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Title: 

ORA Lab Manual Vol. IV Section 16 - Tobacco Product Analysis Page 1 of 21

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Sections in This Document

Introduction............................................................................................................................ 1

Historical Background and Law.............................................................................................. 3

Methodology .......................................................................................................................... 4

3.1. Introduction ................................................................................................................. 4

3.2. Methodology Sources.................................................................................................. 4

Testing Procedures................................................................................................................ 5

4.1. Tobacco Sample Analysis ........................................................................................... 5

4.2. Nicotine in Tobacco Filler (GC-MS) ............................................................................. 7

4.3. NNN and NNK in Tobacco Filler/smokeless (LC-MS/MS)............................................ 8

4.4. Tar, Nicotine and Carbon monoxide (TNCO) by GC-FID/TCD..................................... 9

4.5. Metals Analysis (ICP-MS).......................................................................................... 11

4.6. Carbonyl in Tobacco Filler (LC-UV)........................................................................... 11

4.7. Polymerase Chain Reaction (PCR) ........................................................................... 12

References .......................................................................................................................... 13

Answer Key ......................................................................................................................... 14

6.1. Methodology Sources................................................................................................ 14

6.2. Tobacco Sample Analysis ......................................................................................... 14

6.3. Nicotine in Tobacco Filler (GC-MS) ........................................................................... 14

6.4. NNN and NNK in Tobacco Filler/smokeless (LC-MS/MS).......................................... 15

6.5. Tar, Nicotine and Carbon monoxide (TNCO) by GC-FID/TCD ................................... 16

6.6. Metals Analysis (ICP-MS).......................................................................................... 18

6.7. Carbonyl in Tobacco Filler (LC-UV)........................................................................... 19

6.8. Polymerase Chain Reaction (PCR) ........................................................................... 20

Document History ................................................................................................................ 21

Change History.................................................................................................................... 21

Introduction

The Tobacco analysis training program provides a preliminary training in 

sample preparation techniques, mass spectrometry instrumentation and 

smoke/aerosol analysis of tobacco products. The ORA U New Hire 

Laboratory Analyst Training curriculum (Bingo card) specifies sections of 

the ORA Lab Manual as part of the New Hire Curriculum. The laboratory 
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manual is a basic training document for analysts and smoke technicians in 

the FDA tobacco laboratory area.

The Center for Tobacco Product (CTP) is responsible for establishing the 

assignments and Compliance Program for tobacco product analysis. These 

assignments and Compliance Program documents have analytical 

guidelines and instructions for sample handling and analysis.

This chapter does not include the entire range of methodologies and 

technologies performed in the tobacco laboratory. Most of the technology 

used in the tobacco laboratory will be discussed with the analyst. The 

trainee will be trained in only one of the analytical methods of the tobacco 

laboratory. During training, the trainer will demonstrate the method to the 

trainee, the trainee will run the method with the assistance of the trainer, 

and finally the trainee will be required to perform the method independently 

as a final test. After the training is completed, the employee should be able 

to use any methodology with same technology with just a familiarization. A 

proficiency test will be required after training. The employee can be trained 

in multiple technologies. 

Tobacco Laboratory receives different types of assignments from CTP 

therefore the training should include an evaluation of the sample 

assignment. As part of training, the trainer should consider and discuss 

topics with the trainee that will cultivate a thoughtful and responsible 

approach to each assignment. Topics may include the following:

• Reason for the sample collection: 

- New Tobacco Product Application: This type of sample is related to 

either a Modified-Risk Tobacco Product Application (MRTPA) or 

Pre-Market Tobacco Application (PMTA). 

- Research: Project requested by CTP/ORS for information purposes.

- For-cause Sample: CTP/OCE assignment for a specific sample due 

to an ongoing investigation. 

- Survey Sample: These samples are collected as part of a survey 

program generated by CTP and ORA. 

• Sample not in FACTS: 

- Some official samples are received without FACTS number.

- If required, by assignment, enter the sample in FACTS before 

starting any analysis. Follow STL procedures. 

• Method to be use for analysis: 

- Check assignment or compliance program.

- Check if the methods require a validation or verification before use. 
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Consult with your supervisor.

• Correct sample handling: 

- Assure that the sample contain enough product to perform all 

requested analysis.

- Some assignments require splitting the sample between multiple 

laboratories like SFFL, FCC and/or WEAC. Split the sample before 

starting any analysis. 

- If microbiology testing is required, the sample should be handled by 

a microbiologist first.

After the trainee has completed each exercise, analysis, and worksheet 

preparation, any questions and answers should be discussed thoroughly 

with the trainer. At the end of the training, the analyst should be proficient in 

the analytical technique and instrumentation for the selected method. The 

trainee should also be proficient in sample reporting and the required 

documentation. 

Historical Background and Law

The FDA Center for Tobacco Products (CTP) is responsible for carrying out 

the Family Smoking Prevention and Tobacco Control Act, which Congress 

passed in 2009. This law, commonly called the Tobacco Control Act, gives FDA 

authority to regulate the manufacturing, distribution, and marketing of tobacco 

products. CTP mission is to protect Americans from tobacco-related death and 

disease by regulating the manufacture, distribution, and marketing of tobacco 

products and by educating the public, especially young people, about tobacco 

products and the dangers their use poses to themselves and others.

In 2010, the Southeast Tobacco Laboratory (STL), in Atlanta, GA, was 

established as the first FDA tobacco regulatory laboratory, responsible for 

providing the analytical support for testing Harmful and Potentially Harmful 

Constituents (HPHC’s) in both tobacco and tobacco smoke. STL develops, 

validates, and/or verifies methods for testing in a wide variety of tobacco 

products to support CTP application review, product standard development, 

and compliance & enforcement.
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Methodology

3.1. Introduction

The source for the methodology to regulate the tobacco industry can be STL, 

literature, CDC, CORESTA, ISO and/or Tobacco industry. All methods used in 

STL must be either validated or verified as per STL Method Validation and 

Verification SOP, which is based on CTP Guidance for industry: “Validation 

and Verification of Analytical Testing Methods Used for Tobacco Products” and 

ORA method validation SOP. STL uses the following technologies: Smoking 

Machines, Aerosol Smoking Machine, GC-MS, GC-MS/MS, LC-MS, LCMS/MS, LC-UV, GC-FID, ICP-QQQ, IC (Ion Chromatography), and GCxGCTOF. Assignments and Compliance Programs from CTP will establish 

methodology source.

3.2. Methodology Sources

A. STL (Southeast Tobacco Laboratory)

STL develops, validates, and verifies methods for the analysis of HPHCs in 

tobacco products. STL is validating standard analytical methods (SAM) to 

be used for product analysis. Analytical method validation should be 

performed as per STL method validation SOP. The method validation 

reports are reviewed and approved by Supervisor, Lab Director and QMS. 

Only methodology approved by QA can be used to analyze regulatory 

samples. 

B. CORESTA (Cooperation Centre for Scientific Research Relative to 

Tobacco)

CORESTA is an association founded to promote international cooperation 

in scientific research relative to tobacco and its derived products. They 

have many methodologies for the analysis of constituents in tobacco 

products. All CORESTA methods need either method validation or 

verification before use. CTP will advise which type of method evaluation is 

needed. However, only methodology approved by QA can be used to 

analyze regulatory samples.

C. ISO (International Organization for Standards)

ISO is considered a primary source of official methods. All ISO methods 

must be either validated or verified in STL before use. The method 

validation or verification must be reviewed and approved by QA. CTP will 

advise which type of method evaluation is needed.

D. MRTPA (Modified Risk Tobacco Product Application) or PMTA (PreMarket Tobacco Application)
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The MRTPA/PMTAs submitted by manufacturers may include methods 

used by the manufacturer or a third-party contract laboratory to provide 

application data for the applicant’s tobacco product(s). If the method 

provided is determined to be suitable by CTP, STL may be asked to 

perform a method verification and use the applicant’s method to verify 

select application data. All method verifications and testing must be done 

in accordance with STL Method Validation and Verification SOP. 

All the material provided in the MRTPA, or PMTA submissions are 

confidential and subject to protection. The analyst may not cite MRTPA or

PMTA methods in a publication or discuss them with persons outside of 

STL. The documents require secure storage; the analysts are to be careful 

in how they handle the documents.

E. Other sources (CDC, Literature and Journals)

When methods are not located in the conventional sources mentioned

above, there may be applicable procedures found in the literature. The

Laboratory Information Bulletin (LIB) is a popular source of unofficial 

methodology in FDA published by the Office of Regulatory Science. The 

LIBs are an excellent source for methods. Other good sources for articles 

are PubMed, NCBI, NIH and instrument manufacturer technical 

applications. Any peer-review journal is a good source for analytical 

methods. When using methods from external or alternative sources, the 

methods must be fully validated before use in an ORA laboratory and the 

method validation package must be reviewed and approved by QSM.

F. Questions

1. What do all methodology sources have in common? 

2. What special attention is needed for MRTP and PMTA methodologies?

3. Please list all methodology sources.

4. Look for the HPHC table in the FDA website and mention 10 HPHCs on 

the list. 

Testing Procedures

4.1. Tobacco Sample Analysis

The trainee must perform successfully one of the mentioned methods below. 

In case the method requires smoke collection, the smoke technician is only 

responsible for performing the smoking portion of the sample collection. To 

certify the smoking technician, a qualified analyst will perform the sample 

analysis in the adequate instrument. If the trainee is an analyst, the smoking 

should be performed by a qualified smoke technician. The results must be 
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within acceptable limits. All trainees must pass a proficiency test before 

starting sample analysis in the laboratory. 

The major types of tobacco products are combusted and non-combusted. 

Non-combusted tobacco products consist of smokeless tobacco, primarily 

chewing tobacco and snuff (including snus).; Electronic Nicotine Delivery 

Systems (ENDS); heat-not-burn; and some dissolvable or novel tobacco 

products. Most smokeless tobacco use involves placing the product between 

the gum and the cheek or lip. Electronic nicotine delivery system products use 

an “e-liquid” that may contain nicotine, as well as varying compositions of 

flavorings, propylene glycol, vegetable glycerin, and other ingredients. 

The liquid is heated to create an aerosol that the user inhales. ENDS may be 

manufactured to look like conventional cigarettes, cigars, or pipes. Some 

resemble pens or USB flash drives. Larger devices, such as tank systems or 

mods, bear little or no resemblance to cigarettes. Heat-not-burn tobacco 

products are products in which the tobacco is heated to release nicotine 

without combustion. Combustible tobacco products, such as cigarettes, 

cigars, roll your own (RYO), pipe and waterpipe tobacco are burned and 

inhaled. The tobacco industry is constantly changing and creating new tobacco 

products which may require the adaption of existing tobacco methods or the 

development of new methods (ex. nicotine gel that contains nicotine and can 

be absorbed through the skin).

STL testing includes, but is not limited to, tobacco product filler, smokeless, 

aerosol, e-liquids, and smoke. When analyzing smoke and aerosol products, a 

specialized smoking or aerosol machine is used to generate and collect the 

sample. The operation of these machines requires significant training and 

practice. Smoke technicians must be trained in a method that includes 

smoking. There are different types of smoking machines: linear smoking 

machine, rotary smoking machine, e-vaping machines, cigar smoking 

machine, etc. Smoke technicians and analysts must be trained and be 

proficient in one machine type but must continue training to operate all 

machine types. 

Linear and rotary smoking machines are mainly used for collecting smoke 

constituents of cigarette and cigarette-like products. The sample can be 

collected in either an impinger containing an extraction solution, an 

electrostatic trap, or on a filter pad depending on the type of analyte to be 

analyzed. The linear smoking machines allows the collection of multiple 

individual replicates of mainstream smoke sample at the same time. The 

rotary smoking machine collects smoke from multiple cigarettes on one pad. 

By collecting smoke from multiple cigarettes on one pad, the analyte of interest 

can be concentrated for low level detection. The e-vaping machines is 
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designed to be used with different types of e-cigarettes and vapor devices. A 

cigar smoking machine is used for cigar and cigar-like products with higher 

diameter and length than cigarettes. 

A. Questions:

1. What types of smoking machines can be used to collect smoke from 

cigarettes? 

2. You need to analyze an analyte that is found at low level in a little cigar. 

What would be the appropriate smoking machine to use and why? 

3. What are few of the major tobacco product types? 

4. What collection technique would be appropriate to analyze nicotine in 

smoke? 

5. What does ENDS stand for? 

4.2. Nicotine in Tobacco Filler (GC-MS)

A. Objective 

The analyst will become familiar with the quantitation of nicotine in cigarette 

filler, cigar, little cigars, cigarillo, e-liquid and smokeless products. The 

analysts must follow any of the current SOP for quantitation of nicotine by 

GC-MS. 

B. Reference

1. Any STL SOP for Quantitation of Total Nicotine in Tobacco Filler, 

Smokeless, Smoke, Aerosol or E-liquid using GC-MS 

C. Pre-analysis Questions 

1. What is nicotine’s molecular weight?

2. Why do you need to be careful handling nicotine standards, solutions, 

and samples? 

3. Define CCV, ICV and % RSD. 

4. What GC-MS type will be used for this analysis? What other types of 

GC-MS are available? 

5. What reference materials can be used as control samples? 

6. Why is ISTD added to the sample and standard solutions?

7. Should the samples be analyzed frozen, refrigerated or at room 

temperature? 

8. Sometimes grinding the sample is a requirement for sample 

preparation, why the grinder should be set on pulse instead of 

constant? 

9. What ions will be used for quantitation? 

10.What quality controls are needed for this test?

D. Exercise
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Analyst will perform the analysis of a proficiency sample or SRM (Standard 

Reference Material) following any of the current method for quantitation of 

nicotine in tobacco product filler, e-liquid and smokeless. The sample will 

be reported on the FDA form 431 and 431a.The analyst’s results must be 

within established limits. 

E. Post Analysis Questions

1. Which GC-MS mode is used, SIM or full scan? 

2. What do SIM and MRM stand for? 

3. What was the purity of the nicotine standard? How is this purity used 

within the method? 

4. How can you verify that the instrument used was qualified and ready for 

use?

5. Are the standards and sample solutions labeled correctly? 

4.3. NNN and NNK in Tobacco Filler/smokeless (LC-MS/MS)

A. Objective

N’-Nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-

butanone (NNK), are Tobacco Specific Nitrosamine (TSNA) found in many 

tobacco products. These compounds are classified as group 1 

carcinogens, and they are included in the HPHC list. These products are 

formed during the curing, fermentation, and burning of tobacco. The analyst 

must quantitate these compounds in tobacco filler, e-liquid or smokeless 

product using an LC- MS/MS method.

B. Reference 

1. Any STL SOP Analytical Method for the Quantitation of TSNA in 

Cigarette Filler, E-liquid and Smokeless Products by LC-MS/MS. 

C. Pre-analysis Questions

1. When are NNN and NNK formed in the tobacco manufacturing 

process?

2. Where are TSNAs found in higher amounts?

3. What LC-MS/MS stands for and what is used for? 

4. Explain the most used ionization techniques available for LC-MS? How 

do the ions get ionized in each type of source? 

5. What type of ionization source is used in the TSNA method? 

6. What ions are used for quantitation? What ions are used for 

confirmation? What criteria are used to confirm the molecule? 

7. What is a product ion? 

8. What is the collision energy of the method and what is the collision 

energy for? 

9. What gas is needed in the collision cell and why? 
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10.Why is the column heated? 

11.What are the QC requirements of the method? 

D. Exercise

Analyst will perform the analysis of a proficiency sample or SRM following 

the current method for quantitation of NNN and NNK in cigarette filler and 

smokeless products. The sample results will be reported on the analytical 

worksheet FDA form 431 and 431a. The analyst’s results must be within 

established limits. 

E. Post-analysis Questions

1. What is critical during sample preparation?

2. Should the standards be used frozen, refrigerated or at room 

temperature?

3. Why is system suitability needed?

4. What precautions are needed during the sample and standard 

preparation?

4.4. Tar, Nicotine and Carbon monoxide (TNCO) by GC-FID/TCD 

Note: This method must be used with participation of both training analyst and 

smoke technician. 

A. Objective

Cigarette composition and design determine the amount of “tar”, Nicotine 

Free Dry Particulate Matter (NFDPM), nicotine, and carbon monoxide 

delivered within the smoke. In this method, “tar”, nicotine, Total Particulate 

Matter (TPM), water, and carbon monoxide (CO) are measured in 

mainstream cigarette smoke. Water content and TPM are used to calculate 

the amount of “tar” in the smoke residue. Cigarettes are smoked using a 

linear smoking machine. CO is measured directly by the smoking machine. 

All remaining analytes are collected on a filter pad for subsequent 

extraction and analysis using a gas chromatograph (GC). Smoke 

technicians and analysts must follow a specified smoking regime. STL 

typically performs smoking runs using either the Canadian Intense (CI) or 

ISO smoking regimes. The smoke technician does not perform the 

instrumental analysis for nicotine and water content. 

However, the technician must work with an analyst during the technician’s 

proficiency test to obtain results which will be used to evaluate the 

technician’s performance by comparison to TNCO values from a standard 

reference cigarette. Analysts must also demonstrate proficiency for both 

cigarette smoking, instrumental analysis, and subsequent calculation of 

TNCO parameters. Results must be within specified limits of a standard 

reference cigarette. 
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B. References

1. Quantitation of “Tar”, Nicotine and Carbon Monoxide in Cigarette 

Smoke Using a Linear Smoking Machine and GC/FID and GC/TCD or 

current method 

2. STL SOP for Operation of Linear Smoking Machines or current method. 

C. Pre-analysis Questions

1. What is “Tar”?

2. Why is it important to measure carbon monoxide (CO)?

3. How is water quantitated?

4. What preparations are necessary for cigarettes prior to smoking?

5. What are the Canadian intense (CI) smoking regime parameters?

6. What parameters must be verified in the smoking machine settings prior 

to smoking?

7. In what environmental conditions, should the sample be smoked? 

Where are the required environmental conditions for a smoking 

machine run found?

8. Why are the Cambridge filter pads weighed prior to and immediately 

after performing a smoking machine run?

9. What safety precautions are needed during a smoking machine run?

D. Exercise

A smoking machine run is performed by an analyst or smoke technician 

using a proficiency sample or reference cigarette. An analyst will perform 

the analysis of these samples following the current method for quantitation

of TNCO in cigarette smoke. The sample will be reported on the FDA form 

431 and 431a. Sample results must be within established limits.

E. Post-analysis Questions

1. What is butt length and how is it determined?

2. Describe the appearance of the Cambridge filter pad (CFP) after 

smoking?

3. How many cigarettes are smoked per pad in ISO regime?

4. What is the reference cigarette?

5. Is it a requirement to smoke a reference cigarette with each smoking 

run?

6. What are the special preparations necessary for cigarettes to be 

smoked using the CI regime? Why?

7. What is the GC carrier gas?

8. What does FID mean and how does it work? 

9. What does TCD mean and how does it work?

10.What is the QC of the method and what they measure?
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4.5. Metals Analysis (ICP-MS)

A. Objective

Toxic metals are a concern to the public health. Arsenic and cadmium are 

two of the toxic metals monitored in tobacco products filler. Other metals of 

concern in ENDS aerosol are lead, nickel, iron, and copper. Analysts will be 

trained in a current ICP-MS/MS method and must pass a proficiency test.

B. Reference

1. Any STL SOP Analytical Method for the Quantitation of Metals in any 

Tobacco Product, Aerosol or E-liquid 

C. Pre-analysis Questions

1. What gas is used to generate the plasma?

2. Why are metals monitored in food, drugs, and tobacco?

3. How is the sample prepared before ICP-MS testing?

4. What is digestion?

5. Before starting any sample testing, what needs to be verified in the ICPMS?

6. What are all the types of cones?

7. What is a critical parameter of the nebulizer?

D. Exercise 

The analyst will perform the analysis of a proficiency sample or SRM 

following the current method for quantitation of metals in tobacco filler. The 

sample will be reported on the FDA form 431 and 431a. The analyst’s 

results must be within established limits. 

E. Post-analysis Questions

1. What is ICP and how is it used with MS?

2. Describe the change in appearance of the sample after digestion?

3. Why is hydrofluoric acid used in the digestion?

4. Why is pressure used in the digestion system?

5. Is an internal standard used for metal analysis? If so, why?

6. How is the correct internal standard selected for the analysis?

7. What are the QC requirements of the method? 

4.6. Carbonyl in Tobacco Filler (LC-UV)

A. Objective 

Carbonyl group is a functional group composed of a carbon atom doublebonded to an oxygen atom (C=O). Some carbonyl compounds are 

potentially toxic (carcinogenic), and they can be found in tobacco product 

filler, ENDS, aerosol, and smoke. Some of the most common carbonyl 

compounds found in tobacco products are: acetaldehyde, acetone, 

acrolein, crotonaldehyde and formaldehyde. One of the current STL 
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method requires derivatization of the carbonyl compounds before LC-UV 

detection. The analyst will become familiar with the quantitation of carbonyl 

compounds in tobacco products.

B. Reference

1. Any STL SOP for Quantitation of Carbonyl Compounds in Tobacco 

Product, smoke or ENDS aerosol.

C. Pre-analysis Questions

1. What type of chemical compounds are carbonyls? 

2. Are carbonyls byproducts of tobacco product combustion? 

3. Are carbonyl compounds part of the abbreviated HPHC list? 

4. What is an UV/VIS detector? 

5. What is the HPLC’s column stationary phase? 

6. Why are carbonyls derivatized before analysis? 

7. What is used as reference standard? 

8. Are there other technologies used to quantitate carbonyl compounds? 

D. Exercise

Analyst will perform the analysis of a proficiency sample or SRM following 

the current method for quantitation of carbonyl in tobacco products. The 

sample will be reported on the FDA form 431 and 431a. The analyst’s 

results must be within established limits. 

E. Post-analysis Questions

1. Which two carbonyls elute close to each other? 

2. Why is the composition of the mobile phase important? 

3. What is the purpose of heating the column?

4. Why do the samples need to be neutralized after derivatization? 

5. Why is the gradient increased to a high amount of organic phase at the 

end of the run? 

4.7. Polymerase Chain Reaction (PCR)

A. Objective

Real-time PCR is a technique in molecular genetics that permits the 

analysis of short sequences of DNA or RNA. This technique is so sensitive 

that can detect minute quantities of genetic material. Currently, the realtime PCR is used for the detection of two DNA fragments found in tobacco 

plants. The analyst will become familiar with the analysis and the detection 

of tobacco plant genes in tobacco product by real-time PCR.

B. Reference
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1. Current SOP “Detection of Nicotiana genes Nt1 and NtNir in Tobacco 

and Tobacco Related Products Using Real Time Polymerase Chain 

Reaction” 

C. Pre-analysis Questions

1. How does real-time PCR work? Explain PCR reaction and detection.

2. Why is sample handling so important? 

3. Can the real-time PCR detect the presence of one DNA fragment in the 

sample? 

4. Why does the sample need to be grinded before analysis? 

5. Why does the sample need to be handled first by the analyst performing 

this test? 

6. What are inhibitors and why is it important to be aware of them?

D. Exercise

Analyst will perform the analysis of a proficiency sample or SRM following 

the current method for the detection of tobacco plant DNA in tobacco 

product. The sample will be reported on the FDA form 431 and 431a. The 

analyst’s results must contain satisfactory control and indicate positives 

and/or negatives based on cycle threshold (Ct) values. 

E. Post-analysis Questions

1. How do you know there is tobacco plant DNA in the sample?

2. Which probe dyes are used to target the tobacco genes of interest?

3. What quality controls were used to assure the analysis performed as 

intended?

4. How many sample replicates are needed? What reagent control is used 

to determine if inhibition has occurred during an analysis and what 

steps are taken to rule out false negatives due to inhibition? References

References

A. Cooperation Centre for Scientific Research Relative to Tobacco 

(CORESTA) website: https://www.coresta.org/

B. International Organization for Standardization (ISO): ISO/TC 126 Tobacco 

and Tobacco products.

C. Center of Tobacco Product intranet. 

http://inside.fda.gov:9003/CTP/default.htm

D. Internal References List - upon request
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Answer Key

6.1. Methodology Sources

A. What do all methodology sources have in common? All need either 

validation or verification. 

B. What special attention is needed for MRTPA and PMTA 

methodologies? Confidential, do not distribute. 

C. Please list all methodology sources. STL, literature, LIB, CDC, 

CORESTA, ISO and/or Tobacco industry

D. Look for the HPHC table in the FDA website and mention 10 HPHCs 

on the list. Example: ammonia, acrolein, BaP, benzene, cobalt, furan, 

isoprene, 2-nitropropene, NNN, NDMA, phenol, etc.

6.2. Tobacco Sample Analysis

A. What types of smoking machines can be used to collect smoke from 

cigarettes? Linear and rotary smoking machines

B. You need to analyze an analyte that is found at low levels in a little 

cigar. What is the appropriate smoking machine to use and why?

Rotary smoking machine. Because it can concentrate the analyte of 

interest. 

C. What are the major tobacco product types? Smokeless, ENDS, Smoked 

Tobacco Products, etc.

D. What collection technique is used to analyze nicotine in smoke?

Mainstream smoke collection on a CFP

E. What does ENDS stand for? Electronic nicotine delivery system

6.3. Nicotine in Tobacco Filler (GC-MS)

A. Pre-analysis Questions 

1. What is nicotine’s molecular weight? 162.2 g/mol

2. Why do you need to be careful handling nicotine standards, 

solutions, and sample? Additives are highly toxic. 

3. Define CCV, ICV and RSD. CCV= Continuing Calibration Verification, 

ICV= Initial Calibration Verification, %RSD = % Relative Standard 

Deviation

4. What GC-MS type will be used for this analysis? Single quadrupole 

What other types of GC-MS are available? Example: Triple 

Quadrupole, Time of Flight, Ion Trap, Magnetic Sector, Orbitrap.

5. What reference materials can be used as control samples? 1R6F, 

CRMs, current SRM
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6. Why is ISTD added to the sample and standard solutions? To 

improve the system reproducibility. 

7. Can the sample be analyzed frozen or refrigerated? No, the sample 

must be at room temperature before weighing. 

8. When sample grinding is used, why is the grinder set on pulse 

instead of constant? To prevent sample heating. Nicotine can be 

degraded with heat. 

9. What ions will be used for quantitation? 102 and 133

10.What quality controls are needed for this test? Examples: Linearity 

0.995, SRM accuracy + 10%, CCV accuracy + 10% and %RSD of 

sample triplicate (+ 10%).

B. Post Analysis Questions

1. Which GC-MS mode is used, SIM or full scan? SIM

2. What do SIM and MRM stand for? Single Ion Monitoring and Multiple 

Reaction Monitoring

3. What was the purity of the nicotine standard used? How is this 

purity used within the method? Example 99.9. To correct standard 

concentration. 

4. How can you verify that the pipette and instrument used are 

qualified and ready for use? Pipette and GC will be labeled with 

qualification due date. Instrument qualification paperwork should be 

close to the instrument, or in the instrument’s logbook It should be 

available upon request.

5. How are the standards and sample solutions labeled? Minimum: 

content, date prepared, expiration date, and analyst’s initials.

6.4. NNN and NNK in Tobacco Filler/smokeless (LC-MS/MS)

A. Pre-analysis Questions

1. When are NNN and NNK formed in the tobacco manufacturing 

process? These products are formed during the curing, fermentation 

and burning of tobacco leaves.

2. Where are TSNAs are found in higher amounts? Cigarette Smoke

3. What LC-MS/MS stands for and what is used for? Liquid 

chromatography with triple quadrupole mass spectrometer detector. 

Great for quantitation in MRM mode.

4. Explain the most used ionization techniques available for LC-MS? 

How do the ions get ionized in each type of source? APCI 

(Atmospheric Pressure Chemical Ionization)- the liquid from the LC 

system is pumped through a capillary and there is also nebulization at 

the tip, where a corona discharge takes place. First, the ionizing gas 

surrounding the interface and the mobile phase solvent are subject to 
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chemical ionization at the ion source. Later, these ions react with the 

analyte and transfer their charge, APPI (Atmospheric Pressure 

Photoionization)- this interface is like the APCI ion source, but instead 

of a corona discharge, the ionization occurs by using photons coming 

from a discharge lamp, and ESI (Electrospray Ionization)- the liquid is 

nebulized at the tip of the capillary and a fine spray of charged droplets 

is formed.

5. What type of ionization source is used in the TSNA method? ESI 

(Electrospray Ionization)

6. What ions are used for quantitation? What ions are used for 

confirmation? What criteria are used to confirm the molecule? See 

current method.

7. What is a transition ion? Fragment ion from a parent/precursor 

molecule.

8. What is the collision energy of the method and what is the 

collision energy for? Collision energy is used to accelerate the 

molecule and cause collision with the gas. This will increase the 

molecule fragmentation. 

9. What gas is needed in the collision cell and why? Nitrogen/argon 

because they are inert gases or have triple bond stability and low 

reactivity.

10.Why is the column heated? Peak shape and better resolution.

11.What are the QC requirements of the method? Linearity, CCV, ICV, 

SRM, method blanks etc.

B. Post Analysis Questions

1. What is critical during sample preparation? Prevent contamination, 

not overheat the sample during grinding and sample must be at room 

temperature before handling.

2. Can the standards be used frozen or refrigerated? No, must be at 

room temperature.

3. Why is system suitability needed? To assure that the system is 

working properly before and during sample analysis.

4. What precautions are needed during the sample and standard 

preparation? TSNAs are highly toxic compounds so gloves, lab coat, 

safety glasses are required. Work must be performed under a fume 

hood. See also response to question 1.

6.5. Tar, Nicotine and Carbon monoxide (TNCO) by GC-FID/TCD 

A. Pre-analysis Questions
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1. What is “Tar”? Partially combusted particulate matter produced by the 

burning of tobacco after subtracting nicotine and water content, also 

known as nicotine free dry particulate matter (NFDPM).

2. Why it is important to measure CO? Smoking increases the CO 

content in the blood. CO replaces oxygen in the blood. It is harmful to 

the body in any concentration and can kill you.

3. How is water quantitated in smoke extracts? By GC-TCD (gas 

chromatograph-thermal conductivity detector).

4. What preparations are necessary for cigarettes prior to smoking?

Cigarette samples must be conditioned for a minimum of 48 hours and 

no more than 10 days at 22oC and 60% Relative Humidity before 

smoking. 

5. What are the CI smoking regime parameters? Puff volume=55mL, 

puff once every 30 seconds, and all filter ventilation holes must be 

blocked.

6. What parameters must be verified in the smoking machine settings 

prior to smoking? Puff volume and air velocity. 

7. In what environmental conditions, should the sample be smoked?

22oC and 60% Relative Humidity. Where are the required environmental 

requirements for a smoking machine run found? ISO3308:2000I.

8. Why are CFPs weighed prior to and immediately after performing a 

smoking machine run? Pre and post smoking run weights are used to 

calculate TPM and “Tar” (NFDPM,).

9. What safety precautions are needed during a smoking machine 

run? The precautions are needed to ensure that cigarette smoke is 

pulled into the smoking machine (to minimize personnel contact with 

cigarette smoke and HPHCs). Avoid contact with the high temperature 

(ignited ends) of the cigarette lighters contained within smoking 

machine lighter bar.

B. Post Analysis Questions

1. What is butt length and how is it determined? It is the greatest of 

either 23 mm, length of filter + 8 mm, or length of overwrap + 3mm. This 

value is calculated by determining the mean filter and overwrap lengths 

of 20 cigarettes.

2. Describe the appearance of the CFP after smoking? Covered with a 

brown residue.

3. How many cigarettes are smoked per pad in the ISO regime? 3 

4. What is the reference cigarette? 1R6F

5. Is there a requirement to smoke a reference cigarette with each 

smoking run? Yes, a minimum of one port should be used for a 

reference cigarette.
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6. What special preparations are necessary for cigarettes to be 

smoked using the CI smoking regime? Why? All filter ventilation 

holes must be covered by cellophane tape or by special filter holders 

which guarantee 100% blocking of all filter ventilation holes. Blocking all 

filter ventilation holes is done to maximize the smoke delivery to the 

pad.

7. What is the GC carrier gas? Helium because it is an inert gas.

8. What does “FID” mean and how does it work? Flame Ionization 

Detector. This detector measures organic species ions formed by 

combustion in a hydrogen-air flame.

9. What “TCD” mean and how does it work? Thermal Conductivity 

Detector. This detector senses changes in the thermal conductivity 

when compared to a reference flow of carrier gas. Most compounds 

have a thermal conductivity lower than common carrier gases. This 

detector is considered a universal detector because all compounds 

have a thermal conductivity different from helium (a common carrier 

gas). 

10.What are the QC requirements of the method for both smoking and 

sample analysis? What do the analytical QC requirements 

measure? Smoking QC requirements include measurements of air flow 

and puff volume. Analytical QC requirements include the analysis of 

multiple injections of an ICV standard and CCV standards to ensure 

they fall within the specified ranges, as well as calculation of SRM 

recoveries, and water determination of blanks.

6.6. Metals Analysis (ICP-MS)

A. Pre-analysis Questions

1. What gas is used to generate the plasma? Argon

2. Why are metals monitored in food, drug, and tobacco? Toxic metals 

like lead, arsenic, nickel and cadmium can cause disease in the body 

and can accumulate in tissues to dangerous levels. 

3. How is the sample prepared before ICP-MS testing? The sample is 

digested in acid, heat, and high pressure before analysis.

4. What is digestion? Digestion is the process of using acid, heat, and 

pressure to decompose the organic molecules to CO and release the 

metals from the matrix. 

5. What type of verification is done to the ICP-MS before starting any 

sample testing? The ICP plasma is tested against known parameters 

for operation. This is called tuning. 

6. What is a nebulizer and why is it important to ICP? The nebulizer is 

a device that converts a liquid into an aerosol. ICP instruments need a 

fine aerosol mist to maintain a plasma. 
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B. Post Analysis Questions

1. What is ICP and how is it used with MS? ICP stands for Inductively 

Coupled Plasma. It uses a high intensity radio frequency field to 

convert argon gas into a plasma. The hot plasma creates ions which 

are needed for MS analysis. 

2. Why hydrofluoric acid used in the digestion? To dissolve silicate, 

and therefore to release trapped metals.

3. Why is pressure used in the Ultra wave digestion system? To 

prevent the solution from boiling over. 

4. Why are internal standards used for metal analysis? To correct for 

the high variability during sample introduction in the nebulizer. 

5. How do you select the correct internal standard for your analysis?

Try to match the ionization potential of the internal standard to that of 

the element of interest, then select one close to the mass of the 

element. 

6. What is the QC of the method? See current method

6.7. Carbonyl in Tobacco Filler (LC-UV)

A. Pre-analysis Questions

1. What types of chemical compounds are carbonyls? Aldehydes and 

ketones

2. Are carbonyls byproducts of tobacco product combustion? Yes

3. Are carbonyl compounds in the abbreviated HPHC list? Yes, 

acetaldehyde, acrolein, crotonaldehyde, formaldehyde, others

4. What is an UV/VIS detector? A detector that measures the absorption 

in the ultraviolet-visible spectral region.

5. What is the HPLC’s column stationary phase? C18 stationary phase

6. Why are Carbonyls derivatized before analysis? Unstable due to 

volatility, thermal instability and sensitive to acid environment.

7. What is used as reference standard? 1R6F, NIST3222, or any 

current SRM

8. Are there other technologies used to quantitate carbonyl 

compounds? Yes, GC-LC-MS, LC-MS/MS.

B. Post Analysis Questions

1. Which two carbonyls elute close to each other? Acetone and 

acrolein

2. Why is the composition of the mobile phase important?

Composition of mobile phase determines the separation and retention 

of time of the compounds in the chromatogram. 

3. What is the purpose of heating the column? To improve peak shape 

and sometime resolution.
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4. Why the samples need to be neutralized after derivatization? To 

stop the reaction and prevent the formation of impurities like dimers.

5. Why is the gradient increased to a high amount of organic phase 

at the end of the run? To clean the column from impurities and 

tobacco residues.

6.8. Polymerase Chain Reaction (PCR)

A. Pre-analysis Questions

1. How does the PCR work? Explain PCR reaction and detection.

PCR works by repeating cycles of heating and cooling to denature, 

anneal, extend, or elongate specific DNA fragments according to the 

primers used. This thermal cycling, in the presence of a heat-stable 

DNA polymerase (Taq polymerase), enables temperature dependent 

reactions to occur, specifically, DNA melting and replication.

2. Why sample handling is important? To prevent contamination from 

DNA from the analyst, DNA from other samples in the lab and DNA 

from the DNA ladder used to determine the size of amplified sample, 

reagents, pipette tips etc.

3. Can the PCR detect the presence of 1 DNA fragment in the 

sample? Yes

4. Why does the sample need to be grinded before analysis? To 

release the DNA from the cell.

5. Why the sample need to be handled first by the analyst performing 

this test? To prevent sample contamination

6. What are inhibitors and why is it important to be aware of them?

Inhibitors are various materials or compounds that may be found within 

a sample that interfere with the DNA amplification process. This could 

result in false negative results if inhibitors are present in a sample. 

B. Post Analysis Questions

1. How do you know there is tobacco plant DNA in the sample? As 

the target gene amplifies during the real time-PCR, the fluorescence, 

from the probes used, will grow in intensity. Once the fluorescence 

reading hits the threshold determined by the ABI 7500 the sample will 

be determined positive and a Ct value will be recorded.

2. Which probe dyes are used to target the tobacco genes of 

interest? FAM is used for NT1 and CY5 is used for NtNir. 

3. What quality controls were used to assure the analysis performs 

as intended? Three controls are used:

4. Negative Template Control (NTC): IPC (+) / Targets (-)

5. Negative Amplification Control (NAC): IPC (-) / Targets (-)

6. Positive Tobacco Control (PC): IPC (+) / Targets (+)
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7. How many sample replicates are needed? Three sample replicates to 

show consistent replication and detection (Ct values).

8. What reagent control is used to determine if inhibition has 

occurred during an analysis and what steps are taken to rule out 

false negatives due to inhibition? A TaqMan® Exogenous Internal 

Positive Control (IPC). Reagent is used in the Master Mix to determine if 

inhibitors are present by either not showing any amplification or 

amplifies very late with an abnormal curve (high Ct). If any inhibition 

occurs, samples are to be diluted 1:10 in an either nuclease-free water 

or Tris-EDTA (TE) Buffer. Rerun the original sample template along with 

the diluted template and include the appropriate controls. 

Document History

Revision

# I Date Author Name and Title Approving Official Name and Title

00 I See QMiS LMEB LMEB

01 R See QMiS Javier Vega, Tobacco 

Supervisory Chemist LMEB

* - D: Draft, I: Initial, R: Revision

Change History

Revision 

# Change

00 New Document

01 Revisions made to Sections 1 and 3.1 are highlighted