International Journal of Analytical Mass Spectrometry and Chromatography, 2013, 1, 5-10 Published Online September 2013 (
Mass Spectrometric Structure Elucidation of the Trivalent
and Pentavalent Nitrogen Contaminants of Pholcodine in
the Cough Relief Medical Form Tuxidrin
Ilia Brondz1,2
1Department of Biosciences, University of Oslo, Oslo, Norway
2R&D Department of Jupiter Ltd., Ski, Norway
Received July 5, 2013; revised August 7, 2013; accepted September 1, 2013
Copyright © 2013 Ilia Brondz. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the paper “Supercritical Fluid Chromatography-Mass Spectrometry (SFC-MS) and MALDI-TOF-MS of Heterocyclic
Compounds with Trivalent and Pentavalent Nitrogen in Cough Relief Medical Forms Tuxi and Cosylan” [1], the pre-
sence of morphine and other degradation products of pholcodine in cough relief medical forms of Tuxi are discussed.
Tuxi is recalled from the Norwegian market by Weifa pharmaceutical company, and hence it no longer presents prob-
lems to users and health autho rities there; however, the medical form Tuxidrin, which contains a significan t amount of
pholcodine as the active pharmacolo gical ingredient, is still marketed. In the present paper, Tuxidrin is analyzed to de-
termine the presence of degradation products of pholcodine. The degradation of pholcodine to morphine has been dis-
cussed previously as a factor in the development of addiction to narcotics in young persons. The structures of the con-
taminants in Tuxidrin, such as oxides of pholcodine, are elucidated in the present paper. The toxicity and pharmacology
of oxides of alkaloids have generally not been well studied, and very little is known about the toxicity and pharmaco-
logy of the degradation (oxidation) products of pholcodine: the N-oxide and the N, N'-dioxide of pholcodine. According
to Brondz and Brondz [1], the N-oxide and possibly also the N, N'-dioxide are less toxic than the original alkaloids and
possess greater pharmacological activity, and hence they may be a source of useful new semisynthetic drugs. The ques-
tion of possible addiction to pholcodine oxides has not been studied, and the potential of these substances to provoke
allergies is unclear. The recall of Tuxi from the Norwegian market is mainly based on the fact that pholcodine causes
significantly increased levels of IgE antibodies in sensitized patients. Tuxidrin contains pholcodine and has the same
negative effect as Tuxi, namely provoking allergies or even anaphylactic shock. From this point of view, Tuxidrin has
no advantage over Tuxi. These two medical forms only differ in one respect: Tuxidrin requires a prescription (prescrip-
tion duty medicine), but Tuxi does not (prescription free medicine). This aspect is also discussed in the present paper.
Keywords: High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS); Trivalent Nitrogen;
Pentavalent Nit rogen; Ph ol co dine; Al kaloids; Addic tion to Narcotics; Allergy; Tuxidrin; Morphine;
Pholcodine-N-oxide,10-Hydroxy-pholcodine; IgE Antibodies
1. Introduction
The presence of degradation products such as morphine,
pholcodine-N-oxide, and pholcodine-N,N'-dioxide in the
medical formulation Tuxi is described in [1]. The facts
relating to the degradation of pholcodine to morphine
had been known at the Norwegian pharmaceutical com-
pany Weifa since 1997, when analytical data for the de-
gradation of pholcodine were presented by J. Røe in [2],
and later supported in [1,3]. As early as 1988, Findlay
strongly criticized formulations containing pholcodine
[4]. To date, this situation remains unchanged. Weifa
subsequently recalled Tuxi from the market, but not
The difference between Tuxi and Tuxi Forte, on the
one hand, and Tuxidrin, on the other hand, is that Tuxi
and Tuxi Forte do not contain ephedrine, but Tuxidrin
does. Tuxidrin is a prescription duty medication, mainly
because it contains ephedrine, whereas Tuxi does not re-
quire a prescription. (All forms of medication that con-
tain even small amounts of narcotics must be sold as pre-
scription-duty medications by law.) The necessity of in-
cluding ephedrine as cough relief agent in Tuxidrin is
opyright © 2013 SciRes. IJAMSC
questionable. Ephedrine is a sympathomimetic amine
commonly used as a stimulant, appe tite suppressant, con-
centration aid, and decongestant, and to treat hypotension
associated with anesthesia. It has a similar structure to
amphetamine and methamphetamine. Inclusion of ephe-
drine in the formulation of Tuxidrin was considered ne-
cessary for masking the harm that was observed to be
caused by unrestricted, prescription-free use of Tuxi. The
latter had led to an elevated number of allergy and po ssi-
ble asthma cases in the Norwegian population relative to
the popu lation in n eig hboring Swed en. In Sw eden , wher e
the dominant antitussive medication, Cosylan, was avai-
lable as a prescription-duty medication, the allergic reac-
tivity was six times lower than in Norway.
Furthermore, it is not only opiates that are under regu-
lation. In Ireland, New Zealand, the USA, and most other
countries, the N-oxides of opium alkaloids are covered
under laws and regulations for narcotics control. Phol-
codine in high concentrations in medical forms is also
under narcotics control laws and regulations as a mor-
phine d e r ivati ve.
As stated by Findlay in [4], pholcodine “has been for-
mulated in many combination medications (45)—some
rational and some quite irrational pharmacologically…
Pholcodine has been included in many problematic medi-
cal formulations, especially liquid mixtures with pH le-
vels at which pholcodine quickly degrades. It has been
shown that the opiate in the liquid medical form Cosylan
does not degrade to morphine or oxidize to N-oxide [1],
but in Tuxi, the opiate derivative pholcodine does. The
degradation of pholcodine to morphine is strongly de-
pendent on the pH of the medium [1]. The pH of the
Tuxidrin formulation was optimized (relative to that of
Tuxi) in order to retard the degradation of pholcodine to
morphine; however, its pH was not optimized to retard
the degradation of pholcodine into other oxidation pro-
ducts. It is therefore of interest to examine Tuxidrin for
the presence of contaminants (see Figures 1 and 2).
As is mentioned in [1], and earlier in [5], N-oxides of
some opium alkaloids are less toxic than original alka-
loids, and often have higher specific activity than the
original alkaloids alone. The toxicity of morphine-N-
oxide was studied, as described in [5]. The authors of [5]
stated: “The intravenous and subcutaneous acute toxici-
ties of morphine-N-oxide (mno) in mice were respectively
3.2 and 8 times less than that of morphine. Amiphenazole
or tacrine reduced the acute toxicity of mno but not that
of morphine in mice. The chronic toxicity of mno was
examined in mice and rats. Daily oral doses of 100
mg/kg did no t significantly affect gr owth or condition, or
produce gross or microscopic lesions in mice treated for
3 weeks or rats treated for 3 months. No teratogenic ef-
fect of mno or of bromolysergic acid diethylamide was
observed in rats”.
Figure 1. There is a chromatogram of Tuxidrin. Tuxidrin
(hostedempende, slimløsende) mixture, produced by Weifa
(Norway), was purchased from the Norwegian Medicinal
Depot in Oslo, Norway.The peaks and retention times are as
follows: pholcodine at 6 min, N-oxide of pholcodine at 6.5
min, 10-hydroxy-pholc odine at 11 min, and ephedrine at 14
min. The mass spectra of the substances corresponding to
the peaks were recorded by MS with inline to HPLC. The
spectra of substances related to pholcodine are given below;
the mass spectra of pholcodine, N-oxide of pholcodine, and
10-hydroxy-pholcodine are shown in Figure 4, the spectrum
of ephedrine is not presented. Conditions used for HPLC-
MS are described in the Materials and M ethods section.
Figure 2. The percentage of oxidation products present in,
calculated as a percentage of the total amount of pholcodine.
The blue line is the concentration of pholcodine-N-oxide
and the green line is the concentr ation of 10-hydroxy- phol-
codine. Temperature: 60˚C. Duration of accelerated degra-
dation: 62 days.
The N-oxides of pholcodine mimic quaternary ammo-
nium ions; but, they are not equivalent. However, re-
searchers have not yet studied the tendency of quaternary
ammonium ions of degradation contaminants in medical
formulations containing pholcodine to provoke allergies.
The major allergenic epitopes in IgE-mediated anaphy-
laxis to neuromuscular blocking agents (NMBAs) of de-
gradation contaminants in medical formulations contain-
ing phol codin e were not studied.
10-hydroxy-pholcodine should possess the same al-
Copyright © 2013 SciRes. IJAMSC
lergenic potential as pholcodine. Sensitization toward a
quaternary ammonium ion epitope, which NMBAs share
with many common drugs and chemicals, for example,
morphine, is known as one of the substances.
The presence of degradation products in Tuxidrin,
which are generated during its normal shelf storage time,
has practical significance, because the toxic effects of
pholcodine and its degradation products are not limited
to possible addiction. Expressions of toxicity include
significantly increased levels of IgE antibodies in sensi-
tized patients [6,7], and the provocation of allergies or
even anaphylactic shock. In Norway, sensitivity toward
NMBAs is six times higher than in neighboring Sweden.
This is possibly a result of the monopolization of the
cough relief drug market in Norway by Tuxi, manufac-
tured by Weifa, as a prescription free medication, in con-
trast to the status of Cosylan as a prescription-duty
medication in Sweden.
The generation of degradation products during shelf
storage of the drug pholcodine was measured by simula-
tion, using accelerated degradation under stress condi-
tions (see Figures 2 and 3). The elucidation of the che-
mical structures of the degradation products and other
contaminants was a priority in this study.
2. Materials and Methods
2.1. Instrumentation and Conditions
For HPLC analyses, an Agilent 1100 chromatograph
with a diode array detector and ChemStation software
(Agilent Technologies In c., Colorado Springs, CO, USA)
was used. A 50189-U-Nucleosil HPLC column with the
following parameters was used for enhanced separation:
50189-U Supelco Nucleosil HPLC column, phase C18,
length 25 cm i.d. 4.6 mm, purchased from Sigma-Al-
drich (Sigma-Aldrich, St. Louis, MO, USA). The follow-
ing chromatographic conditions were used. The compo-
sition of the mobile phase was 5.0 mL p.a. quality
triethylamine (Merck, Darmstadt, Germany) and 5.0 mL
p.a. quality trifluoroacetic acid (Merck) added to a 1.0 L
solution of double distilled deionized water containing
2.5% p.a. quality tetrahydrofuran (Merck) and 1.5% p.a.
quality methanol (Merck). Elution was performed iso-
cratic, using a flow rate of 1.2 mL/min. The injection
volume was 20 L. The HPLC system was coupled
inline to a Quattro MS/MS triple quadrupole mass spec-
trometer (Micromass, Altrincham, UK) equipped with a
pneumatically assisted electro-spray ionization source.
Data acquisition and processing wereperformed using a
MassLynx 4.0 SP4 data system (Waters). The effluent
entered the mass spectrometer through an electro-spray
capillary set at 3.0 kV at a source block temperature of
90˚C. The desolvation gas temperature was 150˚C. Ni-
trogen was usedas both drying gas and nebulizing gas at
flow rates of approximately 610 and 80 L/h, respectively.
Figure 3. HPLC-MS analyses of Tuxidrin (experimental
conditions described in the text). The upper chromatogram
shows the Tuxidrin just off the production line, beneath is
the same sample analyzed after 9, 27 and 62 days. The
peaks with their retention times are as follows: pholcodine
at 6 min, pholcodine-N-oxide at 6.5 min, 10-hydroxy-phol-
codine at 11 min, and ephedrine at 14 min.
The ion source parameters were optimized with respect
to the positive molecular ions. The cone voltage was set
at 120 - 210 V. The mass spectra between m/z 50 and m/z
600 were obtained at a scan speed of 200 m/z units/s with
a mass resolution corresponding to 1 unit at half peak
2.2. Materials and Standards
The standards used in the study are described elsewhere
[1]. Tuxidrin (hostedempende, slimløsende) mixture, pro-
duced by Weifa (Norway), was purchased from the Nor-
wegian Medicinal Depot in Oslo, Norway. It was stored
for 3 years at room temperature in the original bottle un-
Copyright © 2013 SciRes. IJAMSC
til the time of analysis. The conditions for accelerated
stress degradation a re described elsewhere [1].
3. Results and Discussion
The importance of antitussive drugs is difficult to overes-
timate [1]; however, uncontrolled consumption of them,
as in the case of Tuxi, is indefensible. The presence of
narcotics and allergenic drugs in medications for con-
sumption by children should be avoided, most especially
if ingestion is a repeated act. The concern here is about
not only the presence of morphine itself in the medical
form, as a degradation product from the active pharma-
cological ingredient (API) in Tuxi and Tuxidrin, but also
the presence of oxidation products and contaminants of
pholcodine. The repeated consumption of opiates or de-
rivatives ofopiates in the early stag es of life has undispu-
table effects on the person’s immune system and psy-
chological development, and can even cause addiction to
narcotics, sensitivity to allergens, asthmatic reactions, or
anaphylactic shock. At the pH of the human stomach,
pholcodine is hydrolyzed to morphine [1], as was well
known by Weifa [2]. At the pH of the human stomach,
pholcodine undergoes accelerated degradation. It is not
always possible to avoid the use of cough relief medi-
cines, but it is possible to avoid the presence of these
additional, and undesirable, narcotic substances as pro-
duction contaminants or degradation products in such
In this context, it was con sidered important to iden tify
the degradation products of pholcodine in Tuxidrin, and
to elucidate the nature of the contaminants generated
during shelf storage and under different pH conditions.
This was the aim of the present study.
Figures 1-3 show the degradation process and the ac-
cumulation of degradation products in Tuxidrin. The
substances were detected at 254 nm. The chromatogra-
phy conditions were described in the previous section.
The peak of the taste correcting substance is not shown
in the chromatogram, as it has a long retention time in
the system used here. There are four significant peaks in
the chromatogram, and their spectra were recorded by
MS: the first peak is pholcodine, the second and third
peaks have m/z 415, and the fourth peak is ephedrine.
Mass spectra are shown in Figure 4 (except the mass
spectrum for ephedrine).
The elucidation of the two different contaminants with
m/z 415 was based on several facts. According to Proksa
[8], 10-hydroxy-morphine is a contaminant of morphine.
The starting product for the synthesis of pholcodine is
morphine, which may be contaminated with 10-hydroxy-
morphine, and one of the resulting products can be 10-
hydrox y-pholcodine [3] ( s ee Figures 5(a) and (b)).
The 10-hydroxy-morp hine is not the natural p roduc t of
biogenesis; it is the oxidation artifact that present in an
Figure 4. The mass spectra recorded for the HPLC chro-
matogram of Tuxidrin. The upper figure shows the ma-
ssspectrum of pholcodine, the middle figure shows the mass
spectrum of pholcodine-N-oxide, and the bottom figure
shows the mass spectrum of a substance tentatively identi-
fied as 10-hydroxy-pholcodine. Conditions used for MS are
described in the M aterials and Methods section.
opium cake and/or as a result of the extraction of mor-
phine from opium cake or poppy plant tissue. Oxygen is
always present in the atmosphere and in extracting liq-
uids; it is the cause of morphine degradation. The phol-
codine in Tuxidrin can also be oxidized at positions other
than the nitrogen atoms [1,3]. The possible appearance in
the Tuxidrin of the structure Figure 5(b) within normal
shelf storage times should be examined and validated.
We therefore exposed Tuxidrin to thermal (60˚C), pH,
and time stress. The results are shown in Figures 2-4.
The concentration of both degradation substances, which
are oxides, increased (Figure 3). This offers the best evi-
dence that the substances are degradation (oxidation)
products. The second substance has an identical retention
time and MS spectrum to that of standard pholcodine-N-
oxide [1].
The MS spectra of the two degradation (oxidation)
Copyright © 2013 SciRes. IJAMSC
Figure 5. Structures: a ispholcodine, and bis10-hydroxy-
substances in chromatogram with m/z 415 do have sig-
nificant differences. The spectrum of substance three
shows fragmentions with m/z 284 (Figure 6(b)). A frag-
ment ion with m/z 284 is theoretically possible for phol-
codine-N-oxide (Figure 6(a)); however, under the HPLC-
MS experimental conditions used here, it is absent. This
fragment ion with m/z 284 is also absent in the mass
spectrum of pholcodine-N-oxide under the SFC-MS ex-
perimental conditions [1].
The oxidation of nitrogen at the morpholine moiety in
pholcodine should yield mass spectra showing ions with
m/z 116 and m/z 130 (Figures 7(a) and (b)).
The presence of ions with m/z 100 and m/z 114 in all
three substances was demonstrated. There were fragment
ions with m/z 100 and m/z 114 in mass spectra (see Fig-
ures 6(c) and (d)). These are fragments of the nonoxy-
genated morpholine moiety. In Figure 4, the ions with
m/z 116 and m/z 130 were absent in the spectra of phol-
codine, pholcodine-N-oxide, and 10-hydroxy-pholcodine.
This is evidence that oxygenation of the pholcodine oc-
curred at the morphine moiety.
4. Conclusions
1) Two degradation products in Tuxidrin with m/z 415
were recorded, and these accumulated during the shelf
storage time.
2) One of these degradation products is 10-hydroxy-
pholcodine, and the other is pholcodine-N-oxide.
3) The source of the degradation product 10-hydroxy-
pholcodinein in Tuxidrin is pholcodine.
4) The pH in Tuxidrin is optimized in order to avoid
the degradation of pholcodine to morphine; however, it is
not adequate to prevent the degradation of pholcodine to
Figure 6. Structures of the ions with m/z 284: (a) an ion
theoretically derived from pholcodine-N-oxide, and (b) an
ion derived from 10-hydroxy-pholcodine and fragment ions
(c) with m/z 100 and (d) with m/z 114.
Figure 7. Structures of the ions with a m/z 116 and bm/z 130
were not recorded in the spectra of degradation products of
pholcodine in Tuxidrin.
other degradation products.
5) Recalling Tuxi from the market is justified , but con-
tinued over-the-counter sale of Tuxidrin is indefensible
because of the fact that the same active pharmacological
ingredient is present in both products, and similar degra-
dation substances are present in both formulations.
5. Recommendations
1) Allergic reactions to 10-hydroxy-pholcodine and
pholcodine-N-oxide should be studied.
2) Tuxidrin should be recalled from the market, as was
done in the case of Tuxi.
6. Acknowledgements
The author is grateful to Jupiter Ltd., Norway, for fi-
Copyright © 2013 SciRes. IJAMSC
Copyright © 2013 SciRes. IJAMSC
nancial support, and to Jon Reierstad at the Technical
Department of the University of Oslo, Oslo Norway, for
technical assistance with the preparation of the figures.
[1] I. Brondz and A. Brondz, “Supercritical Fluid Chroma-
tography—Mass Spectrometry (SFC-MS) and MADI-
TOF-MS of Heterocyclic Compounds with Trivalent and
Pentavalent Nitrogen in Cough Relief Medical Forms
Tuxi and Cosylan,” American Journal of Analytical
Chemistry, Vol. 3, No. 12A, 2012, pp. 870-876.
[2] J. Røe, “Identification of Pholcodine Degradation Prod-
ucts/Determination of Chemical Structures,” The 13th Te-
chnical Conference, Wilmington, September 1997.
[3] O. M. Denk, G. G. Skellern and D. G. Watson, “Impurity
Profiling of Pholcodine by Liquid Chromatography Elec-
trospray Ionization Mass Spectrometry (LC-ESI-MS),”
Journal of Pharmacy and Pharmacology, Vol. 54, 2002,
pp. 87-98. doi:10.1211/0022357021771788
[4] J. W. Findlay, “Pholcodine,” Clinical Pharmacology &
Therapeutics, Vol. 13, No. 1, 1988, pp. 5-17.
[5] M. R. Fennessy and H. J. Fearn, “Some Observations on
the Toxicology of Morphine-N-Oxide,” Journal of Phar-
macy and Pharmacology, Vol. 21, No. 10, 1969, pp. 668-
673. doi:10.1111/j.2042-7158.1969.tb08143.x
[6] E. Florvaag, S. G. O. Johansson, H. Öman, T. Harboe and
A. Nopp, “Pholcodine Stimulates a Dramatic Increase of
IgE in IgE-Sensitized Individuals. A Pilot Study,” Allergy,
Vol. 61, 2006, pp. 49-55.
[7] T. Harboe, S. G. O. Johansson, E. Florvaag and H. Öman,
“Pholcodine Exposure Raises Serum IgE in Patients with
Previous Anaphylaxis to Neuromuscular Blocking Agents,”
Allergy, Vol. 62, No. 12, 2007, pp. 1445-1450.
[8] B. Proksa, “Separation of Morphine and Its Oxidation
Products by Capillary Zone Electrophoresis,” Journal of
Pharmaceutical and Biomedical Analysis, Vol. 20, No.
1-2, 1999, pp. 179-183.