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CLINICAL/FORENSICS ARTICLE

Analyze and Confirm Cannabinoids by LC/MS/MS

Using an Allure® Biphenyl Column

by Kristi Sellers, Clinical/Forensic Innovations Chemist, Becky Wittrig, Ph.D., HPLC Product Marketing Manager, and André Schreiber, Ph.D., Application Chemist, Applied Biosystems
  • Faster sample throughput (short analysis time, no derivatization)
  • Reliable response at 1ng on-column
  • Undisputable identification, using two +MRM transitions

As marijuana is smoked, the main psychoactive component, Δ9-tetrahydrocannabinol (Δ9-THC), is quickly absorbed and metabolized to 11-hydroxy-Δ9-tetrahydrocannabinol (hydroxy-THC), an active metabolite. Hydroxy-THC is further metabolized, rapidly, to 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (carboxy-THC), an inactive metabolite commonly found in urine, blood, hair, and tissues.¹ GC/MS often is used for confirming and quantifying Δ9-THC and carboxy-THC²; however, GC/MS methods require time-consuming steps, like derivatization, to obtain acceptable chromatography. By using HPLC, derivatization can be eliminated, saving time without sacrificing sensitivity.

We developed a quantitative method for analyzing underivatized cannabinoids by HPLC/tandem mass spectrometry. Our goals were threefold; 1) to optimize column selection, 2) to provide a short analysis time, and 3) to obtain reliable confirmation and quantification data in the low nanogram range (< 10ng). We used an Applied Biosystems API 3200 MS/MS detector coupled to a Shimadzu LC20AD Prominence Series chromatograph for optimum chromatographic and detection capabilities.

Figure 1 shows the final product spectra for Δ9-THC and carboxy-THC used to develop the +MRM (multiple reaction monitoring) method.³ We determined the 30mm, 2.1mmID, 3µm Allure® Biphenyl HPLC column to be the best column for this analysis. This column employs a unique separation mechanism, π-π interaction, which greatly improves selectivity and retention, relative to conventional C18 phases. In addition, with the increased retention of the biphenyl phase, higher amounts of methanol can be used in the mobile phase. This noticeably increases sensitivity when using an electrospray interface.

The Allure® Biphenyl column provides good resolution of all compounds in less than 5 minutes — including baseline resolution of Δ9-THC and cannabidiol, which have very similar product ion spectra and +MRM transitions (Figure 2). By using MS/MS detection, we were able to target two +MRM transitions per compound to verify compound identity at approximately 1ng on-column. Table 1 shows the +MRM transitions and the source conditions for approximately 1ng each of several cannabinoid metabolites.

Based on this work, we conclude an Allure® Biphenyl column, coupled with an API MS/MS 3200 detector and a Shimadzu LC20AD Prominence, can be used to quantify low levels of cannabinoid analytes from underivatized sample, and can achieve baseline separation of Δ9-THC and cannabidiol, in less than 5 minutes.


Table 1  MRM transitions for THC and metabolites: multiple transitions are monitored for each compound for definitive identifications.
Analyte
Q1 Mass
Q3 Mass
Time (ms)
DP (V)
EP (V)
CE (V)
CXP (V)
Hydroxy-THC (MRM1)
331.2
313.1
100
36
5
21
10
Hydroxy-THC (MRM2)
331.2
193.1
100
36
5
35
6
Carboxy-THC (MRM1)
345.2
327.0
100
41
4.5
21
10
Carboxy-THC (MRM2)
345.2
299.3
100
41
4.5
25
6
Cannabidiol (MRM1)*
315.2
193.2
100
36
4.5
31
6
Cannabidiol (MRM2)*
315.2
123.2
100
36
4.5
43
6
Cannabinol (MRM1)
311.2
223.0
100
46
8.5
27
8
Cannabinol (MRM2)
311.2
222.5
100
46
8.5
37
10
Δ9-THC (MRM1)*
315.2
193.2
100
41
4.5
33
6
Δ9-THC (MRM2)*
315.2
123.1
100
41
4.5
43
6
Δ9-THC-d3 (MRM1)
318.3
196.3
100
36
4.5
31
6
Δ9-THC-d3 (MRM2)
318.3
123.2
100
36
4.5
43
6
*Note, cannabidiol and Δ9-THC share the same transitions, but are separated chromatographically.
DP — declustering potential, EP — entrance potential, CE — collision energy, CXP — collision cell exit potential


Figure 1  Final product spectra used in developing MRM transitions for compound identification and optimized sensitivity.

Δ9-THC


LC_PH0422

Carboxy-THC


LC_PH0423
For Conditions see Figure 2.


Figure 2  Fast, selective separation of Δ9-THC and its metabolites, using an Allure® Biphenyl HPLC column (extracted ion chromatography).
  1. 11-hydroxy-Δ9-tetrahydrocannabinol
  2. cannabidiol
  3. 11-nor-9-carboxy-Δ9-tetrahydrocannabinol
  4. cannabinol
  5. Δ9-tetrahydrocannabinol
  6. Δ9-tetrahydrocannabinol-d3

LC_PH0424
Sample:
Inj.:
10µL
Conc.:
100ng/mL each component
Sample diluent:
mobile phase
Column:
Allure® Biphenyl
Cat. #:
9166332
Dimensions:
30 x 2.1 mm
Particle size:
3µm
Pore size:
60Å
Conditions:
Mobile phase:
A: 0.1% formic acid in water
B: 0.1% formic acid in methanol
Time (min.)
%B
0.5
2.5
5.0
6.0
6.1
50
90
90
50
—
Flow:
0.5mL/min.
Temp.:
ambient
Det.:
Spectrum Model (Sciex API 3200 Triple Quadrupole)
Interface:
ESI
Ion mode:
positive
Temp.:
500°C
Ion source:
5500V

References

  1. Abbara, C., R. Galy, A. Benyamina, M. Reynaud and L. Bonhomme-Faivre, Development and validation of a method for quantitation of Δ9-tetrahydrocanabinol in human plasma by high performance liquid chromatography after solid phase extraction J. Pharma. Biomed. Anal. 41 (2006) 1011-1016.
  2. Sellers, K. Reliably Confirm Cannabinoids by GC/MS Restek Advantage 2006.04 (2006) 16-17.
  3. Weinmann, W., S. Vogt, R. Goerke, C. Muller and A. Bromberger, Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LS/MS/MS Forens. Sci. Intl. 113 (2000) 381-387.