United States
Environmental Protection
\r ^1 M^k. Agency
Office of Water
www.epa.gov July 1990
Method 548: Determination of
Endothall in Drinking Water by
Aqueous Derivatization,
Liquid-Solid Extraction and
Gas Chromatography with
Electron-Capture Detection
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Note: This method is no longer approved for compliance monitoring
associated with the Safe Drinking Water Act, but it is approved for
Clean Water Act compliance monitoring associated with certain
pesticide active ingredients. See Table IG at 40 CFR Part 136.
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METHOD 548. DETERMINATION OF ENDOTHALL IN DRINKING WATER BV
AQUEOUS DERIVATIZATION, LIQUID-SOLID EXTRACTION, AND
GAS CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
July 1990
J. W. Hodges on
Merlin Bicking (Twi City Test.ing, St. Paul, Minnesota)
W. J. Bashe {Technology Applications, Incorporated)
David Becker {Technology Applications, Incorporated)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 548
DETERMINATION OF ENDOTHALL IN DRINKING WATER BY AQUEOUS DERIVATIZATION,
LIQUID-SOLID EXTRACTION, AND GAS CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
1. SCOPE AND APPLICATION
1.1 This method covers the determination of endoth 11 in drinking water
sources and finished drinking water. The following analyte can be
determined by this method:
Chemical Abstract Services
Analvte Registry Number
Endothall 145-73-3
1.2 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above. When t.his method is
used to analyze unfamiliar samples, compound identification should
be supported by at least one additional qualitative technique. A
gas chromatograph/mass spectrometer (GC/MS) may be used for the
qualitative confirmation of results for endothall using the extract
produced by this method.
1.3 The method detection limit1 (MDL, defined in Section 13) for
endothall is listed in Table 1. The MDL for a specific sample may
differ from the listed value, depending upon the nature of
interferences in the sample matrix and the amount of sample used in
the procedure.
1.4 The endothall-pentafluorophenylhydrazine derivative employed for
chromatographic detection is not available commercially. Thus, this
method employs procedural standards, in which endothall calibration
solutions (9.2.1) are processed through the analysis procedure
(11.2).
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography and in the
interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described in Section 11.
2. SUMMARY OF METHOD
2.1 A 5.0 ml volume of liquid sample is placed in a Kud,erna-Danish tube
and the volume is reduced to less than 0.5 ml using a heating block.
The tube is charged with glacial acetic acid and sodium acetate,
followed by a solution of the derivatization rlaagent, penta-
fluoropjienylhydrazine (PFPH), in glacial acetic acid. After heating
at 150 C for 90 minutes the derivative is extracted by a sol id
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sorbent from the reaction solution, followed by elution with 5.0 ml
of methyl-tert-butyl ether (MTBE). The MTBE extract is analyzed by
gas chromatography with electron capture detection (GC/ECD).
3. DEFINITIONS
3.1 INTERNAL STANDARD - A pure analyte(s) added to a 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 analyte that is not a sample
component.
3.2 SURROGATE ANALVTE - A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of
a surrogate analyte is to monitor method performance with each
sample.
3.3 LABORATORY DUPLICATES (LOI and LD2) - Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LOI .and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures .
3.4 FIELD DUPLICATES (FDI 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 FDI and FD2 give a measure of the precision, associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
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.
3.6 FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 LABORATORY PERFORMANCE CHECK SOLUTION (LPC) - A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria .
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3.8 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
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.
3.10 STOCK STANDARD SOLUTION - A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.11 PRIMARY DILUTION STANDARD SOLUTION - 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.
3.12 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in
Section 10.2.
4.1.1 Glassware must be scrupulously clean2• Clean all glassware
as soon as possible after use by rinsing with the last
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solvent .used in .it. This should be followed by detergent
washing with hot water, and rinses with tap water and
distilled water. It shouldc then be drained dry, and heated
in a laboratory oven at 40 C for several hours before use.
Solvent rinses with methanol may be substituted for the oven
heating. After drying and cooling, glassware should be
stored in a clean environment to prevent any accumulation of
dust or other contaminants.
4. 1.2 The use of high purity reagents and solvents is absolutely
necessary to minimize interference problems. Purification of
solvents by distillation in all-glass systems immediately
prior to use is highly recommended.
4. 2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the matrix being sampled. If significant
interferences occur in subsequent samples, some additional cleanup
may be necessary to achieve the MDL listed in Table I.
4.3 The extent of interferences that may be encountered using gas
chromatographic techniques has not been fully assessed. Although
the GC conditions described allow for a unique resolution of the
specific compound covered by this method, other matrix components
may interfere.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. • From this
viewpoint, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemical specified in
this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additionally references to laboratory safety are
available.
6. APPARATUS AND MATERIALS
6.1 SAMPLING EQUIPMENT (for discrete or composite sampling).
6.1.1 Grab sample bottle - Amber glass fitted with screw caps lined
with Teflon. If amber bottles are not available, protect
samples from light. The cont iner must be washed, rinsed
with methanol, and dried before use to minimize contamination
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6.2 GLASSWARE
6.2.1 Volumetric flasks - 5 ml, 25 ml
6.2.2 Vials - glass, I ml, with Teflon-lined caps
6.2.3 Glass syringes, 250 yL, 500 yL
6.2.4 Pipets - 1 ml, 4 ml
6.3 BALANCE - analytical, capable of accurately weighing 0.0001 g.
6.4 SOLID SORBENT CARTRIDGES - C-18
6.5 Vacuum manifold for extraction using solid sorbent cartridges
Supelco 5-7030 or equivalent
6.6 Kuderna-Danish (K-D) concentrator tubes - 10 or 25 ml graduated
6.6.1 Snyder column, Kuderna-Danish -2- ball micro
6.7 Tube heater for 25 ml K-D tubes
6.8 Boiling chips - carborundum, #12 granules Heat at 400 C for 30
minutes prior to use. Cool and stored in dessicator.
6.9 Gas chromatographic system capable of temperature programming
6.9.1 Autosampler
6.9.2 Electron capture detector
6.9.3 Column 1: Supelco SPB-5, 0.25 mm x 30 m or equivalent
Column 2: J&W DB-I, 0.32 mm x 30 mm or equivalent
6.9.4 Strip-chart recorder compatible with detector. Use of a data
system with printer for measuring and recording peak areas
and retention times is recommended.
7. REAGENTS AND SOLUTIONS
7.1 REAGENT WATER - reagent water is defined as a water of very high
purity, equivalent to distilled in glass solvents
7.2 PENTAFLUOROPHENYLHYDRAZINE (PFPH) - Aldrich
7.3 SODIUM ACETATE - anhydrous
7.4 SODIUM THIOSULFATE
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7.5 ACETIC ACID - glacial
7.6 METHYL-TERT-BUTYL ETHER (MTBE) - distilled ill glass
7.7 ENDOTHALL-PFPH DERIVATIVE - See Appendix for synthesis • procedure
7.8 ENDOSULFAN I
7.9 ENDOTHALL, monohydrate
7.10 STOCK STANDARD SOLUTIONS
7.10.1 Endothall - 10 jig/ml in reagent water
7.10.2 Endothall - 50 jig/ml in reagent water
7.10.3 Stock standard solutions must be replaced after six months,
or sooner, if comparison with check standards indicates a
problem.
7.11 REACTION SOLUTIONS
7.11.1 PFPH solution - 4 mg/ml in glacial acetic acid.
7.11.2 Internal standard stock solution - 10 jig/ml endosulfan I in
MTBE
SAMPLE COLLECTION. PRESERVATION. AND HANDLING
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of Tygon tubing and other potential
sources of contamination.
•a 2 The samples'must be iced or refrigerated at 4 C from the time of
collection until derivatization. The analyte measured here is not
known to be light sensitive, but excessive exposure t light an
heat should be avoided.
8.3 Some samples are likely to be biologically active and the stability
of samples upon storage will be different for each matrix. All
samples should be derivatized within 7 days of collection, and
analysis completed within 1 day of derivatization. If these
criteria are not met, the analyst must demonstrate the stability of
the stored sample by performing suitable holding time studies .
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9. CALIBRATION
9.1 Establish gas chromatographic operating parameters to produce a
retention time equivalent to that indicated in Table 1. The
chromatographic system can be calibrated using the internal standard
technique (Section 9.2).
9.1.1 Due to the complex nature of the sample chromatogram, the
analyst should periodically inject a solution containing only
pure endothall-PFPH (See Appendix) to verify the retention
time of the derivative.
9.2 INTERNAL STANDARD CALIBRATION PROCEDURE:
9.2.1 Use 250 and 500 jiL syringes to add sufficient quantities of
7.10.1 or 7.10.2 stock solutions to reagent water in 25 ml
volumetric flasks to produce endothall standard solutions at
the following concentrations in |ig/L: 500 (250 jiL of 7.10.2
stock), 200 (100 jiL of 7.10.2 stock), 100 (50 jiL of 7.10.2
stock) and 50 (125 jiL of 7.10.1 stock).
9.2.2 Process each standard as per Section 11.2. The internal
standard is added as described in Section 11.2.7. It is
recommended that triplicate samples of each standard be
processed.
9.2.3 Before analyzing matrix samples, the analyst must process a
series of calibration standards to validate elution patterns
and the absence of interferences from reagents.
9.2.4 Analyze each calibration standard and tabulate the ratio of
the area of the endothall-PFPH derivative peak versus that of
the internal standard against endothall concentration. The
results may be used to prepare a calibration curve for
endothall.
9.2.5 The working calibration curve must be verified on each
working day by processing and analyzing one or more
calibration standards. If the response varies from the
previous response by more than ± 20%, the test must .be
repeated using a fresh calibration standard. Should the
retest fail, a new calibration curve must be generated.
10. QUALITY CONTROL
10 .1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. The minimum QC requirements
are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified blanks, laboratory
fortified matrix samples and QC check standards.
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10.2 LABORATORY REAGENT BLANKS. Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
analyzed or reagents are changed, a method blank must be analyzed.
For this method, the method blank is filter d reagent water. If
within the retention time window of an analyte of interest, the
method blank produces a peak which prevents the measurement of that
analyte, determine the source of contamination and eliminate the
interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3 ..1 Select a representative fortified concentration (about 10
times MDL) for endothall. Prepare a concentrate (in reagent
water) containing the analyte at 10 times the selected
concentration. Using a pipet, add 1.00 ml of the concentrate
to each of at least four 10 ml aliquots of reagent water and
analyze each aliquot according to procedures beginning in
Section 11.
10.3. 2 The recovery value should for at least three out of four
consecutively analyzed samples fall in the range of R ± 30%
(or within R ± 3SR, if broader) using the values for Rand SR
for reagent water (Table 2). If the recovery value meets the
acceptance criteria, performance is acceptable and sample
analysis may begin. If the recovery value fails these
criteria, initial demonstration of capability should be
repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples by a
new, unfamiliar method prior to evidencing a basal level of
skill at performing the technique. It is expected that as
•laboratory personnel gain experience with this method the
quality of the data will improve beyond the requirements
stated in Section 10.3.2.
10A The analyst is permitted to modify GC columns, GC conditions, or
detectors to improve separations or lower analytical costs. Each
time such method modifications are made, the analyst must repeat the
procedures in Section 10.3.
10.5 Assessing the Internal Standard - In using the IS calibration
procedure, the analyst is expected to monitor the IS response (peak
area or peak height) of all samples during each analysis day. The
IS response for any sample chromatogram should not deviate from the
calibration standard IS response 'by more than 30%.
10.5.1 If a deviation of greater than 30% is encountered for a
sample, reinject the extract .
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10.5.1.1 If acceptable IS response is achieved for the re-
injected extract, then report the results for that
sample.
10.S. 1.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the sample
should be repeated beginning with Section 11,
provided the sample is still availablE . Otherwise,
report results obtained from the reinjected extract,
but annotate as suspect.
10.5.2 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.5.2.1 If the check standard provides a response factor
(RF) within 20% of the predicated value, then
follow procedures itemized in Section 10.5.1 for
each sample failing the IS response criterion.
10. 5.2.2 If the check standard provides a response factor
(RF) with deviates more than 20% of the predicted
value, then the analyst must recalibrate, as
specified in Section 9.2.
10.6 ASSESSING LABORATORY PERFORMANCE
10.6.1 The laboratory must analyze at least one LFB per sample set
(all samples analyzed within a 24 hour period). The
fortifying concentration in the LFB should be 10 times the
MDL. Calculate accuracy as percent recovery (XJ . If the
recovery falls outside the control limits (See Section
10.6.2), the system is judged out of control, and the source
of the problem must be identified and resolved before
continuing analyses.
10.6.2 Until sufficient LFB data become available, usually a minimum
of results from 20 to 30 analyses, the laboratory should
assess its performance against the control limits described in
Section 10.3.2. When sufficient laboratory performance data
becomes available, develop control limits from the mean
percent recovery (X) and standard deviation (S) of the percent
recovery. These data are used to establish upper and lower
control limits as follows:
Upper Control Limit= X + 3S
Lower Control Limit= X - 3S
After each group of five to ten new recovery measurements,
control limits should be recalculated using only the most
recent 20 to 30 data points.
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10.6.3 It'is •recommended that the laboratory periodically determine
.and document its detection limit capabilities for endothall.
10.6.4 Each quarter the laboratory should analyze QCS (if available).
If criteria provided with the QCS are not met, corrective
. action should be taken and documented.
10.7 ASSESSING ANALYTE RECOVERY
10.7.1 The laboratory must add a known fortified concentration to a
minimum of 10% of the routine samples or one fortified sample
per set, whichever is greater. The fortified concentration,
should not be less than the background concentration of the
sample selected for spiking. The fortified concentration
should be the same as that used for the LFB (Section 10.6).
Over time, samples from all routine sample sources should be
fortified.
10.7.2 Calculate the percent recovery (Rx) for endothall, corrected
for background concentrations measured in the unfortified
sample, and compare these values to the control limits
established in Section 10.6.2 for the analyses of LFBs.
10.7.3 If the recovery falls outside the designated range, and the
laboratory performance for that sample set is shown to be in
control (Section 10.6), the recovery problem encountered with
the dosed sample is judged to be matrix related, not system
related. The result in the unfortified sample must be
labelled suspect/matrix to inform the data user that the
results are suspect due to matrix effects.
11. PROCEDURE
11.1 CLEANUP AND SEPARATION - Cleanup procedures may not be necessary for
a relatively • clean sample matrix. If particular circumstances
demand the use of an alternative cleanup procedure, the analyst must
demonstrate that the recovery of endothall is within the limits
specified by the method.
11.1.1 If the sample is not clea'n, or the complexity is unknown, the
entire sample should be centrifuged ,at 2500 rpm for 10
minutes. The.supernatant is decanted from the centrifuge
bottle and passed through glass fiber filter paper into a
container which can be tightly sealed.
11.1.2 Store all iamples at 4 C.
11.2 SAMPLE EXTRACTION AND ANALYSIS
11.2.1 Measure out a 5.0 ml aliquot of the sample and place it in a
10 or 25 ml K D tube. Add boiling chips .
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11.2.2 Place on tube heater at maximum setting and concentrate
sample to less than 0.5 ml.
11.2.3 Add 4 ml glacial acetic acid, 200 mg sodium acetate and 1 ml
of glacial acetic acid containing 4 mg PFPH. Use glass
stirring rod to break-up the sodium acetate solid. Place a
Micro Snyder column on each K-D tube.
11.2.4 Heat at 150 C for 90 minutes.
11.2.5 Dilute the reaction mixture with reagent water and decant
into a 50 ml beaker or flask. Wash the K-D tube and residue
with aliquots of reagent water and add to the beaker until
the total aqueous volume is 40-45 ml.
11.2.6 Assemble the vacuum manifold. Rinse the solid sorbent
cartridge by passing 5 ml of reagent water though the
cartridge. Discard the water. Extract the aqueous sample
from 11.2.5 by passing the sample through the solid sorbent
cartridge at a rate of 5-6 ml per minute.
11.2.7 Wash the cartridge -with 5 ml reagent water. Elute the
cartridge with two 2 ml aliquots of MTBE. Combine the
eluates with :05 ml of the internal standard stock solution
(7.11.2) and dilute to 5 ml in a volumetric flask with MTBE.
11.2.8 Analyze the eluates by GC/ECD using conditions described in
Table 1. This table includes the retention time and MOL that
were obtained under these conditions. Sample chromatograms
of an endothall standard and a LRB both with internal
standard are represented in Figures 1 and 2. Other columns,
chromatographic conditions, or detectors may be used if the
requirements of Section 10.3 are met
11.3 IDENTIFICATION OF THE ANALYTE
11.3.1 Identify endothall by comparison of its retention time to the
retention time of a reference chromatogram. If the retention
time of the unknQwn compound corresponds, within limits, to
the retention time of a standard endothal 1, then
identification is considered positive. However, positive
identifications should be confirmed by retention time
comparisons on the second GC column, or by using GC/MS.
11.3.2 The width of the retention time window used to make
identifications 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 chromatograms.
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11.3.3 Identification requires expert judgement when sample
components are not resolved chromatographically, that is,
when GC peaks from interferences are present. Any time doubt
exists over the identification of the endothall peak,
appropriate techniques such as use of an alternative detector
which operates on a chemical/physical principle different
from that originally used, e.g., mass spectrometry, or the
use of a second chromatography column must be used.
11.4 If the peak area exceeds the linear range of the calibration curve,
a smaller sample volume should be used. Alternatively, the final
solution may be diluted with MTBE and reanalyzed.
11.5 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
12. CALCULATIONS
12.1 Determine the peak area ratio for endothall in the injected sample.
12.1.1 Calculate the concentration of endothall injected using the
calibration curve in Section 9. 2. The concentration in a
liquid sample can be calculated from Equation 1:
Equation 1 Concentration, jig/L = .
(VS) —
where:
A= Concentration of endothall in extract, in |ig/L
VF= Final volume of MTBE, in ml
VS= Sample volume, in ml
12.2 Report results as micrograms per liter. When duplicate and
fortified samples are analyzed, report all data obtained with the
sample results.
12.3 For samples processed as -part of a set where the 'laboratory
fortified sample recovery falls outside of the control limits
•established in Section 10.6, data must be labeled as suspect.
13. METHOD PERFORMANCE
13 .1 METHOD DETECTION LIMITS - The MDL is defined as the mrnlmum
concentration of a substance that can be measured and reported with
99% confidence that the value is above the background level. The
estimated MDL concentration listed in Table 1 was obtained using
reagent water. Similar results were achieved using representative
matrices.•
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13.2 This method has not been tested for linearity of recovery from
fortified reagent water.
13.3 In a single laboratory using dechlorinated tap and reagent water
fortified matrices, the average recoveries presented in Table 2 were
obtained. The standard deviation of the percent recovery is also
included in Table 2.
14. REFERENCES
1. 40 CFR Part 136, Appendix B.
2. ASTM Annual Book of Standards, Part 31, 03694-78. "Standard
Practices for Preparation of Sample Containers and for Preservation
of Organic Constituents", American Society for Testing and
Materials, Philadelphia, PA.
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TABLE 1. GAS CHROMATOGRAPHY CONDITIONS AND METHOD DETECTION LIMITS
Ana 1 vt.fi Ret. Time fmin. 1 MDL Ijig/T.)
Endothall 42.3 11.5
GC conditions: 0.25 mm x 30 m SPB-5 column; 2 jiL
injection; Ohold one minute at 600C, program to
300 C at 4 C/minute, hold at 300 C for 15 minutes
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TABLE 2. SINGLE OPERATOR ACCURACY AND PRECISION
Analyte
Average Standard Fortified Number
Matrix Percent Deviation Cone. of
Type Recovery (percent) (jig/L) Analyses
Endothall
Reagent
Water
120
108
25.3
15.3
15
150
8
8
Dechlorinated
Tap 84.0
Water 94.0
13.8
13.3
15
150
8
8
100 mg/L sodium thiosulfate (Na2S203) added to prior to fortifying
with endothall
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APPENDIX
Preparation of Endothall-Pentafluorophenylhydrazine
1. Prepare solution A of endothall by dissolving 0.204 g of endothall
monohydrate (1.0 mmol) in 14 ml of methylene chloride and 3. 6 ml of dry
tetrahydrofuran (THF).
2. Prepare solution B of dicyclohexylcarbodiimide (DCC) by dissolving 0.206 g
(1.0 mmol) in 3.4 ml of dry THF.
3. Mix solutions A and B and cover with a watchglass. (Note: a white
precipitate will form in 3 to 5 minutes) .
4. Gently stir the mixture from Step 3 with a magnetic stirrer for 4.5 hours
at ambient temperature.
5. Prepare solution C by dissolving 0.206 g of DCC and 0.198 g of
pentafluorophenylhydrazine (PFPH) in 18 ml of dry THF.
6. Mix solution C with the mixture from step 4, cover with a watchglass and
.stir the mixture overnight (16 hours) at ambient temperature.
7. Filter the mixture and dry the filtrate under reduced pressure to yield a
beige powder.
8. Recrystallize the beige powder with 20 ml of warm (40 C} methanol: H20 {8:2
v/v).
9. Filter the solution from Step 8 to remove the insoluble material.
10. Allow the filtrate from Step 9 to cool to room temperature. A precipitate
will form immediately upon cooling.
11. Filter and wash the precipitate formed in Step 10 with two 1 ml portions of
cold methanol: H20 (8:2). Save the filtrate.
12. Allow the filtrate from Step 11 to stand overnight ,covered with a
watchglass at ambient temperature. A precipitate will form on standing.
13. Filter and wash the precipitate from Step 12 with two 1 ml portions of cold
methanol: H20 (8:2).
14. Recrystallize the off white precipitate from Step 13 with 20 ml of warm
methanol: H20 (8:2). Filter the warm solution and allow the filtrate to
cool, producing a white, crystalline precipitate.
15. Filter the white precipitate from Step 14, wash with two 1 ml portions of
cold methanol: H20 (8:2) and dry under vacuum.
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16. Determine the melting point of the precipitate of Step 15. The melting
point of the endothall-pentafluorophenylhydrazine derivative is 201.0 C.
If the melting point of the precipitate is not within 1.0 C of this melting
point, recrystallize again as per Steps 14 - 15.
98
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19!
II
48
41
m
m
Fffurt 1. Representative chro«atoqra» fr®« Injection of
a 200 ug/L endothal1-PFPH standard
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IMSmPhA
8
i
i—# *-»- -vt—r 1 «¦»""i;§ "f"» ' i ¦§¦ f • r i »—rr'i'T'fT'v t1* 'T*i "» |' a-"*" w'»r-'*> i* l *» t*-i
0«M*n ltmm
41
rifiir® 2. Representative chroaatograia of a laboratory
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