Determination of Ni, Pb, Cd, Cr, As and Se in Mainstream Tobacco Smoke

Health Canada
T-109 December 31, 1999

Table of Contents

1. Scope of Applications
2. Normative references
3. Definitions
4. Method Summary
5. Apparatus and Equipment
6. Reagents and Supplies
7. Preparation of Glassware
8. Preparation of Solutions
9. Preparation of Standards
10. Sampling
11. Tobacco Product Preparation
12. Smoking Machine Preparation
13. Sample Generation
14. Sample Analysis
15. Quality Control
16. Modifications for Intensive Smoking
17. Reference
18. Appendices

1 Scope of Applications

1. This method is applicable to the determination of nickel (Ni), lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), and selenium (Se) in mainstream tobacco smoke by Atomic Absorption Spectroscopy (AAS) or Inductively Coupled Argon Plasma - Atomic Emission Spectroscopy (ICP-AES). The method is designed to quantitate these toxic trace metals in both the particulate phase and gaseous phase of smoke from cigarettes, cigarette equivalents, kreteks, bidis and cigars smoked on a rotary smoking machine.
2. Particulate phase metals are determined as those metals that become part of the mainstream smoke total particulate matter (TPM), trapped on a glass fibre filter disc (pad) or in an electrostatic precipitator.
3. Gaseous phase metals are determined as those metals that may have reacted to form a gaseous species or particulate matter that is not retained in the normal TPM condensate.

2 Normative References

1. American Society for Testing and Materials (ASTM) D 1193-77 - Standard Specifications for Reagent Water, Version 1977.
2. Health Canada Test Method T-115 - Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke, 1999-12-31.

3 Definitions

1. Refer to T-115 for definitions of terms used in this document.

4 Summary of Method

1. Conditioned tobacco product is smoked on a 20-port rotary smoking machine. An electrostatic precipitation generator is utilised to electrostatically precipitate the particulate matter onto a glass electrostatic precipitate (EP) tube. The total particulate matter (TPM) is extracted into 25 mL methanol. The methanol extract is then evaporated using gentle heating while under a constant stream of filtered ultra high purity (UHP) nitrogen. The sample is then subjected to microwave digestion using a mixture of hydrochloric acid, nitric acid and hydrogen peroxide.
2. The gaseous phase metals are trapped by placing an impinger of a 10 % v/v nitric acid solution between the EP tube and the puff drawing mechanism. The impinger solution is added to the same digestion vessel as the EP tube product and subjected to microwave digestion.
3. The digestates are then analysed by flameless atomic absorption spectroscopy (or graphite furnace atomic absorption). This method uses pyrolytic coated partitioned graphite tubes for increased resistivity toward acid, therefore increasing the lifetime of the tube and sensitivity to the analyte.
   
   Note: The analysis of Cd, Pb, Ni, and Cr, can also be achieved by ICP-AES in conjunction with an ultrasonic nebulizer in order to increase sensitivity.
4. Quantitation is achieved by interpolating the relevant calibration curves prepared from solutions of aqueous metal 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. This also requires the digestate to be further diluted resulting in a loss in sensitivity.
5. Important: The electrode tip of the electrostatic precipitator should be made of tungsten or plated with metals that will not interfere with analysis (i.e. Ag, Au etc). The standard EP electrode tip has a Ni plating that, if scratched, yields high background Ni and Cr results.
   
   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.

5 Apparatus and Equipment

1. Equipment needed to perform smoking of tobacco products as specified in T-115.
2. Equipment needed for conditioning as specified in T-115.
3. Equipment needed for marking for butt length as specified in T-115.
4. 70 mL impinger without frit.
5. 1/4" ester grade Tygon tubing or equivalent.
6. 1/4" Nalgene connectors.
7. UHP Compressed Nitrogen.
8. Glass manifold with Teflon stopcocks.
9. Tecator 1015 digestor or equivalent.
10. Heinreich Borgwaldt Central Electrostatic Smoke Trap (EP Unit) with tungsten electrode or equivalent.
11. Heinreich Borgwaldt High Tension Generator, Model 251 or equivalent.
12. 10 mL, 25 mL, 50 mL, 100 mL, 1000 mL volumetric flasks.
13. Pipettor or micro-pipettes for the preparation of working standards.
14. Pipettor (1-5 mL adjustable volume).
15. 125 mL HDPE (high density polyethylene) storage bottles.
16. Atomic Absorption Spectrophotometer.
17. Graphite Tube Atomizer.
18. Varian Partition Tubes (Coated) or equivalent.
19. Hollow Cathode Lamps for : Ni, Pb, Cd, Cr, As, Se and Hg.
20. Microwave Digestion System with temperature and pressure controls or equivalent.
21. Digestion Vessel Assembly (X 2) or equivalent.
22. Alternatively: Varian Axial Vista Simultaneous ICP or equivalent.
23. Cetac U-5000AT⁺ Ultrasonic Nebulizer or equivalent.

6 Reagents and Supplies

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

1. Concentrated hydrochloric acid (HCl) - trace metals analysis grade or equivalent.
2. Concentrated nitric acid (HNO₃ ) - trace metals analysis grade or equivalent.
3. Type I water (meets ASTM D1193 specification).
4. Methanol - distilled-in-glass or equivalent .
5. Hydrogen Peroxide (32 %).
6. Ortho-phosphoric acid - trace metals analysis grade or equivalent.
7. Atomic Absorption Reference Standards - individual standards solutions at 1000 μg/mL.
   
   Note: Reference standards must:
   1. come with a certificate of analysis, and
   2. be NIST traceable.

7 Preparation of Glassware

1. Glassware should be cleaned and dried in such a manner to ensure that 0 contamination from glassware does not occur.
   
   Important: The cleaning of glassware and the cleanliness of the environment in which the analysis is performed, directly effects 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.

8 Preparation of Solutions

1. Nitric Acid Impinger Solution (10 % HNO₃ [v/v] )
   1. Add approximately 500 mL of Type I water to a 1000 mL volumetric flask.
   2. Add 100 mL of conc. HNO₃.
   3. Make solution to volume with Type I water.
      Note: When diluting a concentrated acid solution, always add acid to water.

9 Preparation of Standards

1. Elemental Standards and Required Dilutions
   1. All standards for graphite furnace analysis are made to a 10% HNO₃ (v/v) acid solution.
      Note: For stability purposes, it is desired to dilute the working standards in the same acid as the primary standards.
   2. All purchased standards are in 1000 μg/mL concentrations for stability purposes.
   3. Primary Standard = 1000 μg/mL.
   4. Secondary Standard (As and Se) = 1 mL of Primary Standard to 10 mL = 100 μg/mL.
   5. Mixed Standard, containing:
      100 μL of each Primary Standard (Pb, Ni, Cd). 25 μL Cr Primary Standard. 100 μL As/Se Secondary Standard.
      Make up to 100 mL. Concentrations: Pb, Ni and Cd =1 μg/mL, Cr = 0.25 μg/mL and As/Se = 0.10 μg/mL.
   6. Preparation of working standards (ng/mL):
      
      The following table includes information regarding the five working standards, including the mixed standard volumes and final volumes.
      

      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

10 Sampling

1. The sampling of tobacco products for the purpose of testing shall be as specified in T-115.

11 Tobacco Product Preparation

1. Product is to be conditioned as specified in T-115.
2. Cigarettes, cigarette equivalents, bidis, kreteks and cigars are to be marked for butt length as specified in T-115.
3. Cigarettes to be smoked under intense smoking conditions shall be prepared as specified in T-115.

12 Smoking Machine Preparation

1. Ambient conditions
   1. The ambient conditions for smoking shall be as those specified in T-115.
2. Machine Conditions
3. The machine conditions shall be as those specified in T-115 with the following modifications as detailed below.
   1. Connect the electrostatic smoke trap (EP Unit) with tungsten electrode to 20-Port Smoking Machine, as per manufacturer's specification.
   2. Set the tension generator to 17.5kV for the electrostatic field within the EP unit, and a 0.2 mA current limit.
      Note: ALWAYS TURN OFF THE GENERATOR WHEN MAKING ADJUSTMENTS TO THE EP TUBE AND SMOKE TRAIN.
   3. Set the control panel for smoking machine, for a two second puff duration, one second intermission, resulting in a 60 second puff frequency.
   4. Connect one 70 mL impinger containing 20 mL of 10 % HNO₃ between the EP tube holder and the back-up pad holder of the pneumatics panel using Tygon tubing.
   5. Adjust the pneumatic panel for smoking machine, to a 35 mL (+/- 0.2 mL) puff volume (with the EP tube and impinger in place) and 1.85 second sweep-time.

13 Sample Generation

1. Cigarettes shall be smoked and TPM collected as specified in T-115 with the following changes.
   1. The EP tube is inserted into the back of the EP unit and then the front of the EP tube is inserted into the head block of the smoke machine. Both ends are gently tightened.
   2. The 70 mL impinger, containing 20 mL of fresh 10 % HNO₃ acid, is connected between the EP tube holder and the back-up Cambridge filter cassette on the pneumatics panel using Tygon tubing.
   3. The pad in the back-up filter cassette on the pneumatics panel is replaced.
   4. The set-up is checked for leaks.
   5. Twenty cigarettes are loaded into the ports of the smoking machine using the cigarette load button and positioner.
   6. The EP generator is turned on. When the needle reads 17.5kV, cigarettes are lit with an electric lighter.
   7. Three clearing puffs are performed once smoking is completed to ensure all of the gaseous phase in the dead volume of the system has passed through the impinger and back-up pad.
   8. After smoking is completed, the milli Amp (mA) current on the power supply is recorded.
   9. The EP unit is turned off. Allow 10 seconds pass before proceeding to the next step.
   10. The fittings are unscrewed and the EP tube is removed from unit and end-caps are replaced.
   11. The EP tube is weighed and recorded, and the TPM yield per cigarette is calculated using the formula found in the Calculations section.

14 Sample Analysis

1. Sample Preparation and Digestion
   1. Twenty-five mL (1 × 12 mL and 1 × 13 mL) of methanol is added to the EP tube after smoking, and the tube is shaken to extract the residue.
   2. The extract is transferred to the Teflon microwave digestion vessel (ACV liner).
   3. The methanol is evaporated under a gentle flow of filtered nitrogen and gentle heating in a block digestor or on a hot plate. The residue is then ready to be digested using the microwave digestor.
   4. Six mL of HCl is added to the residue.
   5. Two mL of conc. HNO₃ is added to the sample, swirling in the acid, and allowing the sample to sit until the original frothing subsides and there is no longer evidence of orange/brown fumes (NOx formation).
   6. Eight mL of hydrogen peroxide is added carefully such that there is no excessive effervescence.
   7. Samples are allowed to sit until the effervescence subsides (approximately 10 minutes).
   8. The contents of the impinger are added to the digestion vessel.
   9. The impingers are rinsed with an additional 2 × 5 mL of hydrogen peroxide and the rinses are added it to the digestion vessel.
   10. Samples are allowed to sit until the effervescence subsides (approximately 10 minutes).
   11. The rupture membrane is installed and the digestion vessel is capped.
   12. The digestion vessel is placed into the turntable and locked into position.
   13. The sample with the largest TPM content (the most reactive sample) is chosen as the reference vessel for monitoring the temperature and pressure to control the digestion.
   14. The turntable of samples is loaded into the microwave digestor, and the digestion program is started. See Appendix 2: Microwave Digestion Parameters.
   15. When the digestion is complete, the turntable is removed from the microwave and the samples are allowed to cool to room temperature before opening.
   16. The digestate is inspected. If the digestion appears to be incomplete, the digestion vessels are returned to the microwave for a secondary digestion.
   17. When digestion is complete, the digestate is transferred to a 100 mL volumetric flask and made up to volume by washing the digestion vessel with Type I water.
   18. The contents of the flask are transferred 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.
       Note: As and Se should be analyzed as soon as possible (within 72 hours) due to a loss in response over time.
2. Sample Dilutions required for Individual Elemental Analysis
   1. Samples may require to be diluted so that their absorbances fall within the desired calibration range with a good signal-to-noise ratio and very little matrix effect. Because of minimal matrix effect, standard additions are not required and a standards calibration will suffice.
   2. The analysis of Cd and/or Pb may require a manual dilution prior to analysis by transferring 1000 μL of the digestate to a 10 mL volumetric flask, and making up to volume with Type I water.
      Note: When using an ICP-AES for quantitation, the samples may be analyzed without further dilution for Ni, Cr, Pb and Cd.
      Note: For As and Se, a multiple injection technique may be required for an adequate instrument response.
      Note: These sample dilutions are based on "average" literature values. These dilutions may need to be modified depending on:
      1. the sample's country of origin,
      2. the year in which the sample was grown (environmental factors),
      3. the soil type and conditions in 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).
3. 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.
4. 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.
5. 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 number of cigarettes smoked, will calculate the result in a [ng/cigarette] basis.
      Analytical Result (on a "per cigarette" basis):
      Analyte [ng/cigarette] = (Analytical result [ng/mL] × 100mL × Additional Dilution factor) / No. of Cigarettes (20).
   2. The [ng/cigarette] results can be converted to [μg/cigarette] by dividing this result by 1000.
   3. Total Particulate matter [mg/cigarette] is calculated using the difference in weight of the EP tube before and after smoking and dividing by the number of cigarettes smoked.
      Determination of Total Particulate Matter (TPM):
      TPM [mg/cigarette] = [Wt. of EP tube after smoking (g) - Wt. of EP tube before smoking (g)] × 1000 mg/g / 20.
   4. Analytical Result (on a "per mg TPM" basis), if desired:
      Analyte [ng/mg TPM] = Analyte [ng/cigarette] / TPM [mg/cigarette].

15 Quality Control

1. Metals Smoking Control Process
   1. Each set analysis should contain one of each of the following per day of smoking or batch of up to 24 analyses (20-22 true samples):
      
      Laboratory Reagent Blank (LRB): to determine background contamination from solutions, glassware, or materials used in the analysis process.
      
      Laboratory Fortified Blank (LFB): to determine whether there is any loss of analyte as a result of the analysis process.
   2. Control Sample: to determine the inter-experimental reproducibility of the entire method of analysis
   3. 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 and materials used, it is recommended that a minimum of 10 blanks be analysed using the method in order to establish control parameters for expected levels of background contamination before any samples are analysed. An LRB outside these control limits is an indicator of possible contamination problems or the use of materials and reagents of different lot numbers.
2. Recoveries and Levels of Contamination
   1. Recoveries for a Laboratory Fortified Blank (LFB) for Ni, Pb, Cd, and Cr are normally between 85 and 115 %. Variability in this range is associated to differences in the blanks.
   2. Recoveries for a Laboratory Fortified Blank (LFB) for As and Se range from 60 to 85 %. Lower recoveries result from over-heating of the sample while evaporating the methanol.
   3. Contamination must be monitored with each individual set of samples that are digested and is dependent on the laboratory environment. This ultimately effects the precision of the analysis.
3. 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 10 times as an unknown over several days. The MDL is calculated as three times the standard deviation of these determinations.
         OR
         
      3. As per 2, analyzing a blank a minimum of 10 times.
         
         The MDL (on a ng/cigarette basis) can be calculated by multiplying the determined MDL (ng/mL basis) by the final volume and dividing this by the number of cigarettes smoked.
         The MDL (on a ng/cigarette basis) can be enhanced by varying the amount of cigarettes smoked, however this may affect the amount of background contamination observed.
   2. 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 10 times as an unknown over several days. The LOQ is calculated as 10 times the standard deviation of these determinations.
      3. As per 2, using a blank solution.
         
         The LOQ (on a ng/cigarette basis) can be calculated by multiplying the determined LOQ (ng/mL basis) by the final volume and dividing this by the number of cigarettes smoked.
         
         The effect of modifying the number of cigarettes smoked and the volumes used for extraction and clean-up in the procedure on the LOQ is the same as for the MDL.
4. Stability of Reagents and Samples
   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.

16 Modifications for Intensive Smoking

1. Modifications for intense smoking conditions generally include, but are not limited to, a reduction in the number of cigarettes (or equivalents) smoked.

17 Reference

1. 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.
2. Perinelli, M.A. & Carugno, N. Determination of Trace Metals in Cigarette Smoke by Flameless Atomic Absorption Spectrometry, Beitrage zur Tabakforschung International, Band 9, Heft 4, July 1978, p. 214-217.
3. Bell, Paul & Mulchi, Charles L. Heavy Metal Concentrations in Cigarette Blends, Tobacco Science, Vol. 34, 1990, p. 32-34.
4. NIOSH Method 7300, Elements (ICP), NIOSH Manual of Analytical Methods, Vol. 2, Third Edition, 1984.
5. Varian Analytical Methods for Graphite Tube Atomizers, Varian Australia Pty Ltd, Publication No. 85-100848-00, 1988.
6. Gawalco, et al. 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.

Appendices

Appendix 1: Typical Instrument Parameters

Graphite Furnace Atomic Absorption Analysis of: Ni

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 4
Slit Width (nm): 0.2
Slit Height: Normal
Wavelength: 232.0
Sample Introduction: Sampler Premixed
Measurement Time : 3.1
Replicates: 1
BGD Correction: On

Graphite Furnace Atomic Absorption Analysis of: Pb

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 5
Slit Width (nm): 0.5
Slit Height: Normal
Wavelength: 283.3
Sample Introduction: Sampler Premixed
Measurement Time : 3.0
Replicates: 1
BGD Correction: On

Matrix Modifier: Ortho-phosphoric Acid (1000 µg/mL)

Graphite Furnace Atomic Absorption Analysis of: Cd

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 4
Slit Width (nm): 0.5
Slit Height: Normal
Wavelength: 228.8
Sample Introduction: Sampler Premixed
Measurement Time : 3.1
Replicates: 1
BGD Correction: On

Graphite Furnace Atomic Absorption Analysis of: Cr

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 7
Slit Width (nm): 0.2
Slit Height: Reduced
Wavelength: 357.9
Sample Introduction: Sampler Premixed
Measurement Time : 3.2
Replicates: 1
BGD Correction: Off

Matrix Modifier: Ortho-phosphoric Acid (1000 µg/mL)

Graphite Furnace Atomic Absorption Analysis of: As

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 5
Slit Width (nm): 0.2
Slit Height: Normal
Wavelength: 193.7
Sample Introduction: Sampler Premixed
Measurement Time : 3.0
Replicates: 1
BGD Correction: On
Matrix Modifier : Nickel Nitrate (100 μg/mL)

Graphite Furnace Atomic Absorption Analysis of: Se

Method Parameters:

Instrument Mode: Absorbance
Calibration Mode: Concentration
Measurement Mode: Peak Height

Instrument Parameters:

Lamp Current (mA): 10
Slit Width (nm): 1
Slit Height: Normal
Wavelength: 196.0
Sample Introduction: Sampler Premixed
Measurement Time : 3.0
Replicates: 1
BGD Correction: On

Matrix Modifier: Nickel Nitrate (100 µg/mL)

Appendix 2: Microwave Digestion Parameters

Microwave Digestion Parameters

Manufacturer: CEM
Model: MDS 2100
Digestion Vessel Type: ACV - Advanced Composite Vessels

The following table summarizes the typical method and instrument parameters for the graphite furnace atomic adsorption analysis of various target analytes. These include arsenic, selenium, cadmium, chromium, nickel and lead.

Note: Both pressure and temperature are set as the controlling parameters in this digestion program. If the preset pressure or temperature is not reached, the microwave oven delivers the designated power for the time programmed in the Run Time function.

The following table provides the microwave digestion parameters in 5 stages used for MS Smoke Samples as well as 4 stages of secondary digestion parameters.

Note: These are only suggested parameters as a starting point. The digestion procedure must be optimized for the specific application and instrument used.

Appendix 3: ICP-AES Parameters

Power (kw): 1.20
Plasma Flow (L/minute): 15.0
Auxiliary Flow (L/minute): 1.50
Nebulizer Flow (L/minute): 0.65

The following table provides the ICP-AES analysis Parameters.

Sample Introduction Settings
Sample Uptake Delay(s): 40
Pump rate (rpm): 20
Instrument Stabilization Delay(s): 15
Rinse Time(s): 10

General Settings
Replicates: 3
Replicate Read Time(s): 3.0
Number of Standards Defined: 5

Ultrasonic Nebulizer Set-up
Heater: 140
Cooler: 2