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Elemental Analysis Manual: Section 4.5 Cold Vapor Atomic Absorption Spectrometric Determination of Total Mercury in Seafood Using Microwave Assisted Digestion

Version 1 (June 2008)
Authors: John Cheng
Susan C. Hight

GLOSSARY


4.5.1 SCOPE AND APPLICATION

This method describes procedures for using cold-vapor atomic absorption spectroscopy (CVAAS) and microwave assisted digestion to determine total mercury in seafood. Other matrices may be analyzed by these procedures if performance is demonstrated in the matrix of interest, at the concentration levels of interest. The limits listed in 4.5 Table 1 are intended as a guide and actual limits are dependent on the sample matrix, instrumentation and selected operating conditions.

4.5 Table 1. Analytical Limits

ElementSymbolASDLa (µg/L)LODb (µg/kg)LOQb (µg/kg)
MercuryHg0.00850.866.7

a Based on method blanks.
b Based on 0.5 g analytical portion.

This method should be used by analysts experienced in the use of cold vapor atomic absorption spectrometry, including the interpretation of spectral and matrix interferences and procedures for their correction; and should be used only by personnel thoroughly trained in the handling and analysis of samples for determination of trace elements in food products.

4.5.2 SUMMARY OF METHOD

An analytical portion (0.5 g) is digested with nitric acid in the presence of sodium chloride using multi-step high-pressure microwave heating in a closed-vessel with a feedback program to monitor temperature and pressure. A 50 mL analytical solution containing hydrochloric acid is prepared from the digest. Total mercury is determined in the analytical solution by CVAAS using stannous chloride to reduce mercury (II) ion to mercury (0).

4.5.3 EQUIPMENT AND SUPPLIES

Disclaimer: The use of trade names in this method constitutes neither endorsement nor recommendation by the U. S. Food and Drug Administration. Equivalent performance may be achievable using apparatus and materials other than those cited here.
  1. Mercury analyzer — A self contained unit or assembled apparatus capable of mixing stannous chloride solution with analytical solutions, separating mercury (0) vapor that forms upon mixing from the liquid stream, and sweeping mercury (0) vapor into the path of an atomic absorption detector; consisting of a multichannel peristaltic pump for controlling liquid flow, liquid mixing tee, device for regulating carrier gas flow, gas-liquid separator, device for drying carrier gas, thermally stable double beam optics (temperature controlled sample and reference cells), and ultra stable mercury vapor lamp and detector capable of measuring atomic absorbance at 253.7 nm. Analyzer must have computerized control of operating conditions, an autosampler, and software for calculating mercury concentration in solutions. Analyzer should be capable of determining concentrations in solution ≥0.05 µg/L with precision ≤2% relative standard deviation during a period of 2 to 3 hours. Analyzer with capability to determine 0.0005 µg/L with precision ≤2% relative standard deviation during a period of 2 to 3 hours is preferable.
    Note: Drying carrier gas by diffusing water vapor through a selective membrane such as Nafion® into argon sheath-gas is the preferred method of removing water vapor. Chemical desiccants are not recommended.
  2. Microwave digestion system — Temperature control to 200 °C, pressure control to at least 600 psi, power 0-100% full power (minimum 630 watts, 900 watts for 12 position carousel), programmable in 1% increments. Digestion vessels must be TEM Teflon® lined. In this method, directions on use of microwave digestion equipment are specific to CEM Corporation brand. Use of method with other brands of equipment may require procedural modifications. Vessels designed to vent and reseal can be used provided they vent at pressures <300 psi.
    Safety Note: Microwave digestion systems can be potentially dangerous. Vessels contain concentrated nitric acid at high temperatures and pressures. Analyst must be familiar with manufacturer's recommended safety precautions including connection of the system to an appropriate exhaust system.
  3. Gas supply — Ultra-high purity (99.999%) argon. Use 2-stage regulator to deliver argon at pressure and flow rate recommended by manufacturer of mercury analyzer. Use an additional tank equipped with 2-stage regulator and capillary Teflon® tubing to purge stannous chloride reducing solution with ultra high purity argon flowing at <50 mL/min and <5 psi.
  4. Polypropylene centrifuge tubes for preparation and holding of standard and analytical solutions — 50 mL capacity with screw caps.
    Note: Accuracy of tubes should be periodically checked by randomly selecting several tubes from each case, filling to 50 mark with reagent water of known temperature, and weighing. Calculate volume as V=W/d where V is volume (mL), W is weight (g) of water added to mark, and d is density (g/mL or g/cc) of water at the temperature used. Do not use tubes with volume markings that are more than 1% in error.
  5. Miscellaneous laboratory ware — Use plastic and Teflon® laboratory items and containers whenever possible.

4.5.4 REAGENTS AND STANDARDS

Use reagents with sufficiently high purity and low mercury contamination to ensure that results are accurate and that quality control criteria can be met. Mercury analyzers with higher sensitivity require use of more highly purified reagents than with less sensitive analyzers. Reagents should be checked for contamination before use. Prepare reagents as close in time as possible to the day of use and no longer than 5 days before use. Hold solutions in tightly sealed containers.

Safety Note: Reagents should be regarded as potential health hazards and exposure to these materials should be minimized as much as possible.
  1. Nitric acid (HNO3) — Concentrated, sp gr 1.41, preferably ultra-high purity grade.
  2. Hydrochloric acid (HCl) — Concentrated, sp gr 1.18, preferably ultra-high purity grade.
  3. Reagent water — Water that meets specifications for ASTM Type I water1.
  4. Sodium chloride solution, 1%, (w/v) — Dissolve 0.5 g sodium chloride crystals in 50 mL reagent water in polypropylene tube.
  5. Stannous chloride reducing solution, 10% (w/v) in 7% (v/v) HCl — Mix 800 mL reagent water, 60 mL HCl, and 86 g stannous chloride dihydrate crystals (SnCl2·2H2O) in an acid-cleaned, glass container dedicated for stannous chloride use. Solution must be colorless and particle-free. Discard solutions that indicate presence of stannic ion (are turbid or yellow) or contain particles. Purge with argon and spinning magnetic stir-bar for 3 to 24 hours immediately before use to purge mercury contamination from solution. Left-over solution may be used up to 5 days after preparation if held in tightly sealed container away from light (wrapped in aluminum foil).
  6. Diluent, 10% (v/v) HNO3, 7% (v/v) HCl, 0.02% (w/v) NaCl — Mix approximately 1700 mL reagent water, 200 mL HNO3, 140 mL HCl, and 0.4 g sodium chloride crystals in acid-cleaned, 2-L that has been marked on the outside at the 2-L level. Mix, cool to room temperature, and dilute to 2 L.
  7. Autosampler rinse solution, 1% (v/v) HNO3, 1% (v/v) HCl — Mix 1960 mL reagent water, 20 mL HNO3 and 20 mL HCl in acid-cleaned, 2-L container. Use this solution when determining mercury concentrations ≤1 µg/L.
  8. Alternative autosampler rinse solution, 2% (v/v) HNO3, 2% (v/v) HCl — Mix 1920 mL reagent water, 40 mL HNO3, and 40 mL HCl in acid-cleaned, 2-L container. Use this solution when determining mercury concentrations >1 µg/L.
  9. Hydrochloric acid solution, 7% (v/v) HCl — Mix 93 mL reagent water and 7 mL HCl in acid-cleaned, 100-mL container. Use this solution to prepare intermediate standard solution.
  10. Standard blank — Diluent or 0 concentration mercury standard solution.
  11. Mercury stock standard solution, 1000 mg/L in 2-10% (v/v) HNO3 — Use commercially prepared, single-element solution prepared specifically for spectrometric analysis. A second stock standard solution obtained from a different source (or starting material) should be used to prepare the independent check solution (see below).
  12. Intermediate mercury standard solution, 5 mg/L Hg in 7% (v/v) HCl — Prepare by diluting 250 µL mercury stock standard solution to 50.0 mL with 7% (v/v) HCl. Use 100 µL of this solution to fortify analytical portions for percent recovery determination and various volumes to prepare standard solutions.
  13. Mercury standard solutions (various concentrations in 10% (v/v) HNO3, 7% (v/v) HCl, 0.02% (w/v) NaCl — Prepare standard solutions with concentrations that are appropriate for the sensitivity of the analyzer in 2 steps. (a) Prepare a secondary intermediate solution with appropriate concentration by diluting intermediate mercury standard solution with diluent. (b) Prepare standard blank and at least 4 standard solutions by gravimetrically diluting secondary intermediate solution with diluent.
    Example A (for mercury analyzers with limited sensitivity): Mercury standard solutions: 0, 0.5, 1, 2 and 5 µg/L — Prepare secondary intermediate solution with mercury concentration of 50 µg/L by diluting 500 µL of intermediate mercury standard solution (5 mg/L) to 50.0 mL with diluent. Prepare mercury standard solutions by weighing 0.5, 1, 2 and 5 g portions of 50 µg/L secondary intermediate solution in polypropylene tubes and diluting to 50 g with diluent. Record weights to 4 significant figures.

    Example B (for analyzers with high sensitivity): Mercury standard solutions: 0, 0.05, 0.1, 0.2 and 0.5 µg/L — Prepare secondary intermediate solution with mercury concentration of 5 µg/L by diluting 50 µL of intermediate mercury standard solution (5 mg/L) to 50.0 mL with diluent. Prepare mercury standard solutions by weighing 0.5, 1, 2 and 5 g portions of 5 µg/L solution in polypropylene tubes and diluting to 50 g with diluent. Record weights to 4 significant figures.
  14. Check solution — Use a mercury standard solution approximately mid-range of the standard curve for the check solution.
  15. Independent check solution (ICS) — Prepare a mercury independent check solution with diluent from a different stock solution than that used to prepare the calibration standard solutions. Mercury concentration of ICS should be approximately midpoint of the standard curve.

4.5.5 DIGESTION PROCEDURE

The following operations should be performed in a clean environment to reduce contamination. An exhausting hood must be used when working with nitric acid.

Note: To assist homogenization of the analytical sample, reagent water ≤20% of the mass of seafood may be added if its addition provides a more visually homogenous and easier-to-manipulate material. If reagent water is added to assist homogenization, record to 4 significant figures the weights of edible portion and reagent water that are combined to prepare the analytical sample and apply mass correction factor (MCF) in calculation of concentration of analyte in analytical portion. Reserve a portion of reagent water used for homogenization to prepare method blanks.
  1. Weigh analytical portion into clean vessel liner and determine mass of analytical portion. Generally, weigh 0.5 ± 0.1 g analytical sample. Use 0.5 g reagent water for method blanks (MBKs). Use 0.1 ± 0.01 g for reference materials (RMs).
  2. Pipet 1.0 mL 1% (w/v) sodium chloride solution and 5.0 mL concentrated HNO3 into vessel liner.
  3. Seal vessels, tighten pressure relief nuts and perform the digestion using the program outlined in 4.5 Table 2.
  4. After vessels have cooled to less than 50 °C remove them to an exhausting hood and vent excess pressure slowly.
  5. Place approximately 10 mL reagent water and 3.5 ± 0.1 mL HCl in 50 mL capacity polypropylene tube. Quantitatively transfer digestion solution to the 50 mL tube. Seal tube, shake vigorously and remove cap for approximately 5 minutes to release trapped gas from the warm solution. Dilute to slightly less than 50 mL with reagent water. Reseal container and cool to room temperature. Dilute to 50.0 ± 0.5 mL with reagent water, reseal, and mix. Analyze analytical solutions within 3 days.
Note: Analytical solutions that are clear and colorless to faintly yellow indicate that digestion is acceptably complete. If turbidity, deep color, or charred particles are present indicating incomplete digestion, discard analytical solution and prepare another analytical portion. Do not add hydrogen peroxide to decolorize solutions because it interferes with measurement of mercury.

4.5 Table 2. Microwave Digestion Programa
Digestion Program for CEM MARS 5 with 12-Position Carousel:
800 psi Maximum Pressure with Ramp to Temperature feature

Stage:12
Maximum Power (Watts)3001200
Control Pressure (psi)800800
Ramp Time (min)520
Hold Time (min)03
Control Temperature (°C)130200

a For each stage, power is applied for the Ramp Time minutes or until Control Pressure or Control Temperature is met. If Control Pressure or Control Temperature are met before end of Ramp Time then program proceeds to Hold Time prior to proceeding to next stage. If Ramp Time is met then program proceeds to next stage.

4.5.6 DETERMINATION PROCEDURE

The determination procedure was developed using a CETAC Technologies Quick Trace mercury Analyzer, Model M-75000. 4.5 Table 3 is an example of operating conditions used with this instrument. The optimum conditions must be determined for the equipment used.

4.5 Table 3. Typical Mercury Analyzer Operating Conditions
Conditions for CETAC Quick Trace Mercury Analyzer M-75000

Instrument Conditions
Gas flow (mL/m)125
Sample Uptake (s)25
Rinse (s)65
Read replicates (number)3
Read time (s)1
Pump speed (% of full)80
Wavelength (nm)253.7
Absorption cell temperature
heater set point (°C)
60
Instrumental Zero
Before first sampleno
Periodically (before each calibration)yes
Baseline Correction
#1 Start time (s)15
#1 End time (s)20
#2 Start time (s)not used
#2 End time (s)not used
Calibration Settings
Algorithmlinear
Through blankno
Weighted fitno
Recalibration rate0
Reslope rate0
Reslope standardN/A

Instrument Setup

  1. Setup cold vapor atomic absorption spectrometer according to manufacturer's recommendations and with the following conditions:
    • Set number of read (integration) replicates to 3 per sample uptake.
    • Set the absorption cell heater block set point to a temperature at least 10 °C above ambient.
    • Allow adequate time to warm up instrument and Hg lamp before analyzing solutions.
  2. Optimize operating conditions.
    • Start gas and liquid flows and ensure that liquid flow through uptake tubing, gas-liquid separator, and drain tubing is continuous and pulse free.
    • Analyze a standard solution and follow manufacturer's recommendations for adjusting operating conditions to obtain ideal peak profile.
    • When proper operating conditions are achieved, zero the instrument, then immediately analyze a standard blank, a standard solution with high concentration 3 or more times, and standard blank again. Visually inspect instrument peak profiles and calculate instrument sensitivity (see §3.2.1) and relative standard deviation of the high concentration standard solution. Verify absence of carry-over from high concentration standard to standard blank. Adjust operating conditions and repeat procedure if necessary to meet instrument performance requirements described below (3).
    • Determine baseline correction start and end times from the peak profile. Use a flat region of baseline immediately before start of the mercury signal for the correction.
  3. Check instrument performance
    • Verify sensitivity is within 80-120% of manufacturer specifications.
    • Verify short term precision is less than 5% relative standard deviation.
    • Verify absence of instrument carry-over.

Determination of Analyte Concentration Using Standard Curve

  1. Zero the detector and immediately standardize the instrument using standard blank and standard solutions. Use a linear, least squares calculated intercept curve fit algorithm.
  2. Check standardization performance
    • Correlation coefficient (r) of linear regression (absorbance verses concentration) is ≥ 0.998.
      Note: Optionally, verify that standards have been prepared correctly and are in the linear range by calculating percent relative difference of known concentrations and concentrations calculated from slope, intercept and instrument response of standards. Calculated concentrations that differ ≤2% relative difference from actual concentrations indicates that preparation of standard solutions is acceptable and concentrations are in the linear range. If relative difference is >2%, prepare new standard solutions and re-standardize the instrument.
    • Analyze ICS and standard blank immediately following instrument standardization. Acceptance criteria: ICS recovery within 100 ± 5%, standard blank < ASDL.
  3. Analyze analytical and quality control solutions. Dilute analytical solutions with diluent if concentration is above the highest standard. Interpolate analyte concentration in analytical solution from standard curve using least squares linear regression. A typical sequence for an analytical run is listed in 4.5 Table 4.

    4.5 Table 4. Typical Analytical Sequence

    Auto-
    Sampler
    Tube #
    SolutionPurposeQC Criteria
     standard blankStandardize instrumentr ≥ 0.998, Check for contamination
     standard solution #1Standardize instrumentr ≥ 0.998
     standard solution #2Standardize instrumentr ≥ 0.998
     standard solution #3Standardize instrumentr ≥ 0.998
     standard solution #4Standardize instrumentr ≥ 0.998
    1ICSverify standardization95-105% of expected
    2standard blankverify absence of carry-over<ASDL
    3MBK #1verify absence of contamination≤MBKC
    4MBK #2verify absence of contamination≤MBKC
    5sample #1determine mercury≤5% RSD read (integration) replicates if concentration ≥ASQL
    6sample #2determine mercury≤5% RSD read (integration) replicates if concentration ≥ASQL
    7sample #3determine mercury≤5% RSD read (integration) replicates if concentration ≥ASQL
    8sample #4determine mercury≤5% RSD read (integration) replicates if concentration ≥ASQL
    9sample #4 FAPspike recovery80-120% recovery
    10RMaccuracy80-120% recovery
    11check solutionverify standardization90-110% of expected
    12standard blankverify absence of carry-over<ASDL
  4. Check instrument measurement performance
    • RSD of analytical solution read (integration) replicates is 5% or less for concentrations ≥ ASQL.
    • Check solution (mid-level standard) analyzed at a frequency of 10% and at end of the analytical run has a recovery of 100 ± 10% (continuing calibration verification).
    • Standard blank analyzed following each check solution analysis is <ASDL (to verify absence of carry-over).
    • Measurements are below concentration of highest standard. Dilute analytical solution with diluent if necessary to comply with criteria.
    • Peak profile of analytical solution is comparable to standard solution.

Determination of Analyte Concentration Using Standard Additions

  1. Analyze analytical solutions and quality control solutions using minimum of 2 additional portions of solution with added amounts of analyte at approximately 2 and 5 times, respectively, of the amount of analyte in solution but not less than ASQL. Extrapolate analyte concentration from x-intercept of linear regression curve.
  2. Check standard additions performance
    • Check solution analyzed at a frequency of 10% and at the end of the analytical run has a recovery of 100 ± 10%.
    • Measurements are below upper end of linear working range. Dilute analytical solution with diluent if necessary to comply with criteria.
    • Correlation coefficient (r) of standard additions curve (absorbance verses concentration added) is ≥0.995.
    • Slope of standard additions curve for analytical solution is ±50% of the slope of standard additions curve for a standard blank (or a standard solution without any matrix effect such as the ICS).
    • Peak profile of analytical solution is comparable to standard solution.

4.5.7 CALCULATIONS

Calculate the concentration (mass fraction) of the analyte in the analytical portion according to the formula

EAM Equation 4.5.7 to calculate the concentration (mass fraction) of the analyte in the analytical portion

where
S = concentration of analyte in analytical solution (or diluted analytical solution) (µg/L)
MBKL = laboratory MBK (µg/L)
V = volume (L) of analytical solution (0.050 L)
m = mass of analytical portion (kg)
DF = dilution factor (1 if analytical solution not diluted)
MCF = mass correction factor (1 if no water or other solvent was added to aid homogenization)

Round calculated concentration to at most 3 significant figures. Concentration may be converted to other convenient units (e.g., mg/kg, ng/kg).

4.5.8 METHOD VERIFICATION

The following minimum number of quality control samples are analyzed with each batch of samples: 1 reference material (RM), 1 fortified analytical portion (FAP), and 2 method blanks (MBKs). Replicate analytical portions should be analyzed for each sample whenever analyte nonhomogeneity may be an issue.

A fortified method blank (FMB) checks the accuracy of the fortification procedure without any matrix effects and is an optional quality control sample. Use same fortification level as the FAP.

Reference Material

Control limits for RM Recovery are 100 ± 20% or within concentration uncertainty (converted to percent relative uncertainty) supplied on certificate, whichever is greater. The z-score procedure, which allows for greater deviation and is discussed in §3.5.3, may also be used, although it requires additional calculations. If three or more RMs are analyzed then only two-thirds of an element's RM recovery results must meet the control limit.

FAP Recovery

Control limit for FAP recovery is 100 ± 20%.

Method Blanks (MBK)

Minimum of 2 MBKs analyzed and concentration of both MBKs are ≤MBKC. If 3 or more MBKs are analyzed then at least two-thirds of MBKs are ≤MBKC.

Relative Percent Difference (RPD) of Two Replicate Analytical Portions

Control limit for RPD is 10%.

FMB Recovery (optional)

Control limit for FMB recovery is 100 ± 10%.

4.5.9 REPORT

Report results only when quality control criteria for a batch have been satisfactorily met. Report results that are ≥LOQ as the mass fraction determined followed by the units of measurement. Report results that are ≥LOD and <LOQ as the mass fraction determined followed by the units of measurement and the qualifier that indicates analyte is present at a trace level that is below the limit of reliable quantification (TR). Report results that are <LOD as 0 followed by the units of measurement and the qualifier that indicates analyte is below the level of reliable detection or is not detected (ND).

Example: LOQ = 6.7 µg/kg; LOD = 0.86 µg/kg. Levels found for three different samples were 7.5 µg/kg, 2.1 µg/kg and 0.5 µg/kg.
7.5 µg/kg is ≥LOQ; report 7.5 µg/kg
2.1 µg/kg is ≥LOD but also <LOQ; report 2.1 µg/kg (TR)
0.5 µg/kg is <LOD; report 0 µg/kg (ND)

4.5.10 METHOD VALIDATION

The application of microwave assisted digestion sample preparation to CVAAS determination of total mercury is well documented in scientific literature2. Closed-vessel microwave digestion is fast and contamination is extremely low while modern mercury analyzers are precise and very sensitive.

In-house validation. The method was validated by determining total mercury in 5 reference materials, various seafood products, portions of seafood products fortified with inorganic and organic mercury, and method blanks fortified with inorganic and organic mercury3. Validation results are presented in Appendix A. The average RM recovery for reference materials was 99% with a range of 90 to 103%. The average fortification recovery for seafood products was 101% with a range of 90 to 115%. The average fortification recovery for method blanks was 100% with a range of 95 to 106%.

Uncertainty. A result above LOQ has an estimated combined uncertainty of 10%. Use of a coverage factor of 2 to give an expanded uncertainty at about 95% confidence corresponds with the RM Recovery control limit of ± 20%. A result above LOD but below LOQ is considered qualitative and is not reported with an uncertainty.

A detailed discussion of method uncertainty is presented in §3.3. This method conforms to the information contained in that discussion. Derivation of an estimated uncertainty specific to an analysis is discussed §3.3.2.

Interlaboratory trial. Interlaboratory performance of EAM Method 4.5 was estimated from analytical data and results from laboratory proficiency testing. Further details can be found in Appendix B. FDA laboratories using EAM Method 4.5 demonstrated satisfactory performance, individually and collectively, with an estimated interlaboratory precision of 5 to 12% relative standard deviation (HORRAT of 0.3 to 0.6) for seafood samples containing approximately 0.2 to 0.6 mg/kg total mercury.


REFERENCES

  1. ASTM International (2006) ASTM D 1193-06, "Standard Specification for Reagent Water". ASTM. 
  2. Clevenger, W. L., Smith, B. W., and Winefordner, J. D. (1997) Trace Determination of Mercury: A Review, Crit. Rev. Anal. Chem. 27, 1-26. 
  3. Hight, S. C., and Cheng, J. (2005) Determination of Total Mercury in Seafood by Cold Vapor-Atomic Absorption Spectroscopy after Microwave Decomposition, Food Chem. 91, 557-570.