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906R91103
20480
NETHOD 200.3
SAMPLE PREPARATION PROCEDURE FOR SPECTROCHEMICAL
DETERMINATION OF TOTAL RECOVERABLE ELEMENTS IN BIOLOGICAL TISSUES
William HcQaniel
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Environmental Services Division
Region IV
U. S. Environmental Protection Agency
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Revision 1.0
April 1991
Adapted by:
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
S 23
CO
ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C.2WSO
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METHOD 200.3
SAMPLE PREPARATION PROCEDURE FOR SPECTROCHEMICAL DETERMINATION
OF TOTAL RECOVERABLE ELEMENTS IN BIOLOGICAL TISSUES
I- SCOPE AND APPLICATION
1.1 This method provides sample preparation procedures for the
determination of total recoverable elements in biological tissue
samples.
1.2 This method is applicable to the- following elements:
Chemical Abstract Services
Analvte Registry Numbers (CASRW
7429-90-5
7440-36-0
7440-38-2
7440-39-3
7440-41-7
7440-43-9
7440-70-2
7440-47-3
7440-48-4
7440-50-8
7439-89-6
7439-92-1
7439-93-2
7439-95-4
7439-96-5
7439-97-6
7439-98-7
7440-02-0
7723-14-0
7440-09-7
7782-49-2
7440-22-4
7440-23-5
7440-24-6
7440-28-0
7440-29-1
7440-61-1
7440-62-2
7440-66-6
1.3 Samples prepared by this method can be analyzed by inductively
coupled plasma-atomic emission spectrometry (ICP-AES) Method 200.7,
"Determination of Metals and Trace Elements by Inductively Coupled
Plasma-Atomic Emission Spectrometry," inductively coupled plasma-
mass spectrometry (ICP-MS) Method 200.8, "Determination of Metals
Aluminum
Antimony
Arsenic
Barium
Beryl 1 i urn
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Lithium
Magnesium
Manganese
Mercury
Molybdenum
Nickel
Phosphorus
Potassium
Selenium
Silver
Sodium
Strontium
Thallium
Thorium
Uranium
Vanadium
Zinc
(Al)
(Sb)
(As)
(Ba)
(Be)
(Cd)
(Ca)
(Cr)
(Co)
(Cu)
(Fe)
(Pb)
(Li)
(Mg)
(Mn)
(HG)
(Mo)
(Hi)
(P)
(K)
(Se)
(Ag)
(Na)
(Sr)
(Th)
(U)
(V)
(In)
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and Trace Elements by Inductively Coupled Plasma-Mass Spectrometry,"
and stabilized temperature platform graphite furnace atomic
absorption (STGFAA), Method 200.9, "Determination of Trace Elements
by Stabilized Temperature Graphite Furnace Atomic Absorption
Spectrometry". See analytical methods mentioned for selection of
the appropriate method for determination of a specific analyte.
2. SUMMARY OF METHOD
2.1 Up to 5 g of a frozen tissue sample is transferred to a 125 mL
flask. The tissue is digested with nitric acid, hydrogen peroxide
and heat. This digestion results in a clear solution that 1s then
analyzed by mass or atomic Spectrometry methods. The determined
metal concentration is reported in microgram/gram (vg/g) wet tissue
weight.
3. DEFINITIONS
3.1 TOTAL RECOVERABLE - The concentration of analyte determined to be in
either a solid sample or an unfiltered aqueous sample following
iff treatment by refluxing with hot dilute mineral acid.
fo
fe 3.2 LABORATORY REAGENT BLANK (LRB) - A solution of reagents that is
[ treated exactly as a sample including exposure to all glassware and
[.f equipment that are used with other samples. The LRB is used to
$ determine if method analytes or other interferences are present in
ji! the laboratory environment, reagents, or apparatus.
•---•Jt
K 4- INTERFERENCES
m-
4.1 Chromium contamination of biological samples from the use of
stainless steel has been reported. Use of special cutting
£j implements and dissecting board made from materials that are not of
*y interest is recommended. Knife blades made of titanium with Teflon
-; handles have been successfully used.
4.2 In sample preparation, contamination is of prime concern. The work
area, including bench top and fume hood, should be periodically
cleaned in order to eliminate environmental contamination.
4.3 Chemical interferences are matrix dependent and cannot be predicted.
5. SAFETY
5.1 All personnel handling environmental samples known to contain or to
have been in contact with human waste should be immunized against
known disease causative agents.
5.2 Material safety data sheets for all chemical reagents should be
available to and understood by all personnel using this method.
Concentrated nitric and hydrochloric acids are moderately toxic and
extremely irritating to skin and mucus membranes. Hydrogen peroxide
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Is a strong oxidizing reagent. Use these reagents in a hood
whenever possible and if eye or skin contact occurs, flush with
large volumes of water. Always wear safety glasses or a shield for
eye protection when working with these reagents.
6. APPARATUS AND EQUIPMENT
6.1 LABWARE - For determination of trace levels of elements,
contamination and loss are of prime consideration. Potential
contamination sources include improperly cleaned laboratory
apparatus and general contamination within the laboratory
environment from dust, etc. A clean laboratory work area designated
for trace element sample handling must be used. Sample containers
can introduce positive and negative errors in the determination of
trace elements by contributing contaminants through surface
desorption/leaching, or depleting element concentrations through
adsorption processes. All reusable labware (glass, quartz,
polyethylene, Teflon, etc.), including the sample container, should
be cleaned prior to use or shown to be contaminant free. Labware
should be soaked overnight and thoroughly washed with laboratory-
grade detergent and water, rinsed with water, and soaked for four
hours in a mixture of dilute nitric and hydrochloric acid (1+2+9),
followed by rinsing with water, ASTM type I water and oven drying.
NOTE: Chromic acid must not be used for cleaning glassware.
6.1.1 Glassware - Volumetric flasks, graduated cylinders and 125-mL
Erlenmeyer flasks.
6.1.2 Assorted calibrated pipettes,
6.1.3 Wash bottle - One piece stem, Teflon FEP bottle with Tefzel
ETFE screw closure, 125-mL capacity,
6.2 SAMPLE PROCESSING EQUIPMENT
6.2.1 Balance - Analytical, capable of accurately weighing to
0.1 mg,
6.2.2 Hot Plate - (Corning PC100 or equivalent). An oscillating
hot plate will aid in sample digestion.
6.3 TISSUE DISSECTING EQUIPMENT
6.3.1. Dissecting Board: Polyethylene or other inert, nonmetallic
material, any non-wetting, easy-to-clean or disposable
surface is suitable. Adhesive backed Teflon or plastic
film may be convenient to use.
6.3.2 Forceps: Plastic, Teflon or Teflon coated.
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6.3.3 Surgical Blades: Disposable stainless steel with stainless
steel or plastic handle (Sect. 4.1).
6.3.4 Scissors: Stainless steel.
6.3.5 Plastic bags with watertight seal, metal free.
6.3.6 Label tape: Self-adhesive, vinyl coated marking tape,
solvent resistant, usable for temperatures from +121'C
to -23°C.
6.3.7 Polyvinyl chloride or rubber gloves, talc-free.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Reagents may contain elemental impurities which might affect
analytical data. High-purity reagents should be used whenever
possible. All acids used for this method must be of ultra high-
purity grade.
7.1.1 Nitric acid, concentrated (sp.gr. 1.41).
7.1.2 Hydrochloric acid, concentrated (sp.gr. 1.19).
7.1.3 Hydrogen peroxide (30%)
7.2 WATER - For all sample preparation and dilutions, ASTM type I water
(ASTM D1193) is required. Suitable water may be prepared by passing
distilled water through a mixed bed of anion and cation exchange
resins.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Appropriate individual tissue samples should be taken soon after
collection and must be taken prior to freezing2. If dissection of
the tissue cannot be performed immediately after collection, it
should be placed in a plastic bag (Sect. 6.3.5), sealed and placed
on ice or refrigerated at approximately 4°C.
8.2 Prior to dissection, the tissue should be rinsed with metal-free
water and blotted dry. Dissection should be performed within
24 hours of collection. Each individual tissue sample should also
be rinsed with metal-free water, blotted dry, and frozen at <-20°C
(dry ice).
8.3 Tissue samples of up to 5 g should be taken using a special
implement (Sect. 4.1) and handled with plastic forceps
(Sect. 6.3.2)M.
8.4 A maximum holding time for frozen samples has not been determined.
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9. CALIBRATION AND STANDARDIZATION
9.1 Not applicable. Follow instructions given in the analytical method
selected.
10. QUALITY CONTROL
10.1 Each laboratory determining total recoverable elements is required
to operate a formal quality control (QC) program. The minimum
requirements of a QC program consist of an Initial demonstration of
laboratory capability and analysis of laboratory reagent blanks and
fortified blanks and samples as a continuing check on performance.
The laboratory is required to maintain performance records that
define the quality of data generated.
10.2 Specific instructions on accomplishing the described aspects of the
QC program are discussed in the analytical methods.
11. PROCEDURE
11.1 Sample Preparation - Place up to a 5 g subsample of frozen tissue
into a 125-ml erlenmeyer flask. Any sample spiking solutions should
be added at this time and allowed to be In contact with the sample
prior to addition of acid.
11.2 Add 10 ml of concentrated nitric acid and warm on a hot plate until
the tissue is solubilized. Gentle swirling the samples or use of an
oscillating hot plate will aid in this process.
11.3 Increase temperature to near boiling until the solution begins to
turn brown. Cool sample, add an additional 5 ml of concentrated
nitric acid and return to the hot plate until the solution once
again begins to turn brown.
11.4 Cool sample, add an additional 2 mL of concentrated nitric acid,
return to the hot plate and reduce the volume to 5-10 ml. Cool
sample, add 2 mL of 30% hydrogen peroxide, return sample to the hot
plate and reduce the volume to 5-10 ml.
11.5 Repeat Sect. 11.4 until the solution is clear or until a total of
10 mL of peroxide has been added. NOTE: A laboratory reagent blank
is especially critical in this procedure because the procedure
concentrates any reagent contaminants.
11.6 Cool the sample, add 2 mL of concentrated hydrochloric acid, return
to the hot plate and reduce the volume to 5 mL.
11.7 Allow the sample to cool and quantitatively transfer to a 100-mL
volumetric flask. Dilute to volume with ASTM type I water, mix, and
allow any insoluble material to separate. The sample is now ready
for analysis by either IC"P-AES or ST6FAA. For analysis by ICP-MS an
additional dilution (1+4) is required,
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11.8 Sample Analysis - Use one of the analytical methods listed in
Sect. 1.3.
12. CALCULATIONS
12.1 Not applicable. Discussed in analytical methods listed in Sect.
1.3.
13. PRECISION AND ACCURACY
13.1 Not applicable. Available data Included in analytical methods
listed in Sect. 1.3.
14. REFERENCES
1. Versleck, J., and F. Barbier, "Sample Contamination as A Source of
Error 1n Trace-Element Analysis of Biological Samples," Talanta.
Vol. 29, pp. 973-984, 1982.
2. Ney, J. J., and M. G. Martin, "Influences of Prefreezlng on Heavy
Metal Concentrations in Bluegill Sunfish," Water Res.. Vol. 19,
No. 7, pp. 905-907, 1985.
3. "The Pilot National Environmental Specimen Bank," NBS Special
Publication 656, U. S. Department of Commerce, August, 1983.
4. Koirtyohann, S. R., and H. C. Hopps, "Sample Selection, Collection,
Preservation and Storage for Data Bank on Trace Elements in Human
Tissue," Federation Proceedings, Vol. 40, No. 8, June, 1981,
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