531 1
531 1
Revision 3.1
531.1-1
METHOD 531.1
1.2 This method has been validated in a single laboratory and estimated detection
limits (EDLs) and method detection limits (MDLS) have been determined for
the analytes above. Observed detection limits may vary between ground
waters, depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.4 When this method is used to analyze unfamiliar samples for any or all of the
analytes above, analyte identifications should be confirmed by at least one
additional qualitative technique1.
531.1-2
2.0 SUMMARY OF METHOD
2.1 The water sample is filtered and a 400 µL aliquot is injected into a reverse
phase HPLC column. Separation of the analytes is achieved using gradient
elution chromatography. After elution from the HPLC column, the analytes
are hydrolyzed with 0.05 N sodium hydroxide (NaOH) at 95°C. The methyl
amine formed during hydrolysis is reacted with o-phthalaldehyde (OPA) and
2-mercaptoethanol to form a highly fluorescent derivative which is detected by
a fluorescence detector2. Analytes are quantitated using procedural standard
calibration (Section 3.14).
3.0 DEFINITIONS
3.3 Laboratory Duplicates (LD1 and LD2) -- Two sample aliquots taken in the
analytical laboratory and analyzed separately with identical procedures.
Analyses of LD1 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 (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.
3.5 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.
531.1-3
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.
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.
531.1-4
4.0 INTERFERENCES
4.1.2 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by distillation in
all-glass systems may be required.
4.3 Matrix interference may be caused by contaminants that are present in the
sample. The extent of matrix interference will vary considerably from source
to source, depending upon the water sampled. Analyte identifications must be
confirmed. Positive identification may be made by the 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. A suggested alternative column is described in
Section 6.6.3.
531.1-5
5.0 SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not
been precisely defined; however, each chemical compound must be treated as
a potential health hazard. Accordingly, exposure to these chemicals must be
reduced to the lowest possible level. The laboratory is responsible for
maintaining a current awareness file of OSHA regulations regarding the safe
handling of the chemicals specified in this method. A reference file of material
safety data sheets should also be made available to all personnel involved in
the chemical analysis. Additional references to laboratory safety are available
and have been identified4-6 for the information of the analyst.
6.0 EQUIPMENT AND SUPPLIES (All specifications are suggested. Catalog numbers
are included for illustration only.)
6.1.1 Grab Sample Bottle -- 60 mL screw cap vials (Pierce No. 13075 or
equivalent) and caps equipped with a PTFE-faced silicone septa (Pierce
No. 12722 or equivalent). Prior to use, wash vials and septa as
described in Section 4.1.1.
531.1-6
6.5 Miscellaneous
6.6.2 Column 1 (primary column) - 150 mm long x 3.9 mm I.D. stainless steel
packed with 4 µm NovaPak C18. Mobil Phase is established at 10:90
methanol:water, hold twominutes, then program as a linear gradient to
80:20 methanol:water in 25 minutes. Alternative columns may be used
in accordance with the provisions described in Section 9.4.
6.6.5 Post Column Reactor -- Capable of mixing reagents into the mobile
phase. Reactor should be constructed using PTFE tubing and equipped
with pumps to deliver 0.1-1.0 mL/min. of each reagent; mixing tees;
and two 1.0 mL delay coils, one thermostated at 95°C (ABI URS 051
and URA 100 or equivalent).
531.1-7
7.0 REAGENTS AND STANDARDS
7.1 Reagent Water -- Reagent water is defined as water that is reasonably free of
contamination that would prevent the determination of any analyte of interest.
Reagent water used to generate the validation data in this method was
distilled water obtained from the Magnetic Springs Water Co., 1801 Lone Eagle
St., Columbus, Ohio 43228.
7.3.2 Methanol, HPLC Grade -- Filter and degas with helium before use.
Caution: Stench.
531.1-8
7.7 Stock Standard Solutions (SSS) (1.00 µg/µL) -- Stock standard solutions may be
purchased as certified solutions or prepared from pure standard materials
using the following procedure:
7.7.3 Stock standard solutions should be replaced after two months or sooner
if comparison with laboratory fortified blanks, or QC samples indicate a
problem.
Note: BDMC has been shown to be an effective internal standard for the
method analytes, but other compounds may be used, if the quality control
requirements in Section 9.0 are met.
531.1-9
8.0 SAMPLE COLLECTION, PRESERVATION AND HANDLING
8.2.3 After sample is collected in bottle containing buffer, seal the sample
bottle and shake vigorously for 1 min.
8.2.4 Samples must be iced or refrigerated at 4°C from the time of collection
until analysis is begun. Although preservation results of up to 28 days
indicate method analytes are not labile in water samples when sample
pH is adjusted to 3 or less, and samples are shipped and stored at 4°C,
analyte lability may be affected by the matrix. Therefore, the analyst
should verify that the preservation technique is applicable to the
samples under study.
9.2 Laboratory Reagent Blank (LRB) -- Before processing any samples, the analyst
must demonstrate that all glassware and reagent interferences are under
control. Each time a set of samples is extracted or reagents are changed, a LRB
must be analyzed. If within the retention time window of any analyte of
interest the LRB produces a peak that would prevent the determination of that
analyte, determine the source of contamination and eliminate the interference
before processing samples.
531.1-10
9.3 Initial Demonstration of Capability
9.3.2 For each analyte the recovery value for all of these samples must fall in
the range of ±20% of the fortified amount, and the RSD of the
measurements must be 20% or less. For those compounds that meet
the acceptance criteria, performance is judged acceptable and sample
analysis may begin. For those compounds that fail these criteria, this
procedure must be repeated using four samples until satisfactory
performance has been demonstrated.
9.4 The analyst is permitted to modify HPLC columns, HPLC conditions, and
internal standards to improve separations or lower analytical costs. Each time
such method modifications are made, the analyst must repeat the procedures
in Section 9.3.
9.5.1 When using the internal standard calibration procedure, the analyst
must 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 daily calibration check
standard's IS response by more than 30%.
531.1-11
9.5.2.1 If the reinjected aliquot produces an acceptable internal standard
response, report results for that aliquot.
9.5.3.1 If the check standard provides a response for the IS within 20%
of the predicted value, then follow procedures itemized in
Section 9.5.2 for each sample failing the IS response criterion.
9.6.1 The laboratory must analyze at least one LFB sample with every
20 samples or one per sample set (all samples analyzed within a
24-hour period) whichever is greater. The fortification concentration of
each analyte in the LFB should be 10 times EDL or a concentration in
the middle of the calibration range. Calculate accuracy as percent
recovery (Xi). If the recovery of any analyte falls outside the control
limits (see Section 9.6.2), that analyte is judged out of control, and the
source of the problem must be identified and resolved before
continuing analyses.
9.6.2 Until sufficient data become available from within their own laboratory,
usually a minimum of results from 20-30 analyses, the laboratory
should assess laboratory performance against the control limits in
Section 9.3.2. When sufficient internal performance data becomes
available, develop control limits from the mean percent recovery ( )
and standard deviation (S) of the percent recovery. These data are used
to establish upper and lower control limits as follows:
531.1-12
9.6.3 If acceptable accuracy and method detection limits cannot be achieved,
the problem must be located and corrected before further samples are
analyzed. Data from all field samples analyzed since the last acceptable
LFB should be considered suspect, and duplicate samples should be
analyzed, if they are available, after the problem has been corrected.
LFB results should be added to the on-going control charts to document
data quality.
Since the calibration check sample in Sections 10.2.4 and 10.3.3 and the
LFB are made the same way and since procedural standards are used,
the sample analyzed here may also be used as a calibration check as
described in those sections.
9.7.2 Calculate the percent recovery, P, of the concentration for each analyte,
after correcting the analytical result, X, from the fortified sample for the
background concentration, b, measured in the unfortified sample, i.e.,:
9.7.3 If the recovery of any such analyte falls outside the designated range,
and the laboratory performance for that analyte is shown to be in
control (Section 9.6), the recovery problem encountered with the dosed
sample is judged to be matrix related, not system related. The result
for that analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix effects.
531.1-13
chromatographic performance. LPC sample components and performance
criteria are listed in Table 4. Inability to demonstrate acceptable instrument
performance indicates the need for reevaluation of the instrument system. The
sensitivity requirements are set based on the EDLs published in this method.
If laboratory EDLs differ from those listed in this method, concentrations of the
LPC standard compounds must be adjusted to be compatible with the
laboratory EDLs.
9.9 The laboratory may adopt additional quality control practices for use with this
method. The specific practices that are most productive depend upon the
needs of the laboratory and the nature of the samples. For example, field or
laboratory duplicates may be analyzed to assess the precision of the
environmental measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and storage.
10.2 Internal Standard Calibration Procedure -- The analyst must select one or more
internal standards similar in analytical behavior to the analytes of interest. The
analyst must further demonstrate that the measurement of the internal
standard is not affected by method or matrix interferences. BDMC has been
identified as a suitable internal standard.
531.1-14
where: As = Response for the analyte to be measured
Ais = Response for the internal standard
Cis = Concentration of the internal standard µg/L)
Cs = Concentration of the analyte to be measured µg/L)
10.2.3 If the RF value over the working range is constant (20% RSD or less),
the average RF can be used for calculations. Alternatively, the results
can be used to plot a calibration curve of response ratios (As/Ais) vs. Cs .
531.1-15
11.0 PROCEDURE
11.1.2 Affix the three-way valve to a 10 mL syringe. Place a clean filter in the
filter holder and affix the filter holder and the 7-10 cm syringe needle
to the syringe valve. Rinse the needle and syringe with reagent water.
Prewet the filter by passing 5 mL of reagent water through the filter.
Empty the syringe and check for leaks. Draw 10 mL of sample into the
syringe and expel through the filter. Draw another 10 mL of sample
into the syringe, expel through the filter, and collect the last 5 mL for
analysis. Rinse the syringe with reagent water. Discard the filter.
11.2.1 Section 6.6 summarizes the recommended operating conditions for the
liquid chromatograph. Table 1 lists retention times observed using this
method. Other HPLC columns or chromatographic conditions may be
used if the requirements of Section 9.0 are met.
11.2.3 Inject 400 µL of the sample. Record the volume injected and the
resulting peak size in area units.
11.2.4 If the response for the peak exceeds the working range of the system,
dilute the sample with pH 3 buffered reagent water and reanalyze.
531.1-16
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.
12.0 CALCULATIONS
Use the multi-point calibration established in Section 10.0 for all calculations. Do not
use the daily calibration verification data to quantitate analytes in samples.
13.1 In a single laboratory, analyte recoveries from reagent water were used to
determine analyte MDLs and EDLs and demonstrate method range. Analyte
recoveries and standard deviation about the percent recoveries at one
concentration are given in Table 3.
531.1-17
13.2 In a single laboratory, analyte recoveries from two standard synthetic ground
waters were determined at one concentration level. Results were used to
demonstrate applicability of the method to different ground water matrices.
Analyte recoveries from the two synthetic matrices are given in Table 2.
14.0 REFERENCES
2. Moye, H.A., S.J. Sherrer, and P. A. St. John. "Dynamic Labeling of Pesticides
for High Performance Liquid Chromatography: Detection of
N-Methylcarbamates and o-Phthalaldehyde", Anal. Lett, 10, 1049, 1977.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82, "Standard
Practice for Preparation of Sample Containers and for Preservation", American
Society for Testing and Materials, Philadelphia, PA, p. 86, 1986.
5. "OSHA Safety and Health Standards, General Industry", (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
9. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82, "Standard
Practice for Sampling Water", American Society for Testing and Materials,
Philadelphia, PA, p. 130, 1986.
531.1-18
17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
531.1-19
TABLE 2. SINGLE LABORATORY ACCURACY AND PRECISION FOR
ANALYTES FROM REAGENT WATER AND SYNTHETIC
GROUNDWATERSa
Synthetic Synthetic
Fortified Reagent Water Water 1d Water 2e
Conc.
Analyte µg/L Rb Src R SR R SR
Aldicarb 5.0 115 3.5 106 3.2 102 8.2
Aldicarb Sulfone 10 101 4.0 98 3.9 95 9.5
Aldicarb Sulfoxide 10 97 4.9 105 4.2 94 10.3
Baygon 5.0 106 3.2 96 4.8 97 5.8
Carbaryl 10 97 5.8 94 4.7 104 10.4
Carbofuran 7.5 102 5.1 102 3.1 100 7.0
3-Hydroxycarbofuran 10 102 4.1 98 4.9 101 10.1
Methiocarb 20 94 1.9 102 4.1 112 3.4
Methomyl 2.5 105 4.2 98 3.9 105 9.5
Oxamyl 10 100 4.0 97 2.9 102 10.2
a
Data corrected for amount detected in blank and represent the mean of seven to
eight samples.
b
R = average percent recovery.
c
SR = standard deviation of the percent recovery.
d
Corrected for amount found in blank; Absopure Nature Artesian Spring Water
obtained from the Absopure Water Company in Plymouth, Michigan.
e
Corrected for amount found in blank; reagent water fortified with fulvic acid at the
1 mg/L concentration level. A well-characterized fulvic acid, available from the
International Humic Substances Society (associated with the United States Geological
Survey in Denver, Colorado), was used.
531.1-20
TABLE 3. SINGLE LABORATORY ACCURACY, PRECISION, METHOD
DETECTION LIMITS (MDLs), AND ESTIMATED DETECTION LIMITS
(EDLs) FOR ANALYTES FROM REAGENT WATER
Fortified Recovery RSD MDLb EDLc
a
Analytes µg/L N % % µg/L µg/L
Aldicarb 1.0 8 107 7 0.22 1.0
Aldicarb Sulfone 2.0 8 83 20 1.0 2.0
Aldicarb Sulfoxide 2.0 8 47 21 0.59 2.0
Baygon 1.0 7 101 32 1.0 1.0
Carbaryl 2.0 8 97 23 1.3 2.0
Carbofuran 1.5 7 90 12 0.52 1.5
3-Hydroxycarbofuran 2.0 8 108 29 1.9 2.0
Methiocarb 4.0 8 82 19 1.9 4.0
Methomyl 0.50 7 102 18 0.29 0.50
Oxamyl 2.0 8 82 17 0.86 2.0
a
N = Number of replicates.
b
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.
c
EDL = Estimated detection limit; defined as either MDL (Appendix B to 40 CFR
Part 136 - Definition and Procedure for the Determination of the Method Detection
Limit - Revision 1.11) or a level of compound in a sample yielding a peak in the final
extract with signal-to-noise ratio of approximately five, whichever value is higher.
531.1-21
TABLE 4. LABORATORY PERFORMANCE CHECK SOLUTION
Conc.
Test Analyte µg/mL Requirements
Sensitivity 3-Hydroxycarbofuran 2 Detection of analyte; S/N >3
531.1-23