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METHOD 528 DETERMINATION OF PHENOLS IN DRINKING WATER 

BY SOLID PHASE EXTRACTION AND CAPILLARY COLUMN

GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)

Revision 1.0

J.W. Munch - April 2000

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
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METHOD 528

DETERMINATION OF PHENOLS 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 phenols 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 phenols that are efficiently partitioned from the water sample onto a modified

polystyrene divinylbenzene solid phase sorbent, and sufficiently volatile and thermally

stable for gas chromatography. The method includes the following compounds:

ANALYTE CAS NUMBER

phenol 108-95-2

2-chlorophenol 95-57-8

2-methylphenol (o-cresol) 95-48-7

2-nitrophenol 88-75-5

2,4-dimethylphenol 105-67-9

2,4-dichlorophenol 120-83-2

4-chloro-3-methylphenol 59-50-7

2,4,6-trichlorophenol 88-06-2

2,4-dinitrophenol 51-28-5

4-nitrophenol 93951-79-2

2-methyl-4,6-dinitrophenol 534-52-1

pentachlorophenol 87-86-5
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1.2 Method detection limit (MDL) 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 MDL is compound dependent and is particularly dependent on extraction

efficiency, sample matrix, and instrument performance. MDLs for method analytes range

from 0.02-0.58 :g/L, and are listed in Table 1. The concentration calibration range

demonstrated by this method is 0.1 :g/L to 15 :g/L for most analytes, and approximately

1.0 :g/L to 15 :g/L for 2,4-dinitrophenol, 4-nitrophenol, 2-methyl-4,6-dinitrophenol, and

pentachlorophenol.

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 0.5 g of a modified polystyrene divinyl benzene copolymer. 

The organic compounds are eluted from the solid phase with a small quantity of methylene

chloride. 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. 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. 

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.

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 surrogate

analytes. The CAL solutions are used to calibrate the instrument response with respect to

analyte concentration.
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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 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.

3.5 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.

3.6 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.

3.7 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.

3.8 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.

3.9 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.
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3.10 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, and 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.

3.11 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.

3.12 METHOD DETECTION LIMIT (MDL) -- 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 (Section 9.2.4), and

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

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 PEAK TAILING FACTOR (PTF) -- A calculated value that indicates the amount of

peak tailing exhibited by a chromatographic peak. The value is calculated by dividing the

peak width of the back half of the peak (at 10% peak height), by the peak width of the

front half of the peak (at 10% peak height). The calculation is demonstrated in Figure 4.

3.15 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.

3.16 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 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.
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3.18 SURROGATE ANALYTE (SUR) -- A pure analyte, which is extremely unlikely to be

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

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

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

performance with each sample.

4. INTERFERENCES

4.1 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 the polypropylene cartridge that it is

packed in. Brands and manufacturers 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. Syringes and splitless injection port liners must be cleaned

carefully or replaced as needed. After analysis of a sample containing high concentrations

of compounds, a laboratory reagent blank should be analyzed to ensure that accurate

values are obtained for the next sample.

4.3 Silicone compounds may be leached from autosampler vial septa by methylene chloride. 

This contamination of the extract will be enhanced if particles of the septa are introduced

into standards and sample extracts by the needle used for injection. These silicone

compounds should, in most cases, have no effect on the analysis. However, the analyst

should be aware of this potential problem.

4.4 Airborne phenol may be a source of phenol contamination in samples and sample extracts. 

Samples should not be stored or extracted in areas where phenol is used for other

laboratory operations.

4.5 2,3,4,5-Tetrachlorophenol is used as one of the internal standards for the quantitation of

reactive and thermally labile phenols. Tetrachlorophenol isomers may be present at low

levels (less than 4% total tetrachlorophenol) in pentachlorophenol used as a pesticide and

wood preservative. However, occurrence of pentachlorophenol in U.S. drinking waters is

rare, and measured concentrations are typically 1 :g/L or less. If a matrix interference

with the internal standard is suspected, an alternate internal standard may be selected.
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5. SAFETY

5.1 The toxicity or carcinogenicity of 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. Additional references to

laboratory safety are cited (2-4).

5.2 Some method analytes and solvents, including 2,4,6-trichlorophenol, pentachloro- phenol,

and methylene chloride 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.

6. EQUIPMENT AND SUPPLIES (All specifications are suggested. References to specific

brands or catalog numbers are included for illustration only.)

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

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

solvents, air-drying, and heating (where appropriate) 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. Although specific contamination

problems from bottle caps were not observed during method development, phenolic resin

bottle caps should be avoided. 

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,
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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. 

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. During the course of the development of this method,

two columns were used. Although these are both polyphenylmethylsilicone columns, the

exact phase is slightly different. Information on the exact composition of each phase is

available from the manufacturers. Most of the work was performed with column 1. Any

column which provides analyte separations equivalent to or better than these columns may

be used. Example chromatograms are shown in Figs 1-3. Retention times are presented

in Table 2.

6.9.1. Column 1- 30 m × 0.25 mm id fused silica capillary column coated with a 0.25

:m bonded film of polyphenylmethylsilicone, (J&W DB-5ms).

6.9.2 Column 2- 30 m × 0.25 mm id fused silica capillary column coated with a 0.25

:m bonded film of polyphenylmethylsilicone, (SGE BPX5). 

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 split/splitless injection. The injection system must not allow the analytes to

contact hot stainless steel or other metal surfaces that promote decomposition. 

Other injection techniques such as temperature programmed injections, cold oncolumn injections and large volume injections may be used if QC criteria in

Section 9 and 10 are met. If an alternate injection technique is performed, the

analyst will need to select an instrument configuration which has been specifically

designed for that application. Performance data in Section 17 include data

obtained both by hot, splitless injection and temperature programmed splitless

injection.

6.10.2 The GC/MS interface should allow the capillary column or transfer line exit to be

placed within a few mm of the ion source. Other interfaces, for example the

open split interface, are acceptable if the system has adequate sensitivity.
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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 be

capable of scanning at a minimum from 45 to 450 amu with a complete scan

cycle time (including scan overhead) of 1.0 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 3 when an injection of approximately 5 ng of DFTPP 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 recommended.

6.10.4 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) 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 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 cartridge. All extraction and elution steps must

be the same as in the manual procedure. Extraction and/or elution steps may not be

changed or omitted to accommodate the use of an automated system. 

7. REAGENTS AND STANDARDS

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

7.2 SOLID PHASE EXTRACTION CARTRIDGES -- Varian Bond Elut PPL or equivalent. 

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

not contain plasticizers that leach into the methylene chloride eluant and prevent the

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

volume) are packed with 0.5 g highly cross-linked, and chemically modified styrene divinyl
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benzene copolymer. The packing must have a narrow size distribution and must not leach

interfering organic compounds into the eluting solvent.

7.3 SOLVENTS --

7.3.1 Methylene chloride, acetone, and methanol. High purity pesticide quality or

equivalent. 

7.3.2 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.

7.4 HYDROCHLORIC ACID -- 6 N and 0.05 N.

7.5 SODIUM SULFATE, ANHYDROUS -- (Soxhlet extracted with methylene chloride for

a minimum of 4 h or heated to 400°C for 2 h in a muffle furnace.)

7.6 STOCK STANDARD SOLUTIONS -- Individual solutions of surrogates, internal

standards, and analytes, or mixtures of analytes, may be purchased from commercial

suppliers or prepared from pure materials. To prepare stocks from neat materials, add 10

mg (weighed on an analytical balance to within 0.1 mg) of the pure material to 1.9 mL of

methanol, methylene chloride, or acetone in a 2 mL volumetric flask, dilute to the mark,

and transfer the solution to an amber glass vial. The solvent to be used is dependent upon

the final use of the standard. In general, calibration standards and internal standards are

prepared in methylene chloride, sample fortification solutions are prepared in methanol or

acetone. Follow any specific instructions for each standard or standard mixture. If

compound purity is confirmed by the supplier at >96%, the weighed amount can be used

without correction to calculate the concentration of the solution (5 :g/:L). Store the

amber vials at 0°C or less.

7.7 PRIMARY DILUTION STANDARD SOLUTION -- The stock standard solutions are

used to prepare a primary dilution standard solution that contains multiple method analytes

in methylene chloride. 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, that is, 15 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. Mixtures of method analytes to be used as primary dilution standards may also

be purchased from commercial suppliers.
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7.8 CALIBRATION SOLUTIONS (CAL1 through CAL7) -- Prepare a series of seven

calibration solutions in methylene chloride which contain analytes of interest at suggested

concentrations of 15,10, 5, 2, 1, 0.5, and 0.1 ng/:L, with a constant concentration of

each internal standard in each CAL solution (2-5 ng/:L is recommended). Surrogate

analytes are also added to each CAL solution, and may be added at a constant

concentration or varied concentrations (similar to those for method analytes), at the

discretion of the analyst. CAL1 through CAL7 are prepared by combining appropriate

aliquots of a primary dilution standard solution (Sect. 7.7) and the fortification solution of

internal standards and surrogates (Sect. 7.10). All calibration solutions should contain at

least 80% methylene chloride to avoid gas chromatographic problems due to mixed

solvents. Store these solutions in amber vials at 0°C or less. Check these solutions

regularly for signs of evaporation and/or degradation.

NOTE: Because the MS sensitivity to analytes 9-12 (Table 2) is significantly less than

compounds 1-8, it may be more convenient to prepare calibration solutions in which the

concentrations of analytes 9-12 (Table 2) are higher than the concentrations of analytes 1-

8. Use of this option is at the discretion of the analyst. Calibration requirements are

specified in Sect. 10.

7.9 INTERNAL STANDARD SOLUTION(S) -- This method uses two internal standards:

1,2-dimethyl-3-nitrobenzene (IS#1) and 2,3,4,5-tetrachlorophenol (IS#2). The first

internal standard, 1,2-dimethyl-3-nitrobenzene is used to monitor instrument sensitivity and

is used to quantify analytes 1-8 in Table 2. The second internal standard, 2,3,4,5-

tetrachlorophenol is used to quantify analytes 9-12 (Table 2). IS#2 was selected for its

chemical similarity to these compounds which are susceptible to adsorption and/or thermal

decomposition in the GC inlet. A full explanation of the use of 2,3,4,5-tetrachlorophenol

to quantify these compounds is given in Section 13. If cold, on-column or temperature

programmed injection techniques are used, acceptable performance may be obtained

using only one internal standard (IS#1).

7.9.1 1,2-Dimethyl-3-nitrobenzene (Aldrich) -- 100 :g/mL in methylene chloride. 

Use 25 :L of this solution per 1 mL of sample extract for a final concentration of

2.5 :g/mL.

7.9.2 2,3,4,5-Tetrachlorophenol (Chem Service Inc.) -- 200 :g/mL in methylene

chloride. Use 25 :L of this solution per 1 mL of sample extract for a final

concentration of 5 :g/mL.

7.9.3 The internal standard solutions listed above can be made individually or together

in one solution.
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7.10 SAMPLE FORTIFICATION SOLUTIONS -- 

7.10.1 Surrogate fortification solutions --

7.10.1.1. 2-Chlorophenol-3,4,5,6-d4 (Chem Service Inc.) -- 100 :g/mL in

methanol. Use 20 :L of this solution per 1 L of water sample for a

final concentration of 2 :g/L.

7.10.1.2 2,4-Dimethylphenol-3,5,6-d3 (CDN Isotopes) -- 100 :g/mL in

acetone. Use 20 :L of this solution per 1 L of water sample for a

final concentration of 2 :g/L.

7.10.1.3 2,4,6-Tribromophenol -- 200 :g/mL in methanol. Use 25 :L of this

solution per 1 L water sample for a final concentration of 5 :g/L.

7.10.2 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 :g/mL of each analyte, was used for higher concentration

fortifications, and the other containing10 :g/mL of each analyte in methanol was

used for lower level fortifications.

NOTE: Because the MS sensitivity to analytes 9-12 (Table 2) is significantly

less than analytes 1-8, it may be more convenient to prepare analyte fortification

solutions in which the concentrations of analytes 9-12 are higher than the

concentrations of analytes 1-8. Use of this option is at the discretion of the

analyst.

7.11 GC/MS TUNE CHECK SOLUTION -- Decafluorotriphenylphosphine (DFTPP), 5

:g/mL in methylene chloride. Store this solution in an amber vial at 0°C or less.

 7.12 SODIUM SULFITE, ANHYDROUS -- Reducing agent used to reduce residual chlorine

at the time of sample collection.

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
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should nearly fill the 1 L or 1 qt bottle, but does not need to be headspace free. 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 acidified at the time of collection. Residual chlorine is reduced by

addition of 40-50 mg of sodium sulfite. It may be added as a solid to the sample bottles

before the bottles are transported to the field. It is very important that the sample be

dechlorinated prior to acidification. Wait until sodium sulfite is dissolved before

acidification. Adding sodium sulfite and HCl (together) to the sample bottles prior to

shipping bottles to the sampling site is not permitted. After dechlorination, samples are

acidified to less than pH 2 with 6 N hydrochloric acid. The acid serves as a chemical and

biological preservative. This pH is the same that is used in the sample extraction, and is

required to support the recovery of several method analytes.

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.

8.4 HOLDING TIME -- Results of holding time studies of all method analytes showed that all

compounds are stable for 14 days in water samples when the samples are dechlorinated,

preserved, and stored as described in Sect. 8.2 and 8.3. Therefore, samples must be

extracted within 14 days of collection. Sample extracts may be stored at 0°C or less for

up to 30 days after sample extraction. Data from holding time studies are shown in Tables

7 and 8.

9. QUALITY CONTROL

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

capability (summarized in Table 9) followed by regular analyses of continuing calibration

checks, laboratory performance check standards, laboratory reagent blanks, laboratory

fortified blanks, and laboratory fortified matrix samples. An MDL 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 10.

 
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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 9.

9.2.1 INITIAL DEMONSTRATION OF LOW CARTRIDGE EXTRACTION

BACKGROUND AND SYSTEM BACKGROUND -- Before any samples

are analyzed, or any time a new supply of solid phase extraction 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.

9.2.1.1 A source of potential contamination is the solid phase extraction

cartridge which may contain phthalate esters, silicon compounds, and

other contaminants that could interfere with the determination of

method analytes. Although extraction cartridges 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 5-10 :g/L, or other mid-range concentration. Sample

preservatives described in Sect. 8.2 must be added to these samples. Extract

and analyze these replicates according to the procedure described in Section 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 with the exception of

phenol. The RSD for replicate analyses for phenol must be less than or equal to

30%.

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

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

The average recovery of the replicate values must be within 70-130% of the true

value, except for phenol. Phenol will typically be recovered less effectively than

other method analytes. Because of its higher water solubility some breakthrough
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from the extraction cartridge does occur. The recovery limits for phenol are 50-150%.

9.2.4 MDL 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 at a

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

estimated by selecting a concentration at 2-5 times the noise level. 

Concentrations shown in the example data in Table 1 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 Section 11. Calculate the MDL using the following

equation:

MDL = St( n - 1, 1 - alpha = 0.99)

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 MDL calculations.

9.2.5 The analyst is permitted to modify GC columns, GC conditions, extract

evaporation techniques, internal standards or surrogate compounds. 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 MDL or at a concentration which would yield a response greater than a signal-tonoise 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
528-16

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 DFTPP. 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).

9.7 PEAK TAILING FACTOR (PTF) -- This check consists of calculating the PTF as

described in Sect. 10.2.3.1. and in Figure 4. This check must be performed once every

24 hr of instrument operation.

9.8 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 a 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 lowest CAL point for an analyte must be 50-150% of

the true value for all analytes. Results of LFB analysis from medium and high level

concentrations must be 70-130% of the true value for all analytes except phenol. The

acceptance limit for phenol is 50-150% of the true value.

9.9 INTERNAL STANDARD (IS) --The analyst must monitor the peak area of the 1,2-

dimethyl-3-nitrobenzene (IS#1) in all injections during each analysis day. The IS#1

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#1 area in a chromatographic

run does not meet these criteria inject a second aliquot of that extract.
528-17

NOTE: The peak area of 2,3,4,5-tetrachlorophenol may not be consistent. It may vary

depending upon the composition of the extract or standard being analyzed. See Section

13.2.1 for a detailed explanation.

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

report results for that aliquot.

9.9.2 If a deviation of greater than 30% is obtained for the reinjected extract, when

compared to the most recent CCC, the analyst should check the calibration by

reanalyzing the most recently acceptable calibration standard. If the calibration

standard fails the criteria of Section 10.3.3, recalibration is in order per Section

10. 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.

9.10 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. 

9.10.1 Surrogate recovery criteria are 70-130% of the fortified amount for 2-

chlorophenol-3,4,5,6-d4 and 2,4-dimethylphenol-3,5,6-d3. The criteria for

2,4,6-tribromophenol is 60-130% of the fortified amount. 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. 

9.10.2 If the extract reanalysis meets the surrogate recovery criterion, report only data

for the reanalyzed extract.

9.10.3 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 Section 10.3.3, recalibration is in

order per Section 10. 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 reextract also fails the recovery criterion, report all

data for that sample as suspect.

9.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- Determine that the sample

matrix does not contain materials that adversely affect method performance. This is
528-18

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, matrix independence should be established for each. 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 a LFM for

every 20 samples. If the recovery data for an LFM does not meet the recovery criteria in

Sect. 9.8, 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.

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

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

with an appropriate amount of the fortification solution. The concentrations 5,

10, and 15 :g/L are suggested spiking concentrations. Select the spiking

concentration that is closest to, and at least twice the matrix background

concentration. Use historical data or rotate through the designated

concentrations to select a fortifying concentration. Selecting a duplicate bottle of

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

spiking levels.

9.11.2 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. 9.8).

9.11.3 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 except phenol which should be recovered at 50-150%. If the

accuracy 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
528-19

suspect/matrix to inform the data user that the results are suspect due to matrix

effects.

NOTE: Matrix effects are expected to be more likely with compounds 9-12

(Table 2) than other method analytes.

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

sample should be analyzed in duplicate. Duplicate sample analyses serve as a check on

sampling and laboratory precision. If analytes are not routinely observed in field samples,

duplicate LFMDs should be analyzed to substitute for this requirement. 

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

(FD1 and FD2) as shown below.

9.12.2 Relative percent differences for laboratory duplicates and LFMDs 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.

9.13 QUALITY CONTROL SAMPLE (QCS) -- Each time that new standards 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 a LFB, then the acceptance criteria are the same as for an LFB (Sect.

9.8). 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

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 calibration. A peak tailing factor check is
528-20

required every day that samples are analyzed. In periods of continuous operation, the

peak tailing factor must be performed every 24 hr.

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

DFTPP solution into the GC/MS system. Acquire a mass spectrum that

includes data for m/z 45-450. If the DFTPP mass spectrum does not meet all

criteria in Table 3, 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. 

The tune check may be performed as a separate analysis, or for routine MS tune

verification, DFTPP may be added to one or more of the CAL standards used

for calibration verification, so that the tune check and calibration verification can

be performed in a single analysis. DFTPP elutes shortly after pentachlorophenol

on both of the columns cited in Sect.6.9.

10.2.2 ANALYTE CALIBRATION -- Inject an aliquot of a medium concentration

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

m/z 45-350 with a total cycle time (including scan overhead time) of 1.0 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.

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.

10.2.2.1 The following parameters are suggested GC conditions for hot,

splitless injection: injector temperature 200o C, carrier gas head

pressure 12-15 psi. Inject at an oven temperature of 35o C and hold

in splitless mode for 0.2 min. After 6 min, temperature program the

GC oven at 8o C per min to 250o C. Start data acquisition at
528-21

approximately 10 min. Example chromatograms are shown in

Figures 1 and 2.

10.2.2.2 The following parameters are suggested injection conditions for

temperature programmed splitless injection. Inject with the injector

temperature at 25° C, program the injector at 200° C per min to

200° C. Hold in the splitless mode for 1.0 min. Use the same

column temperature program as listed in Sect. 10.2.2.1. An

example chromatogram is shown in Figure 3.

10.2.3 Performance criteria for the calibration standards. Examine the stored GC/MS

data with the data system software. 

10.2.3.1 PEAK TAILING FACTOR (PTF) -- Peak tailing can be a

problem associated with phenols. The phenols most likely to tail are

those with low acidity constants: 2,4-dinitrophenol, 4-nitro-phenol,

pentachlorophenol and 2-methyl-4,6-dinitrophenol. These

compounds must exhibit a peak tailing factor of 5 or less at a

concentration equivalent to 5-10 :g/L in a water sample (5-10

:g/mL in an extract or calibration standard). For example peak

tailing factor calculations, see Fig.4. Peak tailing factors must be

evaluated for the four analytes listed above each day that samples

are analyzed. In periods of continuous instrument operation, verify

acceptable PTFs every 24 hr. Peak tailing factors may be evaluated

in either a CAL standard, LFB or LFM.

10.2.3.2 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. 11.5).

10.2.4 If all performance criteria are met, inject an aliquot of 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 (0.1

and 0.5 :g/mL). 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
528-22

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. 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,

with a minimum of five calibration concentrations.

 RF 

A Q

A Q

x is

is x

=

( )( )

( )( )

 

 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

point, 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 presented in this method were
528-23

obtained using linear regression (RF vs Ax /Ais). Quadratic fit calibrations

should be used with caution, because the non-linear area of the curve may not be

reproducible.

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 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. 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

1,3-dimethyl-2-nitrobenzene has 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, nondetects may be reported without re-analysis.
528-24

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, 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.

10.3.4.2 Clean or replace the splitless injection liner; silanize a new injection

liner.

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. 

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 CARTRIDGE 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. 11.2). Verify that the sample is at pH 2 or less and

is free of residual chlorine. If the sample is a LRB or LFB, add sodium sulfite
528-25

and acidify following procedures 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 2-5 :g/L. If the

sample is a LFB or LFM, add the desired amount of analyte fortification

solution.

11.1.3 CARTRIDGE CLEAN-UP AND CONDITIONING -- Rinse each cartridge

with three, 3 mL aliquots of methylene chloride. Let the cartridge drain dry after

each flush. Then rinse the cartridge with three, 3mL 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 with three,

3mL aliquots of 0.05 N hydrochloric acid, but before the dilute acid level drops

below the top edge of the packing, turn off the vacuum. Add approximately 3

mL additional 0.05 N hydrochloric acid to the cartridge, attach the transfer tube,

and turn on the vacuum, and begin adding sample to the cartridge. 

11.1.4 Adjust the vacuum so that the approximate flow rate is 20 mL/min (50 min for a

1 L sample). After all of the sample has passed through the SPE cartridge, draw

air or nitrogen through the cartridge for 15-30 min at high vacuum (10-15 in Hg). 

The cartridge packing should appear dry (light tan color) before continuing with

the elution steps. It is important that the cartridge packing be dry, in order to

obtain good recoveries. The drying time may vary, depending upon the strength

of the vacuum source, and the number of cartridges being processed

simultaneously. The color and appearance of the packing is the most reliable

indicator of dryness. During the method development, drying for more than 60

minutes was not observed to have any negative effect upon the sample data.

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.

11.1.5 Rinse the inside of each sample bottle with 8-10 mL methylene chloride and use

vacuum to pull the 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 2-3 mL methylene chloride 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. Small amounts of

residual water from the sample container and the SPE cartridge may form an

immiscible layer with the eluate. Pass the eluate through the drying column (Sect.

6.7), which is packed with approximately 5 to 7 grams of anhydrous sodium
528-26

sulfate, and collect in a clean collection tube. Wash the sodium sulfate with at

least 2 mL methylene chloride and collect 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 losses of analytes. Add the internal standards (Sect 7.9). Adjust

final volume to 1 mL. Make any volume adjustments with methylene chloride.

11.2 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.

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

conditions used for the initial and continuing calibrations (Sect. 10.2.2 and 10.3).

11.4 At the conclusion of data acquisition, use the same software that was used in the

calibration procedure to identify peaks in predetermined retention time windows of

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

the chromatogram. 

11.5 Identification of analytes. 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.

11.5.1 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%. Some ions, particularly the

molecular ion, are of special importance, and should be evaluated even if they

are below 10% relative abundance.
528-27

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

characteristic 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 Section

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 Section 11.2. 

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). Experience indicates that three significant figures may

be used for concentrations above 99 :g/L, two significant figures for

concentrations between 1.0-99 :g/L, and one significant figure for lower

concentrations.

13. METHOD PERFORMANCE 

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

data from both fortified reagent water and fortified matrices using hot, splitless injection

are presented in Tables 4 and 5. Table 6 includes data from 2 matrices using temperature

programmed splitless injection. Method detection limits (MDLs) are presented in Table 1

for both types of injectors used. MDLs were calculated using the formula in Section

9.2.4. Although the calculated MDLs using the two different types are not dramatically

different, for compounds 9-12 (Table 2) the peak shapes are significantly better using

temperature programmed injection, and the peak heights and areas are greater. 
528-28

13.2 POTENTIAL PROBLEM COMPOUNDS –

13.2.1 2,4-Dinitrophenol, 4-nitrophenol, 2-methyl-4,6-dinitrophenol, pentachlorophenol

and 2,4,6-tribromophenol have a tendency to exhibit a chromatographic

phenomenon known as “matrix-induced chromatographic response

enhancement” (5-8). Compounds that exhibit this phenomenon often give

analytical results that exceed 100% recovery. The theory behind this

phenomenon is that these compounds are susceptible to adsorption and/or

thermal degradation in the GC inlet. The “cleaner” the matrix they are injected

in, e.g. clean solvent, the more they degrade. When they are injected in a sample

extract, matrix components in the sample extract “protect” these compounds

from decomposition and a relatively greater response is observed. While most

of the literature references to this phenomenon refer to organophosphate

pesticides in river water and food samples, the effect seen during development of

this method suggests the same type of problem occurs with these acidic phenols.

This method uses 2,3,4,5-tetrachlorophenol as the internal standard for

quantifying these analytes. The chromatographic behavior of 2,3,4,5-

tetrachlorophenol mimics these particular method analytes. Therefore its use as

an internal standard helps maintain accurate measurement of these analytes. It

should be noted however that these particular analytes will probably not be

measured with the same level of precision and accuracy as other method

analytes, but the precision and accuracy requirements should still be achievable.

13.2.2 The same compounds listed in sect. 13.2.1. also have a tendency to tail. QC

criteria for peak tailing factors have been given in Sect. 10.2.3.1. During method

development, significantly less peak tailing was observed using temperature

programmed injection. Other measures shown to minimize peak tailing and

improve peak shape are pressure pulsed injection, and increasing the GC oven

temperature program rate. Pulsed injection is recommended on GCs which have

that option available. A faster GC oven temperature program is recommended if

there are no interferences, and if the minimum number of scans across all

chromatographic peaks can be obtained. This is a function of how fast the MS

can scan.

13.2.3 Phenol is very water soluble compared to other method analytes. Breakthrough

experiments performed during method development indicate that some

breakthrough from the SPE cartridge can be expected. Breakthrough can be

minimized by monitoring the flow of the sample through the cartridge. In general,

slower flow rates will minimize breakthrough. Precision and accuracy

requirements in Sect. 10 should be achievable.
528-29

13.3 HOLDING TIME STUDY RESULTS – 

13.3.1 Holding time studies for aqueous samples were conducted for a period of 35

days. Chlorinated surface water samples fortified with method analytes and

preserved and stored according to requirements in Section 8, were analyzed on

days 0, 7, 10, 15, 23, 28, and 35. Small, but statistically significant losses of 2-

chlorophenol, o-cresol, and 2,4-dimethylphenol were observed beginning

between day 15 and 23. Therefore the aqueous holding time was determined to

be 14 days. Data from these studies are in Table 7.

13.3.2 Holding time studies for sample extracts were conducted for a period of 35

days. A single set of extracts were stored at 0°C, and analyzed on days 0, 14,

23, and 35. No significant losses were observed within this time frame. 

Therefore the extract holding time was established at 30 days. Data from these

studies are in Table 8.

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.

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” also

available from the American Chemical Society at the address in Section 14.2.
528-30

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 2206, (Revised, January 1976).

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

Committee on Chemical Safety, 3rd Edition, 1979.

5. 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.

6. Mol, H.G.J., M. Althuizen, H. Janssen, and C.A. Cramers, “Environmental Applications of

Large Volume Injection in Capillary GC Using PTV Injectors,” J. High Resol. Chromatogr., 19

(1996)69-79.

7. 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.

8. 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.
528-31

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. METHOD DETECTION LIMITS a

Analyte

Hot Splitless Injection b Temperature Programmed

Splitless Injection c

Spiking

Conc. (::g/L)

MDL

(::g/L)

Spiking

Conc. (::g/L)

MDL

(::g/L)

phenol 1.0 0.58 0.1 0.025

2-chlorophenol 0.1 0.020 0.1 0.041

2-methylphenol (o-cresol) 0.1 0.026 0.1 0.028

2-nitrophenol 0.1 0.026 0.1 0.044

2,4-dimethylphenol 0.1 0.026 0.1 0.034

2,4-dichlorophenol 0.1 0.027 0.1 0.046

4-chloro-3-methylphenol 0.1 0.036 0.1 0.042

2,4,6-trichlorophenol 0.1 0.046 0.1 0.024

2,4-dinitrophenol 1.0 0.31 0.5 0.22

4-nitrophenol 1.0 0.42 0.5 0.18

2-methyl-4,6-dinitrophenol 1.0 0.26 0.5 0.092

pentachlorophenol 1.0 0.25 0.5 0.081

a- data obtained using Column 1

b- n=7

c- n=8
528-32

TABLE 2. RETENTION TIMES (RTs) AND SUGGESTED QUANTITATION IONS (QIs)

Cmpd

# a

Analyte RTs (min) RTs (min) QIs

(m/z)

IS

Ref column 1 b

column 2 c

1 phenol 11:00 12:38 94 1

2 2-chlorophenol 11:07 12:50 128 1

3 2-methylphenol (o-cresol) 12:52 14:27 107 1

4 2-nitrophenol 14:36 16:22 139 1

5 2,4-dimethylphenol 15:00 16:35 107 1

6 2,4-dichlorophenol 15:22 17:04 162 1

7 4-chloro-3-methylphenol 17:46 19:27 142 1

8 2,4,6-trichlorophenol 18:56 20:42 97 1

9 2,4-dinitrophenol 21:30 23:30 154,184d 2

10 4-nitrophenol 21:53 23:44 139 2

11 2-methyl-4,6-dinitrophenol 23:09 25:09 121,198d 2

12 pentachlorophenol 25:14 27:12 266 2

13 1,2-dimethyl-3-nitrobenzene (IS#1) 17:43 19:29 134

14 2,3,4,5-tetrachlorophenol (IS#2) 22:09 24:02 232

15 2-chlorophenol-3,4,5,6-d4 (SURR) 11:04 12:47 132 1

16 2,4-dimethylphenol-3,5,6-d3 (SURR) 14:59 16:34 110 1

17 2,4,6-tribromophenol (SURR) 23:37 25:37 330 2

a- Number refers to peak number in Figures 1-3.

b- Column 1- 30 m × 0.25 mm id DB-5ms (J&W), 0.25 :m film thickness.

c- Column 2- 30 m × 0.25mm id BPX5 (SGE), 0.25 :m film thickness.

d- Because the MS response to these compounds is low, and both the listed ions are near 100% ion

abundance, the signal from both ions may be added together to increase sensitivity.
528-33

TABLE 3. ION ABUNDANCE CRITERIA FOR DECAFLUOROTRIPHENYLPHOSPHINE (DFTPP)

Mass (m/z) Relative Abundance Criteria Purpose of Checkpoint a

 51 10-80% of the base peak low mass sensitivity

68 <2% of mass 69 low mass resolution

70 <2% of mass 69 low mass resolution

127 10-80% of the base peak low-mid mass sensitivity

197 <2% of mass 198 mid-mass resolution

198 base peak or >50% of 442 mid-mass resolution and sensitivity

199 5-9% of mass 198 mid-mass resolution and isotope ratio

275 10-60% of the base peak mid-high mass sensitivity

365 >1% of the base peak baseline threshold

441 Present and < mass 443 high mass resolution

442 base peak or >50% of 198 high mass resolution and sensitivity

443 15-24% of mass 442 high mass resolution and isotope ratio

 

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 three resolution checks, which include natural abundance isotope ratios, constitute the next most important

part of the performance test. The correct setting of the baseline threshold, as indicated by the presence of low intensity ions, is the next most

important part of the performance test. Finally, the ion abundance ranges are designed to encourage some standardization to fragmentation

patterns.
528-34

TABLE 4. RESULTS FROM REPLICATE ANALYSES OF REAGENT WATER FORTIFIED WITH METHOD ANALYTES 

AT 1-10 ::g/L a USING HOT SPLITLESS INJECTION

Analyte Concentration= 10 ::g/L b, n=4 Concentration= 5 ::g/L c, n=4 Concentration= 1 ::g/L b, n=7

Mean %

Recovery

RSD (%) Mean %

Recovery

RSD (%) Mean %

Recovery

RSD (%)

phenol 75.9 4.3 103 18 90.8 20

2-chlorophenol 91.3 2.6 86.7 4.3 89.7 7.9

2-methylphenol (o-cresol) 93.3 2.2 86.1 3.6 84.9 7.3

2-nitrophenol 97.4 3.6 97.8 4.8 75.0 11

2,4-dimethylphenol 91.0 1.7 74.7 8.5 76.3 8.0

2,4-dichlorophenol 94.3 2.9 94.4 1.8 88.2 4.8

4-chloro-3-methylphenol 96.5 1.9 92.3 4.2 90.8 8.4

2,4,6-trichlorophenol 89.9 3.4 91.5 4.7 92.9 11

2,4-dinitrophenol 107 3.6 104 6.0 61.8 16

4-nitrophenol 107 3.0 90.5 11 96.3 14

2-methyl-4,6-dinitrophenol 105 3.2 83.0 1.6 91.5 8.9

pentachlorophenol 103 1.9 86.2 9.8 103 7.6

2-chlorophenol-3,4,5,6-d4 (SURR) 93.4 1.8 84.9 6.6 95.9 11

2,4-dimethylphenol-3,5,6-d3 (SURR) 94.0 2.0 82.0 7.2 95.1 13

2,4,6-tribromophenol (SURR) 82.2 1.8 77.1 6.1 98.0 8.6

a- Surrogate concentrations in all sample are 5 :g/L for tribromophenol and 2 :g/L for the deuterated phenols.

b- Data obtained using Column 1.

c- Data obtained using Column 2
528-35

TABLE 5. ACCURACY a AND PRECISION DATA FOR METHOD ANALYTES FORTIFIED AT 10 ::g/L IN THREE MATRICES

HOT SPLITLESS INJECTION

ANALYTE HARD GROUND WATER b, c

n=4

CHLORINATED SURFACE

WATER c n=4

SIMULATED HIGH TOC WATER

c, d n=4

% Rec RSD (%) %Rec RSD (%) %Rec RSD (%)

phenol 77.6 5.1 73.9 4.5 73.7 5.1

2-chlorophenol 91.2 2.6 88.6 3.0 85.8 5.2

2-methylphenol (o-cresol) 93.2 3.4 91.3 2.3 89.2 4.3

2-nitrophenol 102 2.5 99.4 2.1 99.0 2.8

2,4-dimethylphenol 86.3 2.0 86.6 1.9 83.2 5.3

2,4-dichlorophenol 94.8 1.2 93.3 1.4 90.0 5.1

4-chloro-3-methylphenol 98.5 1.6 97.5 1.4 94.6 3.9

2,4,6-trichlorophenol 95.5 3.9 92.7 3.1 89.1 4.1

2,4-dinitrophenol 117 4.2 118 2.3 121 1.0

4-nitrophenol 102 2.4 102 3.8 95.9 2.3

2-methyl-4,6-dinitrophenol 115 2.2 113 1.5 115 0.71

pentachlorophenol 110 4.0 108 5.1 105 1.8

2-chlorophenol-3,4,5,6-d4 (SURR) 93.0 2.4 91.5 1.6 89.1 5.3

2,4-dimethylphenol-3,5,6-d3 (SURR) 88.9 3.0 87.2 2.4 85.4 4.8

2,4,6-tribromophenol (SURR) 74.7 2.6 76.3 5.1 71.0 4.5

a- Accuracy is presented as % recovery

b- Hard municipal chlorinated ground water, 450mg/L hardness measured as calcium carbonate.

c- Data obtained using Column 1.

d- Simulated high organic content (high Total Organic Carbon) sample prepared by adding 10 mg/L humic acid (Fluka Chemical Corp., Milwaukee, WI) to reagent

water.
528-36

TABLE 6. RESULTS OF REPLICATE ANALYSES IN TWO MATRICES USING TEMPERATURE PROGRAMMABLE

INJECTION a

ANALYTE Reagent Water

Concentration= 0.5 ::g/L, n=7

Reagent Water

Concentration= 5 ::g/L, n=4

Chlorinated Surface Water

Concentration= 10 ::g/L, n=5

% Recovery RSD (%) % Recovery RSD (%) % Recovery RSD (%)

phenol 74.5 13 107 21 74.1 7.5

2-chlorophenol 88.3 11 95.5 7.7 94.8 4.3

2-methylphenol (o-cresol) 96.2 9.4 93.0 4.5 103 8.1

2-nitrophenol 89.1 8.9 102 6.8 94.5 7.4

2,4-dimethylphenol 82.0 8.2 85.5 8.8 93.8 9.8

2,4-dichlorophenol 83.0 8.3 100 7.3 93.7 2.9

4-chloro-3-methylphenol 79.0 6.6 101 7.5 96.6 2.9

2,4,6-trichlorophenol 78.9 8.1 102 10 90.7 3.3

2,4-dinitrophenol 63.8 22 95.4 6.6 113 4.7

4-nitrophenol 84.1 14 103 8.1 102 6.8

2-methyl-4,6-dinitrophenol 62.4 9.4 103 5.1 107 4.9

pentachlorophenol 72.0 7.2 88.0 7.2 93.8 6.8

2-chlorophenol-3,4,5,6-d4 (SURR) 79.1 7.6 87.6 3.9 84.7 10

2,4-dimethylphenol-3,5,6-d3 (SURR) 79.9 8.5 91.9 7.8 97.0 8.3

2,4,6-tribromophenol (SURR) 79.6 7.8 86.1 7.8 79.5 12

a- Data obtained using Column 1.
528-37

TABLE 7. RESULTS OF AQUEOUS HOLDING TIME STUDIES FOR METHOD 528 ANALYTES a

ANALYTE DAY 0 DAY 7 DAY 10 DAY 15

% Rec RSD (%) % Rec RSD (%) % Rec RSD (%) % Rec RSD (%)

phenol 74.5 1.9 77.0 3.8 74.3 3.4 74.3 3.5

2-chlorophenol 91.5 1.9 88.8 1.3 88.0 2.9 85.8 4.1

2-methylphenol (o-cresol) 99.9 2.8 96.0 1.2 94.3 2.6 93.0 2.8

2-nitrophenol 99.1 4.3 100 4.0 98.8 3.9 101 3.1

2,4-dimethylphenol 85.5 4.2 80.7 3.9 79.1 8.1 75.2 4.7

2,4-dichlorophenol 94.2 2.2 93.0 2.5 92.3 1.0 91.8 3.3

4-chloro-3-methylphenol 96.5 2.1 95.5 3.5 96.5 2.3 96.5 3.9

2,4,6-trichlorophenol 94.8 3.3 97.1 2.9 96.0 2.8 95.0 3.3

2,4-dinitrophenol 102.8 5.1 102 3.4 107 8.0 112 2.9

4-nitrophenol 100.1 3.2 100 4.2 101 4.9 96.2 3.3

2-methyl-4,6-dinitrophenol 96.1 5.1 109 8.7 113 5.8 115 5.0

pentachlorophenol 94.5 4.1 102 2.1 103 3.5 102 2.3

a- All analytes fortified into a chlorinated surface water at a concentration of 10 :g/L, dechlorinated and acidified according to Section 8, stored

for 48 hr at 10° C, followed by storage at 6° C. For each time point, n=5.
528-38

TABLE 8. RESULTS OF EXTRACT HOLDING TIME STUDIES FOR METHOD 528 ANALYTES a

ANALYTE DAY 0 DAY 14 DAY 23 DAY 35

% Rec RSD (%) % Rec RSD (%) % Rec RSD (%) % Rec RSD (%)

phenol 74.5 1.9 74.2 3.8 72.7 3.4 74.9 3.5

2-chlorophenol 91.5 1.9 89.8 1.3 88.6 2.9 89.1 4.1

2-methylphenol (o-cresol) 99.9 2.8 97.5 1.2 98.4 2.6 96.6 2.8

2-nitrophenol 99.1 4.3 100 4.0 96.4 3.9 101 3.1

2,4-dimethylphenol 85.5 4.2 86.9 3.9 88.0 8.1 88.7 4.7

2,4-dichlorophenol 94.2 2.2 95.4 2.5 96.1 1.0 98.6 3.3

4-chloro-3-methylphenol 96.5 2.1 98.9 3.5 98.1 2.3 102 3.9

2,4,6-trichlorophenol 94.8 3.3 97.1 2.9 97.6 2.8 102 3.3

2,4-dinitrophenol 102.8 5.1 104 3.4 90.9 8.0 99.1 2.9

4-nitrophenol 100.1 3.2 91.1 4.2 89.3 4.9 96.3 3.3

2-methyl-4,6-dinitrophenol 96.1 5.1 108 8.7 98.2 5.8 110 5.0

pentachlorophenol 94.5 4.1 99.1 2.1 100 3.5 105 2.3

a- All extracts were from the Day 0 aqueous holding time samples, and were stored in amber vials at 0° C. For each time point, n=5.
528-39

TABLE 9. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS

Method

Reference

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 below

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 5-10:g/L

RSD must be #20% for all analytes except

phenol which must be # 30%.

Sect. 9.2.3 Initial Demonstration of

Accuracy (IDA)

Calculate average recovery for

replicates used in IDP

Mean recovery 70-130% of true value, except

phenol which is 50-150%

Sect. 9.2.4 Method Detection Limit

(MDL) Determination 

Over a period of three days,

prepare a minimum of 7 replicate

LFBs fortified at a concentration

estimated to be near the MDL. 

Analyze the replicates through all

steps of the analysis. Calculate the

MDL using the equation in Section

9.2.4.

Note: Data from MDL replicates are not

required to meet method precision and

accuracy criteria. If the MDL replicates are

fortified at a low enough concentration, it is

likely that they will not meet precision and

accuracy criteria.
528-40

TABLE 10. QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Reference

Requirement Specification and Frequency Acceptance Criteria

Sect. 8.4 Sample Holding Time 14 days, dechlorinated and acidified

to pH#2

Iced or refrigerated at 10°C or less for up to

48 hours, 6°C thereafter.

Sect. 8.4 Extract Holding Time 30 days Stored at 0°C or less in amber vials.

Sect.9.11 Laboratory Fortified Sample

Matrix (LFM)

Analyze one LFM per extraction

batch (20 samples or less) fortified

with method analytes at a

concentration close to the native

concentration.

Recoveries not within 70-130% of the fortified

amount may indicate a matrix effect.

Sect. 9.12 Field Duplicates Analyze 1 FD for each 20 samples,

or 1 per extraction batch, whichever

is greater.

Suggested RPD±30%.

Sect. 9.13 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 a LFB, those

criteria apply.

Sect 9.2.1 Laboratory Reagent Blank

(LRB)

Daily, or with each extraction batch

of up to 20 samples, whichever is

more frequent.

Demonstrate that all target analytes are below

1/3 the MRL, and that possible interference

from extraction media do not prevent the

identification and quantification of method

analytes.

Sect. 9.8 Laboratory Fortified Blanks

(LFB)

Analyze at least one LFB daily or 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 (except phenol) 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.
528-41

Sect. 9.9 Internal Standard 1,2-dimethyl-3-nitrobenzene (IS#1)

and 2,3,4,5-tetrachloro-phenol

(IS#2) are added to all standards

and sample extracts.

Peak area counts for IS#1 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 9.10 Surrogate Standards Surrogate standards are added to all

standards, samples, LFBs, LFMs,

FDs, LRBs, and LFBs.

Recovery for 2-chlorophenol-3,4,5,6-d4 and

2,4-dimethylphenol-3,5,6-d3 in all standards,

LRB, LFB, LFM, FD and sample extracts

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

for 2,4,6-tribromophenol must be 60-130%.

Sect. 10.2.1 MS Tune Check Analyze DFTPP to verify MS tune

before initial calibration and before

every recalibration.

Criteria are given in Table 3.

Sect.10.2.2 Initial Calibration Use internal standard calibration

technique to generate an average RF

or first or second order calibration

curve. Use at least 5 standard

concentrations that span the

approximate range of 0.1- 15 :g/L.

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. 10.2.3.1 GC Performance-Peak Tailing

Check

Calculate the peak tailing factor for

compounds listed in the referenced

section, at the beginning of each day

during which samples are analyzed. 

In cases of continuous instrument

operation, check peak tailing factors

every 24 hr.

Peak tailing factor of 5 or less. (See Fig.4 for

calculation of PTF.)
528-42

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.

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 IS#1 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.
528-43

FIGURE 1.CHROMATOGRAM OF PHENOL STANDARD MIX (5 ng/ ::L EACH ANALYTE) ON A DB-5ms COLUMN WITH

HOT SPLITLESS INJECTION a.

a- peak

numbers refer to compounds as listed in Table 2.
528-44

FIGURE 2.CHROMATOGRAM OF PHENOL STANDARD MIX (5 ng/ ::L EACH ANALYTE) ON A BPX5 COLUMN WITH

HOT SPLITLESS INJECTION. a

a- peak numbers refer to compounds as listed in Table 2..
528-45

FIGURE 3. CHROMATOGRAM OF PHENOL STANDARD MIX (5 ng/ ::L EACH ANALYTE) ON A DB-5ms COLUMN

WITH TEMPERATURE PROGRAMMED SPLITLESS INJECTION. a

a- peak

numbers refer to compounds as listed in Table 2.
528-46

FIGURE 4. PEAK TAILING FACTOR (PTF) CALCULATION.

Peak Tailing Factor = 

BC

AB

BD = 10 % peak height

Note: the PTF should be calculated from the single ion chromatogram of the quantitation ion.

This example is for the pentachlorophenol peak. The PTF = 2.5.