Solid Phase Extraction and Analysis of THC and Carboxy-THC from Whole Blood usinga Novel Fluorinated SPE Sorbent and Fast LC-MS/MSAlbert Elian*,, Massachusetts State Police Crime Lab,, 59 Horse Pond Rd. Sudbury,y, MA 01776 ,Jeffery Hackett, Northern Tier Research, 1300 Old Plank Road, Mayfield, PA 18433

TIC: from Sample 5 (0.1 std) of 0429.wiff (Turbo Spray)

Max. 3.2e5 cps.
2.53

3.2e5I...

2.01
0.0

0.5
30

1.0
60

1.5
89

2.0
119

2.5
147

3.01773.52064.0236

Time, min
XIC of +MRM (4 pairs): Period 2, 315.2/193.2 Da from Sample 5 (0.1 std) of 0429.wiff (Turbo Spray)

Max. 3516.0 cps.

2.55351603.4
65
Time, min
XIC of +MRM (4 pairs): Period 2, 318.3/196.2 Da from Sample 5 (0.1 std) of 0429.wiff (Turbo Spray)I...

LiLinearityit (r2( 2 >0.995)>0 995) was achieved
hi d fromf0 25 ng/mL
0.25/ L tot 50 ng/mL,/ L (THC/ CarboxyC bTHC) and the limits of detection was determined to be 0.1 ng/mL for both THC andCarboxy-THC respectively. The limits of quantification was 0.25 ng/mL for both THC andCarboxy-THC, respectively. Recoveries were > 85% for THC and > 89% for Carboxy-THC,respectively measured over the calibration range. Intra and inter-day precision was less than
4% and 6%, respectively for THC and less than 6% and 8%, respectively for Carboxy-THC
Ion suppression studies revealed that suppression of monitored ions was less than 6%.

2.4
6

2.5
12

2.6
18

2.7
24

2.8
30

2.9
36

3.0
42

3.1
48

3.2
53

3.3
59

3.6
77

3.7
83

3.8
89

3.9
95

4.0
101

4.11073.571

3.6
77

4.4
124

3.7
83

3.8
89

3.9
95

4.0
101

4.1
107

4.2
112

4.3
118

4.4
124

Max. 912.0 cps.
2.01

I...

1.51

0
0.2
0.4
0.6
0.8
1.0
1.2
1325
3648
60
72
Time, min
XIC of -MRM (4 pairs): Period 1, 346.1/302.3 Da from Sample 5 (0.1 std) of 0429.wiff (Turbo Spray)

5.8
5.5

4.3
118

Max. 1.8e5 cps.

912

Untitled 5 (THC 1): "Linear" Regression ("1 / x" weighting): y = 0.114 x + -0.00295 (r = 0.9996)

4.2
112

2.53

1.8e5
1.0e50.0
2.4
2.5
2.6
2.7
2.8
2.9
3.0
3.1
3.2
3.3
3.4
6
12
18
24
30
36
42
48
53
59
65
Time, min
XIC of -MRM (4 pairs): Period 1, 343.1/299.3 Da from Sample 5 (0.1 std) of 0429.wiff (Turbo Spray)

Fig. 1 Graph of Carboxy-THC extracted from whole blood over the calibration range(0.25 to 50 ng/ mL)

3.5
71

1.63

1.4
83

1.6
95

1.78

2.21

1.8
107

2.0
119

2.25

2.2
130
Max. 6.4e4 cps.

2.02
5.0e4I...

5.0Extraction

0.0

4.5

0.4
25

0.6
360.848

1.060

1.272
Time, min

1.483

1.6
95

1.8
107

2.0119

2.2
130

3.5

3.0

Fig. 4 Chromatogram of THC/ Carboxy-THC (and IS) spiked into whole blood at LOQ (0.25ng/ mL) Rt: THC: 2.57 minutes, THC-D3: 2.55 minutes Carboxy-THC: 2.07, Carboxy-THCD3: 2.06 minutes

2.5

2.0

1.5

1.0

Printing Time: 3:59:33 PM
Printing Date: Monday, March 09, 2009

Polarity/Scan Type: Negative MRM, ...
Analyst Version: 1.4.2

0.5

TIC: from Sample 3 (0.025) of 0112.wiff (Turbo Spray)

Max. 1.6e5 cps.

0.0246810121416182022242628
Analyte Conc. / IS Conc.

30

32

3436

38

40424446

48

50

I...

02.55

1.6e5
1.0e5

2.04

0.0

0.5
30

1.0
60

1.5
89

2.0
119

2.5
147

3.0
177

3.5
206

4.0236

Time, min
XIC of +MRM (4 pairs): Period 2, 318.3/196.2 amu from Sample 3 (0.025) of 0112.wiff (Turbo Spray)

Fig. 2 Graph of THC extracted from whole blood over the calibration range(0.25 to 50 ng/mL)

Max. 1.0e5 cps.

2.55

I...

1.00e50.00

3.465Time, min
XIC of +MRM (4 pairs): Period 2, 315.2/193.2 amu from Sample 3 (0.025) of 0112.wiff (Turbo Spray)

2.46

2.5
12

2.6
18

2.7
24

2.8
30

2.9
36

3.0
42

3.1
48

3.2
53

3.3
59

3.5
71

3.6
77

3.7
83

3.8
89

3.9
95

4.0
101

4.1
107

4.2
112

4.3
118

4.4
124

Max. 1848.0 cps.

Untitled 5 (THC-COOH 1): "Linear" Regression ("1 / x" weighting): y = 0.0899 x + -0.0213 (r = 0.9968)

I...

4.6
4.4

2.55

1848
1000

02.4
2.5
2.6
2.7
2.82.93.0
3.1
3.2
3.3
3.4
61218243036
42485359
65Time, min
XIC of -MRM (4 pairs): Period 1, 346.1/302.3 amu from Sample 3 (0.025) of 0112.wiff (Turbo Spray)

4.2

Instrumentation
Liquid chromatography equipment consisted with a Shimadzu
Prominence (two pumps LC-20AD, autosampler SIL-20AC, and column oven
CTO-20AC), was performed using Imtakt™ C18 column (50x 2.1mm, 5 μm), at
0.55mL/min. flow using a gradient program. The mobile phase program: (A)
0.1% aqueous formic acid) / (B) acetonitrile containing 0.1% formic acid wassstarted
ed at 50%% ((B)) foro 0.5. min,, increasingc e s g too990%% ((B)) ove
over 1.5. minute,u e, anddholding at 90% B for one minute before returning to 50% (B) and equilibrated
for 2 minutes. The total chromatographic run time for each analysis was 4.5
minutes including equilibration time.
MS/MS analysis was conducted using an Applied Biosystems
3200Q Trap instrument equipped with ESI in negative ion mode for CarboxyTHC and D3 and was operated with multiple reaction monitoring (MRM) under
the following conditions: curtain gas 15, collision gas medium, ion sprayvoltage -4500V, temperature 650 °C, ion source gas(1) 50, ion source gas (2) 50.
The following transitions were monitored (quantification ions underlined): m/z
343.1 → 299.3 and 245.3 for Carboxy-THC, and m/z 346.1 → 302.3 and 248.3
for Carboxy-THC-D3. Positive ion mode was employed for THC/ THC-D3
under the following conditions: curtain gas 15, collision gas medium, ion sprayvoltage 5000V,5000V temperature 650 °C
C, ion source gas(1) 50,50 ion source gas (2) 50.
50
The following transitions were monitored (quantification ions underlined): m/z
315.2 → 193.2 and 123.1 for THC, and m/z 318.2 → 196.2 and 123.1 for D3THC.

0.2
134.0

AnalyteArea/ ISArea

In this procedure, after the addition of the internal standards
THC-D3 and Carboxy-THC-D3) to 1 ml of whole blood, 2 mL of ice coldacetonitrile were added dropwise whilst mixing. The samples were allowed tostand for 10 minutes, after which the samples were centrifuged (10 minutes at3000 rpm). Each supernatant was decanted into a clean tube and evaporated toabout 200 μl. Then to each tube was added 5 mL of pH 7 phosphate buffer (0.1M) prior to solid phase extraction. The SPE columns (Fluoro-C10 (UCT Inc.,))(6mL, 200 mg) were conditioned with methanol, pH 7 buffer (3, 3, mL,respectively) after which, the samples were loaded. The SPE columns weregwith 3 mL hexanes thenwashed with 3 mL DI water, dried, and washed again
dried again for 5 minutes under full vacuum. Following elution ofTHC/Carboxy-THC with 2 mL of hexane: ethyl acetate (1/1) with 2% acetic
acid, the eluents were collected and evaporated to dryness. The residue wasreconstituted with 50 µl of the methanol solution prior to the chromatographic
analysis.

4.0
3.8

3.5
71

3.6
77

3.7
83

3.8
89

3.9
95

4.0
101

4.1
107

4.2
112

4.3
118

4.4
124

Max. 3.4e4 cps.

3.6

2.04

3.4e4

3.4
3.2

I...

Cannabis (marijuana) is one of the most widespread and frequently useddrugs in the world. It is generally smoked as a cigarette or in a pipe or waterpipe (“bong”). The active ingredient, Δ9-THC, induces feelings of euphoria
and relaxation, but also impairs perception and cognizance. Although manyarguments have been made for the legalization of marijuana, it remains an illicit
drug in most countries. Because of its widespread use, it is often necessary toanalyze biological samples, usually urine or blood, for cannabinoid use. Use of
these drugs is confirmed by detection and/ or quantitation of THC and its major
metabolite, 11-nor-9-carboxy- Δ9-tetrahydrocannabinol ((THC-COOH)
Carboxy-THC)
Currently, screening for these compounds is often accomplished byimmunoassay followed by GC/MS for confirmation and/or quantitation..
However, due the availability of LC/MS/MS , many frequently used
procedures including THC are being developed away from the traditional GCMS onto this instrument. LC/MS/MS is a complementary technique to GC/MSand the very compounds that are challenging to analyze by GC/MS, i.e. polar,amines, and semi-volatile compounds, are ideal candidates for LC/MS/MS
analysis. Furthermore, sample preparation is often greatly simplified as thefinal step of sample preparation i.e. “derivatization” can be omitted. With theaddition of fast LC, each run can be performed in under 5 minutes.

THC and Carboxy-THC and their deudurated analogs, werepurchased from Cerilliant. Formic acid (99%) was from Acros Organics, allother solvents were HPLC grade or better and were obtained from Fisher
Scientific.
A standard stock reference solution of THC and CarboxyTHC(1.0 μg/ml) was prepared in acetonitrile. THC and Carboxy-THC standards
were prepared by spiking drug free blood at the following concentrations: 0.25,
1.0, 2.0, 5.0, 10 and 50 ng/mL. Another stock reference solution was prepared in
acetonitrile (1.0 μg/ml) and was used for the preparation of controls at 4.0 and
15.0 mg/L. THC and Carboxy-THC concentrations in the controls blood were
calculated from linear regression of the standard responses based on the peakarea ratio.

0.0
0.2
0.4
0.6
0.8
1.0
1.2
1325
36
48
6072
Time, min
XIC of -MRM (4 pairs): Period 1, 343.1/299.3 amu from Sample 3 (0.025) of 0112.wiff (Turbo Spray)

3.0
2.82.62.4

1.4
83

1.6
95

1.8
107

2.0
119

2.2
130
Max. 960.0 cps.

2.2

2.07

960

2.0

I...

INTRODUCTION

RReagents

AnalyteArea/ ISArea

In this study,study solid phase extraction (SPE) is described using a novel
fluorinated (Heptadecafluorotetrahydrodecyl (C10H4F17)) phase to isolate
THC and its primary metabolite Carboxy-THC from whole blood samples. SPE
was performed in hydrophobic mode after samples of whole blood wereprecipitated with acetonitrile. After applying the sample to the SPE column inaqueous phosphate buffer (pH 7) the sorbent was washed with deionized water,phosphate buffer (pH 7), and dried. The SPE column was eluted with a solvent
consisting of ethyl acetate/ hexanes (50:50) containing 2% acetic acid. Theeluate was collected, evaporated to dryness and dissolved in mobile phase (50μL) for analysis by fast LC-MS/MS in positive/ negative MRM mode.
Chromatography was performed in gradient mode employing a C18 column
and a mobile phase consisting of acetontitrile (containing 0.1% formic acid)and 0.1% aqueous formic acid. The total run time for each analysis was lessthan 5 minutes.
minutes
The limits of detection/ quantification for this method were determined to be0.1 ng/ mL and 0.25 ng/ mL, respectively. The method was found to be linear
from 0.25 ng/ mL to 50 ng/ mL (r2≥0.995). Recoveries of the individual
cannabinoids were found to be greater than 85%. In this report, results ofauthentic samples analyzed for THC and Carboxy-THC are reported using thisnew methodology.

Fig. 3 Chromatogram of THC/ Carboxy-THC (and IS) at LOD 0.1 ng in whole blood (1 mL)
Rt: THC: 2.57 minutes, THC-D3: 2.55 minutes Carboxy-THC: 2.07, Carboxy-THC-D3: 2.06minutes

RESULTS AND DISCUSSION

MATERIALS AND METHODSI...

ABSTRACT

1.8

01.6
1.4
1.2

*POST PM BY RHH

0.2
130.4
25

0.6
36

0.8
48

1.0
60

1.2
72
Time, min

1.4
83

1.6
95

1.8
107

2.0
119

2.2
130

*API 3200 QTRAP S/N AF011810604

1.0
0.8
0.6
0.4
0.2
0.0
02

4

68

10

12

14

16

18

20

2224
26
28
Analyte Conc. / IS Conc.

3032343638

40

4244

464850

CONCLUSIONSThis poster describes a procedure, where a novel fluorinated SPE sorbent extraction
methodology coupled to fast LC-MS/MS, a highly efficient technique has been developed. The
use of this process should greatly assist analysts in the field of drug related driving cases
quickly resolve the issues involved in reporting values for both THC and its primarymetabolite (Carboxy-THC) in whole blood samples. Comparison of re-analyzed cases with
their previous historical data has shown the effectiveness of this new, improved methodology.