LC Technical Note

Analysis of Cannabinoid in Urine by LC/MSLT122
GL Sciences Inc.

Detection and determination of 11-nor-9-carboxy-Δ9-concentration in urine is in ppb level.

carboxylic acid (THCA), which is a metabolite of principal

In this note, MonoSpin, which provides high recovery

psychoactive constituent of cannabis plant, is necessary toeven from sample of less than 100 µL, was used for theconfirm cannabis use from urine. Sample pretreatment

sample pretreatment, and the purified solution wasmethod suitable for handling low volume and low

injected into LC/MS/MS system. The results showed goodconcentration sample has been desired for thelinearity, recovery, and reproducibility.

determination because THCA

(Y. Yui and S. Ota)

Evaluation of purification by MonoSpin C18-CX (analyzed by HPLC-UV system)＜After purification＞＜Before purification＞
1. Carboxy-THC (10 μg/mL)

10.0

0.0

Volts

Volts

102Conditions
ColumnEluent:Flow rate: Col.
Temp.:
Detection:
Injection Vol.:
Sample:46Time [min]810120246Time [min]81012

Chemical Structure: Inertsil ODS-3 (5 μm, 150 × 4.6 mm I.D.): A) 0.1 % HCOOH in H2OB) 0.1 % HCOOH in CH3CN
A/B = 60/40 - 10 min - 0/100, v/v: 1.0 mL/min: 40 ℃: UV 214 nm: 10 μL: (±)-Carboxy-THC 10 μg/mL

OOHOH

O

(±)-11-nor-9-Carboxy-Δ9-THC(THC-COOH)
Structures are created using Chemistry 4-D Draw which is provided by ChemInnovation Software, Inc.

What is MonoSpin ?
MonoSpin is a series of spin columns for solid phase extraction (SPE). Owing to the high permeability of monolithic silica disk
packed into the spin column, the procedures, such as conditioning, sample loading, washing, and elution can be carried out onlyby centrifuging the column. It is also the advantage that the elution volume is only 200 µL.

An Example of Procedures

Centrifuge

Centrifuge

Centrifuge

Centrifuge

ElutionGL Sciences LC Technical Note
Sample Pretreatment using MonoSpin C18‐CXMonoSpin C18‐CX has octadecyl group and cation‐exchange group on the surface of its silica monolithic support. In this case,acidic buffer was used for sample loading. THC‐COOH, which hardly dissociates under acidic condition, could be retained withhydrophobic interaction.

Sample solution
0.1 % formic acid in water
Sample matrix
Standard solution*490 μL
490 μL
10 μL

1. Conditioning（Equilibrating）+) Solution of internal standard* 10 μL

Centrifuge**

Wash with 300 μL of mthenol and300 μL of
0.1 % formic acid in water1000 μL

2 min each

* Their concentration is described in the results.

2. Sample loading

Centrifuge

Load 200 μL of samplesolution

2 minCentrifuge

3. Wash
Wash with 300 μL of 0.1% formic acid in water

2 min

4. Elution
Centrifuge
Elute with100 μL of 5%ammonia in methanol

5. HPLC Analysis
1 min** All the centrifugation was operated at 3,000 xg.

Evaluation of Recovery by Standard Addition
THCA
I.S.***

3.0e4

Conditions;
System: 0.5 ng/mL: 2.5 ng/mL

Column2.5e4

Intensity [cps]Eluent
2.0e4

1.5e4

Flow Rate
Col. Temp.
Detection
Injection Vol.

1.0e4
4.07

5000.00.0

1.031.35
0.82

1.0: LC800 (GL Sciences) API3000(AB SCIEX): InertSustain C18(2 μm, 100 × 2.1 mm I.D.): A) 0.1% HCOOH in H2OB) 0.1% HCOOH in CH3CN A/B =35/65 - 6 min - 5/95- 4 min - 5/95 - 0.1 min- 35/65 - 5 min – 35/65, v/v: 200 μL/min: 40 ℃: MS/MS (ESI, Nega, MRM)
: 5 μL

2.022.225.32.56

2.0

3.0

4.0
5.0
Time [min]

6.0

7.0

8.0

*** Deuterium labeled compound ((±)-11-nor-9-Carboxy-Δ9-THC-D9,
THC-COOH-d9) was used as internal standard.
Scan Parameters

AnalytePrecursor Ion(m/z)

Product Ion(m/z)(±)-Carboxy-THC

343.0

299.0

(±)-Carboxy-THC-d9

352.1

308.1

GL Sciences LC Technical Note
Calibration Curves
Calibration curve was plotted 4 times by conducting same experiment on different days. All the calibration
curve showed good linear response (r2 = 0.9998～1.000).

1st day

2nd day

3.0

3.0

Analyte Area / IS Area

Analyte Area / IS Arear2 =0.9999
2.0

1.0

0.0
0.5 1.0

5.0
Analyte Conc. / IS Conc.

r2=0.9999
2.0

1.0

0.0
10.0

0.5 1.0

3rd day

10.0

5.0
Analyte Conc. / IS Conc.

10.0

4th day3.0

3.0

r2=0.9998

Analyte Area / IS Area

Analyte Area / IS Area

5.0
Analyte Conc. / IS Conc.

2.0

1.0

r2=1.000
2.0

1.0

0.0

0.0
0.5 1.0

5.0
Analyte Conc. / IS Conc.

10.0

0.5 1.0

Recovery, Reproducibility, and Accuracy
From the chromatograms, recovery, reproducibility, and accuracy were calculated for each day. Almost all results
were satisfactory.

Recovery (%)
0.5 ng/mL
1.0 ng/mL
10 ng/mL

1st day
86.8
99.0
88.1

2nd day
94.4
100.8
91.1

3rd day
87.0
101.4
91.6

4th day
88.5
101.7
92.8

Inter-day average
89.2
100.7
90.9

Reproducibility (%)
0.5 ng/mL
1.0 ng/mL
10 ng/mL

1st day
2.67
1.00
1.14

2nd day
0.74
2.85
1.05

3rd day
2.17
2.79
1.79

4th day
3.20
0.57
0.80

Inter-day average
2.2
1.8
1.2

Accuracy (%)
0.5 ng/mL
1.0 ng/mL
10 ng/mL

1st day
101.0
99.2
99.9

2nd day
105.3
97.1
99.9

3rd day
101.0
99.5
99.9

4th day
101.7
98.9
100.1

Inter-day average
102.2
98.7
100.1

GL Sciences LC Technical Note
The series of MonoSpin
MonoSpin SAX

MonoSpin C18(CH2)17CH3

Monolith

Monolith

Octadecyl group is chemicallybonded, and non-polar compounds
can be retained because of its
hydrophobic interaction. It can beused for extraction or desalting.

MonoSpin NH2
Aminopropyl group is bonded.
It is suitable for extarction ofhydrophilic compounds, such as
sugar chain.

NH2(CH3)3NH
B(OH)2

Phenylboronic acid ischemiaclly bonded. Componds
containing cis-diol group canbe retained with highselectivity.

MonoSpin TiO

MonoSpin SCX(CH2)3

SO3--bonded. It offers strong cationexchange and hydrophobicinteraction. It is suitable forextarction of basic drugs.

Monolith

MonolithPropylbenzenesulfonic acid is

MonoSpin Amide

Monolithic silica is coated withtitanium dioxide. It is suitablefor extraction of phoshatecontaining compounds.

TiO2

MonoSpin CBANH2（used in this note）

MonoSpin C18-CX-OOH

Carboxyl group is bonded tosilica monolith. It is suitable forextraction of basic compounds
through weak cation-exchangeinteraction.

MonoSpin Trypsin
Monolith

Monolith

Functional groups of both C18
and SCX are bonded. C18-CX isoften superior to C18 or SCX inpurification and clean-up of basic
drugs in serum and urine.

(CH2)17 C 3 HSO3

Monolith

Monolith

Amide group is bonded. Widevariety of hydrophilic compounds,
such as sugar chain, can beretained and extracted in HILIC
mode.

O(CH2)3(CH2)3 N+

Trimethylaminopropyl groupis bonded. It offers strong
anion- exchange and weakhydrophobic interaction. It is
suitable for extarction of acidicdrugs.

MonoSpin PBA
Monolith

Monolith(CH2)3Cl

Trypsin

Trypsin is immobilized on
the surface of silica
monolithic support. It enables
rapid protein- digesting.

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly. We reserve theright to amend this information or data at any time and without any prior announcement.

GL Sciences, Inc. JapanGL Sciences B.V.

GL Sciences (ShangHai) Ltd.

GL Sciences, Inc. USA

22-1 Nishishinjuku 6-Chome
Shinjuku-ku, Tokyo,163-1130, Japan
Phone: +81-3-5323-6620
Fax: +81-3-5323-6621
Email: world@gls.co.jp
Web: www.glsciences.com

De Sleutel 95652 AS Eindhoven
The Netherlands
Phone: +31 (0)40 254 95 31
Email: info@glsciences.eu
Web: www.glsciences.eu

Tower B, Room 2003,
Far East International Plaza,NO,317 Xianxia Road,
Changning District.
Shanghai, China P.C. 200032
Phone: +86 (0)21-6278-2272
Email: contact@glsciences.com.cn
Web: www.glsciences.com.cn

4733 Torrance Blvd. Suite 255
Torrance, CA 90503
Phone: 310-265-4424
Fax:
310-265-4425
Email: info@glsciencesinc.com
Web: www.glsciencesinc.com

International Distributors
Visit our Website at:https://www.glsciences.com/company/distributor.html