QUANTITATIVE ANALYSIS OF THC AND RELATED CANNABINOIDS IN MULTIPLE MATRICES

USING SIMPLIFIED SOLID PHASE EXTRACTION WITH UPLC/MS/MS

Xin Zhang, Jonathan P. Danaceau, Kim Haynes and Erin Chambers
Waters Corporation, Milford, MA, USA

Matrix-Specific Protocol Guide

Recovery and Matrix Effects Calibration Curves for THC-COOH
INTRODUCTION SAMPLE PREPARATION RESULTS
Cannabis continues to be a highly abused recreational drug. In Urine samples: Pretreatment: Glucuronide hydrolysis: 40 µL internal
addition, the increasing number of states legalizing it for medical standard mix was added to 2 mL spiked human urine sample in a glass
Chromatography R2 = 0.998
use, combined with the trend towards legalization for recreational vial, followed by 2.4 mL 0.1M potassium phosphate buffer (pH 6.8)
Urine
purposes means than analytical methods for the quantification of Δ- containing 10 µL β-Glucuronidase. Vials were capped, vortex mixed,
9-tetrahydrocannabinol (THC), its metabolites and related and incubated at 37°C for 16 hours. 150 µL of 10M NaOH was then 20160401_BEHC18_39 1: MRM of 6 Channels ES+
added, and samples were hydrolyzed in a dry for 30 min at 70 °C. Once 3.18 TIC
cannabinoids continue to be necessary in clinical research. Among 3.41e6
drugs of abuse, natural cannabinoids present some unique the samples had cooled, 850 µL glacial acetic acid was added to
neutralize the samples. Solid Phase Extraction: 500 µL pretreated THC-COOH
analytical challenges. Excreted THC and related compounds are THC-COOH
highly glucuronidated, requiring efficient deconjugation before sample (equivalent to 180 µL urine) was directly applied to the Oasis THC
PRiME μElution plate. All wells of the SPE plate were then washed 2.45
analysis. In addition, the highly hydrophobic nature of natural
with 2 x 300 μL aliquots of 25% methanol. The samples were then
cannabinoids makes them exceptionally susceptible to loss via non- eluted with 2 x 25 μL aliquots of 60:40 ACN:IPA and diluted with 50
specific binding, meaning that care must be taken with sample μL of water.
handling and processing of prepared extracts. Finally, matrix R2 = 0.998

%
effects can be a challenge to control for these compounds, and can 2.41 Plasma
Plasma samples: Pretreatment: 200 µL 0.1% FA in ACN was
vary significantly in different biological matrices. THC-COOH
added to 100 µL spiked plasma to precipitate out the protein in a micro
This work uses a novel reversed-phase solid phase extraction centrifuge tube. Then the mixture was vortexed for 5 seconds and
(SPE) sorbent, Oasis PRiME HLB, which has been developed to THC-OH
centrifuged for 5 min at 7000 rcf. The supernatant was then diluted
enable simpler and faster SPE protocols, while at the same time with 400 µL water. Solid Phase Extraction: The entire pre-treated
generating cleaner extracts than other sample preparation sample was directly loaded on to the Oasis PRiME HLB µElution plate 0 Time
methods. 3 step load-wash-elute SPE protocols, eliminating without conditioning or equilibration. All wells were then washed with 2 x 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50
conditioning and equilibration, were successfully employed to 250 μL aliquots of 25:75 methanol:water. All the wells were then
extract THC, THC-OH and THC-COOH from multiple matrices, eluted with 2 x 25 μL aliquots of 90:10 ACN:MeOH and diluted with R2 = 0.999
including plasma, oral fluid (OF), whole blood and urine. 50 μL of water prior to analysis.
Figure 1. Chromatography of OH-THC, COOH-THC
Whole Blood
and THC analyzed on an ACQUITY UPLC® BEH C18
Blood samples: Pretreatment: 100 µL spiked whole blood was column (1.7 µm; 2.1 x 50 mm). Retention times for THC
METHODS added to 25 µL of a solution of 0.1 M zinc sulfate/ammonium acetate, -OH, THC-COOH and THC were 2.41, 2.45, and 3.18
THC-COOH
and the mixture was vortexed for 5 seconds to lyse the cells. All minutes, respectively. 0.1-100 ng/mL
samples were then precipitated by adding 375 µL 0.1% formic acid in
INSTRUMENTAL CONDITIONS ACN. The entire sample was vortexed for 10 seconds and centrifuged
UPLC System: ACQUITY UPLC® I-Class-FL for 5 min at 7000 rcf. The supernatant was then diluted with 800 µL Phospholipid Removal from Plasmsa
water. Solid Phase Extraction: The entire pretreated sample was
MS: XEVO TQ-S directly loaded on to the Oasis PRiME HLB µElution plate in 2 aliquots 20160401_BEHC18_03 2: MRM of 11 Channels ES+
TIC
without conditioning or equilibration. All wells were then washed with 2 x R2 = 0.999
Column: ACQUITY UPLC® BEH C18, 1.7 100 7.58e8

µm; 2.1 x 50 mm
250 μL aliquots of 25:75 methanol:water. All the wells were then A THC-COOH Oral Fluid

Mobile Phase A (MPA) 0.1% Formic Acid in Water

eluted with 2 x 25 μL aliquots of 90:10 ACN:IPA and diluted with 50
μL of water. THC
THC-COOH
DISCUSSION

%
Mobile Phase B (MPB) 0.1% Formic Acid in ACN Oral fluid samples: Pretreatment: Oral fluid samples were THC-OH Different matrices require different pretreatment protocols, such as
collected with Quantisal collection device from Immunalysis. The deconjugation for urine samples. Oral fluid required an addition of
Column Temp: 40 ˚C7
collection applicator was saturated with spiked oral fluid, and then 2.32 ACN for complete extraction of THC from the collection device.
Sample Temp: 10 ˚C placed in a collection vial, which contained 3.0 mL of sample 2.23 3.04
Figure 4. Calibration curves for THC-COOH in urine, plasma, Blood and plasma require PPT to disrupt protein binding and release
0
stabilization buffer. Per Quantisal instruction, this was claimed to be the
Strong Needle Wash 2% Formic Acid in equivalent of collecting 1.0±0.1 mL of sample. 1 mL acetonitrile was
1.00
20160401_BEHC18_29
2.00 3.00 4.00 5.00
2: MRM of 11 Channels ES+
whole blood and oral fluid, respectively. In all cases R2 values were all analytes. Once the samples were done with pretreatment, the SPE
70:30 ACN:Water >0.998 indicating excellent linearity procedure with Oasis PRiME HLB is as simple and straightforward as
then added to the collection vial to help improve THC extraction. The 100
5.60 TIC
collection kit was stored in a refrigerator overnight. 500 µL aliquots of 7.58e8 LOAD, WASH and ELUTE! With the unique µElution plate, non-
Weak Needle Wash 10% ACN
buffer stabilized oral fluid samples (equivalent to 100 µL oral fluid) were B specific binding were minimized with no evaporation or reconstitution.
pre-treated by adding 200 µL 4% H3PO4 and 10 µL of working IS mixture Accuracy and Precision—Whole Blood Samples
Solid Phase Extraction: The entire pre-treated sample was directly THC-OH (0.1- THC-COOH (0.1- THC (0.05-100ng/
Table 1. MS Parameters for Cannabinoid analysis N=6
%

loaded on to the Oasis PRiME HLB µElution plate without conditioning 2.33 2.58 4.99 5.34 100ng/mL) 100ng/mL) mL)
or equilibration, followed by washing with 2 x 250 μL 5% NH4OH in 2.67
2.72 4.66 QC Level
MS parameters for all analytes and internal standards 25:75 methanol:water. All the wells were then eluted with 2 x 25 μL %Acc. %RSD % Acc. %RSD % Acc. %RSD
90:10 ACN:MeOH and diluted with 50 μL of water. A CORTECS C18 (ng/mL) CONCLUSIONS
Cone column was used to minimize matrix effects. 0 Time 0.375 97.9 0.6% 105.8 8.1% 108.2 3.0%
Analyte MRM transitions (m/z) Coll V 1.00 2.00 3.00 4.00 5.00
(V)
2 96.0 3.7% 94.7 2.3% 100.5 3.7%  Optimized protocols for urine, plasma, whole blood and oral
331.3>313.1 40 18 Figure 2. Chromatogram of phospholipid removal from fluid
THC-OH 7.5 100.0 2.7% 98.9 2.8% 98.9 1.4%
331.3>193.1 40 30 plasma samples. A. Residual phsopholipids (black trace)  A novel SPE sorbent extraction that is easy to perform, fast
THC-OH-d3 334.3>316.1 40 18 and cannabinoids (Orange trace) from an plasma sample Figure 3. Recovery and Matrix Effects from Urine (A), 20 99.3 3.2% 100.2 2.1% 97.8 1.2% and clean
345.3>327.3 50 20 extracted using Oasis PRiME HLB. B. Residual plasma (B), whole blood (C), and oral fluid (D). With  Elimination of phospholipids from SPE extracts
THC-COOH 37.5 96.5 2.2% 101.2 3.0% 94.2 0.7%
345.3>299.3 50 25 phospholipids remaining in a plasma sample prepared by one exception, recoveries of all analytes in all  Demonstrated linear. accurate and precise data in the analysis
precipitation with 2:1 ACN:plasma. Phospholipid traces in A matrices were >75% and all %CVs were less than 8%. Mean 98 2% 100 4% 99.9 2%
THC-COOH-d3 348.3>330.3 50 20 of THC and its metabolites in multiple matrices.
315.1>193.2 40 25
and B are at the same scale to demonstrate the removal by In all cases, matrix effects for the final methods were  Elimination of evaporation and reconstitution steps minimizes
THC SPE. The cannabinoid traces show the potential co-elution negligible, even without IS correction, demonstrating Table 2. Quality Control results for cannabinoids in whole blood. In
315.1>135.1 40 25 the risk of non-specific binding during reconstitution
with residual plasma phospholipids if they are not removed the high degree of cleanliness achieved with Oasis most all cases, accuracies were within 5% and %RSDs were less
THC-d3 318.1>196.2 40 25
PRiME HLB. than 5%. Similar results were seen with the other matrices.
prior to analysis.

For research use only. Not for use in diagnostic procedures

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