The Stability of Acid Phytocannabinoids Using Electrospray Ionization LC–MS in Positive and Negative Modes

April 10, 2020
Volume: 
3
Issue: 
3
Figure 1: Decarboxylation of phytocannabinoids. THCA (top left) spontaneously decarboxylates to THC upon exposure to heat. Similarly, CBDA (lower left) spontaneously decarboxylates to CBD.
Table I: UHPLC mobile phases and quaternary gradient
Table II: LC-TOF parameters
Abstract / Synopsis: 

In this study, we evaluated the stability of carboxylated phytocannabinoids analyzed with positive and negative electrospray (ESI+ and ESI-, respectively) liquid chromatography–mass spectrometry (LC–MS) as a function of drying gas temperature. We used both chromatographic and flow injection analysis (FIA) conditions. Post-chromatographic (in-source) acid decarboxylation was not observed over a drying gas temperature range of 75 °C through 300 °C in ESI+ mode under chromatographic conditions. FIA experiments further demonstrated no correlation between in-source acid decarboxylation in ESI+ mode over a drying gas temperature range of 100 °C through 350 °C. Conversely, chromatographic experiments in ESI- mode did reveal a decrease in tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) relative abundances and concomitant increases in the relative abundances of tetrahydrocannabinol (THC) and cannabidiol (CBD) as a function of drying gas temperatures. These data highlight a potential concern for the quantitation of acid phytocannabinoids using ESI- mode LC–MS technologies 

The Cannabis spp. genome encodes synthase enzymes (1) for the synthesis of carboxylated phytocannabinoids such as Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabigerolic acid (CBGA). In the living plant, psychoactive Δ9-tetrahydrocannabinol (THC) does not exist at significant concentrations nor does cannabidiol (CBD)—another cannabinoid that has garnered interest in recent years. THC and CBD are formed through decarboxylation of THCA and CBDA, respectively, through exposure to light, heat, and curing processes (2). Figure 1 illustrates the acid to neutral decarboxylation of THCA and CBDA. The reaction order, ko, and the activation energy (EA) of acid -> neutral decarboxylation has been determined over specific temperatures and times in C. sativa flowering buds (3). Chemical testing can also influence cannabinoid acid decarboxylation if the testing system exposes the sample to heat (4) as in the injection port of a gas chromatography (GC) system. Liquid chromatography–mass spectrometry (LC–MS) systems also expose the acid phytocannabinoids to heat in the electrospray ionization (ESI) source region. To our knowledge, the effect on acid decarboxylation as a function of temperature in the ESI source region has not been reported. In fact, a PubMed query of “phytocannabinoid decarboxylation in the ESI source” and related queries yielded zero results. One study evaluated the changes in chemical profiles of extracts at ambient temperature versus decarboxylation at a series of temperature ranges (5). Our study used ESI LC–MS in both positive and negative modes to determine the effect of electrospray source temperature on the stability of acid phytocannabinoids in neat solutions. (See upper right for Figure 1, click to enlarge.)

Experimental
We make the following assumption: under the conditions of a high performance liquid chromatography (HPLC) method, decarboxylation is mitigated and acid phytocannabinoids are stable under the time frame of the analysis as they transport across the column stationary phase. The null hypothesis states the acid phytocannabinoids are equally stable and remain relatively unchanged in the ESI source. To reject the null hypothesis, we must observe a decrease in the relative abundance of the carboxylated species with a commensurate increase in the relative abundance of the decarboxylated analogues. To make this determination, we used electrospray ionization in both positive and negative modes under chromatographic and flow injection analysis (FIA) conditions. In the various experiments, the drying gas temperature was varied from 75 °C through 350 °C and we monitored (M+H)+ and (M-H)- pseudomolecular ions and (M+Na)+ adducts specific to the carboxylated and decarboxylated chemical species.

Standards and Reagents
THCA, THC, CBDA, and CBD were obtained from Cerilliant. All cannabinoid reference standards were obtained in U.S. Drug Enforcement Administration (DEA) exempt format at 1.0 mg/mL in organic solvent. LC–MS grade methanol, acetonitrile, and formic acid were obtained from Agilent Technologies. Millipore de-ionized (DI) water was used for the aqueous mobile phase.

Analytical Instrumentation
Low Resolution LC–MS Experiments
An LC–MS system (Agilent Technologies) equipped with a diode array detector (DAD) and a nebulizer ESI source was used. The mass spectrometer was operated in ESI+ and ESI- modes over the mass range of 100–500 m/z. For each experiment, the drying gas temperatures were 100 °C, 150 °C, 200 °C, 250 °C, and 300 °C. The LC–MS conditions are shown below.

LC Conditions:
Column: Agilent InfinityLab Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm
Mobile Phase: A) 0.1% (V/V) formic acid aqueous phase; B) 0.05% (V/V) formic acid organic phase
Flow rate: 1.0 mL/min
Column temperature: 50 °C
Injection volume: 5 µL
Diode array detector: 230 nm and 270 nm

iQ LC/MSD Parameters:
Drying gas flow: 13 L/min
Nebulizer: 55 psig
Vcap: 3500

LC Mobile Phase Gradient:
Time (min)                    %B
0                                   60
1.0                                60
7.0                                70
8.2                                95

High Resolution Accurate Mass (HRAM) FIA Experiments
A quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies) equipped with a dual ESI source was used. The QTOF was operated in ESI+ mode over the mass range of 100–500 m/z. Samples were directly infused into the electrospray source with a syringe pump at a rate of 200 µL/h. The drying gas temperature was incremented stepwise at 50 °C intervals from 100 °C through 350 °C. The QTOF parameters and drying gas temperature program are shown below. 

QTOF LC–MS Parameters:
Drying gas flow: 12 L/min
Nebulizer:30 psig
Vcap: 4000
Fragmentor: 150
Skimmer: 65
Acquisition rate: 1 Hz

Drying Gas Temperature Program:
0.0 to 1.5 min, Hold 100 °C
1.5 to 2.5 min, Gradient to 150 °C
2.5 to 3.5 min, Hold 150 °C
3.5 to 4.5 min, Gradient to 200 °C
4.5 to 5.5 min, Hold 200 °C
5.5 to 6.5 min, Gradient to 250 °C
6.5 to 7.5 min, Hold 250 °C
7.5 to 8.5 min, Gradient to 300 °C
8.5 to 9.5 min, Hold 300 °C
9.5 to 10.5 min, Gradient to 350 °C
10.5 to 11.5 min, Hold 350 °C

HRAM Chromatographic Confirmation Experiments
An ultrahigh-pressure liquid chromatography (UHPLC) system and time-of-flight (LC/TOF) mass spectrometer (Agilent Technologies) equipped with a dual ESI source was used. The column was an Agilent Poroshell 120 EC-18 3.0 mm x 50.0 mm, 2.7 µm particle size (PN 699975-302). The flow rate was 0.5 mL/min. The injection volume was 5 µL. The TOF mass spectrometer was operated in ESI- mode over the mass range of 100–1000 m/z with reference mass on. For each experiment, the drying gas temperatures were 75 °C, 100 °C, 175 °C, 200 °C, and 250 °C. The LC/TOF conditions are given in Tables I and II. (See upper right for Table I and Table II, click to enlarge.)

References: 
  1. H. van Bakel, et al., Genome Biol. 12, R102 (2011).
  2. H. Perrotin-Brunel, et al., J. Molecular Structure 987, 67–73 (2011).
  3. M. Wang, et al., Cannabis and Cannabinoid Research 1.1, 262–271 (2016).
  4. C. Lanz, et al., PLoS One 11(1), e0147286 (2016).
  5. M.M. Lewis, et al., ACS Omega 2, 6091−6103 (2017).

Peter J.W. Stone, Sue D’Antonio, Nikolas C. Lau, and Wendi A. Hale are with Agilent Technologies in Santa Clara, California. Anthony Macherone is with Agilent Technologies and The Johns Hopkins School of Medicine in Baltimore, Maryland. Direct correspondence to: [email protected]

How to Cite this Article

P.J.W. Stone, S. D’Antonio, N.C. Lau, W.A. Hale, and A. Macherone, Cannabis Science and Technology 3(3), 34–40 (2020).