract blank values when performing DL calculations.

9.2.5 The analyst is permitted to modify GC columns or GC conditions. Each

time such method modifications are made, the analyst must repeat the

procedures in Sect. 9.2.1 through 9.2.4.

9.3 MINIMUM REPORTING LEVEL (MRL) -- The MRL is the threshold

concentration of an analyte that a laboratory can expect to accurately quantitate in an

unknown sample. The MRL should be established at an analyte concentration either

greater than three times the DL or at a concentration which would yield a response

greater than a signal-to-noise ratio of five. Although the lowest calibration

standard for an analyte may be below the MRL, the MRL for an analyte must

never be established at a concentration lower than the lowest calibration

standard for that analyte.

9.4 LABORATORY REAGENT BLANKS (LRB) -- With each extraction batch,

analyze a laboratory reagent blank to determine the background system

contamination. If, within the retention time window of any analyte, the LRB

produces a peak that would prevent the determination 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. Background from method analytes or contaminants that interfere with

the measurement of method analyses should be # 1/3 the MRL. Any time a new

batch of SPE cartridges is received, or new supplies of other reagents are used,

repeat the demonstration of low background described in Sect. 9.2.1.

9.5 CONTINUING CALIBRATION CHECK (CCC) -- This calibration check is

required at the beginning of each day that samples are analyzed, after every ten field

samples, and at the end of any group of sample analyses. See Sect. 10.3 for

concentration requirements and acceptance criteria.

9.6 MS TUNE CHECK -- This performance check consists of verifying the MS tune

using the mass spectrum of BFB. A complete description of the check is in Sect. 

10.2.1. This check must be performed each time a major change is made to the mass

spectrometer, and each time analyte calibration is performed (i.e., average RFs are

calculated, or first or second order calibration curves are developed).
529-17

9.7 LABORATORY FORTIFIED BLANK (LFB) -- With each extraction batch, extract

and analyze an LFB containing each analyte of concern. If more than 20 field

samples are included in a batch, analyze an LFB for every 20 samples. The fortified

concentration of the LFB should be rotated between low, medium, and high

concentrations from day to day. The low concentration LFB must be as near as

practical to the MRL. Results of LFB analyses corresponding to the low fortification

concentration for an analyte must be within 50-150% of the true value. Results of

LFB analysis from medium and high level concentrations must be 70-130% of the

true value for all analytes. 

9.8 INTERNAL STANDARD (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 should not deviate from the response in the most recent CCC

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

9.8.1 If the reinjected aliquot produces an acceptable internal standard response,

report results for that aliquot.

9.8.2 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 Sect. 10.3.3, recalibration is in order

per Sect. 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. Alternatively, collect a new sample and re-analyze.

9.9 SURROGATE RECOVERY -- The surrogate standards are fortified into all

calibration standards, samples, LFBs, LFMs, FDs, FRBs and LRBs. The surrogate is

a means of assessing method performance from extraction to final chromatographic

measurement. 

9.9.1 Surrogate recovery criteria are 70-130% of the fortified amount for all

method surrogates. When surrogate recovery from a sample, blank, or CCC

does not meet these criteria, check : (1) calculations to locate possible

errors, (2) standard solutions for degradation, (3) contamination, and (4)

instrument performance. Correct any problems that are identified. If these

steps do not reveal the cause of the problem, reanalyze the extract. 

9.9.2 If the extract reanalysis meets the surrogate recovery criterion, report only

data for the reanalyzed extract.
529-18

9.9.3 If the extract reanalysis fails the recovery criterion, the analyst should check

the calibration by reanalyzing the most recently acceptable calibration

standard. If the calibration standard fails the criteria of Sect. 10.3.3,

recalibration is in order per Sect. 10.2. If the calibration standard is

acceptable, it may be necessary to extract another aliquot of sample if

sample holding time has not been exceeded. If the sample re-extract also

fails the recovery criterion, report all data for that sample as suspect, or

analyze a duplicate sample.

9.10 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- Determine that the

sample matrix does not contain materials that adversely affect method performance. 

This is accomplished by analyzing replicates of laboratory fortified matrix samples

and ascertaining that the precision, accuracy, and method detection limits of analytes

are in the same range as obtained with laboratory fortified blanks. If a variety of

different sample matrices are analyzed regularly, for example, drinking water from

groundwater and surface water sources, LFM data should be collected for each

matrix. Over time, LFM data should be documented for all routine sample sources

for the laboratory. A laboratory fortified sample matrix should be extracted and

analyzed for each extraction batch. If more than 20 samples are processed in a

batch, extract and analyze an LFM for every 20 samples. If the recovery data for an

LFM does not meet the recovery criteria in Sect. 9.7, and LFBs show the laboratory

to be in control, then the samples from that matrix (sample location) are documented

as suspect due to matrix effects.

9.10.1 Within each extraction batch, a minimum of one field sample is fortified as

an LFM for every 20 samples analyzed. The LFM is prepared by spiking a

sample with an appropriate amount of the fortification solutions described

in Sect. 7.12.2 and 7.12.3. Select the spiking concentration that is at least

twice the matrix background concentration. Selecting a duplicate bottle of a

sample that has already been analyzed, aids in the selection of appropriate

spiking levels. If this is not possible, use historical data or rotate through

low, medium and high calibration concentrations to select a fortifying

concentration.

9.10.2 Calculate the percent recovery (R) for each analyte, after correcting the 

measured fortified sample concentration, A, for the background

concentration, B, measured in the unfortified sample, i.e.,

where C is the fortified concentration. Compare these values to

control limits for LFBs (Sect. 9.7).
529-19

9.10.3 Recoveries may exhibit a matrix dependence. For samples fortified at or

above their native concentration, recoveries should range between 70 and

130% for all method analytes. If the accuracy (percent recovery) of any

analyte falls outside the designated range, and the laboratory performance

for that analyte is shown to be in control, the accuracy problem encountered

with the fortified sample is judged to be matrix related, not system related. 

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.

9.11 FIELD DUPLICATES (FD) -- Within each extraction batch, a minimum of one field

sample should be analyzed in duplicate. If more than 20 samples are extracted in a

batch, analyze a FD for each 20 samples. Duplicate sample analyses serve as a

check on sampling and laboratory precision. If analytes are not routinely observed in

field samples, duplicate LFMs should be analyzed to substitute for this requirement.

Refer to Sect. 9.10.1 for guidance on spiking concentrations.

9.11.1 Calculate the relative percent difference (RPD) for duplicate measurements

(FD1 and FD2) as shown below.

9.11.2 Relative percent differences for laboratory duplicates and duplicate LFMs

should fall in the range of ± 30 %.

NOTE: Greater variability may be observed for target analytes with

concentrations at the low end of the calibration range.

9.12 QUALITY CONTROL SAMPLE (QCS) -- Each time that new PDSs are prepared,

analyze a QCS from an external source. If standards are prepared infrequently,

analyze a QCS at least quarterly. The QCS may be injected as a calibration standard,

or fortified into reagent water and analyzed as an LFB. If the QCS is analyzed as a

calibration check standard, then the acceptance criteria are the same as for the CCC

(Sect. 10.3.3). If the QCS is analyzed as an LFB, then the acceptance criteria are the

same as for an LFB (Sect. 9.7). If measured analyte concentrations are not of

acceptable accuracy, check the entire analytical procedure to locate and correct the

problem. 

10. CALIBRATION AND STANDARDIZATION

10.1 Demonstration and documentation of acceptable mass spectrometer tune and initial

calibration is required before any samples are analyzed. After initial calibration is
529-20

successful, a continuing calibration check is required at the beginning and end of

each period in which analyses are performed, and after every tenth sample. 

Verification of mass spectrometer tune must be repeated each time a major

instrument modification or maintenance is performed, and prior to analyte

recalibration. 

10.2 INITIAL CALIBRATION

10.2.1 MS TUNE -- Calibrate the mass and abundance scales of the MS with

calibration compounds and procedures prescribed by the manufacturer with

any modifications necessary to meet tuning requirements. Inject 5 ng or less

of BFB into the GC/MS system. Acquire a mass spectrum that includes

data for m/z 45-250. If the BFB mass spectrum does not meet all criteria in

Table 1, the MS must be retuned and adjusted to meet all criteria before

proceeding with calibration. A single spectrum at the apex of the

chromatographic peak, or an average of the three spectra at the apex of the

peak, or an average spectrum across the entire GC peak may be used to

evaluate the performance of the system. Background subtraction is

permitted. 

NOTE: Although BFB is most commonly used as the tune check for

volatile compound analysis, it was selected for use in this method for

semivolatile analytes because the ions used for the BFB tune check are

similar to the analyte quantitation ions in this method.

10.2.2 ANALYTE CALIBRATION FOR FULL SCAN MASS

SPECTROMETRY -- Inject an aliquot, usually 1-2 μL (unless a large

volume injector is used), of a medium to high concentration calibration

solution. For example, 2-10 :g/mL, and acquire and store data from m/z

45-250 with a total cycle time (including scan overhead time) of 0.7 sec or

less. Cycle time must be adjusted to measure at least five or more scans

during the elution of each GC peak. Seven to ten scans across each GC peak

are recommended. 

NOTE: On some instruments that cannot scan quickly, e.g. ion traps, the

scan time and the chromatographic peak width may need to be carefully

evaluated to make sure that this requirement is met.

Chromatographic conditions used during method development are outlined

below. These conditions were found to work well on the instrumentation

used. Since some of the method analytes are vulnerable to active sites and

thermal decomposition, optimum chromatographic conditions may vary

with individual instrument design. Although the following conditions are
529-21

recommended, GC conditions may be modified, if all performance criteria

in Sections 9 and 10 are met.

10.2.2.1 PTV Splitless Injection, Full Scan Mass Spectrometry -

Inject with the injector temperature at 60° C, program the

injector at 200° C per min to 250° C. Hold in the splitless mode

for 1.0 min. Use an initial GC oven temperature of 45° C for 3.3

minutes. Program the oven temperature at 7° C per min to 210°

C. Then program at 20° C per min to 250° C. An example

chromatogram obtained under these conditions is shown in

Figure 1.

10.2.2.2 Cold On-Column Injection, Full Scan Mass Spectrometry –

Inject with the injector temperature at 50° C, program the

injector at 150° C per min to 220° C. Hold in the splitless mode

for 2.0 min. Use an initial GC oven temperature of 50° C for 3

minutes. Program the oven temperature at 7° C per min to 210°

C. Then program at 20° C per min to 250° C. 

10.2.3 Performance criteria for the calibration standards. Examine the stored

GC/MS data with the data system software. The GC/MS/DS peak

identification software should be able to recognize a GC peak in the

appropriate retention time window for each of the compounds in the

calibration solution, and make correct identifications (Sect. 11.8, 11.9).

10.2.4 If all performance criteria are met, inject an appropriate volume (usually 1-2

:L unless a large volume injector is used) of each of the other CAL

solutions using the same GC/MS conditions. 

10.2.4.1 Some GC/MS systems may not be sensitive enough to detect

some of the analytes in the two lowest concentration CAL

solutions. If this is the case, it is acceptable to calibrate using

the remaining higher concentration points, as long as a minimum

of 5 calibration points are used to generate the calibration curve

or average response factor (RF) for each analyte. In addition,

some GC/MS systems might reach signal saturation at the

highest calibration concentration. If this is the case, it is

acceptable to drop the highest point and calibrate on the

remaining points, as long as at least 5 calibration concentrations

are used to generate the calibration curve or average RF for each

analyte. Points in the middle of the calibration range may not be

dropped. Use a minimum of 3 standards for a calibration range

of 1 order of magnitude, and at least 5 standards for 2 orders of
529-22

magnitude. Data outside of the established calibration range

should never be reported.

10.2.5 Concentrations may be calculated through the use of average response

factor (RF) or through the use of a calibration curve. Average RF

calibrations may only be used if the RF values over the calibration range are

relatively constant (<30% RSD). 

Average RF is determined by calculating the mean RF of each calibration

point.

 

 where:

Ax = integrated abundance (peak area) of the quantitation ion

of the analyte.

Ais = integrated abundance (peak area) of the quantitation ion

internal standard.

 Qx = quantity of analyte injected in ng or concentration units.

 Qis = quantity of internal standard injected in ng or

concentration units.

10.2.6 As an alternative to calculating average RFs and applying the RSD test, use

the GC/MS data system software to generate a linear regression or quadratic

calibration curve. The analyst may choose whether or not to force zero, to

obtain a curve that best fits the data. Examples of common GC/MS system

calibration curve options are: 1) Ax

 /Ais vs Qx

 /Qis and 2) RF vs Ax

 /Ais.

10.2.7 Acceptance criteria for the calibration of each analyte is determined by

calculating the concentration of each analyte and surrogate in each of the

analyses used to generate the calibration curve or average RF. Each

calibration standard, except the lowest point, for each analyte must calculate

to be 70-130% of its true value. The lowest point must calculate to be 50-

150% of its true value. If this criteria cannot be met, reanalyze the

calibration standards, or select an alternate method of calibration. The data

in this method were generated using both linear and quadratic fits,

depending upon the analyte. Quadratic fit calibrations should be used with

caution, because the non-linear area of the curve may not be reproducible.

10.2.8 ANALYTE CALIBRATION FOR SIM MASS SPECTROMETRY –

Collect data for a mid to high level standard in the full scan mode as

directed in Sect. 10.2.2 above. 
529-23

Chromatographic conditions used during method development are outlined

below. These conditions were found to work well on the instrumentation

used. Since some of the method analytes are vulnerable to active sites and

thermal decomposition, optimum chromatographic conditions may vary

with individual instrument design. Although the following conditions are

recommended, GC conditions may be modified, if all performance criteria

in Sections 9 and 10 are met. 

10.2.8.1 PTV Splitless Injection, SIM Mass Spectrometry -

Inject with the injector temperature at 60° C, program the

injector at 150° C per min to 220° C. Hold in the splitless mode

for 2.0 min. Use an initial GC oven temperature of 45° C for 3

minutes. Program the oven temperature at 7° C per min to 210°

C. Then program at 20° C per min to 250° C. 

10.2.9 Divide the chromatogram into retention time windows, each of which

contains one or more chromatographic peaks. During SIM analysis a small

number of selected masses corresponding to dominant ions for the analytes

in that window will be scanned within each window. The number of

windows used and the selection of the retention times for each window will

depend upon the type of instrumentation used. Highest sensitivity can be

expected when a minimum number of ions are scanned in a window, i.e.

one analyte per window. However, if analytes are closely eluting, it may be

necessary to include more than one analyte per window.

CAUTION: When acquiring SIM data, GC operating conditions must be

carefully reproduced for each analysis to provide reproducible retention

times. If this is not done, the correct ions will not be monitored at the

appropriate times. As a precautionary measure, the chromatographic peaks

in each window must not elute too close to the edge of the time window. 

As a minimum, there should be at least 5 sec between the edge of the time

window and the beginning or end of an analyte peak.

10.2.10 Using the GC/MS software, create an acquisition method such that 2 or 3

identifying ions are scanned for each chromatographic peak. Three ions are

recommended, but in some cases, only 2 ions may be used if only 2

abundant ions are present in the mass spectrum, e.g. RDX. Suggested

quantitation ions and secondary confirmation ions are listed in Table 2. Reanalyze the calibration standard using the SIM acquisition method. 

Continue with calibration as described in Sect. 10.2.3 through 10.2.7.

10.3 CONTINUING CALIBRATION CHECK (CCC) -- The minimum daily calibration

verification is as follows. Verify the initial calibration at the beginning and end of

each group of analyses, and after every tenth sample during analyses. (In this
529-24

context, a “sample” is considered to be a field sample. LRBs, LFMs, LFBs and

CCCs are not counted as samples.) The beginning CCC each day should be at or

near the MRL in order to verify instrument sensitivity prior to any analyses. If

standards have been prepared such that all low CAL points are not in the same CAL

solution, it may be necessary to analyze two CAL solutions to meet this requirement.

Alternatively, it may be cost effective to obtain a customized standard to meet this

criteria. Subsequent CCCs can alternate between a medium and high concentration

standard. 

10.3.1 Inject an aliquot of the appropriate concentration calibration solution and

analyze with the same conditions used during the initial calibration.

10.3.2 Determine that the absolute areas of the quantitation ions of the internal

standard have not changed by more than 30% from the areas measured in

the most recent continuing calibration check, or by more than 50% from the

areas measured during initial calibration. If this area has changed by more

than these amounts, adjustments must be made to restore system sensitivity. 

These adjustments may include cleaning of the MS ion source, or other

maintenance as indicated in Sect. 10.3.4. Major instrument maintenance

requires recalibration. Control charts are useful aids in documenting system

sensitivity changes.

10.3.3 Calculate the concentration of each analyte and surrogate in the check

standard. The calculated amount for each analyte for medium and high level

CCCs must be within 70-130% of the true value. The calculated amount

for the lowest calibration point for each analyte must be within 50-150% of

the true value. If these conditions do not exist, remedial action should be

taken which may require recalibration. Any field sample extracts that have

been analyzed since the last acceptable calibration verification should be

reanalyzed after adequate calibration has been restored, with the following

exception. If the continuing calibration check in the middle or at the

end of an analysis batch fails because the calculated concentration is

>130% of the true value, and field sample extracts showed no detection

of method analytes, non-detects may be reported without re-analysis.

10.3.4 Some possible remedial actions are listed below. This list is not meant to

be all inclusive. Major maintenance such as cleaning an ion source,

cleaning quadrupole rods, replacing filament assemblies, replacing the

electron multiplier, changing the GC column, etc. require returning to the

initial calibration step (Sect. 10.2).

10.3.4.1 Check and adjust GC and/or MS operating conditions; check the

MS resolution, and calibrate the mass scale.
529-25

10.3.4.2 Replace the GC injection port liner; cleaning and deactivating

used liners is not recommended.

10.3.4.3 Flush the GC column with solvent according to manufacturer's

instructions.

10.3.4.4 Break off a short portion (about 1 meter) of the column from the

end near the injector, or replace GC column. This action will

cause a change in retention times. A change in retention times

may require a change in the SIM scan time windows.

10.3.4.5 Prepare fresh CAL solutions, and repeat the initial calibration

step. 

10.3.4.6 Clean the MS ion source and rods (if a quadrupole).

10.3.4.7 Replace any components that allow analytes to come into

contact with hot metal surfaces.

10.3.4.8 Replace the MS electron multiplier, or any other faulty

components.

11. PROCEDURE

11.1 SOLID PHASE EXTRACTION

11.1.1 This procedure may be performed manually or in an automated mode (Sect.

6.11) 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. 

11.1.2 Mark the level of the sample on the outside of the sample bottle for later

sample volume determination (Sect. 11.5). Verify that the sample is

between pH 5 and 7, and is free of residual chlorine. If the sample is an

LRB or LFB, add sample preservatives specified in Sect. 8.2. Add an

aliquot of the surrogate fortification solution(s), and mix immediately until

homogeneous. The resulting concentration of these compounds in the water

should be 1-5:g/L. Generally, SIM analyses should be performed with

surrogate concentrations at the low end of this range. If the sample is an

LFB or LFM, add the desired amount of analyte fortification solution.
529-26

11.2. CARTRIDGE EXTRACTION

11.2.1. CARTRIDGE CLEAN-UP AND CONDITIONING -- Rinse each cartridge

with three, 5 mL aliquots of ethyl acetate. Let the cartridge drain dry after

each flush. Then rinse the cartridge with three, 5 mL aliquots of methanol,

but DO NOT allow the methanol to elute below the top of the cartridge

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

Rinse the cartridge with two, 10 mL aliquots of reagent water, stopping the

flow when there is approximately 4 cm of reagent water in the column.

Attach the transfer tube, and turn on the vacuum, and begin adding sample

to the cartridge. 

11.2.2. Adjust the vacuum so that the approximate flow rate is 10-15 mL/min. 

After all of the sample has passed through the SPE cartridge, draw air or

nitrogen through the cartridge for 10-15 min at high vacuum (10-15 in. Hg). 

The drying time may vary, depending upon the strength of the vacuum

source, and the number of cartridges being processed simultaneously. Do

not dry the cartridge for more than 10-15 min. Additional time is not likely

to increase dryness, and excess drying times have been shown to decrease

recoveries of nitrobenzene (10).

NOTE: Samples with a high level of hardness and/or high TOC may exhibit

a lower flow rate than “cleaner” samples at the same vacuum setting. This

may be due to partial plugging of the solid phase. Fortified sample matrices

of these types showed no loss of method performance.

11.2.3. Place a collection tube in the vacuum chamber underneath each extraction

cartridge. Rinse the inside of each sample bottle with 5 mL ethyl acetate

and use vacuum to pull the rinse solvent through the transfer tube and

through the cartridge, collecting the solvent in a collection tube. Remove

the transfer tubing from the top of the cartridge. Add another 5 mL of ethyl

acetate to the top of the cartridge with a disposable pipette. Pull this solvent

through the cartridge at low vacuum, such that the solvent exits the

cartridge in a dropwise fashion. Combine this second eluate with the first

one. Dry and concentrate the combined eluate as directed in Sect. 11.4.

11.3. DISK EXTRACTION

11.3.1 DISK CLEAN-UP AND CONDITIONING -- Rinse each disk with three, 5

mL aliquots of ethyl acetate. Let the disk dry after each rinse. Then rinse

the disk with three, 5 mL aliquots of methanol, but DO NOT allow the

methanol to elute below the top of the disk. From this point, do not allow

the disk to go dry. Rinse the disk with two, 10 mL aliquots of reagent

water, stopping the flow when there is approximately 3-4mm of reagent
529-27

water on top of the disk. Turn on the vacuum, and begin adding sample to

the disk. 

11.3.2. The sample can be passed through the disk at high vacuum (10-15 in. Hg). 

After the entire sample has been extracted, dry the disk at full vacuum for 5-

10 min. Do not dry the disk for more than 10 min. Additional time is not

likely to increase dryness, and excess drying times have been shown to

decrease recoveries of nitrobenzene (10).

11.3.3. Place a collection tube in the vacuum manifold beneath the filtration

glassware setup for each extraction disk. Rinse the inside of each sample

bottle with 5 mL ethyl acetate and use this rinse solvent to elute each disk,

collecting the solvent in a collection tube. Add another 5 mL of ethyl

acetate to the top of the disk with a disposable pipette, using the solvent to

wash down the sides of the filtration glassware. Pull this solvent through

the disk. Add another 3 mL of ethyl acetate to the top of the disk with a

disposable pipette, using the solvent to wash down the sides of the filtration

glassware. Pull this solvent through the disk. Dry and concentrate the

combined eluates as directed in Sect. 11.4.

11.4. Small amounts of residual water from the sample container and the SPE cartridge

may form an immiscible layer with the eluate. To eliminate residual water, pass the

eluate through the drying column (Sect. 6.7). The drying column is packed with

approximately 5 to 7 grams of anhydrous sodium sulfate, and is pre-wetted with a

small volume of ethyl acetate prior to passing the eluate through it. Collect the dried

eluate in a clean collection tube. After passing the eluate through the drying tube,

wash the sodium sulfate with at least 3 mL ethyl acetate and collect it in the same

tube. Concentrate the extract to approximately 0.9 mL in a warm (40° C) water bath

under a gentle stream of nitrogen. Do not concentrate the extract to less than 0.5

mL, as this will result in loss of analytes. Add the internal standard (Sect. 7.12.1).

Adjust final volume to 1 mL. Make any volume adjustments with ethyl acetate.

11.5 Fill the sample bottle to the volume mark noted in Sect.11.1.2. with tap water. 

Transfer the tap water to a 1000 mL graduated cylinder, and measure the sample

volume to the nearest 10 mL. Record this volume for later analyte concentration

calculations. As an alternative to this process, the sample volume may be

determined by the difference in weight between the full bottle (before extraction)

and the empty bottle (after extraction). Assume a sample density of 1.0 g/mL.

11.6 Analyze an aliquot of the sample extract with the GC/MS system under the same

conditions used for the initial and continuing calibrations.

11.7 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
529-28

interest. Use the data system software to examine the ion abundances of

components of the chromatogram. 

11.8 IDENTIFICATION OF ANALYTES FOR FULL SCAN MASS SPECTROMETRY -- 

Identify a sample component by comparison of its mass spectrum (after background

subtraction) to a reference spectrum in the user-created data base. The GC retention

time of the sample component should be within 1-2 sec of the retention time observed

for that same compound in the most recently analyzed continuing calibration check

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 a compound. However, the experience of the analyst should

weigh heavily in the interpretation of the chromatogram.

11.8.1 In general, all ions that are present above 10% relative abundance in the mass

spectrum of the standard should be present in the mass spectrum of the sample

component and should agree within absolute 20%. For example, if an ion has

a relative abundance of 30% in the standard spectrum, its abundance in the

sample spectrum should be in the range of 10 to 50%. 

11.9 IDENTIFICATION OF ANALYTES FOR SIM MASS SPECTROMETRY -- 

Identify a sample component by comparison of its selected ions to a reference

spectrum in the user-created data base. The GC retention time of the sample

component should be within 1-2 sec of the retention time observed for that same

compound in the most recently analyzed continuing calibration check 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

identification window size for a compound. However, the experience of the analyst

should weigh heavily in the interpretation of the chromatogram.

NOTE: Because less spectral information is being acquired for analyte identification

when SIM is used, retention times of the analyte peaks are an even more important

factor in analyte identification than in full scan data analysis.

11.9.1 The apex of the chromatographic peak for the quantiation ion and

confirmation ions for each analyte must be within ± 1 scan of one another.

11.9.2 The integrated ion current for each quantitation and confirmation ion must

be at least three times background noise, and must not have saturated the

detector.

11.9.3 All ions monitored in the standard should be present in the SIM mass

spectrum of the sample component and should agree within absolute 20%. 
529-29

For example, if an ion has a relative abundance of 30% in the standard

spectrum, its abundance in the sample spectrum should be in the range of 10

to 50%. 

12. DATA ANALYSIS AND CALCULATIONS

12.1 Complete chromatographic resolution is not necessary for accurate and precise

measurements of analyte concentrations if unique ions with adequate intensities are

available for quantitation. Identification is hampered when sample components are

not resolved chromatographically and produce mass spectra containing the same ions

contributed by more than one analyte. When GC peaks obviously represent more

than one sample component (i.e., broadened peak with shoulder(s) or valley between

two or more maxima), appropriate analyte spectra and background spectra can be

selected by examining plots of characteristic ions for each tentatively identified

component. When analytes coelute (i.e., only one GC peak is apparent), the

identification criteria can be met but each analyte spectrum will contain extraneous

ions contributed by the coeluting compound. In validating this method,

concentrations were calculated by measuring the quantitation ions listed in Table 2. 

Other ions may be selected at the discretion of the analyst. If the response of any

analyte exceeds the calibration range established in Sect. 10, dilute the extract, add

additional internal standard, and reanalyze. The resulting data should be

documented as a dilution, with an increased MRL.

12.1.1 Calculate analyte and surrogate concentrations, using the multipoint

calibration established in Sect. 10. Do not use daily calibration verification

data to quantitate analytes in samples. Adjust final analyte concentrations

to reflect the actual sample volume determined in Sect. 11.5. 

12.1.2 Calculations should utilize all available digits of precision, but final

reported concentrations should be rounded to an appropriate number of

significant figures (one digit of uncertainty). 

13. METHOD PERFORMANCE 

13.1 PRECISION, ACCURACY AND DLs-- Single laboratory accuracy and precision

data from both fortified reagent water and fortified matrices are presented in Tables

3-20. Detection limits (DLs) are presented in Tables 3, 9 and 15 for the three

techniques demonstrated.

13.1.1 Data presented in Tables 3-8 and 21-22 were obtained on a Varian Saturn 4

GC/MS (ion trap) system, using PTV splitless injection and full scan mass

spectrometry. Gas chromatographic conditions are given in Sect. 10.2.2.1.
529-30

NOTE: Selected ion storage was evaluated on this instrument. Sensitivity

was not significantly improved relative to full scan monitoring.

13.1.2 Data presented in Tables 9-14 were obtained on a Schimadzu QP5050A

GC/MS (quadrupole) system, using PTV splitless injection and SIM mass

spectrometry. A high pressure injection (14.5 psi) was employed on this

instrument. Other gas chromatographic conditions are given in Sect.

10.2.8.1.

13.1.3 Data presented in Tables 15-20 were obtained on a Varian Saturn 4 GC/MS

(ion trap) system, using cold on-column injection and full scan mass

spectrometry. Gas chromatographic conditions are given in Sect. 10.2.2.2.

13.2 EVALUATION OF ADDITIONAL EXPLOSIVES – Nitroglycerin (NG), octahdro1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX ) and pentaerythritoltetra-nitrate (PETN)

were evaluated for inclusion into this method. HMX was never observed in any of

the chromatograms. Precision and accuracy for NG and PETN were not sufficient to

reliably meet the QC criteria in the method. The method may be suitable for

qualitative identification of these two compounds.

13.3 Data were not obtained for 2,4-dinitrotoluene with the on-column technique. The

mass spectrum of this compound was observed to change with concentration. At

low concentrations, an unusual spectrum was observed. It was determined that this

resulted from reactions of 2,4-dinitrotoluene with residual solvent in the ion trap. 

This is more likely to happen with on-column techniques than splitless techniques

because more solvent is transferred to the column. Although the on-column

technique was not demonstrated with a quadrupole instrument, it is suspected that

this observation was an ion trap phenomenon.

13.4 Data were not obtained for 3,5-dinitroanaline with the on-column technique. 

Because a commercial standard mixture was used, where the concentration of this

analyte was five times lower than the other analytes, many of the demonstration

samples contained it at a concentration too low to be accurately quantified.

13.5 Some matrix effects may be observed. Some method analytes extracted from some

water matrices may exhibit “matrix induced chromatographic response

enhancement” (11-13). This phenomenon has been widely reported for the analysis

of thermally labile pesticides in water matrices, and may be applicable to some of

these method analytes as well. Compounds that exhibit this phenomenon may give

analytical results that exceed 100% recovery in fortified matrix samples, especially

at low concentrations. It has been proposed that thermally labile compounds are

susceptible to decomposition in the GC injection port and/or while in the GC

column. Co-extracted matrix components help shield and protect these compounds

through the GC system, thus resulting in a higher response than when clean
529-31

standards are analyzed. This effect can usually be minimized by reducing the

residence time of method analytes in the GC system by using a relatively high linear

velocity and a short column. The use of clean, deactivated injection port liners is

also advised. 

13.6 ANALYTE STABILITY STUDIES -- 

13.6.1 SAMPLES - Chlorinated surface water samples, fortified with method

analytes at 5.0 μg/L, were preserved and stored as required in Sect. 8. The

average of replicate analyses (n=5) conducted on days 0, 7, 11, 14, and 19

are presented in Table 21. These data document the 14 day holding time.

13.6.2 EXTRACTS - Extracts from the day 0 holding time study described above

were stored at 0 °C, and analyzed in replicate (n=5) on days 0, 17, 15, 21,

28, and 35. The results of these analyses are presented in Table 22, and

document the 30 day holding time.

14. POLLUTION PREVENTION

14.1 This method utilizes SPE technology to remove the analytes from water. It requires

the use of very small volumes of organic solvent and very small quantities of pure

analytes, thereby minimizing the potential hazards to both the analyst and the

environment when compared with 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.

15. WASTE MANAGEMENT

15.1 The analytical procedures described in this method generate relatively small amounts

of waste since only small amounts of reagents and solvents are used. The matrices

of concern are finished drinking water or source water. However, the Agency

requires that laboratory waste management practices be conducted consistent with all

applicable rules and regulations, and that laboratories protect the air, water, and land

by minimizing and controlling all releases from fume hoods and bench operations. 

Also, compliance is required with any sewage discharge permits and regulations,

particularly the hazardous waste identification rules and land disposal restrictions. 

For further information on waste management, consult “The Waste Management

Manual for Laboratory Personnel” available on-line from the American Chemical
529-32

Society at http://chemistry.org/portal/Chemistry?PID=acsdisplay.html&DOC=

government%5Cpublications%5Ceip_hazwaste.html.

16. REFERENCES

1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for

Wastewaters," Environ. Sci. Technol., 15 (1981) 1426-1435.

2. "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.

3. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational

Safety and Health Administration, OSHA, (Revised, July 1, 2001).

4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,

Committee on Chemical Safety, 6th Edition, available from the ACS Office of Society

Services by e-mail at OSS@acs.org.

5. “Drinking Water Standards and Health Advisories,” United States Environmental

Protection Agency, Office of Water, EPA 822-B-00-001, 2000.

6. “Method 8095, Explosives by Gas Chromatography,” United States Environmental

Protection Agency, Office of Solid Waste, 2000.

7. Walsh, M.E. and T.A. Ranney, “Determination of Nitroaromatic, Nitramine, and Nitrate

Ester Explosives in Water Using Spe and GC-ECD,”, U.S. Army Corps of Engineers, Cold

Regions Research and Engineering Laboratory, Special Report 98-2.

8. Jenkins, T.F., P.G. Thorne, E.F. McCormick, and K.F. Myers, “Preservation of Water

Samples Containing Nitroaromatics and Nitramines,” U.S. Army Corps of Engineers, Cold

Regions Research and Engineering Laboratory, Special Report 95-16, 1995.

9. Winslow, S.D., B.V. Pepich, M.V. Bassett, S.C. Wendelken, D.J. Munch, and J.L.

Sinclair, “Microbial Inhibitors for U.S. EPA Drinking Water Methods for the

Determination of Organic Compounds,” Environ. Sci. Technol., 35 (2001) 4103-4110.

10. Winslow, S.D., B. Prakash, M.M. Domino, B.V. Pepich and D.J. Munch, “Method 526,

Determination of Selected Semivolatile Organic Compounds in Drinking Water by Solid

Phase Extraction and Capillary Column Gas Chromatography/ Mass Spectrometry

(GC/MS),” In Methods for the Determination of Organic and Inorganic Compounds in

Drinking Water, Vol. 1, EPA 815-R-00-014, 2000.
529-33

11. Erney, D.R., T.M. Pawlowski, C.F. Poole, “Matrix Induced Peak Enhancement of

Pesticides in Gas Chromatography,” J. High Resol. Chromatogr., 20 (1997) 375-378.

12. Hajslova, J., k. Holadova, V. Kocourek, J. Poustka, M. Godula, P. Cuhra, M. Kempny,

“Matrix Induced Effects:A Critical Point in the Gas Chromatographic Analysis of Pesticide

Residues,” J. Chromatogr., 800 (1998) 283-295.

13. Erney, D.R., A.M. Gillespie, D.M. Gilvydis, and C.F. Poole, “Explanation of the MatrixInduced Chromatographic Response Enhancement of Organophosphorous Pesticides

During Open Tubular Column Gas Chromatography with Splitless or Hot On-column

Injection and Flame Photometric Detection,” J. Chromatogr., 638 (1993) 57-63.
529-34

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

Table 1. Ion Abundance Criteria for BFB

Mass

(m/z)

Relative Abundance Criteria Purpose of Checkpoint a

50 15-40% of mass 95 Low-mass sensitivity

75 30-80% of mass 95 Low-mass sensitivity

95 Base peak, 100% relative abundance Low-mass resolution and sensitivity

96 5-9% of mass 95 Low-mass resolution and isotope ratio

173 < 2% of mass 174 Mid-mass resolution

174 > 50% of mass 95 Mid mass resolution and sensitivity

175 5-9% of mass 174 Mid-mass resolution

176 > 95% but < 101% of mass 174 Mid-mass resolution and isotope ratio

177 5-9% of mass 176 Mid-mass resolution

 

a All ions are used primarily to check the mass measuring accuracy of the mass spectrometer and

data system, and this is the most important part of the performance test. The resolution checks,

which include natural abundance isotope ratios, constitute the next most important part of the

performance test. Finally, the ion abundance ranges are designed to encourage some

standardization to fragmentation patterns.
529-35

Table 2. Retention Times and Suggested Quantitation and Confirmation Ions for

Explosive Analytes a,b

Analyte Retention

Time

(min)

Quantitation

Ion

(m/z)

Confirmation

Ions

(m/z)

nitrobenzene (NB) 7.72 51 or 77 51, 77, 123

2-nitrotoluene (2NT) 9.40 65 91, 120

3-nitrotoluene (3NT) 10.18 91 65, 137

4-nitrotoluene (4NT) 10.57 65 or 91 65, 91, 137

1,3-dinitrobenzene (DNB) 15.13 50 or 75 50, 75, 76

2,6-dinitrotoluene (2,6DNT) 15.32 165 63, 89

2,4-dinitrotoluene (2,4DNT) 16.60 165 63, 78

1,3,5-trinitrobenzene (TNB) 19.35 75 or 213 74, 75, 213

2,4,6-trinitrotoluene (TNT) 19.43 210 63, 89

RDX 21.97 46 75, 128

4-amino-2,6-dinitrotoluene (4AmDNT) 22.81 180 78, 104

3,5-dinitroanaline (DNA) 23.00 183 or 64 63, 64, 183

2-amino-4,6-dinitrotoluene (2AmDNT) 23.47 180 or 78 52, 78, 180

Tetryl 24.77 194 or 77 51, 77, 194

3,4-dinitrotoluene (IS) 17.42 63 or 182 63, 78, 182

nitrobenzene-d5 (SUR) 7.66 82 54, 128

1,3,5-trimethyl-2-nitrobenzene (SUR) 12.33 148 or 91 91, 93, 148

1,2,4-trimethyl-5-nitrobenzene (SUR) 14.72 148 or 91 77, 91, 148

a

 Even when instruments are tuned to BFB criteria, differences in instrumentation can cause 

slight variations in mass spectra, e.g. quadrupole vs. ion trap. The ions listed here are suggested

ions because they were successfully used during method development. 

b Two or three ions from the Quantitation Ion and Confirmation Ion columns were used for SIM

monitoring. 
529-36

Table 3. Detection Limits Obtained Using Programmed Temperature Vaporizing (PTV) 

Splitless Injection and Full Scan Mass Spectrometry

Analyte DL (:g/L) a

 [fortified conc. for DL

determination]

DL (:g/L) b

 [fortified conc. for DL

determination]

nitrobenzene 0.039 [0.10] 0.18 [0.50]

2-nitrotoluene 0.11 [0.50] 0.041 [0.10]

3-nitrotoluene 0.083 [0.50] 0.091 [0.50]

4-nitrotoluene 0.13 [0.50] 0.029 [0.10]

1,3-dinitrobenzene 0.021 [0.10] 0.076 [0.50]

2,6-dinitrotoluene 0.029 [0.10] 0.059 [0.50]

2,4-dinitrotoluene 0.11 [0.50] 0.11 [0.50]

1,3,5-trinitrobenzene 0.037 [0.50] 0.042 [0.50]

2,4,6-trinitrotoluene (TNT) 0.084 [0.50] 0.046 [0.50]

RDX 0.082 [0.50] 0.12 [0.50]

4-amino-2,6-dinitrotoluene 0.065 [0.50] 0.072 [0.50]

3,5-dinitroanaline 0.052 [0.20] 0.011 [0.10]

2-amino-4,6-dinitrotoluene 0.075 [0.50] 0.053 [0.50]

Tetryl 0.18 [1.0] 0.058 [1.0]

a

 Data from replicate disk extractions, n=8.

b

 Data from replicate cartridge extractions, n=8.
529-37

Table 4. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Disk Extraction, PTV

Splitless Injection and Full Scan Mass Spectrometry (n=8)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.10 70.6 18

2-nitrotoluene 0.50 102 7.1

3-nitrotoluene 0.50 114 4.9

4-nitrotoluene 0.50 81.4 11

1,3-dinitrobenzene 0.10 126 5.4

2,6-dinitrotoluene 0.10 83.6 12

2,4-dinitrotoluene 0.50 101 7.5

1,3,5-trinitrobenzene 1.0 111 4.5

2,4,6-trinitrotoluene 1.0 136 4.5

RDX 0.50 103 5.3

4-amino-2,6-dinitrotoluene 0.50 130 3.4

3,5-dinitroanaline 0.20 130 6.3

2-amino-4,6-dinitrotoluene 0.50 127 3.9

 Tetryl 1.0 134 4.2
529-38

Table 5. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Cartridge

Extraction, PTV Splitless Injection and Full Scan Mass Spectrometry (n=8)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.5 79.2 16

2-nitrotoluene 0.5 82.4 15

3-nitrotoluene 0.5 82.0 7.4

4-nitrotoluene 0.1 110 9.0

1,3-dinitrobenzene 0.5 99.1 5.1

2,6-dinitrotoluene 0.5 109 3.6

2,4-dinitrotoluene 0.5 99.3 7.1

1,3,5-trinitrobenzene 0.5 122 2.3

2,4,6-trinitrotoluene 0.5 85.2 3.6

RDX 0.5 131 6.3

4-amino-2,6-dinitrotoluene 0.5 108 4.4

3,5-dinitroanaline 0.1 133 2.6

2-amino-4,6-dinitrotoluene 0.5 108 3.3

Tetryl 1.0 124 1.6
529-39

Table 6. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at 5.0 :g/L, Using PTV Splitless Injection and Full Scan

Mass Spectrometry

Analyte Disk Extraction (n=5) Cartridge Extraction (n=5)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 87.8 7.8 97.3 9.3

2-nitrotoluene 87.0 5.6 95.0 12

3-nitrotoluene 85.9 7.9 95.8 5.9

4-nitrotoluene 90.4 1.4 99.6 5.0

1,3-dinitrobenzene 99.7 8.5 92.3 6.3

2,6-dinitrotoluene 92.2 7.5 88.1 5.1

2,4-dinitrotoluene 93.8 3.7 97.5 4.8

1,3,5-trinitrobenzene 102 4.4 98.5 4.9

2,4,6-trinitrotoluene 111 3.2 101 6.0

RDX 97.1 2.5 102 8.1

4-amino-2,6-dinitrotoluene 100 7.5 101 4.8

3,5-dinitroanaline 105 2.8 97.5 4.4

2-amino-4,6-dinitrotoluene 109 3.5 102 3.1

Tetryl 104 5.4 82.7 5.4

nitrobenzene-d5 (SUR) ND a ND 90.2 8.3

1,3,5-trimethyl-2-

nitrobenzene (SUR)

94.7 3.8 96.0 2.7

1,2,4-trimethyl-5-

nitrobenzene (SUR)

106 9.3 102 6.0

a

 ND- Not determined; These data were collected before this surrogate was added to the method.
529-40

Table 7. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Surface Source) Fortified with Method Analytes at 5.0 :g/L, Using PTV

Splitless Injection and Full Scan Mass Spectrometry a

Analyte Disk Extraction (n=5) Cartridge Extraction (n=5)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 96.3 5.1 88.0 11

2-nitrotoluene 84.5 6.0 85.6 11

3-nitrotoluene 81.2 8.3 91.1 3.8

4-nitrotoluene 98.1 6.3 87.5 7.1

1,3-dinitrobenzene 103 8.7 87.1 11

2,6-dinitrotoluene 96.1 2.3 82.8 7.1

2,4-dinitrotoluene 101 5.8 94.3 11

1,3,5-trinitrobenzene 111 5.5 101 9.0

2,4,6-trinitrotoluene 109 8.2 97.9 7.1

RDX 111 4.8 107 6.2

4-amino-2,6-dinitrotoluene 108 4.8 100 6.0

3,5-dinitroanaline 114 1.3 97 6.1

2-amino-4,6-dinitrotoluene 115 1.7 101 6.2

Tetryl 116 4.5 87.6 8.9

nitrobenzene-d5 (SUR) ND b ND 87.5 2.2

1,3,5-trimethyl-2-

nitrobenzene (SUR)

109 9.9 90.6 10

1,2,4-trimethyl-5-

nitrobenzene (SUR)

112 4.0 86.6 5.0

a The sample matrix was dechlorinated and preserved as described in Section 8 prior to

fortification with method analytes.

b

 ND- Not determined; These data were collected before this surrogate was added to the method.
529-41

Table 8. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Ground Source with a High Hardness Level a

) Fortified with method

Analytes at 5.0 :g/L, Using PTV Splitless Injection and Full Scan Mass

Spectrometry 

Analyte Disk Extraction (n=5) Cartridge Extraction (n=5)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 92.1 17 92.5 12

2-nitrotoluene 85.9 12 88.4 4.1

3-nitrotoluene 87.8 5.4 93.7 7.7

4-nitrotoluene 95.1 9.7 91.8 7.7

1,3-dinitrobenzene 102 3.4 90.3 9.0

2,6-dinitrotoluene 96.3 4.3 89.2 4.3

2,4-dinitrotoluene 103 6.9 97.7 4.7

1,3,5-trinitrobenzene 106 8.2 105 6.6

2,4,6-trinitrotoluene 118 8.0 106 4.8

RDX 93.1 9.7 114 11

4-amino-2,6-dinitrotoluene 106 6.8 103 4.1

3,5-dinitroanaline b115 8.3 103 4.5

2-amino-4,6-dinitrotoluene 112 7.4 110 6.4

Tetryl 107 16 92.9 7.9

nitrobenzene-d5 (SUR) ND c ND 84.7 15

1,3,5-trimethyl-2-

nitrobenzene (SUR)

96.0 8.4 95.0 10

1,2,4-trimethyl-5-

nitrobenzene (SUR)

107 7.8 96.8 6.2

a

 Hardness level measured at 310 and 360 mg/L as calcium carbonate, for disk and cartridge 

samples, respectively. The sample matrix was dechlorinated and preserved as described in

Section 8 prior to fortification with method analytes.

b

 3,5-Dinitroanaline was fortified at 0.2 :g/L.

c

 ND - Not determined; These data were collected before this surrogate was added to the method.
529-42

Table 9. Detection Limits Obtained Using Programmed Temperature Vaporizing (PTV)

Splitless Injection and Selected Ion Monitoring Mass Spectrometry

Analyte DL (:g/L) a

 [fortified conc. for DL

determination]

DL (:g/L) b

 [fortified conc. for DL

determination]

nitrobenzene 0.009 [0.05] 0.010 [0.05]

2-nitrotoluene 0.008 [0.05] 0.003 [0.05]

3-nitrotoluene 0.005 [0.05] 0.004 [0.05]

4-nitrotoluene 0.005 [0.05] 0.006 [0.05]

1,3-dinitrobenzene 0.006 [0.05] 0.003 [0.05]

2,6-dinitrotoluene 0.004 [0.05] 0.004 [0.05]

2,4-dinitrotoluene 0.004 [0.05] 0.008 [0.05]

1,3,5-trinitrobenzene 0.018 [0.05] 0.012 [0.05]

2,4,6-trinitrotoluene (TNT) 0.004 [0.05] 0.008 [0.05]

RDX 0.006 [0.05] 0.010 [0.05]

4-amino-2,6-dinitrotoluene 0.005 [0.05] 0.019 [0.05]

3,5-dinitroanaline 0.008 [0.01] 0.029 [0.01]

2-amino-4,6-dinitrotoluene 0.020 [0.05] 0.030 [0.05]

 Tetryl 0.090 [0.10] 0.14 [0.25]

a

 Data from replicate disk extractions, n=8.

b

 Data from replicate cartridge extractions, n=7.
529-43

Table 10. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Disk Extraction, PTV

Splitless Injection and Selected Ion Monitoring Mass Spectrometry (n=8)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.05 87.1 6.5

2-nitrotoluene 0.05 79.7 6.6

3-nitrotoluene 0.05 112 2.8

4-nitrotoluene 0.05 123 2.5

1,3-dinitrobenzene 0.05 124 3.3

2,6-dinitrotoluene 0.10 89.2 2.2

2,4-dinitrotoluene 0.10 95.7 3.2

1,3,5-trinitrobenzene 0.05 120 9.4

2,4,6-trinitrotoluene 0.10 104 1.9

RDX 0.10 133 3.2

4-amino-2,6-dinitrotoluene 0.25 98.4 3.2

3,5-dinitroanaline 0.02 107 10

2-amino-4,6-dinitrotoluene 0.10 119 3.1

 Tetryl 0.25 113 10
529-44

Table 11. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Cartridge

Extraction, PTV Splitless Injection and Selected Ion Monitoring Mass

Spectrometry (n=7)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.05 107 5.7

2-nitrotoluene 0.05 110 1.8

3-nitrotoluene 0.05 114 2.0

4-nitrotoluene 0.05 119 3.2

1,3-dinitrobenzene 0.05 128 1.6

2,6-dinitrotoluene 0.10 102 3.1

2,4-dinitrotoluene 0.10 109 5.4

1,3,5-trinitrobenzene 0.10 108 8.9

2,4,6-trinitrotoluene 0.10 110 4.3

RDX 0.10 114 8.9

4-amino-2,6-dinitrotoluene 0.25 103 3.7

3,5-dinitroanaline 0.05 95.6 9.4

2-amino-4,6-dinitrotoluene 0.10 134 5.6

 Tetryl 0.25 102 17
529-45

Table 12. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at 1.0 :g/L, Using PTV Splitless Injection and Selected

Ion Monitoring Mass Spectrometry

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 91.3 5.8 86.5 3.4

2-nitrotoluene 89.7 1.8 91.9 3.0

3-nitrotoluene 91.3 1.7 89.7 4.8

4-nitrotoluene 90.5 2.0 89.8 4.1

1,3-dinitrobenzene 99.0 2.8 96.2 5.9

2,6-dinitrotoluene 98.1 2.7 98.8 5.1

2,4-dinitrotoluene 102 3.3 100 4.9

1,3,5-trinitrobenzene 106 2.3 96.9 6.2

2,4,6-trinitrotoluene 110 2.6 104 5.1

RDX 100 2.8 108 6.4

4-amino-2,6-dinitrotoluene 116 2.8 117 4.7

3,5-dinitroanaline a104 2.6 107 8.6

2-amino-4,6-dinitrotoluene 113 1.9 111 5.7

 Tetryl 122 3.2 111 8.9

nitrobenzene-d5 (SUR) 88.5 2.3 86.5 2.2

1,3,5-trimethyl-2-

nitrobenzene (SUR)

84.9 2.3 84.4 1.6

1,2,4-trimethyl-5-

nitrobenzene (SUR)

87.4 3.3 88.2 2.4

a

 3,5-Dinitroanaline was fortified at 0.2 :g/L.
529-46

Table 13. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Surface Source) Fortified with Method Analytes at 1.0 :g/L, Using PTV

Splitless Injection and Selected Ion Monitoring Mass Spectrometry a

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 71.5 3.4 85.8 4.8

2-nitrotoluene 79.8 2.7 93.2 4.2

3-nitrotoluene 78.9 2.6 91.2 3.6

4-nitrotoluene 74.9 2.6 89.8 4.1

1,3-dinitrobenzene 80.7 2.1 102 2.4

2,6-dinitrotoluene 81.2 2.3 101 2.2

2,4-dinitrotoluene 88.7 2.6 108 2.4

1,3,5-trinitrobenzene 82.3 1.6 104 3.3

2,4,6-trinitrotoluene 91.1 2.5 116 3.8

RDX 85.2 1.3 111 3.8

4-amino-2,6-dinitrotoluene 103 2.0 127 2.5

3,5-dinitroanaline b96.5 2.3 119 1.8

2-amino-4,6-dinitrotoluene 97.4 0.8 123 2.5

 Tetryl 99.5 3.2 137 3.8

nitrobenzene-d5 (SUR) 87.4 3.4 88.4 5.1

1,3,5-trimethyl-2-

nitrobenzene (SUR)

93.3 2.8 83.2 4.6

1,2,4-trimethyl-5-

nitrobenzene (SUR)

100 3.1 89.7 4.1

a The sample matrix was dechlorinated and preserved as described in Section 8 prior to

fortification with method analytes.

b

 3,5-Dinitroanaline was fortified at 0.2 :g/L.
529-47

Table 14. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Ground Source with a High Hardness Level a

 ) Fortified with Method

Analytes at 1.0 :g/L, Using PTV Splitless Injection and Selected Ion

Monitoring Mass Spectrometry 

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 95.6 8.6 84.2 2.4

2-nitrotoluene 102 4.6 85.1 3.8

3-nitrotoluene 94.6 3.3 85.0 2.4

4-nitrotoluene 94.6 5.1 85.5 3.6

1,3-dinitrobenzene 86.1 5.1 104 1.0

2,6-dinitrotoluene 91.7 2.4 104 0.8

2,4-dinitrotoluene 90.1 5.9 109 1.5

1,3,5-trinitrobenzene 88.1 4.2 109 2.1

2,4,6-trinitrotoluene 94.1 9.4 113 1.2

RDX 102 3.4 124 6.9

4-amino-2,6-dinitrotoluene 105 7.1 117 1.2

3,5-dinitroanaline b106 5.6 98.5 3.3

2-amino-4,6-dinitrotoluene 106 4.6 113 2.8

Tetryl 121 4.1 120 5.9

nitrobenzene-d5 (SUR) 97.1 5.5 87.6 4.5

1,3,5-trimethyl-2-

nitrobenzene (SUR)

89.2 6.5 84.7 4.3

1,2,4-trimethyl-5-

nitrobenzene (SUR)

86.0 3.1 92.2 4.5

a

 Hardness level measured at 380 and 390 mg/L as calcium carbonate, for disk and cartridge 

samples, respectively. The sample matrix was dechlorinated and preserved as described in

Section 8 prior to fortification with method analytes.

b

 3,5-Dinitroanaline was fortified at 0.2 :g/L.
529-48

Table 15. Detection Limits Obtained Using On-Column Programmed Temperature

Vaporizing (PTV) Injection and Full Scan Mass Spectrometry

Analyte DL (:g/L) a

 [fortified conc. for DL

determination]

DL (:g/L) b

 [fortified conc. for DL

determination]

nitrobenzene 0.031 [0.10] 0.024 [0.05]

2-nitrotoluene 0.013 [0.10] 0.010 [0.05]

3-nitrotoluene 0.019 [0.05] 0.007 [0.05]

4-nitrotoluene 0.010 [0.05] 0.012 [0.05]

1,3-dinitrobenzene 0.012 [0.05] 0.016 [0.05]

2,6-dinitrotoluene 0.005 [0.05] 0.005 [0.05]

2,4-dinitrotoluene ND c ND

1,3,5-trinitrobenzene 0.026 [0.05] 0.050 [0.05]

2,4,6-trinitrotoluene (TNT) 0.010 [0.05] 0.007 [0.05]

Hexahydro-1,3,5-trinitro-1,3,5-

triazine (RDX)

0.040 [0.10] 0.024 [0.05]

4-amino-2,6-dinitrotoluene 0.027 [0.10] 0.007 [0.05]

3,5-dinitroanaline ND ND

2-amino-4,6-dinitrotoluene 0.05 [0.25] 0.039 [0.25]

2,4,6-trinitrophenylmethylnitramine

(Tetryl)

0.28 [1.0] d ND

a

 Data from replicate disk extractions, n=8.

b

 Data from replicate cartridge extractions, n=7.

c

 ND - Not determined; see Sect. 13.3 and 13.4.

d

 n=6
529-49

Table 16. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Disk Extraction, On

Column PTV Injection and Full Scan Mass Spectrometry (n=8)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.25 68.5 12

2-nitrotoluene 0.25 64.4 5.9

3-nitrotoluene 0.10 85.1 6.7

4-nitrotoluene 0.10 81.0 5.5

1,3-dinitrobenzene 0.10 77.6 5.6

2,6-dinitrotoluene 0.10 104 3.8

2,4-dinitrotoluene ND a

1,3,5-trinitrobenzene 0.10 114 16

2,4,6-trinitrotoluene 0.25 88.6 4.3

RDX 0.10 102 13

4-amino-2,6-dinitrotoluene 0.25 92.8 5.8

3,5-dinitroanaline ND

2-amino-4,6-dinitrotoluene 1.0 107 7.1

 Tetryl 1.0 111 5.2

a

 ND - Not determined; See Sect. 13.3 and 13.4.
529-50

Table 17. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at Low Concentrations, Using SPE Cartridge

Extraction, On-Column PTV Injection and Full Scan Mass Spectrometry (n=8)

Analyte Fortified

Concentration

(:g/L)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 0.10 77.0 11

2-nitrotoluene 0.10 74.9 10

3-nitrotoluene 0.05 114 4.0

4-nitrotoluene 0.05 116 6.6

1,3-dinitrobenzene 0.25 92.1 2.8

2,6-dinitrotoluene 0.10 114 3.7

2,4-dinitrotoluene ND a

1,3,5-trinitrobenzene 0.25 126 11.5

2,4,6-trinitrotoluene 0.25 116 19

RDX 0.10 117 10

4-amino-2,6-dinitrotoluene 0.25 101 7.4

3,5-dinitroanaline ND

2-amino-4,6-dinitrotoluene 1.0 106 9.6

 Tetryl 1.0 108 7.1

a

 ND - Not determined; see Sect. 13.3 and 13.4.
529-51

Table 18. Precision and Accuracy Data Obtained from Reagent Water Samples Fortified

with Method Analytes at 1.0 :g/L, Using On-Column PTV Splitless Injection

and Full Scan Mass Spectrometry

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 95.3 2.6 95.0 1.7

2-nitrotoluene 92.3 2.5 91.5 3.4

3-nitrotoluene 93.3 1.8 91.1 1.3

4-nitrotoluene 91.2 3.6 88.7 1.7

1,3-dinitrobenzene 92.8 4.0 98.7 8.0

2,6-dinitrotoluene 101 2.8 104 4.1

2,4-dinitrotoluene ND a ND

1,3,5-trinitrobenzene 110 6.5 105 14

2,4,6-trinitrotoluene 123 2.0 117 6.3

RDX 112 1.6 122 5.9

4-amino-2,6-dinitrotoluene 125 4.0 123 6.8

3,5-dinitroanaline ND ND

2-amino-4,6-dinitrotoluene 107 7.1 106 9.6

 Tetryl 111 5.2 108 7.1

nitrobenzene-d5 (SUR) 90.4 3.1 90.7 2.6

1,3,5-trimethyl-2-

nitrobenzene (SUR)

92.8 1.4 88.5 2.2

1,2,4-trimethyl-5-

nitrobenzene (SUR)

96.8 1.5 98.0 3.0

a

 ND - Not determined; See Sect. 13.3 and 13.4.
529-52

Table 19. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Surface Source) Fortified with Method Analytes at 1.0 :g/L, Using OnColumn PTV Injection and Full Scan Mass Spectrometry 

a

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 78.6 4.1 95.1 2.8

2-nitrotoluene 77.1 3.2 90.0 2.2

3-nitrotoluene 78.8 2.8 91.7 2.5

4-nitrotoluene 76.9 3.3 88.5 3.1

1,3-dinitrobenzene 83.6 2.8 95.9 1.8

2,6-dinitrotoluene 86.7 2.8 105 2.1

2,4-dinitrotoluene ND b

1,3,5-trinitrobenzene 107 6.7 110 2.2

2,4,6-trinitrotoluene 112 2.8 131 1.8

RDX 99.3 11 127 7.8

4-amino-2,6-dinitrotoluene 107 2.7 128 3.8

3,5-dinitroanaline ND

2-amino-4,6-dinitrotoluene 95.4 5.5 107 3.1

 Tetryl 103 2.7 123 6.5

nitrobenzene-d5 (SUR) 90.0 5.0 93.8 2.8

1,3,5-trimethyl-2-

nitrobenzene (SUR)

97.2 3.8 86.2 3.3

1,2,4-trimethyl-5-

nitrobenzene (SUR)

104 3.6 96.7 1.8

a The sample matrix was dechlorinated and preserved as described in Section 8 prior to

fortification with method analytes.

b

 ND - Not determined; see Sect. 13.3 and 13.4.
529-53

Table 20. Precision and Accuracy Data Obtained from Tap Water (Chlorinated Water

from a Ground Source with a High Hardness Level a

) Fortified with Method

Analytes at 1.0 :g/L, Using On-Column PTV Injection and Full Scan Mass

Spectrometry 

Analyte Disk Extraction (n=4) Cartridge Extraction (n=4)

Accuracy

(% recovery)

Precision

(%RSD)

Accuracy

(% recovery)

Precision

(%RSD)

nitrobenzene 94.1 2.4 91.4 3.5

2-nitrotoluene 88.5 2.7 90.0 2.4

3-nitrotoluene 92.2 3.0 89.9 2.6

4-nitrotoluene 89.0 2.5 89.6 2.6

1,3-dinitrobenzene 96.4 4.0 107 6.3

2,6-dinitrotoluene 101 2.4 105 1.2

2,4-dinitrotoluene ND b

1,3,5-trinitrobenzene 129 5.3 123 8.8

2,4,6-trinitrotoluene 133 3.0 136 1.7

RDX 126 5.6 150 3.7

4-amino-2,6-dinitrotoluene 128 4.4 140 3.9

3,5-dinitroanaline ND

2-amino-4,6-dinitrotoluene 110 2.4 121 6.5

Tetryl 132 5.6 135 5.3

nitrobenzene-d5 (SUR) 89.1 4.4 88.2 3.2

1,3,5-trimethyl-2-

nitrobenzene (SUR)

91.4 4.3 92.1 6.0

1,2,4-trimethyl-5-

nitrobenzene (SUR)

96.8 4.6 97.3 5.3

a

 Hardness level measured at 380 and 390 mg/L as calcium carbonate, for disk and cartridge 

samples respectively. The sample matrix was dechlorinated and preserved as described in

Section 8 prior to fortification with method analytes.

b

 ND - Not determined; See Sect. 13.3 and 13.4.
529-54

Table 21. Aqueous Holding Time Data for Surface Water Samples Fortified with Method

Analytes at 5 μg/L and Preserved and Stored as Directed in Section 8.

Analyte Day 0

Amount a

(μg/L)

Day 7

Amount

(μg/L)

Day 11

Amount

(μg/L)

Day 14

Amount

(μg/L)

Day 19

Amount

(μg/L)

nitrobenzene 4.2 4.4 4.3 4.1 4.2

2-nitrotoluene 3.9 4.1 4.0 3.8 3.6

3-nitrotoluene 3.8 3.9 3.7 3.6 3.6

4-nitrotoluene 4.0 4.1 4.0 3.9 3.6

1,3-dinitrobenzene 4.1 4.3 4.2 4.1 4.0

2,6-dinitrotoluene 3.8 3.7 3.6 3.6 3.5

2,4-dinitrotoluene 3.9 4.0 3.9 3.6 3.6

1,3,5-trinitrobenzene 4.8 5.4 5.3 5.2 5.1

2,4,6-trinitrotoluene 4.4 4.5 4.2 3.9 4.0

RDX 4.7 4.8 4.4 4.4 4.4

4-amino-2,6-dinitrotoluene 4.8 5.1 4.9 4.7 4.8

3,5-dinitroanaline b 1.0 1.1 1.1 1.1 1.1

2-amino-4,6-dinitrotoluene 4.6 4.8 4.5 4.5 4.6

Tetryl 4.5 4.9 4.9 4.4 5.1

a

 Each concentration represents the mean of 5 replicate measurements. The RSD of each set of 

 replicates is less than 5.2%.

b

 3,5-Dinitroanaline was fortified at 1μg/L.
529-55

Table 22. Extract Holding Time Data for Surface Water Samples Fortified with Method

Analytes at 5 μg/L, Preserved and Stored as Directed in Section 8.

Analyte Day 0

Amount a

(μg/L)

Day 7

Amount

(μg/L)

Day 15

Amount

(μg/L)

Day 21

Amount

(μg/L)

Day 28

Amount

(μg/L)

Day 35

Amount

(μg/L)

nitrobenzene 4.4 4.6 4.4 4.2 4.2 4.3

2-nitrotoluene 4.1 4.2 4.1 3.8 3.9 4.2

3-nitrotoluene 4.0 4.0 3.8 3.6 3.8 4.0

4-nitrotoluene 4.2 4.2 4.1 3.7 3.7 3.9

1,3-dinitrobenzene 4.2 4.2 4.2 4.0 4.9 4.3

2,6-dinitrotoluene 3.9 4.0 3.8 3.5 3.6 3.7

2,4-dinitrotoluene 4.0 4.2 3.7 3.6 3.7 3.8

1,3,5-trinitrobenzene 4.7 5.2 5.0 5.0 5.2 5.2

2,4,6-trinitrotoluene 4.4 4.6 4.2 4.0 4.1 4.2

RDX 4.6 5.1 4.6 4.5 4.5 4.4

4-amino-2,6-

dinitrotoluene

4.7 5.1 4.8 4.7 4.9 4.9

3,5-dinitroanaline b1.0 1.1 1.1 1.1 1.1 1.1

2-amino-4,6-

dinitrotoluene

4.6 4.8 4.4 4.5 4.5 4.7

Tetryl 4.4 4.7 4.7 4.6 4.7 4.7

a

 Each concentration represents the mean of 5 replicate measurements. The RSD of each set of 

replicates is less than 15% except for 2-nitrotoluene on day 15 which was 23%.

b

 3,5-Dinitroanaline was fortified at 1μg/L.
529-56

Table 23. Initial Demonstration of Capability (IDC) Requirements (Summary)

Method Ref. Requirement Specification and Frequency Acceptance Criteria

Sect. 9.2.1 Initial Demonstration of

Low Method

Background

Analyze LRB prior to any other IDC

steps. 

Demonstrate that all target analytes are

# 1/3 the MRL, and that possible

interferences from extraction media do

not prevent the identification and

quantification of method analytes.

Sect. 9.2.2 Initial Demonstration of

Precision (IDP)

Analyze 4-7 replicate LFBs fortified at 2-

5 :g/L

RSD must be #20% for all analytes.

Sect. 9.2.3 Initial Demonstration of

Accuracy (IDA)

Calculate average recovery for replicates

used in IDP

Mean recovery 70-130% of true value.

Sect. 9.2.4 Detection Limit

(DL) Determination 

Over a period of three days, prepare a

minimum of 7 replicate LFBs fortified at

a concentration estimated to be near the

DL. Analyze the replicates through all

steps of the analysis. Calculate the DL

using the equation in Section 9.2.4.

NOTE: Data from DL replicates are not

required to meet method precision and

accuracy criteria. If the DL replicates are

fortified at a low enough concentration, it

is likely that they will not meet precision

and accuracy criteria.
529-57

Table 24. Quality Control Requirements (Summary)

Method Ref. Requirement Specification and Frequency Acceptance Criteria

Sect. 8.1 -

Sect 8.4

Sample Collection,

Preservation, and

Holding Time

14 days, with addition of Trizma buffer

and copper sulfate

Iced or refrigerated at 10° C or less for up

to 48 hours to allow time for shipping;

refrigerated at 6° C or less after arrival at

the laboratory.

Sect. 8.4 Extract Holding Time 30 days Stored at 0° C or less in amber vials.

Sect. 9.10 Laboratory Fortified

Sample Matrix (LFM)

Analyze one LFM per extraction batch

(20 samples or less) fortified with method

analytes at approximately 2 times the

native concentration.

Recoveries not within 70-130% of the

fortified amount may indicate a matrix

effect.

Sect. 9.11 Field Duplicates Analyze 1 FD for each 20 samples, or 1

per extraction batch, whichever is greater.

Suggested RPD±30%.

Sect. 9.12 Quality Control Sample

(QCS)

Analyze QCS whenever new standards

are prepared, or at least quarterly.

If analyzed as a calibration sample, CCC

criteria apply. If analyzed as an LFB,

those criteria apply.

Sect 9.4 Laboratory Reagent

Blank (LRB)

One with each extraction batch of up to

20 samples.

Demonstrate that all target analytes are 

# 1/3 the MRL, and that possible

interference from extraction media do not

prevent the identification and

quantification of method analytes.
529-58

Method Ref. Requirement Specification and Frequency Acceptance Criteria

Sect. 9.7 Laboratory Fortified

Blanks (LFB)

Analyze at least one LFB daily or one for

each extraction batch of up to 20 field

samples. Rotate the fortified concentration

between low, medium and high amounts.

Results of LFB analyses must be 70-

130% of the true value for each analyte

and surrogate for all fortified

concentrations greater than the lowest

CAL point. Results of LFBs

corresponding to the lowest CAL point

must be 50-150% of the true value.

Sect. 9.8 Internal Standard The internal standard, 3,4-dinitrotoluene,

is added to all calibration standards and

extracts. 

Peak area counts for the IS in LFBs,

LRBs and sample extracts must be within

70-130% of the peak area in the most

recent CCC, and 50-150% of average area

in the initial calibration.

Sect. 9.9 Surrogate Standards Surrogate standards (nitrobenzene-d5;

1,3,5-trimethyl-2-nitro-benzene; and

1,2,4-trimethyl-5-nitro-benzene) are

added to all calibration standards,

samples, LFBs, LFMs, FDs, and LRBs. 

Recovery for all surrogates in all

calibration standards, LRB, LFB, LFM,

FD and sample extracts must be 70-130%

of the true value. 

Sect. 10.2.1 MS Tune Check Analyze BFB to verify MS tune before

initial calibration and before every

recalibration.

Criteria are given in Table 1.
529-59

Method Ref. Requirement Specification and Frequency Acceptance Criteria

Sect. 10.2.2-

Sect. 10.2.10

Initial Calibration Use internal standard calibration

technique to generate an average RF or

first or second order calibration curve. 

Use a minimum of 3 standards for a

calibration range of 1 order of magnitude,

and at least 5 standards for 2 orders of

magnitude.

When each calibration standard is

calculated as an unknown using the

calibration curve, the result must be 70-

130% of the true value for all but the

lowest standard. The lowest standard

must be 50-150% of the true value.

Sect. 10.3 Continuing Calibration

Check

Verify initial calibration by analyzing a

calibration standard prior to analyzing

samples, after every 10 samples, and after

the last sample. Always analyze a low

concentration (near the MRL) CCC at the

beginning of the analysis period. Rotate

through low, medium, and high 

concentration calibration standards to

meet every 10 sample requirement.

The result for each analyte and surrogate

must be 70-130% of the true value for all

concentrations except the lowest CAL

point for each analyte. The lowest CAL

point for each analyte must be 50-150%

of the true value.

The peak area of the IS must be within

70-130% of the peak area in the most

recent CCC, and 50-150% of the average

peak area calculated during initial

calibration.
529-60