I',

c !annabinoid Chromatography 101

Chromatography is often called separation science since it 'is a technique for separating
compounds in a matrix, In the study presented here we are separating cannabinoids from
a matrix of plant material, including chlorophyll, fats, protein and various other related
compounds including the essential oils,
Cannabinoid chromatography focuses on five compounds: Delta 8 and Delta 9
Tetrahydrocannabinol (/\8, 9-THC), Cannibinol (CBN), Cannabidiol (CBD) and
Canabinolic Acid (THC-A). The most important being the THC-A in Liquid Phase
Chromatography as is the practice here, as opposed to gas chromatography, which is
practiced in 99% of other laboratories. The vital difference between gas and liquid phase
chromatography is that in gas chromatography the sample is heated to greater than 200
degrees centigrade to perform the analysis in the gas phase, In heating the sample the
THC-A is converted 1:1 to /\9-THe. Understanding this principle is key to making oral
cannabis medicines.
Each of the five cannabinoids has a distinct chemical structure, that is, the carbon,
hydrogen and oxygen atoms that make up the different cannabinoids differ in number and
spatial arrangement giving them distinct chemical properties,
Two of these chemical properties are exploited by LC Chromatography, the first being
their retention properties on a silica based, hydrocarbon coated column on which they are
separated, Because of the slightly differing chemistries of the individual cannabinoids
they will travel along this column at different rates carried by an organic solvent in which
they are dissolved, They therefore enter the detector at different times from the beginning
of the run, Subsequently, they enter a beam ofUV light, which they absorb, again
differently, depending on their chemical structures, This UV absorption is also
characteristic of the particular compound and can be used for identification purposes,
In order to confirm which cannabinoid is which and how much of each one there is in our
test solution we must run pure standards on the Le. These are the individual
cannabinoids in pure form made up to a specific known concentration, These standard
runs are used to calibrate the Le. That is, we can use these standards to tell the computer
the concentration of each peak made by each compound, The peaks are made by the
absorption ofUV light by the compound and the area under the peak is directly
proportional to the concentration of the test solution, Therefore, knowing the peak area of
the standard allows us to calculate the concentration of a sample from its peak area,
In summary, by using pure standards ofthe individual cannabinoids we can quantifY
cannabinoids in samples and therefore determine their concentration, Furthermore, the
pure standards allow us to identifY the individual cannabinoids based on their retention
times (time from injection to entering the detector) and their UV spectra.

In the chromatograms on the following pages a few points are in order:

The total THC potential refers to the percentage of THC which will become available on
heating, for example smoking or baking into brownies. To calc~late THC potential we
have to add the % Amount values next to the names /\9-THC and THC-A The total THC
potential is then realized. Note that the /\9-THC in unburned samples is often less than
1% and that the THC-A can be as high as 25%* CBN and CBD remain constant on
heating. /\8-THC is often present in small amounts and also being psychoactive is
included with the /\9-THC, both chromatographically and hence in calculations.
CBD//\9 ratios are provided on a number of chromatograms, this value is given for the
'ground state' cannabis, before it is heated. Obviously, this value will change
dramatically after heating the sample, approaching infinity . The ground state value is
used to provide an indication of the CBD content, since this compound is an important
modulator of the /\9-THC effect.

* We had one sample in our lab that measured out at 25% THC-A and 1% /\9-THC
giving it a total. THC potential of26%. Samples with this level ofTHC potential are rare.
"This will be deducted from your share in paradise".
Dr. J. J. Moreau

A Paul Hornby, Ph.D.

June 29, 2003

Cover: 3D Plot of Steve's Budder sample demonstrating different cannabinoids and their
chromatographic and UV properties.

 Data File C:\HPCHEM\1\DATA\MJ-0408\BD-4 . D

Cannabinoids. 100mg/10ml . 2.5 ul.
Injection Date
Sample Name
Acq. Operator

Sample Name: Budder Green

4 /8/0 4 1:31:12 PM
Budder Green
Felipe

Vial

Inj Volume
2.5 ILl
C:\HPCHEM\1\METHODS \ MJ-1A.M
4 / 8 /0 4 2:41:05 PM by Felipe
(modified after loading )
Analysis Method
C: \ HPCHEM \ 1 \ METHODS \MJ-1A.M
Last changed
4/21/04 8:58:23 AM by Felipe
(modified after loading)
Method for Cannabinoid Analysis: New Zorbax SBC18 Donated by Tom T.
Isocratic. Detection by Diode Array at 219 nm.
Acq. Method
Last changed

=========== = =========================================================
DAD1 A, 8ig=219,4 Ref-450,BO (MJ-0408I8D-4.D)

Norm.

0
I

~
,0

1000
500 .
0

~N

~~

=
.
~~

'"N
~~ ::
"":00

'JfA~
~

<

,"",

d;

'"
0
<D

'"

<0'"

.u:I

'"

NN

2D

,J'""\

o~

'"

10

<D

",,""

\
40

30

50

60

External Standard Report

=====================================================================

Sorted By
Calib. Data Modified
Multiplier
Dilution

Retention Time
4 / 21 / 04 8:58 : 19 AM
0.2000
200.0000

Signal 1: DADI A, Sig=219,4 Ref=450,80

RetTime Sig Type
[min]

Area
[mAU*s]

Amt / Area

Amount

Grp

Name

[%]

-------1--1------1------ ---- 1------- --- 1---------- 1--1---- --- ---- ---15.717

16.541
26.317
36.736
50.889

1
1
1
1
1

VB
VB
BBA
BBA
BBA

4403.82715
1126.55017
713.08832
1.12777e4
8.7864ge4

2.56423e-5
4 . 51698
2.61517e-5
1 . 17845
2.56423e-5 7.31410e - 1
2.56423e-5
11.56747
2.56423e-5
90.12243

Totals :

CBDA
CBD
CBN
-9-THC
THC-A

108.11673

Results obtained with enhanced integratorl

1 Warnings or Errors :
Warning : Calibration warnings (see calibration table listing)

*** End o f Report ***

Instrument 1 4/21/04 8:59:23 AM Felipe

Page 1 of 1

mi

Sample Name: Budder Green

Data File C:\HPCHEM\1 \ DATA\ MJ-0408 \ BD-4 . D

Purity results peak 15 at 36 .736 min.

2 1 9 ,4 Re f =45 o , 80 (MJ - 0 4 08\ BD-4.D

19na 1 DAD 1 A , S 19=
mAU

1-
I I
I I
I
I I
I I
I I
I I
I I
I I
I I
I I

1400
1200 c
1000 c

800
600
400

iAi

.t

200
0

10

I
I

20

I
I

50

40

30

60

250

->

300

:J5o

400

450

500

550

nrr

mi

36

37

38 mi

The purity factor is within the threshold limit. <-

Purity factor
Threshold
Reference
Spectra

999 .36 2 (3 of 3 spectra are within the threshold

limit. )
970.000
(Set by user )
Peak start and end spectra (integrated) (35.782 /
37.781)
3
(Selection automatic, 3)

*** End of Report ***

Instrument 1 4/21/04 9:00 : 35 AM Felipe

Page 1 of 1

Data File C:\HPCHEM \ 1\DATA\MJ-0413\BD-1.D

Cannabinoids.
Injection Date
Sample Name
Acq. Operator

Sample Name: BD 1k ppm

4/13/04 6:19:03 PM
BD 1k ppm
Felipe

Vial
Inj Volume

o
5 Jil

Acq. Method
Last changed

C:\HPCHEM\l\METHODS\MJ-IA.M
4 / 13 /0 4 7 : 24:03 PM by Felipe
(modified after loading)
Analysis Method
C: \ HPCHEM \ 1\METHODS \ MJ-1A.M
Last changed
4 / 21 / 04 8:54:37 AM by Felipe
(modified after loading )
Method for Cannabinoid Analysis: New Zorbax SBC18 Donated by Tom T.
Isocratic. Detection by Diode Array at 219 nm.
DADl A, 5ig=219,4 Ref=450,5O (MJ-0413IBD1 .D)

Norm. c

alO
Oal

400
300 c

100
0

N"/FI

<Xl

!If

0=;

-'""
...,'

,~

'"

..,~

:A:!

N
20

10

~
A

2>

'"0

,0

ZOO c

0
I
I-

40

30

50

60

Area Percent Report

=====================================================================

Sorted By
Calib. Data Modified
Multiplier
Dilution

Retention Time
Wednesday, April 21, 2004 8:53:44 AM
1.0000
1. 0000

Signal 1: DAD1 A, Sig=219,4 Ref=450,80

Peak RetTime Sig
#
[min]

Type

Area
[mAU*s]

Area

Name

1041 . 45386
266.85358
135.87231
2865.33057
2 . 17004e4

3.9194
1.0043
0.5113
10.7835
81.66 83

CBDA
CBD
CBN
A9-THC
THC-A

----1-------1---1------1-- --- -----1--------1------------------ -- --- -1

2
3
4
5

13 . 711
14 .679
21.572
29.611
39.338

Totals :

1
1
1
1
1

PBA
PBA
PBA
BBA
BBA

2.60100e4

Results obtained with enhanced integrator!

*** End of Report ***

Instrument 1 4 /2 1/04 8:54:37 AM Felipe

Page 1 of 1

mi

Data File C:\HPCHEM\1\DATA\ MJ-0408\BD-4.D

Sample Name: Budder Green

purity results peak 16 at 50.889 min.

S~gna 1

2 1 9 ,4 Re f =45 o , 80 (MJ - 0408 \ BD -4. D)

DADl A, S ~q=

mAU _

~-,

I
I
I
I
I
I
I
I
I
I
I
I

1400 1200
1000
800

600
400
200

10

250

->

1\

,l,

300

350

20

400

450

500

550

1 \i
40

30

I
I
I
I
I
I
I

50

50

The purity factor is within the threshold limit.

purity factor
Thresho ld
Reference
Spectra
Warning

60

52

mi

mi

<-

999.920 (3 of 3 spectra are within the threshold

limit.)
970.000
(Set by user )
Peak start and end spectra (integrated) (49.714 /
52.718)
3
(Selection automatic, 3)
Spectral absorbances > 1000 rnAU (see help for more
information)

*** End of Report ***

Instrument 1 4/21/04 8:58:38 AM Felipe

Page 1 of 1

Mariceuticals
Natural Medications
Attention: Steve

April 29, 2004

Certificate of Analysis
Budder

nd

Parameter Tested
PesticidelHerbicide Screen
not detected (detection llffilt I ppm).

Methodology: HPLCIDAD

Thank you for using AA

Dr. 0

Value (ppm)
nd < 1.0

Certificate of Analysis
Number: 03-S-44
To:

Date of Report: May 3, 2004

Steve

Sample: "Budder"

Submitted: April 27, 2003

Results of analysis:
(I) Plate Count (cfu/g)
(2) Total colifonns (MPN/g)
(3) Fecal colifonns (MPN/g)
(2) E. coli !lOg
(3) Staphylococcus aureus (cfu/g)
(4) Salmonella/25g
(5) Yeast (cfu/g)
(5) Mold (cfu/g)

1000
<3 (ND)
<3 (ND)
<3 (ND)
< 10
Negative
<4
<4

cfu - colony forming unit

MPN - Most Probable Number
ND - not detected

Methodology:
Analyses were conducted on a day of arrival according to: Compendium of Analytical
Methods. HPB Methods ofMicrobiological Analysis of Food, (I) - MFHPB-18, (2) MFHPB-19, (3) - MFHPB-21, (4) -MFHPB-20, (5) -MFHPB-22,
Thank you for using AA
Dr. 0

Sample Name: Budder

' Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D

Pesticide/Herbicide Screen.
Injection Date
Sample Name
Acq. Operator

4 / 28 / 04 1:2 8 :57 PM
Budder
Felipe

Vial

Inj Volume
10 pI
Method
C:\HPCHEM \ 1 \ METHODS \ PEST- 1B.M
Last changed
5 / 2/ 04 11:12:00 AM by Felipe
Method for detecti on o f Pesitcides / Herbicides uses C18 column and
P04 aqueous an d acetonltrl'1 e organlc PJh ases. Detectlon at 245 nm .
DADl A, Sig-245,4 Ref- 450,80 (PES-0428\8D-2.D)

~"

mAU

1~

10
5

_l

0
-5
-10

20

10

30

40

50

=====================================================================

External Standard Report

================================= = === = ====== = == = =====================

Sorted By
Calib. Data Modified
Multiplier
Dilution

Retention Time
5/2/04 11:11:48 AM
1.0000
1. 0000

Signal 1: DAD 1 A, Sig=245,4 Ref=450,80

Signal 2: NEW,
not found
Signal 3: NEW,
not found
Ret Time Sig Type
[min]

Area

Amt / Area

Amount
[ppm]

Grp

Name

--- - ---/--/------/----------/----------/----------/--/--------------7.000
7.200
8 . 100
B.400
9.200
9 _500
9 . 600
9.700
9.730
9. BOO
11.600
12 . 000
12 . 600
14 . 600
15.600
17 . 900
19 . 400

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

Instrument 1 4/ 28 / 04 2:44:32 PM Felipe

Dirun
Desethylatrazine
Benzthiazuron
Hexazinon
Metoxuron
Simazine
Methabenzthiazuron
Nonlinuron
Atrazine
Isoproturon
Metobromuron
Metzachlor
Propazine
Setbutylazine
Tertbutylazine
Linuron
Metalochlor
Page 1 of 4

mi

Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D

RetTime Sig Type
[min)

Area

Amt/Area

Sample Name: Budder

Amount
[ppm)

Grp

Name

-------1-- 1------1----------1----------1----------1--1--------------20.100

Avid

0.00000

Totals :

Signal 1: DAD1 A, Sig=245,4 Ref=450,80

Unca1ibrated Peaks
compound name not specified
Signal 2: NEW,
not found
Uncalibrated Peaks
compound name not specified
Signal 3: NEW,
not found
Uncalibrated Peaks
compound name not . specified
. RetTime Sig Type
Area
Amt/Area
Amount
Grp
Name
[min)
[ppm)

-- ---- -1--1------1--- ----- --1----------1-- ------- -1--1- -------------4.934

5.767

1 PBA
1 PBA

68.61839
233.40512

0.00000
0 . 00000

Uncalib. totals

0.00000
0.00000

?
?

0.00000

Results obtained with enhanced integrator!

2 warnings or Errors :
Warning
Calibration warnings (see calibration table listing)
Warning : Calibrated compound(s) not found
=====================================================================
=====================================================================

Area Percent Report

=====================================================================

Sorted By
Calib . Data Modified
Multiplier
Dilution

Retention Time
5 /2/0 4 11:11:48 AM
1.0000
1.0000

Signal 1: DAD1 A, Sig=245,4 Ref=450,80

Peak Ret Time Sig
#
[min]

Type

Area

Area
%

Name

----1------ -1--- 1------ 1----------1--------1- -----------------------1

2
3

0.000
0 . 000
0.000

2
3
2

0.00000
0.00000
0.00000

0.0000 Avid
0.0000 Avid
0.0000 Metalochlor

0.000

0.00000

0.0000 Metalochlor

5
6
7
8
9
10
11
12
13
14

0.000
0.000
0.000
0.000
0.000
0.000
0 . 000
0.000
0.000
0.000

2
3
2
3
2
3
2
3
2
3

0.00000
0.00000
0.00000
0.00000
0 . 00000
0.00000
0.00000
0.00000
0.00000
0.00000

0.0000
0.0000
0.0000
0 . 0000
0 . 0000
0.0000
0.0000
0.0000
0.0000
0.0000

15

0.000

0.00000

0.0000 Metobromuron

Instrument 1 4/28/04 2:44:32 PM Felipe

Linuron
Linuron
Tertbutylazine
Tertbutylazine
Setbutylazine
Setbuty lazine
Propazine
Propazine
Metzachlor
Metzachlor
Page 2 of 4

Sample Name: Budder

Data File C:\HPCHEM\1\DATA\PES-0428\BD-2 . D

Peak RetTime Sig
#
[min]

Type

Area

Area
%

Name

----1-------1---1------1----7-----1--------1------------------------16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54

0.000
0.000
0 . 000
0.000
0 . 000
0 . 000
0 . 000
0 . 000
0.000
0.000
0 . 000
0.000
0 . 000
0 . 000
0.000
0.000
0.000
0.000
0.000
0.000
0 . 000
7.000
7 . 200
8.100
8.400
9.200
9.500
9.600
9 . 700
9.730
9.800
11.600
12 . 000
12.600
14 . 600
15.600
17.900
19.400
20.100

3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
2
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

0.00000
0.00000
0.00000
0.00000
0.00000
0 . 00000
0.00000
0 . 00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0.00000
0 . 00000
0 . 00000
0.00000
0.00000
0.00000
0.00000
0.00000
0 . 00000
0.00000
0 '. 00000
0 . 00000
0.00000
0.00000
0.00000
0.00000
0 . 00000
0 . 00000
0 . 00000
0 . 00000
0 . 00000

Totals :

0.00000

Uncalibrated Peaks:
Peak RetTime Sig Type
#
[min]

Area

0.0000
0.0000
0.0000
0.0000
0.0000
0 . 0000
0.0000
0 . 0000
0.0000
0.0000
0 . 0000
0.0000
0.0000
0.0000
0 . 0000
0 . 0000
0.0000
0.0000
0.0000
0.0000
0 . 0000
0.0000
0.0000
0.0000
0 . 0000
0.0000
0.0000
0.0000
0.0000
0.0000
0 . 0000
0 . 0000
0.0000
0 . 0000
0.0000
0.0000
0.0000
0 . 0000
0.0000

Metobromuron
Isoproturon
Isoproturon
Atrazine
Atrazine
Nonlinuron
Nonlinuron
Methabenzthiazuron
Methabenzthiazuron
Simazine
Simazine
Metoxuron
Metoxuron
Hexazinon
Hexazinon
Benzthiazuron
Benzthiazuron
Desethylatrazine
Desethylatrazine
Dirun
Dirun
Dirun
Desethy1atrazine
Benzthiazuron
Hexazinon
Metoxuron
Simazine
Methabenzthiazuron
Nonlinuron
Atrazine
Isoproturon
Metobromuron
Metzachlor
Propazine
Setbutylazine
Tertbutylazine
Linuron
Metalochlor
Avid

Area
%

Name

----1-------1---1------1----------1--------1------------------------1
2

4.934
5 . 767

1
1

Uncalib . totals

PBA
PBA

68.61839
233.40512

22.7196?
77.2804?

302.02351

Results obtained with enhanced integrator!

2 Warnings or Errors :
Instrument 1 4/28/04 2:44:32 PM Felipe

Page 3 of 4

" Data File C:\HPCHEM\1\DATA\PES-0428\BD-2.D

Warning
Warning

Sample Name: Budder

Calibration warnings (see calibration table listing)

Calibrated compound{s) not found

=====================================================================

*** End of Report ***

Instrumeilt 1 4/28/04 2:44:32 PM Felipe

Page 4 of 4