Selgitustaotlus

ATT00002.htm

Arguments - 6-methylnicotine.docx

Arguments questioning the statement that 6-methylnicotine (CAS: 13270-56-9) is an alkaloid.
According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), which standardizes symbols, nomenclature and physical quantity standards used by chemists around the world, alkaloids are defined as a group of naturally occurring basic compounds organic, mainly heterocyclic, mostly of plant origin containing nitrogen [1]. Cleary alkaloids as chemical compounds produced by plants are also defined in the book titled: Alkaloids . Biochemistry , ecology and medicine applications [2]. The constitutive feature of an alkaloid is not whether a given type of compound is produced by the plant or not. For example, nicotine, cotinine , nornicotine and other alkaloids produced by the tobacco plant will be alkaloids regardless of whether they were synthesized in a laboratory or extracted. This is due to the fact that these compounds are produced by plants. DK Semwal understands this concept in a similar way, in his book "The essential guide to alkaloids” [3] states that alkaloids are “naturally occurring organic substances” containing, among others: nitrogen and having an alkaline nature. They do not have to be of plant origin because they can be synthesized by other living organisms, including some animals. Nevertheless, their inherent feature is their origin from biosynthesis. Accordingly, if a given compound is not produced by living organisms, it is not an alkaloid, and there are many examples of compounds that contain nitrogen, have an alkaline environment, but are not produced by a plant and are therefore not considered an alkaloid. To date, there are no reports that the chemical compound known as 6-methylnicotine (systematically 2-methyl-5-[(2S)-1-methylpyrrolidin-2-yl]pyridine) occurs in nature in its pure form. Due to the above and due to the great interest of researchers in the use of nicotinic acetylcholinergic receptors due to their therapeutic potential, various methods have been developed for obtaining 6-substituted nicotine analogues by chemical synthesis, including 6-methylnicotine [4]. One of the first and most common synthetic methods of obtaining 2-methyl-5-[(2S)-1-methylpyrrolidin-2-yl]pyridine is the method involving the reaction of basic nicotine with methyllithium [5], which is a strong nucleophile in one of the organic solvents (e.g. toluene), which leads to the isolation of 6-methylnicotine with small amounts of 4-methylnicotine and 2-methylnicotine as shown in Figure 1.

Fig. 1. Reaction of nicotine with methyllithium in hot toluene.
Another method of synthetically obtaining 6-methylnicotine is to use 6-methylnicotinic acid methyl ester and -butyrolactone as starting raw materials and carry out ester condensation reactions, ring opening, reduction, and ring closing amination reactions [6].
The canonical (boundary) structures of the pyridine ring in the nicotine molecule are shown in Fig. 2

Fig. 2. Canonical (boundary) structures of the pyridine ring in the nicotine molecule
The structure and nature of the pyridine ring causes it to easily undergo electrophilic addition to the lone electron pair of the nitrogen atom, but it is very difficult to undergo electrophilic substitution in the ring. The alkylation reaction, i.e. the attachment of a methyl (alkyl) group to a ring, is an example of such a substitution. This group can be transferred in the form of a radical, carbanion or carbocation, which is the basis for the systematics of these reactions. Electrophilic substitution at the carbon atom of the pyridine ring occurs difficult due to the significant reduction of electron density by the highly electronegative nitrogen atom. Therefore, the probability that such a reaction may occur in the natural environment decreases significantly and indicates the need to use chemical synthesis methods. Moreover, there are numerous scientific reports referring to 6-methylnicotine as a synthetic compound, not derived from tobacco or nicotine. According to the above-mentioned arguments, the compound 6-methylnicotine cannot be considered an alkaloid.
Literature:
[1] Moss, G. P., Smith, P. A. S., & Tavernier, D. (1995). Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC Recommendations 1995). Pure and applied chemistry , 67 (8-9), 1307-1375.
[2] Rodger, M. F., & Wink , M. (1998). Alkaloids Biochemistry , ecology and medicine applications .
[3] Semwal , D. K., (2023). The Essential Guide to Alkaloids . Nova Science Publishers , Inc.
[4] Dukat, M., Dowd, M., Damaj, M. I., Martin, B., El-Zahabi, M. A., & Glennon, R. A. (1999). Synthesis, receptor binding and QSAR studies on 6-substituted nicotine derivatives as cholinergic ligands. European journal of medicinal Chemistry , 34 (1), 31-40.
[5] Seeman, J. I., Secor, H. V., Howe, C. R., Chavdarian, C. G., & Morgan, L. W. (1983). Organometallic methylation of nicotine and nicotine N-oxide. Reaction pathways and racemization mechanisms. The Journal of Organic Chemistry , 48 (25), 4899-4904.
[6] SHENZHEN ZINWI BIO-TECH CO LTD(Shenzhen Zhenwei Biotechnology Co., Ltd.) , 06 May 2022, Patent Application.

E-kiri.eml

Hi,

My assistant asked you to:

â€œPlease confirm that the 6-methylnicotine compound cannot be considered an alkaloid within the meaning of the IUPAC definition.â€

You replied that you could not provide such information.

But what if we want to introduce liquids with this substance to your market?

Then you will also not provide us with an answer to the question whether they should be registered?

So who can we ask?

Karina Grabowska PhD

Emergency Management Director

Aroma King Global sp. z o.o.

M +48 535 284 800

E k.grabowska@aromakingglobal.com

ul. Owsiana 60, 40-780 Katowice

This email and any attachments are confidential and may be legally privileged and protected by copyright. If you are not the intended recipient of this email you should not copy it or disclose its contents to anyone. If you have received this email in error, please notify the sender immediately and delete the email. Views or opinions in this email are solely those of the author. Unencrypted Internet communications are not secure and the sender does not accept responsibility for interception of this message by third parties. This communication is not intended to form a binding contract unless expressly indicated to the contrary and properly authorized. The recipient should scan this email and any attachments for the presence of viruses. The sender accepts no liability for any viruses transmitted in this email.

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ATT00002.htm

Arguments - 6-methylnicotine.docx

Arguments questioning the statement that 6-methylnicotine (CAS: 13270-56-9) is an alkaloid.
According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), which standardizes symbols, nomenclature and physical quantity standards used by chemists around the world, alkaloids are defined as a group of naturally occurring basic compounds organic, mainly heterocyclic, mostly of plant origin containing nitrogen [1]. Cleary alkaloids as chemical compounds produced by plants are also defined in the book titled: Alkaloids . Biochemistry , ecology and medicine applications [2]. The constitutive feature of an alkaloid is not whether a given type of compound is produced by the plant or not. For example, nicotine, cotinine , nornicotine and other alkaloids produced by the tobacco plant will be alkaloids regardless of whether they were synthesized in a laboratory or extracted. This is due to the fact that these compounds are produced by plants. DK Semwal understands this concept in a similar way, in his book "The essential guide to alkaloids” [3] states that alkaloids are “naturally occurring organic substances” containing, among others: nitrogen and having an alkaline nature. They do not have to be of plant origin because they can be synthesized by other living organisms, including some animals. Nevertheless, their inherent feature is their origin from biosynthesis. Accordingly, if a given compound is not produced by living organisms, it is not an alkaloid, and there are many examples of compounds that contain nitrogen, have an alkaline environment, but are not produced by a plant and are therefore not considered an alkaloid. To date, there are no reports that the chemical compound known as 6-methylnicotine (systematically 2-methyl-5-[(2S)-1-methylpyrrolidin-2-yl]pyridine) occurs in nature in its pure form. Due to the above and due to the great interest of researchers in the use of nicotinic acetylcholinergic receptors due to their therapeutic potential, various methods have been developed for obtaining 6-substituted nicotine analogues by chemical synthesis, including 6-methylnicotine [4]. One of the first and most common synthetic methods of obtaining 2-methyl-5-[(2S)-1-methylpyrrolidin-2-yl]pyridine is the method involving the reaction of basic nicotine with methyllithium [5], which is a strong nucleophile in one of the organic solvents (e.g. toluene), which leads to the isolation of 6-methylnicotine with small amounts of 4-methylnicotine and 2-methylnicotine as shown in Figure 1.

Fig. 1. Reaction of nicotine with methyllithium in hot toluene.
Another method of synthetically obtaining 6-methylnicotine is to use 6-methylnicotinic acid methyl ester and -butyrolactone as starting raw materials and carry out ester condensation reactions, ring opening, reduction, and ring closing amination reactions [6].
The canonical (boundary) structures of the pyridine ring in the nicotine molecule are shown in Fig. 2

Fig. 2. Canonical (boundary) structures of the pyridine ring in the nicotine molecule
The structure and nature of the pyridine ring causes it to easily undergo electrophilic addition to the lone electron pair of the nitrogen atom, but it is very difficult to undergo electrophilic substitution in the ring. The alkylation reaction, i.e. the attachment of a methyl (alkyl) group to a ring, is an example of such a substitution. This group can be transferred in the form of a radical, carbanion or carbocation, which is the basis for the systematics of these reactions. Electrophilic substitution at the carbon atom of the pyridine ring occurs difficult due to the significant reduction of electron density by the highly electronegative nitrogen atom. Therefore, the probability that such a reaction may occur in the natural environment decreases significantly and indicates the need to use chemical synthesis methods. Moreover, there are numerous scientific reports referring to 6-methylnicotine as a synthetic compound, not derived from tobacco or nicotine. According to the above-mentioned arguments, the compound 6-methylnicotine cannot be considered an alkaloid.
Literature:
[1] Moss, G. P., Smith, P. A. S., & Tavernier, D. (1995). Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC Recommendations 1995). Pure and applied chemistry , 67 (8-9), 1307-1375.
[2] Rodger, M. F., & Wink , M. (1998). Alkaloids Biochemistry , ecology and medicine applications .
[3] Semwal , D. K., (2023). The Essential Guide to Alkaloids . Nova Science Publishers , Inc.
[4] Dukat, M., Dowd, M., Damaj, M. I., Martin, B., El-Zahabi, M. A., & Glennon, R. A. (1999). Synthesis, receptor binding and QSAR studies on 6-substituted nicotine derivatives as cholinergic ligands. European journal of medicinal Chemistry , 34 (1), 31-40.
[5] Seeman, J. I., Secor, H. V., Howe, C. R., Chavdarian, C. G., & Morgan, L. W. (1983). Organometallic methylation of nicotine and nicotine N-oxide. Reaction pathways and racemization mechanisms. The Journal of Organic Chemistry , 48 (25), 4899-4904.
[6] SHENZHEN ZINWI BIO-TECH CO LTD(Shenzhen Zhenwei Biotechnology Co., Ltd.) , 06 May 2022, Patent Application.

Report_2023_11_0005_EN.pdf

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 1 of 5

ANALYSIS REPORT

Particulars of the Client* Description Order number

SYMETRICUS sp. z o.o. ul. Stężycka 107 m. 1

80-174 Gdańsk

Quantitative determination of aerosol components derived from e-liquid, according to agreed

specification. Quantitative determination of nicotine in e-liquid.

ZO 2023/11/000007

Sample identification:

Sample signature Sample designation* Sample collection method Additional information

2023/11/0005/001 Blueberry Ice Sample collected

and delivered by the client

Date of delivery: 06.11.2023

Object of analysis: Disposable e-cigarette

Sample condition: no objection

*Information provided by the Client.

Results:

1. Sample mass and puffs number for nicotine, propylene glycol, glycerin and volatile organic compounds determination.

Sample signature Subject of determination Method identification** The result of the

analysis

Standard

Deviation Unit

2023/11/0005/001

Sample mass at 40 puffs.

vaping process to

methanol

SL/2020/036

Ed. 1 of 03.09.2020, NA 0,2368 - g

** Determination method: A-accredited, NA-non-accredited, AS- accredited subcontractor, NAS – non-accredited subcontractor.

2. Sample mass and puffs number for tobacco-specific nitrosamines, aldehydes and ketones determination.

Sample signature Subject of determination Method identification** The result of the

analysis Standard Deviation

Unit

2023/11/0005/001

Sample mass at 40 puffs.

vaping process to acetonitrile.

SL/2020/036 Ed. 1 of 03.09.2020, NA

0,2198 - g

3. Sample mass and puffs number for heavy metals determination.

Sample signature Subject of determination Method identification** The result of the

analysis Standard Deviation

Unit

2023/10/0016/001 Sample mass at 40 puffs. Vaping process to water.

SL/2020/036 Ed. 1 of 03.09.2020, NA

0,2270 - g

The analyses have been conducted by: Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. Routine Analyses Dept.

Date of commencement of analyses 07.11.2023

Date of completion of the analyses 16.11.2023

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 2 of 5

ANALYSIS REPORT

4. Results of heavy metals determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Content of lead Pb

SL/2020/042 Ed. 2 of 28.04.2023, NA

<LOQ -

µg/g

Content of cadmium Cd <LOQ -

Content of arsenic As <LOQ -

Content of chrome Cr <LOQ -

Content of nickel Ni <LOQ -

Content of copper Cu <LOQ -

Content of aluminum Al <LOQ -

Content of tin Sn <LOQ -

Content of iron Fe <LOQ -

5. Results of volatile organic compounds determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Average content of benzene

SL/2020/037 Ed. 2 of 08.05.2023, NA

<LOQ -

µg/g

Average content of xylenes <LOQ -

Average content of toluene <LOQ -

Average content of isoprene <LOQ -

Average content of 1,3-butadiene <LOQ -

Average content of ethylene glycol

<LOQ -

Average content of diethylene glycol

<LOQ -

6. Results of aldehydes and ketones determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Average content of formaldehyde

SL/2020/040 Ed. 1 of 03.09.2020, NA

11,64 0,46

µg/g

Average content of acetaldehyde 5,42 0,29

Average content of acrolein 10,18 0,15

Average content of crotonaldehyde

<LOQ -

Average content of isovaleraldehyde

<LOQ -

Average content of o,m,p- tolualdehyde

<LOQ -

Average content of hexaldehyde <LOQ -

Average content of diacetyl SL/2020/037

Ed. 2 of 08.05.2023, NA

<LOQ -

Average content of acetyl propionyl

<LOQ -

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 3 of 5

ANALYSIS REPORT

7. Results of tobacco-specific nitrosamines determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001 Average content of NNK SL/2020/039

Ed. 2 of 22.10.2020, NA

<LOQ - µg/g

Average content of NNN <LOQ -

8. Results of nicotine, propylene glycol and glycerin determination after heating e-liquid.

Sample signature Subject of determination Method identification** The result of the analysis

Uncertainty Unit

2023/11/0005/001

Average content of propylene glycol

SL/2020/038 Ed. 3 of 05.05.2023, NA

484,2 53,3

mg/g*** Average content of glycerin SL/2020/038

Ed. 3 of 05.05.2023, NA 215,9 23,7

Average content of nicotine SL/2020/038

Ed. 3 of 05.05.2023, A <LOQ -

Average number of puffs NA

40 - -

Average nicotine dose per puff <LOQ - mg/puff

9. Results of nicotine determination in liquid.

Sample signature Subject of determination Method

identification** The result of the analysis

Uncertainty Unit

2023/11/0005/001 Average content of nicotine SL/2022/020 ed. 2 of

06.07.2023, A <LOQ - mg/g

** Determination method: A-accredited, NA-non-accredited, AS- accredited subcontractor, NAS – non-accredited subcontractor. *** amount of nicotine [mg] per 1 g of vaped liquid; LOQ – limit of quantification.

Additional information:

I.Sampling conditions: Samples of aerosols were taken in the SMOKY-LAB apparatus. Sampling parameters: - The air flow through the system was 1,1 L/min. - The test consists of 3 sec. puff and 27 sec. relaxation time interval.

II.Heavy metals determination method: The aerosol was collected into the ultrapure water with nitric acid (trace analysis quality) in the absorber. The samples were analyzed directly on Agilent ICP-OES VDV 5100 System in the axial mode. The cyclon chamber and glass nebulizer was used. The RF Power was 1,20 kW and the plasma flow of argon was 12 L/min.

Table 1.The Limits of quantification of heavy metals.

Subject of designation Unit Limit of quantification

Content of lead Pb µg/g 10,00

Content of cadmium Cd µg/g 10,00

Content of arsenic As µg/g 10,00

Content of chrome Cr µg/g 10,00

Content of nickel Ni µg/g 10,00

Content of copper Cu µg/g 10,00

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 4 of 5

ANALYSIS REPORT

Subject of designation Unit Limit of quantification

Content of aluminum Al µg/g 10,00

Content of tin Sn µg/g 10,00

Content of iron Fe µg/g 10,00

III. Volatile Organic Compounds determination method: The aerosol was collected to methanol in the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with mass spectrometry Shimadzu GCMS-QP2010 SE System. The quantitative analysis were performed in split injection mode by gradient temperature program and SIM detector mode. The Zebron WAX column was used with parameters: 30 m length; 0,25 I.D. mm and 0,25 µm of film thickness.

Table 2. The limits of quantification of volatile organic compounds.

Subject of designation Unit Limit of quantification

Content of benzene µg/g 50,0

Content of xylenes µg/g 50,0

Content of toluene µg/g 50,0

Content of isoprene µg/g 50,0

Content of 1,3-butadiene µg/g 50,0

Content of ethylene glycol µg/g 250,0

Content of diethylene glycol µg/g 50,0

IV.Aldehydes and ketones determination method: The aerosol was collected to acetonitrile in the absorber. The analytes were derivatized in acetonitrile solution by 2,4-DNPH (dinitrophenylhydrazine in phosphoric acid). Analysis of the standard solutions and the samples was performed using ultraperformance liquid chromatography with diode-array detector coupled with tandem mass spectrometry UHPLC-PDA/MS/MS Shimadzu Nexera X2 8040. The Luna Omega column (1.6 um; C 18; 100 A LC Column 100x2.1 mm) was used for the determinations.

Table 3. The limits of quantification of aldehydes and ketones.

Subject of designation Unit Limit of quantification

Content of formaldehyde µg/g 3,02

Content of acetaldehyde µg/g 4,15

Content of acrolein µg/g 5,01

Content of crotonaldehyde µg/g 5,92

Content of isovaleraldehyde µg/g 6,83

Content of o,m,p- tolualdehyde µg/g 8,45

Content of hexaldehyde µg/g 7,55

****Content of diacetyl µg/g 50,0

****Content of acetyl propionyl µg/g 25,0

****The aerosol was collected to methanol in the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with mass spectrometry Shimadzu GCMS-QP2010 SE System. The quantitative analysis were performed in split injection mode by gradient temperature program and SCAN and SIM detector mode. The Zebron WAX column was used with parameters: 30 m length; 0,25 I.D. mm and 0,25 µm of film thickness.

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 5 of 5

ANALYSIS REPORT

V.Tobacco-specific nitrosamines determination method: The aerosol was collected to acetonitrile into the absorber. Analysis of the standard solutions and the samples was performed using ultraperformance liquid chromatography with diode-array detector coupled with tandem mass spectrometry UHPLC-PDA/MS/MS Shimadzu Nexera X2 8040. The Luna Omega column (1.6 um; C 18; 100 A LC Column 100x2.1 mm) was used for the determinations.

Table 4. The limits of quantification of tobacco-specific nitrosamines.

Subject of designation Unit Limit of quantification

Content of TSNA: 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK)

µg/g

2,5

Content of TSNA: N-nitrosonornicotine (NNN)

2,5

VI.Nicotine, propylene glycol and glycerin determination method: The aerosol was collected to methanol into the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with flame ionization detector Shimadzu GC2010 Plus System. The quantitative analysis were done in split injection mode by isothermal and gradient temperature program. The Zebron ZB-624 column was used with parameters: 30 m length; 0,32 I.D. mm and 1,8 µm of film thickness

Table 5. The limits of quantification of nicotine.

Subject of designation Unit Limit of quantification

Content of nicotine in aerosol after heating

mg/g 2,5 *****

Average nicotine dose per puff mg/puff 0,03125

Content of nicotine in e-liquid mg/g 2,77

*****LOQ per 0,5g off loss mass

Authorization of the results

and report

END OF REPORT

The laboratory gives measurement uncertainty when it is relevant to the validity of the analyses results or for compliance with the specified limit values and at the Client's request. The report may not be published, in whole or in part, without the written consent of Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. The report may not be reproduced or distributed, in part, without the prior written permission of the Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. The obtained results apply only to the tested (collected and delivered by the client) samples. The laboratory is not responsible for information provided by the Client and for the collection and transport of the sample if the sample has been collected and provided by the Client. The tests results do not include the sampling stage.

image001.png

Report_2023_11_0005_EN.pdf

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 1 of 5

ANALYSIS REPORT

Particulars of the Client* Description Order number

SYMETRICUS sp. z o.o. ul. Stężycka 107 m. 1

80-174 Gdańsk

Quantitative determination of aerosol components derived from e-liquid, according to agreed

specification. Quantitative determination of nicotine in e-liquid.

ZO 2023/11/000007

Sample identification:

Sample signature Sample designation* Sample collection method Additional information

2023/11/0005/001 Blueberry Ice Sample collected

and delivered by the client

Date of delivery: 06.11.2023

Object of analysis: Disposable e-cigarette

Sample condition: no objection

*Information provided by the Client.

Results:

1. Sample mass and puffs number for nicotine, propylene glycol, glycerin and volatile organic compounds determination.

Sample signature Subject of determination Method identification** The result of the

analysis

Standard

Deviation Unit

2023/11/0005/001

Sample mass at 40 puffs.

vaping process to

methanol

SL/2020/036

Ed. 1 of 03.09.2020, NA 0,2368 - g

** Determination method: A-accredited, NA-non-accredited, AS- accredited subcontractor, NAS – non-accredited subcontractor.

2. Sample mass and puffs number for tobacco-specific nitrosamines, aldehydes and ketones determination.

Sample signature Subject of determination Method identification** The result of the

analysis Standard Deviation

Unit

2023/11/0005/001

Sample mass at 40 puffs.

vaping process to acetonitrile.

SL/2020/036 Ed. 1 of 03.09.2020, NA

0,2198 - g

3. Sample mass and puffs number for heavy metals determination.

Sample signature Subject of determination Method identification** The result of the

analysis Standard Deviation

Unit

2023/10/0016/001 Sample mass at 40 puffs. Vaping process to water.

SL/2020/036 Ed. 1 of 03.09.2020, NA

0,2270 - g

The analyses have been conducted by: Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. Routine Analyses Dept.

Date of commencement of analyses 07.11.2023

Date of completion of the analyses 16.11.2023

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 2 of 5

ANALYSIS REPORT

4. Results of heavy metals determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Content of lead Pb

SL/2020/042 Ed. 2 of 28.04.2023, NA

<LOQ -

µg/g

Content of cadmium Cd <LOQ -

Content of arsenic As <LOQ -

Content of chrome Cr <LOQ -

Content of nickel Ni <LOQ -

Content of copper Cu <LOQ -

Content of aluminum Al <LOQ -

Content of tin Sn <LOQ -

Content of iron Fe <LOQ -

5. Results of volatile organic compounds determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Average content of benzene

SL/2020/037 Ed. 2 of 08.05.2023, NA

<LOQ -

µg/g

Average content of xylenes <LOQ -

Average content of toluene <LOQ -

Average content of isoprene <LOQ -

Average content of 1,3-butadiene <LOQ -

Average content of ethylene glycol

<LOQ -

Average content of diethylene glycol

<LOQ -

6. Results of aldehydes and ketones determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001

Average content of formaldehyde

SL/2020/040 Ed. 1 of 03.09.2020, NA

11,64 0,46

µg/g

Average content of acetaldehyde 5,42 0,29

Average content of acrolein 10,18 0,15

Average content of crotonaldehyde

<LOQ -

Average content of isovaleraldehyde

<LOQ -

Average content of o,m,p- tolualdehyde

<LOQ -

Average content of hexaldehyde <LOQ -

Average content of diacetyl SL/2020/037

Ed. 2 of 08.05.2023, NA

<LOQ -

Average content of acetyl propionyl

<LOQ -

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 3 of 5

ANALYSIS REPORT

7. Results of tobacco-specific nitrosamines determination.

Sample signature Subject of determination Method identification** The result of the analysis

Standard Deviation

Unit

2023/11/0005/001 Average content of NNK SL/2020/039

Ed. 2 of 22.10.2020, NA

<LOQ - µg/g

Average content of NNN <LOQ -

8. Results of nicotine, propylene glycol and glycerin determination after heating e-liquid.

Sample signature Subject of determination Method identification** The result of the analysis

Uncertainty Unit

2023/11/0005/001

Average content of propylene glycol

SL/2020/038 Ed. 3 of 05.05.2023, NA

484,2 53,3

mg/g*** Average content of glycerin SL/2020/038

Ed. 3 of 05.05.2023, NA 215,9 23,7

Average content of nicotine SL/2020/038

Ed. 3 of 05.05.2023, A <LOQ -

Average number of puffs NA

40 - -

Average nicotine dose per puff <LOQ - mg/puff

9. Results of nicotine determination in liquid.

Sample signature Subject of determination Method

identification** The result of the analysis

Uncertainty Unit

2023/11/0005/001 Average content of nicotine SL/2022/020 ed. 2 of

06.07.2023, A <LOQ - mg/g

** Determination method: A-accredited, NA-non-accredited, AS- accredited subcontractor, NAS – non-accredited subcontractor. *** amount of nicotine [mg] per 1 g of vaped liquid; LOQ – limit of quantification.

Additional information:

I.Sampling conditions: Samples of aerosols were taken in the SMOKY-LAB apparatus. Sampling parameters: - The air flow through the system was 1,1 L/min. - The test consists of 3 sec. puff and 27 sec. relaxation time interval.

II.Heavy metals determination method: The aerosol was collected into the ultrapure water with nitric acid (trace analysis quality) in the absorber. The samples were analyzed directly on Agilent ICP-OES VDV 5100 System in the axial mode. The cyclon chamber and glass nebulizer was used. The RF Power was 1,20 kW and the plasma flow of argon was 12 L/min.

Table 1.The Limits of quantification of heavy metals.

Subject of designation Unit Limit of quantification

Content of lead Pb µg/g 10,00

Content of cadmium Cd µg/g 10,00

Content of arsenic As µg/g 10,00

Content of chrome Cr µg/g 10,00

Content of nickel Ni µg/g 10,00

Content of copper Cu µg/g 10,00

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 4 of 5

ANALYSIS REPORT

Subject of designation Unit Limit of quantification

Content of aluminum Al µg/g 10,00

Content of tin Sn µg/g 10,00

Content of iron Fe µg/g 10,00

III. Volatile Organic Compounds determination method: The aerosol was collected to methanol in the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with mass spectrometry Shimadzu GCMS-QP2010 SE System. The quantitative analysis were performed in split injection mode by gradient temperature program and SIM detector mode. The Zebron WAX column was used with parameters: 30 m length; 0,25 I.D. mm and 0,25 µm of film thickness.

Table 2. The limits of quantification of volatile organic compounds.

Subject of designation Unit Limit of quantification

Content of benzene µg/g 50,0

Content of xylenes µg/g 50,0

Content of toluene µg/g 50,0

Content of isoprene µg/g 50,0

Content of 1,3-butadiene µg/g 50,0

Content of ethylene glycol µg/g 250,0

Content of diethylene glycol µg/g 50,0

IV.Aldehydes and ketones determination method: The aerosol was collected to acetonitrile in the absorber. The analytes were derivatized in acetonitrile solution by 2,4-DNPH (dinitrophenylhydrazine in phosphoric acid). Analysis of the standard solutions and the samples was performed using ultraperformance liquid chromatography with diode-array detector coupled with tandem mass spectrometry UHPLC-PDA/MS/MS Shimadzu Nexera X2 8040. The Luna Omega column (1.6 um; C 18; 100 A LC Column 100x2.1 mm) was used for the determinations.

Table 3. The limits of quantification of aldehydes and ketones.

Subject of designation Unit Limit of quantification

Content of formaldehyde µg/g 3,02

Content of acetaldehyde µg/g 4,15

Content of acrolein µg/g 5,01

Content of crotonaldehyde µg/g 5,92

Content of isovaleraldehyde µg/g 6,83

Content of o,m,p- tolualdehyde µg/g 8,45

Content of hexaldehyde µg/g 7,55

****Content of diacetyl µg/g 50,0

****Content of acetyl propionyl µg/g 25,0

****The aerosol was collected to methanol in the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with mass spectrometry Shimadzu GCMS-QP2010 SE System. The quantitative analysis were performed in split injection mode by gradient temperature program and SCAN and SIM detector mode. The Zebron WAX column was used with parameters: 30 m length; 0,25 I.D. mm and 0,25 µm of film thickness.

ZP-309 wyd. z dn. 27.03.2023

Al. Zwycięstwa 96/98; 81-451 Gdynia

office@spark-lab.pl

www.spark-lab.pl; +48 782 811 350

NIP: 586 228 03 65

Report number:

2023/11/0005/EN

Page 5 of 5

ANALYSIS REPORT

V.Tobacco-specific nitrosamines determination method: The aerosol was collected to acetonitrile into the absorber. Analysis of the standard solutions and the samples was performed using ultraperformance liquid chromatography with diode-array detector coupled with tandem mass spectrometry UHPLC-PDA/MS/MS Shimadzu Nexera X2 8040. The Luna Omega column (1.6 um; C 18; 100 A LC Column 100x2.1 mm) was used for the determinations.

Table 4. The limits of quantification of tobacco-specific nitrosamines.

Subject of designation Unit Limit of quantification

Content of TSNA: 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK)

µg/g

2,5

Content of TSNA: N-nitrosonornicotine (NNN)

2,5

VI.Nicotine, propylene glycol and glycerin determination method: The aerosol was collected to methanol into the absorber. Analysis of the standard solutions and the samples was performed with gas chromatography combined with flame ionization detector Shimadzu GC2010 Plus System. The quantitative analysis were done in split injection mode by isothermal and gradient temperature program. The Zebron ZB-624 column was used with parameters: 30 m length; 0,32 I.D. mm and 1,8 µm of film thickness

Table 5. The limits of quantification of nicotine.

Subject of designation Unit Limit of quantification

Content of nicotine in aerosol after heating

mg/g 2,5 *****

Average nicotine dose per puff mg/puff 0,03125

Content of nicotine in e-liquid mg/g 2,77

*****LOQ per 0,5g off loss mass

Authorization of the results

and report

END OF REPORT

The laboratory gives measurement uncertainty when it is relevant to the validity of the analyses results or for compliance with the specified limit values and at the Client's request. The report may not be published, in whole or in part, without the written consent of Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. The report may not be reproduced or distributed, in part, without the prior written permission of the Laboratorium Analiz Chemicznych Spark-Lab Sp. z o.o. The obtained results apply only to the tested (collected and delivered by the client) samples. The laboratory is not responsible for information provided by the Client and for the collection and transport of the sample if the sample has been collected and provided by the Client. The tests results do not include the sampling stage.

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