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El-ahajBabiker Mohamed et al. Int. Res. J. Pharm.

2013, 4 (7)

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY


ISSN 2230 8407

www.irjponline.com
Research Article

STABILITY OF CODEINE: CHARACTERIZATION OF OXIDATION PRODUCTS OF CODEINE FORMED


IN AQUEOUS SOLUTION
El-ahajBabiker Mohamed1, ShehabNaglaa Ahmed2 and Ali Heyam Saad3*
1
Department of Pharmaceutical Chemistry, Ajman University of Science and Technology, Fujairah, UAE
2
Department of Pharmacognosy and Phytochemistry, Dubai Pharmacy College, Dubai, UAE
3
Department of Pharmaceutics, Dubai Pharmacy College, Dubai, UAE
*Corresponding Author Email: heyam57@hotmail.com
Article Received on: 19/03/13 Revised on: 01/04/13 Approved for publication: 25/05/13
DOI: 10.7897/2230-8407.04711
IRJP is an official publication of Moksha Publishing House. Website: www.mokshaph.com
All rights reserved.
ABSTRACT
The aim of the present study was to characterize some compounds resulting from storing codeine aqueous solutions under different conditions in search of an
impurity observed in some codeine preparations and in the urine of their users. Acidic, neutral and basic aqueous solutions of codeine were stored in the dark
or exposed to sunlight for one month. Aliquots of the solutions were withdrawn at 3-day intervals, extracted with organic solvent at basic pH and analyzed by
GC-MS before and after trimethylsilylderivatization; where applicable separated compounds were identified by database mass spectral libraries. Other
compounds were tentatively characterized from GC-MS data and reaction mechanistic interpretations. At neutral pH, codeine was partially oxidized to
norcodeine and codeinone while at basic pH only the former compound was formed. In both cases, light had no effect. In the acidic solutions exposed to light,
codeine was autoxidized to two products which were tentatively characterized as epimeric forms of 10-hydroxycodeine. None of the three products was
detected in pharmaceutical preparations of codeine or in opium. The nature of the oxidation products of codeine in aqueous solution is determined by the state
of protonation of the molecule as well as by the presence or absence of photo effects. The results indicated that codeine aqueous pharmaceutical preparations
should always be protected from light. On the other hand the stability of codeine in opium is most probably due to the co-presence of compounds with
antioxidant properties.
Keywords: Codeine; Oxidation; Norcodeine; Codeinone; Autoxidation; GC-MS

INTRODUCTION
Codeine (Figure 1) is a narcotic analgesic drug, which is used
in the alleviation of minor to moderate pain and as an
antitussive. Its pharmaceutical preparations (analgesic tablets,
capsules and antitussive syrups) are subject to abuse1 and are
sometimes used, in large doses, by heroin addicts to obtain a
rewarding effect or to justify the presence of morphine,
resulting from heroin, in their urine2. Codeine is prepared
either by the methylation of morphine or extraction from the
natural product opium3. During routine confirmation of
immunoassay-opiate-positive urine by GC-MS, we had
detected the opium alkaloid thebainone-A (Figure 2) in the
urine of codeine pharmaceutical preparation users. In
investigative experiments designed to trace the source of
thebainone-A in those preparations, aqueous solutions of
codeine at different pH values were exposed to sunlight or
kept in the dark and then analyzed by GC-MS. Thebainone-A
was not detected in any of the test solutions, however, other
compounds were, differing in nature according to the pH of
the solutions and the presence or absence of photo effects.
The main aim of this study was to characterize those
compounds and investigate their possible formation in
aqueous pharmaceutical preparations as they might be a
source of toxicity. Furthermore, as opium forms an important
source of codeine, it will be investigated for codeine-related
products that might have been formed during processing and
storage.
MATERIALS AND METHODS
Materials
Reference standard of codeine phosphate was a kind gift from
Pharmacare Pharmaceuticals, Dubai, UAE. Opium samples
were obtained as seizures submitted to our laboratory by the
Drug Enforcement Department of Sharjah Police (UAE).
Syrups containing codeine, pseudoephedrine and triprolidine

were obtained from local pharmacies. Bis (trimethylsilyl)


trifluoroacetamide
(BSTFA)
containing
1%
trimethylchlorosilane (TMCS) was purchased from Sigma
Aldrich, GmbH, Deisenhofen, Germany. Dichloromethane,
isopropanol, ethyl acetate, methanol, concentrated ammonia,
concentrated hydrochloric acid, sodium hydroxide and
sodium sulphate anhydrous, all of analytical grades, were
purchased from BDH Chemicals Ltd, Poole, England.
Methods
Codeine aqueous solutions: preparation, storage and GCMS analysis
Two sets of three 100 mg/ml aqueous solutions of codeine
phosphate were prepared in water, 0.1M HCl and 0.01M
NaOH, in screw-capped Pyrex glass test tubes. The solutions
of one set were exposed to the morning sunlight (at ~2530oC) for 100 hours in 30 consecutive days, while those of
the other set were kept in the dark at room temperature
(25oC). At 3-day intervals, the pH of 1 ml portions of all the
solutions was adjusted to ~10 with either 2 M HCl or 2 M
NaOH, as appropriate and each of the solutions was
extracted,
first
with
a
9/1
mixture
of
dichloromethane/isopropanol (3 ml) and then with ethyl
acetate (3 ml). After evaporation of the combined solvent
extracts under nitrogen at 45oC, each of the residues was
dissolved in methanol (1 ml) and a 2-mlaliquot was injected
into the GC-MS. The methanol was then evaporated off each
of the remaining solutions and the residues were separately
heated with BSTFA/1% TMCS (100 ml) at 60oC for 10 min.
Each reaction mixture was then diluted to 1 ml with ethyl
acetate and a 2 ml aliquot was injected into the GC-MS.

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Codeine-containing syrups: extraction of codeine and
GC-MS analysis
The pH of 5 ml of syrup containing codeine phosphate,
pseudoephedrine and triprolidine (10 mg/5 ml) was adjusted
to ~10 with conc. ammonia. The solution was extracted with
a 9/1 mixture of dichloromethane/isopropanol (10 ml). A 100
ml aliquot of the organic layer was diluted to 1 ml with the
dichloromethane/isopropanol mixture and a 2 ml aliquot was
injected into the GC-MS. The solvent was then evaporated
off the remaining solution under nitrogen at 45oC and the
residue was heated with BSTFA/1% TMCS (100 ml) at 60oC
for 10 min. The reaction mixture was diluted to 1 ml with
ethyl acetate and a 2 ml aliquot was injected into the GC-MS.

GC-MS instrumentation and analytical conditions


GC-MS analyses were conducted on an Agilent (HP) series
6980 GC interfaced with a 5973 HP quadruple mass selective
detector (MSD) (Palo Alto, CA, USA) using a HP 5MS
capillary column (Cross-linked 5% methyl phenyl silicone,
30 m x 0.25 mm i.d., 0.25 mm film thickness; HewlettPackard, Palo Alto, CA, USA). Analytical conditions: The
injector port and transfer line were maintained at 250oC and
280oC, respectively. Helium was used as the carrier gas at a
flow-rate of 1 ml/min. A 2-mlsplitless injection was
performed with the following oven program: an initial
temperature of 100oC held for 2 min and then ramped at
20oC/min to 280oC, with a final hold time of 7 min. The total
run time was 18 min. Electron impact (EI) analysis was
performed with the ionization energy set at 70eV. Data were
acquired in the scan mode over a range of m/z 50-550 at a
rate of 2 scans/second. The ion source temperature was
230oC.

Opium sample solutions: preparation and GC-MS


analysis
The solutions were prepared assuming that the opium
seizures contained ~1% w/w of codeine. A suspension of
powdered dry opium or of resinous opium cut into small
pieces (1 g) in 10% ammonia solution (10 ml) was shaken
with a 9/1 mixture of dichloromethane/isopropanol (10 ml)
for 10 min. After centrifugation at 3000 rpm for 5 min, the
organic layer was separated and extracted with 1M
hydrochloric acid (10 ml). The aqueous layer was separated
and basified to pH 10 with conc. ammonia and then extracted
with dichloromethane/isopropanol (10 ml). The organic layer
was then separated and filtered through a phase separator
filter paper containing anhydrous sodium sulphate. After
evaporation of the solvent from a 100 mL aliquot under
nitrogen at 50oC, BSTFA/1% TMCS (100 ml) was added and
the reaction mixture was heated at 60oC for 10 min; a 2 ml
aliquot was injected into the GC-MS.

Database mass spectral libraries


Three database mass spectral libraries were available for
characterization of unknown compounds: Wiley7N.l, Nist98.l
and PMW_Tox3.l.
NCH3

OH

C H 3O

Figure 1: codeine. Figure 2: Thebainone-A

Abundance
TIC: CODPHN_1.D
2800000
2600000

2400000
2200000
2000000
1800000

codeine

1600000
1400000
1200000
1000000
800000
600000

norcodeine

400000
200000
0

6.00

7.00

8.00

9.00

10.00

11.00

Codeinone

12.00

13.00

Time-->

14.00

15.00

Abundance
Scan 1239 (12.304 min): CODPHN_1.D

297

40000

35000

30000

25000

20000

42

15000
190
10000

240

115
81

5000

128

99

55

40

60

165

80

100

120

140

228

212

254

178

140

68
0

152

160

180

200

220

240

260

268

282

280

300

m/z-->

Figure 3: A: Total ion chromatogram obtained from a neutral-pH solution of codeine exposed to sunlight or in dark.

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El-ahajBabiker Mohamed et al. Int. Res. J. Pharm. 2013, 4 (7)


B: Mass spectrum of codeinone

Abundance
Ion 315.00 (314.70 to 315.70): CODPH1_1.D
300000
280000
260000

240000
220000
200000
180000
160000
140000

120000
100000
80000
60000

II

40000
20000
0

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

Time-->

Abundance
Scan 1354 (12.973 min): CODPH1_1.D

315

300000
280000

260000
44

240000
220000

112

200000

180000
160000
140000
120000
100000

126

80000
70

60000
40000

83

57
0

40

189
162

96

20000
60

80

100

120

204
239

139
140

160

258

225

175
180

200

286

272

220

240

260

280

298
300

320

m/z-->

Abundance
Scan 1419 (13.351 min): CODPH1_1.D

44

315

110000
100000

90000
112
80000
70000
60000

II

50000
40000
30000
70

189

20000

94

10000

204

162

128

239

146

258

40

60

80

100

120

140

160

180

200

220

298
282

223
0

240

260

280

387
300

320

340

360

380

m/z-->

Figure 4: A: Ion chromatogram at m/z 315, obtained from aqueous acidic-pH solution of codeine exposed to sunlight
B and C: Mass spectra of compounds I and II as per labels

Abundance

Ion 459.00 (458.70 to 459.70): HOCOD_2.D


500000
450000

III

400000
350000
300000
250000
200000
150000

IV

100000
50000
0

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

16.00

17.00

Time-->

B
III

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El-ahajBabiker Mohamed et al. Int. Res. J. Pharm. 2013, 4 (7)


Abundance
Scan 1406 (13.126 min): HOCOD_2.D

73
600000
550000
500000
450000

196

400000
459

350000
300000
250000
200000
150000
94

100000

256

234
50000
0

40

60

80

100

120

370

171

129

55

152
140

160

180

200

220

260

280

431

342

294
240

402

312

275

216

300

320

340

360

380

400

420

440

460

m/z-->

Abundance
Scan 1454 (13.400 min): HOCOD_2.D

73
550000
500000
450000

196

400000
350000

459
300000
250000
200000
150000
100000

94
256

234

50000
0

129

55
40

60

80

100

120

171

152
140

275

216
160

180

200

220

240

260

280

300

402

370

312

431

342

294
320

340

360

380

400

420

440

460

m/z-->

Figure 5: A: Extractedion chromatogram at m/z 459, obtained from aqueous acidic-pH solution of codeine exposed to sunlight
B and C: Mass spectra of compounds III and IV as per labels. The GC-MS analysis was carried out after TMS derivatization
Abundance
Ion 459.00 (458.70 to 459.70): OPNEO2.D
300000
280000

260000
240000
220000
200000
180000
160000
140000
120000
100000
80000
60000

40000
20000

6.00

7.00

8.00

9.00

10.00

11.00

12.00

13.00

14.00

15.00

Time-->

Abundance
Scan 1263 (12.444 min): OPNEO2.D
229
800000
700000
600000
500000
400000
73

459

300000

200000
370
100000
45
0

50

103
100

164

132
150

188

207
200

254
250

328

284
309
300

350
350

403
400

431
450

m/z-->

Figure 6: A: Extractedion chromatogram at m/z 459, obtained from TMS-derivatized basic-pH extract of opium.
B: mass spectrum of the compound V

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El-ahajBabiker Mohamed et al. Int. Res. J. Pharm. 2013, 4 (7)


N C H

16

C o d e in e
M W

= 299

C H

9
14

11

a u t o x id a t io n
( a c id ic s o lu t io n ,
s u n lig h t )

15
7

13

12
3

17

10

O H
o x id a t io n
( n e u t r a l s o lu t io n )

N - d e m e t h y la t io n
( n e u t r a l o r b a s ic s o lu t io n )

N C H

N H

C H

C H

O H

N C H

H O

N o r c o d e in e
M W = 285

C o d e in o n e
M W = 297

N C H

H O

+
C H

C H

O H

M W
M W

O H

A lp h a - 1 0 - h y d r o x y c o d e in e

B e t a - 1 0 - h y d r o x y c o d e in e
= 315

o f d i- T M S d e r iv a t iv e = 4 5 9

Figure 7: Chemical structures of oxidation and autoxidation products of code

RESULTS AND DISCUSSION


Codeine
In aqueous solution, codeine exhibited different behaviors
according to the state of protonation of the molecule, as
governed by the pH of the solution, and the presence or
absence of photo effects. The results are depicted in Figure 3,
4 and 5. In neutral/basic-pH solutions, exposed to sunlight or
kept in the dark, codeine was N-demethylated to norcodeine
(Figure 3 and 7), which is a known product of microbial
oxidation4,5. In the neutral solution, exposed to light or kept
in the dark, codeine was oxidized to codeinone (Figure 3 and
7), which is a known product of chemical andmicrobial 6
oxidation of codeine. Both norcodeine and codeinone have
been characterized by database mass spectral libraries. At the
end of the reaction time, i.e. 100 hours, the two degradation
products were estimated to have been formed in
approximately 17% concentrations of that of codeine as
measured from peak heights in the corresponding
chromatogram (Figure 3). In acidic solutions kept in the dark,
codeine did not undergo any change; however, in the acidic
solutions exposed to sunlight, it behaved in a different way to
the neutral/basic solutions. At the end of the reaction time,
i.e. 100 hours, almost all of the codeine was oxidized to a
mixture of two hydroxycodeines of identical mass spectra
(compounds I and II, Figure 4). The formation of the
hydroxycodeines has been judged from: (a) increase of the
molecular weight of codeine by 16 (i.e. the atomic weight of
oxygen) giving rise to both compounds I and II (Figure 4)
and (b) formation of di-trimethylsilyl derivatives of
compounds I and II (compounds III and IV respectively,
Figure 5). The identical mass spectra of compounds I and II
(Figure 4) and of their corresponding TMS derivatives,
compounds III and IV (Figure 5) indicate that the two
compounds are diastereomeric. Hydroxylation of candidate
compounds exposed to light for a long time is an autoxidation
reaction7. The role of light is to generate free radicals to
which molecular oxygen adds to form hydroperoxides
(ROOH), which will react further to give alcohols8.
Mechanistically, the two most favoured auto hydroxylation
sites in the codeine molecule are the secondary-benzylic C10
and the tertiary allylic C14 (Figure 1) which is due to their
high reactivity towards free radical formation. The latter
carbon was excluded as the site of hydroxylation in

compounds I and II based on observed differences in the


mass spectra: the m/z of the base-peak ions of the TMS
derivatives of compounds I and II and that reported for 14hydroxycodeine8 were 196, 196 and 229, respectively.
Therefore, compounds I and II could be designated as 10hydroxycodeine formed in two diastereomeric (a and
bepimeric) forms (Figure 7). Epimerization is a process that
takes place in compounds in aqueous solutions whereby a
functional group at a chiral carbon undergoes partial
inversion of configuration9. Usually, epimeric forms of a
substance in aqueous solution exist in equilibrium10. It may
be of interest to note that chemical hydroxylation of codeine
by hydrogen peroxide had been reported11 to take place at the
aromatic ring; however, the authors did not give evidence of
this nor did they specify the position of the hydroxy group.
Analytical artifactual formation of the codeine degradation
and autoxidation products under the experimental conditions
used in this study was ruled out by analyzing fresh solutions
of codeine phosphate; none of the degradation products was
detected. Furthermore, contrary to previous reports12, none of
the degradation products found in this study was an isomer of
codeine. Neither norcodeine nor codeinone was detected in
syrups of codeine that had been stored in the dark for more
than two years suggesting that other ingredients present in the
syrups had played an antioxidant role. Although it is unlikely
that codeine pharmaceutical preparations will be formulated
at the acidic conditions that favor autoxidation in the
presence of light, catalysis by excipients may not be
completely ruled out and should be investigated.
Opium
As early as 1911, opium was reported to contain a
hydroxycodeine13. The data shown in Figure 6 confirm this
and almost certainly indicate the presence of 14hydroxycodeine upon comparing the mass spectrum with that
reported in the literature8. Since position 14 in opium is
allylic and represents an active site for autoxidation, 14hydroxycodeine in opium may be an autoxidation product the
formation of which is influenced by the natural matrix of
opium. Generally, the processing of opium involves, in its
early stages, sun drying of the latex, which is an aqueous
suspension.
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Morphine
As a matter of interest, the same experiments performed with
codeine were repeated with morphine. No oxidation products
of morphine analogous to those of codeine found in this work
were detected for morphine.
CONCLUSION
Light and pH of aqueous solutions are two important
parameters to consider in stability investigations of medicinal
substances in their pharmaceutical preparations and
processed natural sources. Generally, the presence of
compounds with antioxidant properties will help avert
oxidation and autoxidation of pharmacologically active
ingredients in pharmaceutical dosage forms and natural
products. It is recommended that stability studies should
involve investigations of antioxidants with the aim of their
inclusion in pharmaceutical formulations of the subject
substance. The dependence of the chemical nature of
degradation of a compound on the state of protonation of the
molecule in aqueous solution is an interesting subject for
exploration.
REFERENCES
1. Sweetman S. Martindale: The Complete Drug Reference. 33rd ed.
Sweetman SC, the Pharmaceutical Press, London; 2002. p. 25
2. Al Amri AM. Marker compounds in urine for opiate product use. A
Doctoral thesis, Department of Chemistry, Loughborough University,
England; 2004.
3. Thomas N. Encyclopedia of alcohol and drug abuse. Brown Walker
Press, Parkland, Florida, USA; 2002.
4. Garrido JMPJ, Delerue Matos C, Borges F, Macedo TRA, Oliveira Brett
AM. Voltammetric Oxidation of Drugs of Abuse II Codeine and

5.

6.
7.

8.
9.

10.

11.

12.
13.

Metabolites. Electroanalysis 2004; 16(17): 1427-1433. http://dx.doi.org/


10.1002/elan.200302967
Long MT, Hailes AM, Kirby GW, Bruce NC. Transformation of
morphine alkaloids by Pseudomonas putida M10. Applied and
Environmental Microbiology 1995; 61(10): 3645-3549. PMid:7487001
PMCid:PMC167664
Findlay SP, Small LF. The preparation and properties of codeinone. J
Am Chem Soc 1950; 3247. http://dx.doi.org/10.1021/ja01163a126
Kunz DA, Reddy GS, VatvarsA. Identification of transformation
products arising from bacterial oxidation of codeine by Strepromyce
sgriseus. Applied and Environmental Microbiology 1985; 831-836.
PMid:3936418 PMCid:PMC291756
Smith MB, March J. Marchs Advanced Chemistry, Reactions,
Mechanisms and Structure. 5thed, John Wiley and Sons, Inc., New York;
2000. p. 770-773 PMid:11147435
Bruce NC, Wilmot CJ, Jordan KN, Stephens LD, Lowe CR. Microbial
degradation of the morphine alkaloids. Purification and characterization
of morphine dehydrogenase from Pseudomonas putida M10. Biochem J
1991; 274: 875-80. PMid:2012614 PMCid:PMC1149991
Cerami J, Gala D, Hou D, Kalliney S, Mas JL, Nyce P, Peer L, Wu G.
Dilevalol via resin-mediated epimerization: A case study. Reaction
mechanism to reactor design to a viable process. Org Proc Res Dev
2000; 4: 107-121. http://dx.doi.org/10.1021/op990185y
Tanaka R, Iio S, Chinaka N, Takayama KH. Analysis of reaction
products of morphine and codeine with hydrogen peroxide by high
performance liquid chromatography/mass spectrometry. Analytical
Science 2003; 19: 163-165. http://dx.doi.org/10.2116/analsci.19.163
PMid:12558043
Gundermann P, Pohloudek Fabini R. Analysis and the stability of
codeine Phosphate. Pharmazie 1983; 38: 92-94. PMid:6856666
Opium, B.P. in the British Pharmaceutical Codex, The British
Pharmaceutical Society of Great Britain, London, 1911.
www.ibiblio.org/herbmed/electric/bpc1911/papaver.html

Cite this article as:


El-ahajBabiker Mohamed, ShehabNaglaa Ahmed and Ali Heyam Saad.
Stability of codeine: Characterization of oxidation products of codeine
formed in aqueous solution. Int. Res. J. Pharm. 2013; 4(7):49-54
http://dx.doi.org/10.7897/2230-8407.04711

Source of support: Nil, Conflict of interest: None Declared

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