Rapid and Simple Confirmation and

Quantification of 11-Nor-∆ 9

-Tetrahydrocannabinol-9-Carboxylic Acid
(THC-COOH) in Human Urine by 2D-LC-MS/MS
Berit P Jensen, Mark Lewis & Grant Moore
Toxicology, Specialist Cluster, Canterbury Health Laboratories, Christchurch, New Zealand.
Email: berit.jensen@cdhb.health.nz

INTRODUCTION RESULTS
Example chromatograms:
• Cannabinoids are among the most commonly detected compounds in toxicology
screening programs and workplace drug testing.
• Urine is tested for the 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) External cut-off control (15 ng/ml)
metabolite along with its glucuronides.
• For workplace drug testing, a positive confirmation requires THC-COOH >15ng/ml and the
ratio of 2 qualifier ions within 30% of those of the cut-off control.
• Traditionally this assay involved hydrolysis, solid phase extraction, evaporation of solvents,
derivatisation and finally detection by GC-MS, a time- and labour-intensive process.

AIM
• To develop a rapid and simple 2D-LC-MS/MS method for THC-COOH in human urine to be
used in routine medical screening and workplace confirmations adhering to AS/NZS
4308:2008 standards.

Patient sample (745 ng/ml)

METHODS
Sample preparation directly in vial:

175 µl urine + 25 µl 1 µg/ml D9-THC-COOH + 25 µl 2M NaOH

Heat 15min @ 500C

Add 125 µl 2% formic acid

Inject 20 µl on 2D LC-MS/MS

Online sample clean-up (back flush):

Position 1 (Load) Position 2 (Elute)
Example standard curve:
Column A Column B Column A Column B THC - A - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 6 QCs
9 y = 0.008344 - χ -0.021793
8.5 R 2 = 0.99739378
Pump 1 Pump 1 8 Type: Linear, Origin: Ignore, Weight: 1/χ
7.5
Relative responses

Pump 2 7
ALS ALS Pump 2 6.5
6
5.5
22 11 22 11 5
4.5
Waste 33 66 Waste 33 66 4
44 55 44 55 3.5
3
2.5
2
1.5
1
Mass Spec Mass Spec 0.5
0
-0.5
-50 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 9501000 1050
Concentration (THC-COOH (ng/ml))
LC conditions
Trapping column Zorbax Eclipse Plus-C18, 2.1 x 12.5 mm, 5 µm, room temp.
Validation:
Analytical column Poroshell 120 EC-C18, 2.7 µm, 3.0 x 50 mm, 50°C
Mobile phase A 0.2% formic acid + 10mM ammonium formate in H2O
Range in human urine: 5-1000 ng/ml THC-COOH
Mobile phase B Acetonitrile
Calibration curve: Linear weighted 1/x, r2≥0.996
Time Pump 1 (loading pump) Pump 2 (Eluting pump) Valve Position Limit of detection: 0.5 ng/ml
(min) B (%) Flow rate (ml/min) B (%) Flow rate (ml/min)
0.0 35 0.1 85 0.6 Load Matrix effect: 95-105%
0.1 35 2.0 85 0.6 Load Recovery: 100%
0.35 35 2.0 85 0.6 Elute Hydrolysis of glucuronides: 100%
0.4 35 2.0 85 0.6 Elute
Carry-over <0.3%
0.5 35 0.1 95 0.6 Elute
Intra-and interday Precision and bias <5% (<10% at LLOQ)
0.9 35 0.1 95 0.6 Elute
1.0 35 0.1 85 0.6 Elute External QC program Results within ± 10%
1.24 35 0.1 85 0.6 Load Corr. with previous assay r2=0.997, deviation <12% (n=115)
Autosampler stability Stable at 4°C for >3 days

MS conditions
Instrument Agilent 6460 QQQ LC/MS with Agilent JetStream
Ion mode Negative
MRM Quantifier Qualifier 1 Qualifier 2 CONCLUSION
THC-COOH 343.2/299.2 343.2/245.1 343.2/191.1
An assay for confirmation and quantification of THC-COOH in human urine was developed:
D9-THC-COOH 352.2/208.2 353.2/254.1 353.2/194.1

• Simple sample preparation due to alkaline hydrolysis directly in the injection vial.
COOH COOH • 2 min total runtime using robust online sample clean-up (2D-LC-MS/MS).
OH OH
• Excellent correlation with previous method and external QC samples.
H3C D3C
H3C O CH3 D3C O CD3
The assay is to be implemented in routine use analysing >80 samples per day.
11-nor-9-carboxy- -THC9
11-nor-9-carboxy- -THC-D9
9