Analysis of Vape Fluids and CBD Products Using Direct Sample Introduction (DSI) GC-MS/MS Sahar Caravan, Dr. Huang, and Dr. Bisceglia Chemistry Dept., Hofstra University, Hempstead NY
CBD Products CBD Products
Vape Fluids
Abstract
Results 9.00E+09
B
60/40 PG/VG Mock Vape Fluid
PG and VG
8.00E+09
Method
6.00E+09
4.00E+09
Nicotine and Vanillin
61 m/z
Low Temp
2.6 4.4 6.5 +/- +/- 6.1 +/- +/- 1.5 +/0.003 0.006 0.003 0.14 0.03
7.9 4.9 +/- +/0.09 0.09
9.8 +/0.98
High Temp
5.4 10.8+ 23.4 +/- /+/0.11 0.24 0.42
28 +/- 2.8 +/0.7 0.028
17 +/0.255
22 +/0.264
43 +/0.65
32 +/0.05 8.3 +/8 0.11
12 +/0.2
18 +/0.34
23 +/0.28
Increased Hold 1.5 29 Time Low +/- +/- 38 +/Temp 0.02 0.07 0.1
2.00E+09
A 0.00E+00
0
2
6
8 Retention Time (min)
10
12
14
DSI 58 Fragment Quantification (n=3)
C
2.50E+00
4
Arnica Muscle Rub samples were injected using the DSI method in order to have a programmable heating mechanism. 0.0020-0.0040 g of muscle rub were placed into glass microtubules. These tubules were placed in Eppendorf tubes and centrifuged for 30 seconds at 8,000 RPM. The glass microtubules were removed from the Eppendorf tubes using Teflon tweezers and then placed into the chromatophore, middle injection port. The acquisition method was a full scan detecting fragments from 50 m/z to 400 m/z for 29 minutes. The injection temperature was varied as it started at 220 °C and ramped to 300 °C at 40 °C/min and held for one minute. The split ratio was set to 1:300. The column oven temperature and mobile phase flow rate was identical to the aforementioned vape parameters. This method was also developed in order to optimize separation of high molecular weight, buttery components like muscle rub.
3.50E+10
DSI 56 Fragment Quantification (n=3)
D
3.50E-01
B
DSI TIC of CBD Muscle Rub
Solvents
4.00E+10
16
Sample
Calculated CBD (mg/mL)
Reported CBD (mg/mL)
3.00E+10
y = 0.0618x - 1.5073 R² = 0.9995
3.00E-01
2.50E-01
y = 0.0118x - 0.4154 R² = 0.9984
2.00E-01
2.50E+10
2.00E+10
Flavorings
1.50E+10
1.00E+10
1.50E-01
Possible Cannabinoids
CBD (20.5 min )
CBC (19.8 min) 5.00E+09
1.00E-01 5.00E-01
CBD MD
29.5 +/- 3.09
25
Natural CBD Full Spectrum
6.47 +/- 1.03
100
Arnica CBD Muscle Rub
6.69 +/- 0.54
6
0.00E+00 0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Time (Minutes)
-H2O 0.00E+00 40
45
50
55
Percentage Propylene Glycol
-2 H2O 58 m/z
Figure 5. (A.) Total Ion Chromatograph of CBD Muscle Rub. (B.) Tabulation of quantified CBD content in commercial fluid.
0.00E+00 35
60
65
70
35
40
45
50
55
60
65
70
Percentage Glycerin
Figure 4. (A.) Total Ion Chromatograph of Mock 60/40 PG/VG E-cigarette Fluid. (B.) Tabulation of Heating Methods and Thermal Degradants Quantified. (C.) Thermal Degradant Intensity Intensity of 56 m/z fragment with increasing VG ratio. (D.) Intensity of 58 m/z fragment with increasing PG ratio
-H2O Acrolein
Discussion
56 m/z
Figure 2. Proposed mechanism for forming propylene glycol and glycerin degradants
The DSI method GC-MS/MS clearly demonstrates how simulating the heating temperatures that are used by vape users affects the quantity of thermal degradants. For the low temperature method, an increase in propylene glycol had an increase in breakdown into the 58 m/z fragment (Figure 4C). This fragment is unique to propylene oxide and is not present in the original propylene glycol (Figure 2). The higher temperature method yielded less of the 58 m/z fragment and has minimal spectra to draw from (Figure 4C). It is possible that higher heating parameters degrade the sample beyond identifiable fragments. Since users tend to range their e-cig heating temperatures from 150 to 300 °C, the temperature ranges were manipulated to best simulate these conditions (Table 1). However, literature exploring e-cigs often runs e-cig fluid samples at higher temperature methods of 300+ °C. This is done to allow greater resolution and separation on chromatograms. The DSI method allows for real time heating of sample that can be customized. Since e-cig fluid contains VG as well as PG, the VG degradants with the low temperature method was also explored. Similar to the PG results, an increase in Vegetable Glycerin Ratio had a consistent increase in 56 m/z thermal degradant (Figure 4A). The 56 m/z fragment is unique to the thermal degradant and is not present in the original VG sample (Figure 4D). Based on our research, both PG and VG have possible thermal degradants that arise with high and low heating temperatures. Upon addition of Vanillin, a common flavoring agent, PG degradant and VG degradant increased significantly (Table 4B).
Methodology In order to explore different compositions of vape fluids degradation, 1.5 µL of each sample was first prepared. Samples were mock e-cig fluid consisting of PG/VG and an internal standard of nicotine. This sample was placed into a glass micro vial and into a plastic Eppendorf tube. These tubes were centrifuged for 30 seconds at 8,000 RPM. The vial was removed with Teflon tweezers and placed into the ChromatoProbe injector (Figure 3).
Method Low Temperature High Temperature Low Temperature, High Hold.
Method
Results
5.00E-02
Three methods were created (Table 1). Two manipulating heating protocol, and one changing the hold time.
Figure 5. Structure of CBD (left) and THC (right)
1.00E+09
Ratio of 58 m/z Peak Area to Nicotine Peak Area
Glycerin
Vape 56 m/z
3.00E+09
1.00E+00
45 m/z
VG VG 50% 60%
5.00E+09
1.50E+00
Figure 1. Vape Fluid Delivery Device(ENDS) (left) and Juul Pods with Cartridge (right)2
VG 30%
7.00E+09
2.00E+00
Atomizer and Cartridge
Vap PG PG PG e 58 30% 50% 60% m/z
CBD oil is growing immensely in popularity. Stores like TJ-Maxx and Marshalls have sections of hemp oil derived products. Companies market their CBD containing products as stress-relieving and pain-healing compounds.5 However, there is little substantiated evidence to their claim. Some items are marked as CBD containing, but are full spectrum. This means they contain other cannabis derivatives such as Cannabichromene (CBC), Cannabigerol (CBG), Δ-9-tetrahydrocannabinol (THC) and Tetrahydrocannabivarin (THCV). These compounds are structurally similar and are therefore difficult to separate (Figure 5). There is also minimal research and quality control behind these CBD containing products. The quality control measures are not well regulated. The DEA recently announced that since THC components are reported to be less than 0.1%, regulatory measures are not strict.6 The problem is the lack of regulations results in unsubstantiated claims on labels and minimal quality control. This project aims to determine the other CBD spectrum components in commercial CBD products, and quantify the amount of CBD in them. The purpose of this experiment is to explore different types of CBD containing products such as muscle rub, roll-on oil, and medical grade oil. Often, it is difficult to separate and quantify CBD full spectrum products due to the similarity in structure. However, the aim is to optimize methods using GCMS/MS to best separate and analyze the components.
Intensity (GCps)
Battery
A
Ratio of 56 m/z Peak Area to Nicotine Peak Area
Electronic Cigarettes, or E-cigs, are devices that heat vape fluid to mimic the cigarette-smoking feel. E-cigs are marketed as healthier alternatives to traditional cigarettes and often contain appealing sweet flavors to attract a younger demographic. Discrete vaping devices like Juul and other compact ecigarette devices have resulted in a spur of e-cig smoking (Figure 1). Vape fluid mainly consists of propylene glycol(PG), vegetable glycerin(VG), flavoring, and nicotine. Many of these compounds degrade upon heating (Figure 2). Due to the growing market among young adults, it is important to research possible harmful products generated in the vaping process besides nicotine. These degradants are not extensively documented, and the goal of this project is to show Direct Sample Introduction(DSI) Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS) is a viable method for detection and quantification of thermal degradants. The DSI method allows immediate, customizable heating of sample. This is a better simulation of how users would vape on their e-cig devices or Juul. This project aims to optimize the heating parameters to simulate the actual heating conditions of an e-cig device. E-cig fluids are a customizable market. Users can choose between many flavors and ratios of PG/VG. Higher PG ratios are for a deeper “throat hit” 1, while higher VG ratios are for a sweeter and thicker vape clouds. Juul fluids (Figure 1) are approximately 40/60 PG/VG.2 The heating parameters for e-cig devices are customizable with a heating range between 150°C and 300°C.3 Our research explores the quantity of degradants as the simulated smoking conditions are varied.
GCps
Abstract
Figure 3.
Chromato-Probe4
Heating Range in PTV 50°C -225 °C, 2 Minute Hold Time 60°C- 300 °C, 2 Minute Hold Time 50°C -225 °C, 4 Minute Hold Time
Table 1. Comparison of high temperature method to low temperature method. Both methods were using a Bruker Scion 456-GC with TQ MS with Helium as the carrier gas. The GC column was kept constant as a Restex Rxi-35Sil MS, 30 m, 0.25 mm ID, and 0.25 mm thickness.
Conclusion Vape users often customize their e-cig fluid solvent ratios to develop a personalized ‘hit’ when smoking. Compared to other methods of injection, the DSI method GC/MS-MS allows for programmable immediate heating. Our research shows that an increase in solvent ratio results in an increase in thermal degradants (Figure 4). Exploring commercial e-cig fluids for possible toxic thermal degradants with DSI can provide great insight for vape users and companies. Users can become more informed about the breakdown products that emerge when heating their vape fluid. The companies can gauge more reasonable heating parameters for the ENDS devices.
Discussion The DSI method GC-MS/MS method showed the most promising results as the separation was better, CBC was detected, and quantification error was only 10%. The DSI method proved successful for quantifying CBD but not THC. The dilution of 1:20 necessary for autosampler injection made detection more difficult. Of the three unknown samples, the CBD MD unknown had the least hits using the AMDIS software. It also had the least peaks, and greatest separation. CBD analysis is important due to the rise in CBD market value and increased presence in brick-and-mortar stores. There is little regulation into CBD containing products and many claims go unsubstantiated. The findings of this project show that GCMS/MS is a great alternative to CBD product analysis. However, the methods of injection must be optimized to ensure greater resolution and separation.
Future Work • Determine THC content in commercial fluids • Evaluate the range of THC and CBD spectrum products that can present in commercial products • Determine thermal degradants in the oils and flavorings
Conclusion Commercial CBD products can be analyzed and quantified using DSI GCMS/MS. This is evidenced by results for separation that are comparable to those seen in the autosampler and other current methods used. The use of DSI is novel in that allows for viscous compounds like butters can be successfully analyzed with minimal sample prep.
References 1. 2. 3. 4.
5. 6.
PG vs VG Eliquid and how it affects Vaporizer performance, https://veppocig.com/pg-vs-vgeliquid/, (accessed May 6, 2021). JUULpods and E-Liquid FAQs - JUUL Support, https://support.juul.com/home/learn/faqs/juulpodbasics, (accessed May 6, 2021). Bowen, A.; XING, C. Nicotine Salt Formulations for Aerosol Devices and Methods Thereof. CA2909967A1, November 13, 2014. Čajka, T., Maštovská, K., Lehotay, S. J., and Hajšlová, J. (2005) Use of automated direct sample introduction with analyte protectants in the GC–MS analysis of pesticide residues. Journal of Separation Science 28, 1048–1060. Why Is CBD Everywhere? https://www.nytimes.com/2018/10/27/style/cbd-benefits.html (accessed April 10 2021). CBD Quality Control Measures https://www.news-medical.net/life-sciences/CBD-Quality-Control-Measures.aspx (accessed April 10 2021).
Acknowledgments • • • •
Hofstra University Honor’s College Research Assistantship Program Donors Lister Fellowship Chemistry Department Dr. Ling Huang, Dr. Kevin Bisceglia