Determination of Ni, Pb, Cd, Cr, As, Se and Hg in Whole Tobacco

Health Canada
T-306 December 31, 1999

Scope of Applications
Normative References
Method Summary
Apparatus and Equipment
Reagents and Supplies
Preparation of Glassware
Preparation of Solutions
Preparation of Standards
Sampling
Sample Preparation
Sample Analysis
Atomic Absorption Analysis
Quality Control
References
Appendix

1 Scope of Applications

Applicable to the determination of the toxic trace metals: nickel (Ni), lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), selenium (Se) and mercury (Hg) in whole tobacco. The method is designed to quantitate these toxic trace metals from a single digestion, of which aliquots are taken for independent analysis.
Different digestion and analytical techniques that are specifically designed for one particular element, may be less labour intensive and should be considered if the analysis of a single element is desired.

2 Normative References

American Society for Testing and Materials (ASTM) D1193-77 - Standard Specification for Reagent Water, Version 1977.
Health Canada Test Method T-402 - Preparation of Cigarettes, Cigarette Tobacco, Cigars, Kreteks, Bidis, Packaged Leaf, Pipe and Smokeless Tobaccos for Testing, 1999-12-31.

3 Method Summary

This method is a microwave digestion method used to prepare samples for atomic absorption spectroscopy (AAS) analysis. Freeze dried tobacco is macerated and placed into the microwave digestion vessel. The tobacco is then treated with a mixture of hydrochloric acid, nitric acid and hydrogen peroxide. The vessel is then sealed and placed into the microwave digestor for dissolution. When digestion is complete, the vessel is removed from the digestor, allowed to cool, and transferred to a volumetric flask where it is made to volume with Type I water.
Aliquots of the digestate are analyzed for Ni, Pb, Cd, Cr, As, and Se by flameless atomic absorption spectroscopy (or graphite furnace atomic absorption). This method uses pyrolytic coated partition tubes for increased resistivity toward acid therefore increasing the lifetime of the tube and sensitivity to the analyte. Quantitation is achieved by interpolating the relevant calibration curves prepared from standard metal solutions of aqueous standards in the same acid concentration to minimize matrix affects. For some metals, the use of a matrix modifier is required to prevent loss of analyte during the analysis.

Note: Arsenic and selenium may also be analyzed by hydride generation using sodium borohydride. Extreme care, and a secondary digestion procedure, must be used to ensure these metals are in the proper oxidation state for the hydride reaction to quantitatively occur.

Note: Ni, Pb, Cd, and Cr may also be analyzed by Inductively Coupled Argon Plasma (ICP). With the use of HCl in the digestion procedure, Atomic Emmission Spectroscopy (AES) is used.
An aliquot of the digestate is analyzed by using cold vapour atomic absorption spectroscopy for Hg. This method uses a continuous flow vapour generator to react the mercury with stannous chloride. A peristaltic pump pushes the reducing agent and sample through a mixing coil to a gas liquid separator. Nitrogen gas carries the mercury vapour into a flow cell positioned in the burner compartment.

Note: The reaction is very sensitive to fluctuations in temperature so the response must be checked frequently against standards.

Note: The testing and evaluation of certain products against this test method may require the use of materials and or equipment that could potentially be hazardous and this document does not purport to address all the safety aspects associated with its use. Anyone using this test method has the responsibility to consult with the appropriate authorities and to establish health and safety practices in conjunction with any existing applicable regulatory requirements prior to its use.

4 Apparatus and Equipment

Lyophilizer.
20 mm × 150 mm disposable borosilicate culture tubes.
15 mm × 125 mm culture tube for macerating.
10 mL, 50 mL, 100 mL volumetric flasks.
Pipettor or micro-pipettes for the preparation of analytical run standards.
Eppendorf pipette (1-5 mL adjustable volume) or equivalent.
125 mL HDPE storage bottles.
20 mL scintillation vials with aluminum lined caps.
Atomic Absorption Spectrophotometer.
Programmable Sample Changer.
Vapour Generation Assembly.
Mercury Flow Through Cell.
Graphite Tube Atomizer.
Alternatively: Varian Axial Vista simultaneous ICP or equivalent.
Varian Partition Tubes (Coated) - Part # 63-100012-00.
Hollow Cathode Lamps for: Ni, Pb, Cd, Cr, As, Se and Hg.
Microwave Digestion System.
Advanced Composite Vessel (ACV) Digestion Vessel Assembly (X 2) or equivalent.

5 Reagents and Supplies

Note: All reagents shall be, at the least, recognized as analytical reagent grade in quality

Concentrated hydrochloric acid (HCl) - trace metals analysis grade or equivalent.
Concentrated nitric acid (HNO₃) - trace metals analysis grade or equivalent.
Type I water (meets ASTM D 1193 specification).
Ortho-phosphoric Acid - trace metals analysis grade or equivalent.
Stannous Chloride.
Hydrogen Peroxide (32 %).
Laboratory tissues.
Atomic Absorption Reference Standards - individual standards solutions at 1000 µg/mL.

Note: Reference standards must:
1. come with a certificate of analysis.
2. be NIST traceable.

6 Preparation of Glassware

Glassware should be cleaned and dried in such a manner to ensure that contamination from glassware does not occur.

Important: The cleaning of glassware and the cleanliness of the environment in which the analysis is performed, has a direct effect on the accuracy and precision of the method. In order to achieve accurate results, all glassware and digestion vessels must be cleaned immediately prior to use with dilute HCl (1+1) and then rinsed with Type I water.

7 Preparation of Solutions

Stannous Chloride Solution (25 % w/v SnCl2 in 20 % v/v HCl)
1. Weigh 125 g of stannous chloride into an acid washed 500 mL volumetric flask.
2. Add 100 mL of conc. HCl to completely dissolve the solid material.
  
  Note: Gentle heating may be applied in order to speed up this process.
3. Allow solution to cool before carefully adding Type I water to make up to the 500 mL volume.
4. Mix well and transfer the contents to the 500 mL bottle for the reducing agent channel of the vapour generation assembly.
  
  Note: If any precipitate appears in the bottle or flask, discard the solution and prepare fresh. It is necessary to keep the stannous chloride in solution as well as contaminant free as possible.

8 Preparation of Standards

Elemental Standards Stocks and Required Dilutions

All standards for graphite furnace analysis or ICP-AES are made to a 10 % (v/v) acid solution.
Note: For stability purposes, it is desired to dilute the analytical run standards in the same acid as the stock solution was purchased in.
All purchased standards are in 1000 µg/mL concentrations for stability purposes.
Primary Standard = 1000 µg/mL.
Secondary Standard (As and Se) = 1 mL of Primary Standard to 10 mL acid solution = 100 µg/mL.
Mixed Standard: 100 µL of each Primary Standard (Pb, Ni, Cd); 25 µL Cr Primary Standard and 100 µL As/Se Secondary Standard to 100 mL acid solution. Concentration: Pb, Ni and Cd = 1 µg/mL; Cr = 0.25 µg/mL; As/Se = 0.10 µg/mL.

Preparation of Working Standards (ng/mL)

The following table displays the preparation of working standards, including the volume of the mixed standard and the final volume.

Standard #	Mixed Std (µL)	Final Volume (mL)
0	0	100
1	250	100
2	500	100
3	1500	100
4	3000	100
5	5000	100

For Hg :

Hg standards for cold vapour analysis must be prepared in the same acid concentration and ratio as the samples are in. Using the recommended digestion procedure, that would be 8 % HCl (v/v) and 5 % HNO₃ (v/v).
The purchased stock standard is in a 10 % (v/v) HNO₃ acid solution at a concentration of 1000 µg/mL for stability purposes.
In order to make the proper dilutions, it is necessary to prepare a secondary stock standard at a concentration of 1 µg/mL also in a 10 % (v/v) HNO₃ acid solution. This secondary stock solution is considered to be stable for one week.
Primary Standard = 1000 µg/mL.
Secondary Standard = 100 µL of Primary Stock to 100 mL = 1 µg/mL.

Preparation of Working Standards (ng/mL).

The following table displays the preparation of working standards including the volume of secondary standards, final volume and the standard concentration.

Standard #	Secondary Std (µL)	Final Volume (mL)	Std Concentration [ng/mL]
1	30	100	0.3
2	50	100	0.5
3	150	100	1.5
4	300	100	3.0
5	500	100	5.0

9 Sampling

The sampling of tobacco products for the purpose of testing shall be as specified in T-402.

10 Sample Preparation

Cigarette tobacco (or little cigar, fine-cut and, chewing tobacco) is removed from its packaging to make a composite sample.
Prepare tobacco as specified in T-402.

11 Sample Analysis

Determination of Moisture Content
1. Transfer approximately 3 g of tobacco or tobacco type product into the pre-weighed 20 mL scintillation vial with aluminum lined cap.
2. Accurately weigh and record the total weight of the capped scintillation vial with tobacco.
3. Place samples in the freezer, prior to placing them in the lyophilizer (freeze-dryer), for a minimum of one hour.
4. Place samples into the freeze-dryer with the caps loosened to allow the moisture to be driven off of the tobacco.
5. After a minimum 48 hours, remove the vials and tighten the caps immediately to prevent re-absorption of moisture from the air.
6. Accurately weigh and record the total weight of the capped scintillation vial with tobacco after freeze-drying and calculate the % Moisture Content.
  
  Note: A minimum of three blank scintillation vials should be used to monitor any changes in weight as a result of external moisture.
Grinding of Samples
1. Macerate tobacco by placing a 15 mm × 125 mm centrifuge tube directly into the scintillation vial containing the dried tobacco and using the tube to grind the sample, as with a mortar and pestle.
2. After macerating the tobacco, wipe off the remaining tobacco particles from the tube using a laboratory tissue before proceeding to the next sample.
  Note 1: Contamination or cross-contamination from one sample to another is negligible.
  Note 2: This technique of grinding does not necessarily create a uniform sample, but does result in an easier and more reproducible digestion.
Digestion of Sample
1. Accurately weigh 1 g (+/- 0.1 g) of ground tobacco into the liner of the ACV digestion vessel.
  Note: Increasing sample weight to 2 g will enhance method detection limits, but may require longer digestion times.
2. Add 8 mL of conc. HCl and gently swirl to ensure all the tobacco is covered in the acid.
3. Add 3 mL of conc. HNO₃ to the sample, swirling in the acid, and allow the vessel to sit until the original frothing subsides and there is no longer evidence of orange/brown fumes (NOx formation).
4. Add 10 mL of 10% HNO₃.
5. Carefully add 2 mL of hydrogen peroxide such that there is no excessive effervescence.
6. Allow samples to sit until the effervescence subsides (approximately 30 minutes).
7. Add 10 mL of 10% HNO₃.
8. Install the rupture membrane and cap the digestion vessel.
9. Place the digestion vessel into the turntable and lock into position.
10. Choose the sample with the largest sample weight (as the most reactive sample) as the reference vessel for monitoring pressure and temperature to control the digestion.
11. Load the turntable of samples into the microwave digestor, and digest. See Appendix 2: Microwave Digestion Parameters.
12. When the digestion is complete, remove the turntable from the microwave and allow the samples to cool to room temperature before opening.
13. Inspect the digestate. If the digestion appears to be incomplete, carefully add 2 to 4 mL of hydrogen peroxide and return to the microwave for a secondary digestion.
14. Transfer the digestate to a 100 mL volumetric flask and make to volume using the washings of the digestion vessel with Type I water.
15. Hg determination is to be performed within 24 hours. In order to prevent Hg contamination, the Hg analysis is performed prior to the transfer of flask contents.
16. Transfer the contents of the flask into a 125 mL HDPE storage bottle.
  Note: Samples should be stored in the highest concentration of both analyte and acid for stability purposes. Manual dilutions of the digestate should only take place at the time of analysis.
Sample Dilutions required for Individual Elemental Analysis
1. Samples may be required to be diluted so their absorbances fall within the desired calibration range with a good signal-to-noise ratio and very little matrix effect. Because of little matrix effect, standard additions is not required and a standards calibration will suffice.
  Note: This dilution must be accounted for when calculating the results in a ng/g basis.
  Note: For As and Se, a multiple injection technique may be required for adequate instrument response.
  Note: When using an ICP for quantitation, the samples may be analyzed without further dilution for Ni, Cr, Pb and Cd , however the concentration range may be required to be increased if larger samples sizes are used.
2. The analysis of Hg requires no dilution.
  Note: Dilutions are based on "average" literature values. These dilutions may need to be modified depending on:
  1. the samples' country of origin,
  2. the year in which the sample was grown (environmental factors),
  3. the soil type and conditions which the sample was grown,
  4. the type of tobacco used for the sample,
  5. the stalk position of the tobacco used for analysis (if not a blended, finished product).

12 Atomic Absorption Analysis

Analysis of Ni, Pb, Cd, Cr, As, and Se by Graphite Furnace Atomic Absorption
1. Samples are analysed using the suggested parameters in Appendix 1: Instrument Parameters.
  Note: Parameters may differ between instruments and must be optimized for the particular instrument used.
Analysis of Ni, Pb, Cd and Cr by ICP-AES
1. Samples are analysed using the suggested parameters in Appendix 3: ICP Parameters.
  Note: Parameters may differ between instruments and must be optimized for the particular instrument used.
Analysis of Hg by Cold Vapour Atomic Absorption
1. Samples are analysed using the parameters in Appendix 1: Instrument Parameters.
2. It is important to analyse the samples for Hg within 48 hours (24 hours desired) of completing the digestion.
3. If samples are not analysed within this time frame, the digestate should be returned to the digestion vessel and the secondary digestion procedure performed.
Calculations
1. Results reported by the instrument software are expressed as [ng/mL] in solution. This result multiplied by the dilution of the sample and divided by the original sample weight being digested will calculate the result in a [ng/g] basis.
2. The [ng/g] results can be converted to [µg/g] by dividing this result by 1000.
3. All results are expressed on a "dry matter" basis. These may be expressed on an "as received" basis using the appropriate moisture result.
  Note: Representative calculations are as follows:
  Analytical Result (on a "dry matter" basis):
  
  Analyte [ng/g] = (Analytical result [ng/mL] × 100 mL × Additional Dilution) / Sample Weight (g).
  
  Conversion to an "as received" basis:
  
  Analyte [ng/g]"as received" = Analyte [ng/g] "dry matter" × (1- (% Moisture / 100)).
  
  where the % Moisture is determined by freeze drying in Samples Analysis - Determination of Moisture Content.

13 Quality Control

Control Processes
1. Each set analysis should contain one of each of the following per batch of up to 24 analysis (20-22 true samples):
  1. Laboratory Reagent Blank (LRB): to determine background contamination from solvents or glassware used in the analysis process.
  2. Laboratory Fortified Blank (LFB): to determine whether there is any loss of analyte as a result of the analysis process.
  3. Control Tobacco Sample: to determine the inter-experimental reproducibility of the entire method of analysis
  4. Duplicate Sample: to determine the reproducibility of the procedure within the same experiment or batch on analysis.
    Note: As an initial evaluation of the method for specific equipment and the environment of analysis, it is recommended that the method be used to analyse a reference material of a similar nature that has certified values for comparison.
Method Detection Limit (MDL) / Limit of Quantitation (LOQ)
Note: Individual instruments will have different MDL's and LOQ's depending on the optimization of the instrument.
1. The MDL is defined as:
  1. The concentration of analyte that yields an absorbance of 0.004 units (the characteristic mass).
    OR
  2. Determined by analyzing the lowest standard level a minimum of ten times as an unknown over several days. The MDL is calculated as three times the standard deviation of these determinations.
    OR
  3. As per two, analysing a blank a minimum of 10 times.
2. The MDL (on a ng/g basis) can be calculated by multiplying the determined MDL (ng/mL basis) by the final volume and dividing this by the sample weight used.
3. The MDL (on a ng/g basis) can be enhanced by varying the sample weight used, however this may affect the digestion parameters and the amount of potential interference.
4. The LOQ is either:
  1. The lowest standard used in the preparation of the calibration curve (excluding a blank)
    OR
  2. Determined by analyzing the lowest standard level a minimum of ten times as an unknown over several days. The LOQ is calculated as 10 times the standard deviation of these determinations.
    OR
  3. Same as two, using a blank solution.
5. The LOQ (on a ng/g basis) can be calculated by multiplying the determined LOQ (ng/mL basis) by the final volume and dividing this by the sample weight used.
6. The effect of modifying the sample weight used and the volumes used for extraction and clean-up in the procedure on the LOQ is the same as for the MDL.
Stability of Reagents and Samples
1. For all analytes other than Hg :
  1. Secondary and Mixed Standards are stable for one week.
  2. Working standards must be prepared every other day.
  3. All samples must be analyzed within one week of the digestion or samples will have to be re-digested.
2. For Hg Analysis:
  1. All samples and analytical run standards must be analyzed within 48 hours of the digestion (24 hours desired).
  2. All solutions for the analysis (i.e. hydride generation solution) are stable for only two weeks because of probable contamination problems.

14 References

Environmental Carcinogens - Selected Methods of Analysis, Vol. 8 - Some Metals: As, Be, Cd, Cr, Ni, Pb, Se, Zn. IARC Scientific Publication No.71, 1986. p. 129-138.
Perinelli, M.A. & Carugno, N., 1978. Determination of Trace Metals in Cigarette Smoke by Flameless Atomic Absorption Spectrometry, Beitrage zur Tabakforschung International, Band 9, Heft 4, Juli 1978. p. 214-217
Westcott, D.T. & Spincer, D., 1974. The Cadmium, Nickel and Lead Content of Tobacco and Cigarette Smoke, Beitrage zur Tabakforschung International, Band 7, Heft 4, April 1974. p. 217-221.
Varian Instruments at Work: Rapid Determination of Mercury in Fish Tissue, a Rapid, Automated Technique for Routine Analysis, No. AA-60, May 1986.
Varian Instruments at Work: Automated Cold Vapor Determination of Mercury: EPA Stannous Chloride Methodology, No. AA-51, September 1985.
Gawalco Et Al, 1997. Comparison of Closed-Vessel and Focused Open-Vessel Microwave Dissolution for Determination of Cadmium, Copper, Lead and Selenium in Wheat, Wheat Products, Corn Bran, and Rice Flour by Transverse- Heated Graphite Furnace Atomic Absorption Spectrometry, Journal of AOAC International, Vol. 80, No. 2, 1997. p. 379-387.