THC-COOH Quantification in Urine UsingDilute and Shoot LC ...
[Pages:4]Application Note: 548
THC-COOH Quantification in Urine Using Dilute and Shoot LC-MS/MS Method for Forensic Toxicology
Samuele Scurati, Thermo Fisher Scientific, Rodano, Italy Maura Brambilla, Ludovico D'Amato, Paolo Brambilla, University Department of Laboratory Medicine, Hospital of Desio, Desio, Italy
Key Words
? TSQ Quantum Access MAX
? Accela Pump
? Cannabinoids
? Forensic Toxicology
Introduction
Cannabis sativa is a widely used drug of abuse. Tetrahydrocannabinol (THC) is the major psychoactive chemical compound in the cannabis plant. After smoke inhalation, THC is absorbed and distributed in blood. Subsequently, it is rapidly metabolized to THC-COOH, conjugated with glucuronic acid, and excreted through urine. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered a useful tool to establish the consumption of cannabis by the assessment of THC-COOH in urine for forensic toxicology purposes.
Goal To develop a reliable and fast analytical method for the quantitative determination of THC-COOH in urine using a Thermo Scientific TSQ Quantum Access MAX triple stage quadrupole mass spectrometer.
Experimental
Sample Preparation A urine sample was hydrolyzed with 10M NaOH and heated at 60 ?C for 15 minutes. The pH was restored with Fisher Chemical acetic acid. Hydrolyzed samples as well as calibrators were diluted 1:10 in Fisher Chemical water/acetonitrile (1:1). Then, 10 ?L were directly injected. Quantitative analysis was performed on the basis of calibration curves prepared in urine, ranging from 7.8 to 1000 ng/mL. Calibrators were injected in duplicate.
UHPLC conditions Liquid chromatography separation was performed using a Thermo Scientific Accela autosampler and pump. The sample was injected directly on a Thermo Scientific Hypersil GOLD column (50 ? 2.1 mm, 1.9 m). A gradient LC method used mobile phases A (0.1% aqueous formic acid) and B (Fisher Chemical Optima LC/MS acetonitrile) at a flow rate of 300 L/min. The run time was 6 minutes.
Mass Spectrometry MS analysis was carried out on a TSQ Quantum Access MAXTM triple stage quadrupole mass spectrometer equipped with a Thermo Scientific Ion Max source with a heated electrospray ionization (HESI) probe. The MS conditions were as follows:
Scan type:
SRM
Divert valve:
2 - 4 min to source
Selected ions for quantification: m /z 343 299 + 245 for THC-COOH in negative mode
Results and Discussion Figures 1 and 2 show the ion chromatograms of the lowest and highest calibration points. Excellent linearity (r2 = 0.99) fits for the calibration curve were observed over the range of 7.8-1000 ng/mL urine, with a Coefficient of Variation (%CV) at the lower end of 6.5%. The limit of quantitation (LOQ) was established as 7.8 ng/mL in urine.
Figure 4 reports an ion chromatogram of a real urine sample positive for cannabinoids (225 ng/mL urine), analyzed as described.
To examine the difference between hydrolyzed and non-hydrolyzed urine, we analyzed the same urine sample without the hydrolysis step. When urines were not hydrolyzed, the portion excreted as free THC-COOH was detected at 3.06 minutes, while THC-COOH-glucuronide was detected at 2.58 minutes (Figure 5). The precursor ion m/z 343 was generated as result of an in-source fragmentation and a consequent loss of glucuronic acid.
Because THC-COOH is mainly excreted as glucuronic acid conjugate, it is always necessary to perform urine hydrolysis before the LC-MS analysis to obtain an accurate quantification of THC-COOH.
THC-COOH neg
RT: 2.00 - 4.00 SM: 3G 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5
[M-H]-
RT: 3.06
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)
Figure 1. Ion chromatogram of 7.8 ng/mL urine calibration standard
THC-COOH neg
RT: 2.00 - 4.00 SM: 3G 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5
[M-H]-
RT: 3.06
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)
Figure 2. Ion chromatogram of 1000 ng/mL urine calibration standard
THC-COOH neg
Area
THC-COOH_neg Y = 1368.24+1286.83*X R ^2 = 0.9955 W : 1/X 1400000
1200000
1000000
800000
600000
400000
200000
0
0
500
1000
ng /m L
Figure 3. Calibration curve of THC-COOH in negative ionization mode
RT: 2.00 - 4.00 SM: 3G 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5
[M-H]-
RT: 3.07
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)
Figure 4. Ion chromatogram of urine sample containing 225 ng/mL. Sample was hydrolyzed and diluted 1:10 before the analysis.
RT: 2.00 - 4.00 SM: 3G
100
95
RT: 2.58
90
85
80
75
70
65
THC-COOH neg
60 THC-COOH 55 Glucoronide
50
45
40
35
30
25
20
15
10
5
RT: 3.06
THC-COOH free portion in urine
2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min)
Figure 5. Ion chromatograms of urine sample containing 225 ng/mL. Sample was diluted 1:10 before the analysis; no hydrolysis was performed.
Conclusion A robust 6-minute method for the quantification of THCCOOH with a dynamic range of 7.8-1000 ng/mL urine has been developed using the TSQ Quantum Access MAX mass spectrometer for forensic toxicology purposes.
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