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Stability Study of Mixed Neutral and Acidic Cannabinoid Standards

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Abstract

This study examines the stability of working solutions prepared by combining neutral and acidic cannabinoid standards. Stability was assessed for 16 cannabinoids across 30 days for solutions prepared in commonly used diluents and stored under typical lab conditions.

Introduction

To ensure accurate reporting, it is essential that the stability of cannabinoid standards be studied. For example, if improperly manufactured or stored, tetrahydrocannabinolic acid (THCA) will convert to its methoxy counterpart, tetrahydrocannabinol (THC), giving invalid results for both analytes. To extend stability and simplify the preparation of working standards, Restek has formulated two certified reference materials (CRMs) comprised of 16 commonly analyzed cannabinoids. These standards are formulated in two high-concentration (1000 µg/mL) solutions: a cannabinoids acids 7 mix (cat.# 34144) and a cannabinoids neutrals 9 mix (cat.# 34132). Using these two CRMs instead of 16 single-compound standards saves labs time and money, and minimizes preparation errors. More important, because the acids and neutrals are formulated separately in appropriate diluents instead of as a single solution, long-term stability can be increased by preventing decarboxylation of the acids.

Stability studies on sealed ampuls of both the cannabinoid acids and neutrals CRMs have been performed by Restek under accelerated conditions for standard stability for up to four weeks. However, to assess the stability of working standards, the current study was conducted by combining the cannabinoid CRMs in commonly used diluents and storing the mixed solutions under typical lab conditions.

Experimental

The components of each cannabinoid CRM are given below. To measure the stability of a combined solution of Restek’s cannabinoids acids 7 standard (cat.# 34144) and cannabinoids neutrals 9 standard (cat.# 34132), duration, temperature, and diluent parameters (Figure 1) were selected to represent use in a typical production lab.  Peak response (area) data was then collected and compared. Data was normalized to Day 0 results, and the acceptance criterion was defined as ±5% of the original response on Day 0. 

Cannabinoids Acids 7 Standard (cat.# 34144)
1000 µg/mL, acetonitrile with 1% DIPEA and 0.05% ascorbic acid

  • Cannabichromenic acid (CBCA) (185505-15-1)
  • Cannabidiolic acid (CBDA) (1244-58-2)
  • Cannabidivarinic acid (CBDVA) (31932-13-5)
  • Cannabigerolic acid (CBGA) (25555-57-1)
  • Cannabinolic acid (CBNA) (2808-39-1)
  • Tetrahydrocannabinolic acid (THCA-A) (23978-85-0)
  • Tetrahydrocannabivarinic acid (THCVA) (39986-26-0)

Cannabinoids Neutrals 9 Standard (cat.# 34132)
1000 µg/mL in purge-and-trap methanol

  • Cannabichromene (CBC) (20675-51-8)
  • Cannabicyclol (CBL) (21366-63-2)
  • Cannabidiol (CBD) (13956-29-1)
  • Cannabidivarin (CBDV) (24274-48-4)
  • Cannabigerol (CBG) (25654-31-3)
  • Cannabinol (CBN) (521-35-7)
  • d8-Tetrahydrocannabinol (d8-THC) (5957-75-5)
  • d9-Tetrahydrocannabinol (d9-THC) (1972-08-3)
  • Tetrahydrocannabivarin (THCV) (31262-37-0)

 

Figure 1: Experimental Parameters for Testing the Stability of Prepared Cannabinoid Standards (50 ppm Mixtures of Acids and Neutrals)
Test periods are days 0, 10, 15, and 30. Storage conditions are -20, 0, 10, and 25 degrees Celsius. Diluents are acetonitrile, methanol, and acetonitrile:water (75:25).

 

Standard Solution Preparation

Prior to dilution, storage and handling of both cannabinoid CRMs followed their certificates of analysis (CoA), including the recommended storage condition of -20 ⁰C and sonication prior to use. Using aliquots from the same ampuls for each CRM, mixed standards were prepared at 50 ppm in each diluent to a final volume of 1 mL following the procedure in Figure 2. Each working standard was prepared in a 2 mL amber vial, capped, and then stored under one of the test conditions described in Figure 1. 

Figure 2: Sample Preparation of 50 ppm Working Standards (Combined Cannabinoid Acids and Neutrals CRMs)
Instructions for mixing 50 microliters of each standard together along with 900 microliters of diluent for a final volume of 1 milliliter working standard.

 

Analytical Conditions

Analytes were monitored using the following chromatographic conditions. On each test day, the prepared working standards were removed from their respective storage conditions, analyzed, recapped, and then returned to their storage conditions after analysis. 

Column: Raptor ARC-18 2.7 µm 150 mm x 4.6 mm (cat. # 9314A65)
Injection volume: 5 µL
Mobile phase A: Water, 5 mM ammonium formate, 0.1% formic acid
Mobile phase B: Acetonitrile, 0.1% formic acid
Flow rate: 1.5 mL/min
Instrument: Shimadzu Nexera X2
Detector: Shimadzu DAD @ 228 nm
Temperature: 30 °C
Gradient: Isocratic, 75% B
Run time: 11 minutes

Results and Discussion

The example chromatogram in Figure 3 shows good peak shape and separation for all 16 neutral and acidic cannabinoids on a Raptor ARC-18 column. Each working standard was injected in triplicate, and the area values for each cannabinoid were averaged and normalized to the average Day 0 area values to produce the relative response results presented in Tables I and II and Figures 4-10. Overall, the data indicate that the cannabinoids in the mixed acids and neutrals working standards were stable across most diluent and storage condition combinations. Responses for all analytes were within the ±5% criteria, except for CBDV, CBG, and CBD in room temperature storage (25 ⁰C) on day 30. In addition, it was observed that the acetonitrile:water (75:25) diluent data showed an increasing response, which suggests evaporation of the diluent.

Figure 3: Representative Chromatogram for a 50-ppm Mixed Standard Blended from Acidic and Neutral Cannabinoid CRMs (Room Temperature, Day 30).

cgarm-img
LC_GN0702
ColumnRaptor ARC-18 (cat.# 9314A65)
Dimensions:150 mm x 4.6 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Temp.:30 °C
Standard/SampleCannabinoids acids 7 standard, 1000 µg/mL, acetonitrile with 1% DIPEA and 0.05% ascorbic acid (cat.# 34144)
Cannabinoids neutrals 9 standard, 1000 µg/mL, P&T methanol (cat.# 34132)
Diluent:Acetonitrile
Conc.:50 ppm
Inj. Vol.:5 µL
Mobile Phase
A:Water, 5 mM ammonium formate, 0.1% formic acid
B:Acetonitrile, 0.1% formic acid
Time (min)Flow (mL/min)%A%B
0.001.52575
11.001.52575
DetectorShimadzu DAD @ 228 nm
InstrumentShimadzu Nexera X2
Sample PreparationTo prepare the working standards, 50 µL of the cannabinoids acids 7 standard (cat.# 34144); 50 µL of the cannabinoids neutrals 9 standard (cat.# 34132); and 900 µL of acetonitrile were aliquoted into 2 mL, screw-thread vials (cat.# 21143), capped with short-cap, screw vial closures (cat.# 24498), and stored at room temperature for 30 days.

 

Table I: Stability of Neutral Cannabinoids in Mixed Working Standards Stored Under Test Conditions (% Response Relative to Day 0). Green Indicates Result Passes the ±5% Acceptance Criteria, Yellow Indicates a Borderline Result, Red Indicates a Failing Result.

Storage Temperature - 20 ⁰C
0 ⁰C
Diluent  Acetonitrile
Methanol
Acetonitrile
Day 10 15 30 10 15 30 10 15 30
Cannabidivarin 103 101 102 98 99 98 102 102 104
Cannabigerol 103 102 103 99 98 98 102 103 103
Cannabidiol 103 102 102 98 98 98 102 102 103
Tetrahydrocannabivarin 103 102 102 98 99 98 104 102 103
Cannabinol 103 102 102 98 98 97 103 102 103
∆9-Tetrahydrocannabinol 104 103 102 98 98 97 101 101 101
∆8-Tetrahydrocannabinol 103 101 102 98 99 98 103 102 104
Cannabicyclol 104 103 102 99 99 97 102 102 103
Cannabichromene 104 102 102 98 98 97 102 102 103

 

Storage Temperature 10 ⁰C
25 ⁰C
Diluent  Acetonitrile
Methanol
Acetonitrile:
Water

Acetonitrile 
Day 10 15 30 10 15 30 10 15 30 10 15 30
Cannabidivarin 99 100 100 98 98 97 102 102 102 101 98 88
Cannabigerol 99 102 101 98 99 99 102 103 103 99 95 87
Cannabidiol 99 101 101 97 98 98 102 103 103 102 99 93
Tetrahydrocannabivarin 99 101 101 97 98 98 102 103 104 103 102 103
Cannabinol 99 102 102 96 98 98 103 103 104 103 102 104
∆9-Tetrahydrocannabinol 99 102 102 96 98 98 102 103 104 103 102 103
∆8-Tetrahydrocannabinol 99 101 101 97 98 98 103 103 104 104 102 105
Cannabicyclol 99 102 102 96 98 98 102 103 104 104 103 104
Cannabichromene 99 102 102 96 98 98 102 103 104 104 103 105

 

Table II: Stability of Acidic Cannabinoids in Mixed Working Standards Stored Under Test Conditions (% Response Relative to Day 0). Green Indicates Result Passes the ±5% Acceptance Criteria, Yellow Indicates a Borderline Result, Red Indicates a Failing Result

Storage Temperature - 20 ⁰C 0 ⁰C
Diluent Acetonitrile Methanol Acetonitrile
Day 10 15 30 10 15 30 10 15 30
Cannabidivarinic acid 102 100 100 98 99 97 100 100 101
Cannabidiolic acid 102 100 100 98 98 97 101 101 101
Cannabigerolic acid 102 100 100 98 97 97 100 100 101
Tetrahydrocannabivarinic acid 102 100 101 98 98 97 100 100 101
Cannabinolic acid 101 101 101 98 98 97 100 101 101
Tetrahydrocannabinolic acid 102 104 100 98 98 97 100 100 101
Cannabichromenic acid 102 102 101 98 98 97 101 101 101

 

Storage Temperature 10 ⁰C 25 ⁰C
Diluent Acetonitrile Methanol Acetonitrile:
Water
Acetonitrile
Day 10 15 30 10 15 30 10 15 30 10 15 30
Cannabidivarinic acid 97 99 99 96 98 97 99 100 101 101 99 95
Cannabidiolic acid 98 100 100 96 98 97 101 102 102 101 99 96
Cannabigerolic acid 98 99 99 96 97 97 101 102 103 101 98 95
Tetrahydrocannabivarinic acid 98 99 100 96 97 98 100 101 102 101 99 99
Cannabinolic acid 99 100 101 96 97 98 100 102 103 102 101 104
Tetrahydrocannabinolic acid 99 100 100 96 98 97 100 101 102 101 99 100
Cannabichromenic acid 100 101 101 97 98 98 101 102 103 103 101 103

 

Figure 4: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Acetonitrile, Stored at -20 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in acetonitrile and stored at -20 degrees Celsius.

 

Figure 5: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Acetonitrile, Stored at 0 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in acetonitrile and stored at 0 degrees Celsius

 

Figure 6: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Acetonitrile, Stored at 10 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in acetonitrile and stored at 10 degrees Celsius.

 

Figure 7: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Acetonitrile, Stored at 25 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in acetonitrile and stored at 25 degrees Celsius.

 

Figure 8: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Methanol, Stored at 10 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in methanol and stored at 10 degrees Celsius.

 

Figure 9: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Methanol, Stored at -20 ⁰C, and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in methanol and stored at -20 degrees Celsius.

 

Figure 10: Percent Response Results Relative to Day 0 for 16 Cannabinoids at 50 ppm in a Mixed Standard Prepared in Acetonitrile:Water (75:25); Stored at 10 ⁰C; and Analyzed on Days 0, 10, 15, and 30
chart showing relative responses of 16 cannabinoids for working standard prepared in acetonitrile:water (75:25) and stored at 10 degrees Celsius.

Conclusion

A certificate of analysis for a CRM gives the expiration date of a properly stored, unopened ampul. Once opened, the solutions should be transferred to an appropriate vial and stored under recommended conditions between use. Like all analytes, cannabinoid stability is affected by the environmental and handling conditions to which the CRM and working standards are exposed. While Restek has completed stability studies on both sealed CRM ampuls and mixed working standard containing both acidic and neutral cannabinoids, it is strongly advised that labs conduct their own stability studies and implement SOPs to address the use and handling of CRMs and the preparation of working standards. During the current study, the cannabinoids were generally stable across the test conditions; however, for the most accurate results we recommend, especially when combining multicomponent ampuls, that standards be prepared fresh daily to prevent degradation. In addition, having technicians who prepare samples also prepare their own QC samples and calibration curves is a best practice because it can reduce variation. More information about Restek reference standard manufacturing and testing can be found at https://www.restek.com/articles/restek-reference-standards.

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