Transcript Slide 1

Investigation of the Impurities in Dronabinol Samples by LC/MS
Authors: Huahua Jian, Isil Dilek, Uma Sreenivasan,
Kenan Yaser
Introductio
n
Chromatogram
s
HPLC Chromatogram:
Mass Spectra of Selected Impurities
0
0
10
20
30
40
50
60
RRT 0.78 - Cannabinol
RRT 0.91 -- Cis 9-THC
RRT 1.06 – Dihydrocannabinol
75.221
68.043
57.149
58.976
55.205
51.178
45.702
43.461
39.438
35.355
36.894 - Impurity A
28.309
29.579
29.957
2
25.171
26.314
4
8.193
8.899
9.403
10.578
11.580
12.075
13.081
14.027
15.869
17.211
17.876 - Cannabidiol
19.145
19.658
20.310
21.801
22.219
23.166
6
5.106
Dronabinol is a light yellow to amber glassy
material. It is sensitive to light, heat, and oxygen
(air). The impurities in commercial Dronabinol
drugs may come from either the synthetic process
or through product degradation. Identification of
these impurities is required by FDA and ICH
guidelines for pharmaceuticals.
8
33.339
Tetrahydrocannabinol (9-THC) is a psychoactive
substance found in cannabis plants (“Medical
Marijuana”). Synthetic 9-THC, also called
Dronabinol, was approved by US FDA as a drug to
treat pain, anorexia and nausea related to
chemotherapy and other disorders.
31.858 - Cannabinol
mAU
47.379 - delta8-THC
40.824 - Dronabinol
VWD1 A, Wavelength=228 nm (LC70108\J0122817.D)
RRT 0.30 –
Hydroxydihydrocannabinol
70
80
min
TIC Chromatogram:
A comparison of impurities in Dronabinol from a
variety of sources was performed by HPLC and
LCMS.
Dronabinol
CH3
OH
H3C
H3C
O
CH3
Materials and Methods
Materials
• Austin Pharma API, Dronabinol, USP
• Capsules manufactured from Austin Pharma API
• Marinol ® (RLD)
• Generic Dronabinol manufactured by Par
Pharmaceuticals
Methods
HPLC method conditions are based on USP 29
monograph for Dronabinol. LCMS run conditions
were adapted from the HPLC method using
ammonium formate as the mobile phase additive
for MS detection.
Capsules were extracted based on USP Dronabinol
capsule monograph.
Instrument
Column
Agilent G6410 Series Triple Quad
(QQQ) LC/MS/MS
Phenomenex Luna 3u C18(2)
150x4.6 mm
Gradient
Flow Rate
authentic references: RRT 0.78 – Cannabinol; RRT 0.91 -- Cis 9-THC; RRT
1.15 -- 8-THC.
• The proposed structures of other impurities were based on LCMS results.
Extracted Ion Chromatogram (EIC) confirmed the relative retention time of
these impurities. MRM studies did not provide additional information as the
impurities are similar in structure.
Comparison of Impurity Profiles of Dronabinol
RRT
m/z
M.W.
Generic
Brand
Capsule
Made from Marinol®
AustinPharm
a
API
Par
Generic
Austin
Pharma
API
Sample
Stressed
API
Sample
Enriched
with
Impurities
CH3
0.23
329
328
+
+
+
+, a
+
Hydroxydihydrocannabinol
0.25
361
360
+
+
+
+
+
Trihydroxydihydrocannabinol
0.26
345
344
+
+
+

+
Dihydroxydihydrocannabinol
0.28
329
328
+, a
+, a
+, a

+, a
Hydroxydihydrocannabinol
0.30
329
328
+
+
+
+
+
Hydroxydihydrocannabinol
0.36
345
344
+
+
+

+, a
Dihydroxydihydrocannabinol
0.38
345
344
+
+
+
+
+
315
314





Cannabidiol
0.47
327
326

+
+
+
+
Hydroxycannabinol
0.63
313
312
+
+
+
+
+
329
328
+
+
+


Hydroxydihydrocannabinol
0.71
313
312
+
+
+
+
+
Dihydrocannabinol
311
310
+
+
b
+
+
312
+, b
+, b
+, b
+
+, b
Dihydrocannabinol
0.91
315
314
+
+
+
+
+
Cis 9-THC
71:24:5 v./v. MeOH/H2O/THF, with 5
mM ammonium formate
1.00
315
314
NA
NA
NA
NA
NA
Dronabinol, API
1.06
313
312
+
+
+

+
Dihydrocannabinol
Isocratic
1.10
313
312
+
+
+
+, a
+
Dihydrocannabinol
1.15
315
314
+
+

+
+
8-THC
Injection Volume
5-20 µL
UV wavelength
228 nm
Polarity, scan type
Positive scan
Ionization Source
Electrospray Ionizaion (ESI)
Mass scan range
300-400 Da
+:
:
a:
b:
Present in sample.
Not detected in sample or peak is too small to be extracted.
Mass spectrum is compromised because the impurity is low in the sample.
The mass spectra of RRT 0.82 for fresh API sample is 312. For the other four samples, it is a mixture
of two components, with molecular weights 312 and 328. The ratio of the two peaks varies, but m/z
313 (M+H+) and m/z 329 (M+16+H+) are all evident. The m/z 329 peaks is oxygen adduct of m/z 313
peak. The structure of this impurity is proposed to be an isomer of Dihydrocannabinol and its oxygen
adduct.
CH3
OH
CH3
 -THC
C21 H30 O 2 ; F.W . 314; m/ z 315
8
H3C
H3C
O
OH
CH3
9
Cis- -THC
C21 H30 O 2 ; F.W . 314; m/ z 315
CH3
H3C
H3C
O
CH3
Cannabinol
C21 H26 O 2 ; F.W . 310; m/ z 311
CH3
CH3
HO
OH
OH
OH
OH
HO
H3C
H3C
O
CH3
Dihydrocannabinol
C21 H28 O 2 ; F.W . 312; m/ z 313
CH3
HO
Cannanbidiol (cis- or trans-)
C21 H30 O 2 ; F.W . 314; m/ z 315
CH3
CH3
H3C
H3 C
HO
OH
HO
Cannabinol
313
1 mL/min
O
Dihydrocannabinol
0.68
0.78
H3C
H3C
Dihydroxydihydrocannabinol
0.45
CH3
OH
Identification
0.82
Column Temperature 20ºC
Mobile Phase
Structures of Dronabinol
Impurities
• Identification of the specified impurities were confirmed by comparison to
H3 C
H3 C
O
CH3
Trihydroxydihydrocannabinol
C21 H28 O 5 ; F.W . 360; m/ z 361
HO
CH3
OH
OH
HO
O
CH3
Hydroxycannabinol
C21 H26 O 3 ; F.W . 326, m/ z 327
H3 C
H3 C
O
CH3
Hydroxydihydrocannabinol
C21 H28 O 3 ; F.W . 328; m/ z 329
H3 C
H3C
O
CH3
Dihydroxydihydrocannabinol
C21 H28 O 4 ; F.W . 344; m/ z 345
Conclusions
•
Impurities in Dronabinol Samples were identified.
• Specified impurities were Cannabinol, cis- 9-THC and 8-THC.
• Unspecified impurities were typically oxidative in nature. Structures were
proposed for the observed unspecified impurities based on LCMS results.
• Similar impurity profiles were observed in all three capsules and stressed
API, with an increase in number and amount of impurities.
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