Isolation and characterization of cetirizine degradation product: Mechanism of cetirizine oxidation

Article abstract of DOI:10.1007/s11095-010-0114-x

Purpose: The goal of the study was to isolate and analyze a cetirizine degradation product, formed within a PEG-containing formulation and to elucidate the mechanism of oxidation of cetirizine. Methods: Cetirizine, formulated in PEG-containing matrix, was subjected to forced degradation conditions in the pH range from 3 to 10, and the product was analyzed by HPLC and LC-MS/MS. Additionally, pure cetirizine was subjected to selective oxidization by hydrogen peroxide and sodium percarbonate. The reaction mixture was purified, and the isolated material was analyzed by ¹H NMR. Results: Oxidation process was investigated in order to model the degradation of cetirizine in PEG-containing formulation. Site of oxidation is proposed based on correlation of the results of forced degradation with ionization scheme of cetirizine. The finding was verified by spiking of cetirizine degradation sample with cetirizine N-oxide reference standard. Conclusions: Degradation of cetirizine in polyethylene glycol arose from the reaction between the drug and the reactive peroxide intermediates such as peroxyl radicals formed through oxidation of PEG. Selective oxidation of cetirizine and isolation/characterization of the oxidation product allowed the identification of the oxidation product as cetirizine N-oxide. The mechanism of oxidation is proposed.

Full text of DOI:10.1007/s11095-010-0114-x

Pharm Res (2010) 27:1318–1324
DOI 10.1007/s11095-010-0114-x
RESEARCH PAPER
Isolation and Characterization of Cetirizine Degradation
Product: Mechanism of Cetirizine Oxidation
Tatyana Dyakonov & April Muir & Hassen Nasri & Dana Toops & Aqeel Fatmi
Received: 20 May 2009 /Accepted: 24 November 2009 /Published online: 31 March 2010
#
Springer Science+Business Media, LLC 2010
ABSTRACT
INTRODUCTION
Purpose The goal of the study was to isolate and analyze a
cetirizine degradation product, formed within a PEG-containing
formulation and to elucidate the mechanism of oxidation of
cetirizine.
Cetirizine hydrochloride (cetirizine), the active component
of ZYRTEC® tablets and syrup, is an orally active and
selective H1-receptor antagonist used for the treatment of
allergy symptoms. Soft gelatin capsules (Softgels) offer the
possibility of delivering a liquid in a solid oral dosage form.
A softgel can therefore contain the active ingredient in
solution, suspension or emulsion, which may inherently
lead to better absorption of the active ingredient compared
to the delivery in a tablet or powder. Many problems asso-
ciated with tableting, including poor compaction and lack of
content or weight uniformity, can be eliminated if drug is
incorporated into this dosage form. Softgel dosage forms are
perceived by customers as easier to swallow, help to mask the
taste of the medicine and provide faster onset of action (1). In
order to develop this highly water soluble and sensitive
compound in soft gelatin dosage form, cetirizine was
formulated in PEG-containing matrix. However, cetirizine
formulated in PEG-containing solution for Softgels was
found to degrade during manufacture and storage.
Our preliminary results, supported by the literature
reports (2), showed that cetirizine does not degrade under
acidic or basic conditions. It is known that cetirizine is
susceptible to oxidation under chemical (hydrogen peroxide),
biochemical (enzymatic) and electrochemical conditions (3).
The metabolism of levocetirizine (R enantiomer of cetirizine)
was studied (3) and found to proceed through the metabolic
pathways: hydroxylation, O-dealkylation, N-oxidation and N-
dealkylation (Fig. 1).
Methods Cetirizine, formulated in PEG-containing matrix,
was subjected to forced degradation conditions in the pH
range from 3 to 10, and the product was analyzed by HPLC
and LC-MS/MS. Additionally, pure cetirizine was subjected to
selective oxidization by hydrogen peroxide and sodium
percarbonate. The reaction mixture was purified, and the
1
isolated material was analyzed by H NMR.
Results Oxidation process was investigated in order to model
the degradation of cetirizine in PEG-containing formulation. Site
of oxidation is proposed based on correlation of the results of
forced degradation with ionization scheme of cetirizine. The
finding was verified by spiking of cetirizine degradation sample
with cetirizine N-oxide reference standard.
Conclusions Degradation of cetirizine in polyethylene glycol
arose from the reaction between the drug and the reactive
peroxide intermediates such as peroxyl radicals formed
through oxidation of PEG. Selective oxidation of cetirizine
and isolation/characterization of the oxidation product allowed
the identification of the oxidation product as cetirizine N-oxide.
The mechanism of oxidation is proposed.
.
.
KEY WORDS cetirizine oxidation PEG
:
:
:
:
T. Dyakonov (*) A. Muir H. Nasri D. Toops A. Fatmi
Banner Pharmacaps,
Excipients containing polyoxyethylene chains, for example
polyethylene glycol (PEG) 400 and polysorbate, are known to
contain some residual peroxides, which accumulate during
storage and could potentially affect the stability of oxidation-
4125 Premier Drive,
High Point, North Carolina 27265, USA
e-mail: tadyakonov@banpharm.com

Mechanism of Cetirizine Degradation
1319
HO
chromatograms were acquired using Chemstation LC 3D
Rev A 10.02 software (Agilent Technologies). The detector
wavelength was set at 230 nm, and the injection volume was
25μL. The HPLC column was Xterra-RP C18, 5 µm, 250×
4.6 mm (Waters Corporation). Column temperature was
maintained at 25°C. A binary mixture of potassium phos-
phate buffer: acetonitrile 70:30 (v/v) (solvent A) and potassium
phosphate buffer: acetonitrile 35:65 (v/v) (solvent B), both at
pH 2.5, was pumped at the flow rate of 1.0 mL/min.
O
N
N
O
N
N
O
O
O
OH
OH
Cl
Cl
(a)
(b)
N
N
OH
OH
LC-MS/UV and LC-MS/MS/UV Method
O
Cl
Cl
LC-MS/UV and LC-MS/MS-UV data were obtained
using an Agilent 1200 series HPLC system, equipped with
UV detector, HTS PAL autosampler and MDS Sciex/
Applied Biosystems QTRAP 4000 mass spectrometer with
Analyst 1.4.2 software. The flow rate was 1.0 mL/min, and
the injection volume was 10 μL. The analytical column
used was Xterra-RP C18, 5 µm, 250×4.6 mm (Waters
Corporation). Column temperature was maintained at 25°C.
The mobile phase consisted of 0.1% trifluoroacetic acid and
acetonitrile in the ratio of 75:25 (v/v)–0.1% trifluoroacetic
acid and acetonitrile in the ratio of 25:75 (v/v).
(c)
(d)
Fig. 1 Metabolic products of levocetirizine oxidation produced by (a)
Hydroxylation (b) N-oxidation (c) O-dealkylation (d) N-dealkylation (3).
sensitive pharmaceuticals (4,5). It was reported (5,6) that
auto-oxidation can progress through formation of hydro-
peroxides and peroxyl radicals in polyoxyethylene linkages
containing substances such as polysorbate surfactants and
PEG. Since the cetirizine soft gelatin capsule matrix contains
PEG, we believe that this may be the main cause for
cetirizine oxidation. In order to identify cetirizine degrada-
tion products, caused by PEG-containing formulations, we
selectively oxidized cetirizine. The resulting degradation
product was isolated and analyzed. Hence, the aim of this
work was to identify a degradation product, investigate, and
establish the mechanism of the degradation.
Synthesis and Purification of Cetirizine Degradation
Product
Method 1
Cetirizine was oxidized by aqueous hydrogen peroxide. For
this reaction, cetirizine hydrochloride (1 g) was exposed to
30% aqueous solution of hydrogen peroxide for 5 h at 70–
80°C, then the reaction mixture was extracted with ethyl
acetate. Organic phase was separated and concentrated.
The crude material was purified by preparatory TLC, as
described by Hamburger et al. (5) with some modifications.
Spotting was done on precoated silica gel aluminum
plate 60 F254. The solvent system consisted of ethyl
MATERIALS AND METHODS
Reagents and Chemicals
Cetirizine hydrochloride was supplied by Matrix Laboratories
Limited (Lot CTP 0171106). Cetirizine N-oxide was obtained
from LGC GmbH (Lot 380.13.07.03). PEG 400 was obtained
from BASF. Ethyl acetate (Fisher Scientific), methanol (EMD),
ammonia (JT Baker) and acetonitrile (EMD and Burdick &
Jackson) were of the HPLC grade. Potassium phosphate
monobasic monohydrate, magnesium sulfate, hydrogen per-
oxide, and sodium percarbonate were of the ACS grade and
were purchased from JT Baker. Phosphoric acid (NF FCC)
was also purchased from JT Baker. Trifluoroacetic acid and
NMR solvent were purchased from Sigma-Aldrich.
O
N
N
O
O
OH
Cl
HPLC/UV Chromatographic Method
m/z 201
An Agilent 1200 series, calibrated HPLC system equipped
with a binary pump and UV detector was used. The
Fig. 2 Estimated structure of degradation product (m/z 405/407).

1320
Dyakonov et al.
acetate-methanol-ammonia (80:15:5, v/v/v) mixture.
Scraped spots were extracted with ethyl acetate, dried over
magnesium sulfate, concentrated and analyzed by HPLC/UV
and 1H NMR.
Method 2
Cetirizine was selectively oxidized with sodium percarbon-
ate. The kinetics of oxidation was monitored by HPLC/
Fig. 3 1H NMR spectra of cetirizine (a) and degradation product (b).

Mechanism of Cetirizine Degradation
1321
Cetirizine
700
650
600
550
500
450
400
350
300
250
200
150
100
Cet N-oxide
Cetirizine
Cet N-oxide
(a)
(b)
(c)
31.59
40.50
31.00
16.72
Peg Esters
Peg Esters
50
2.88
23.28
11.97
6.31
7.93 11.05
10
15.74
15
33.23
34.29
35
41.94
35.81
38.29
0
5
20
25
30
40
Retention time
Fig. 4 Chromatograms of cetirizine (a), cetirizine N-oxide standard (b), and stressed cetirizine solution, spiked with cetirizine N-oxide (c).
UV. The reaction mixture was purified by preparatory
TLC, as described in the Method 1.
samples were then incubated for 48 days at 30, 60 and
80°C. Aliquots of the solutions were also stored at room
temperature as controls. Samples were withdrawn peri-
odically and analyzed by HPLC for cetirizine and related
impurities. All solutions were purged with nitrogen each
time the vials were opened.
1H NMR
The NMR measurements were carried out on a 400 MHZ
Varian spectrometer. The NMR spectra were measured in
methanol-d4 and D2O. Chemical shifts are reported on δ
scale (ppm) by assigning the residual solvent peak to 3.30
for methanol.
70
60
50
40
30
20
10
0
pH–Stability Profile
A cetirizine solution was prepared by dissolving 8 g of the
drug in 8.6 g of DI water and 260 g of PEG 400. The
solution was stirred for 1 h at room temperature then
transferred into 10 separate vials. The pH of the solution
in each vial was adjusted individually to 3.0, 4.0, 4.5,
5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 10.0 using NaOH.
Triplicate aliquots of each solution were transferred into
separate scintillation vials and purged with nitrogen; the
2
4
6
8
10
pH
2 days
6 days
16 days
28 days
48 days
Fig. 5 The effect of pH on the oxidation of Cetirizine in PEG solution
under forced degradation conditions.

1322
Dyakonov et al.
Initiation
X -
H2X+
H3X++
HX
R
+ H
RH + light or catalist
A
B
Propagation
C
N
N
+ O2
R
ROO
O
O
OH
ROOH + R
+ H
+ RH
ROO
Cl
ROOH
ROO
Fig. 8 Cetirizine zwitterionic form HX (predominant specie between pH
3 to 8).
Termination
inactive products
inactive products
2RO2
+
RO2
R
preparing the N-oxide of tertiary amines were considered.
All methods used hydrogen peroxide in different media
(acetic acid, water) and different catalysts (sodium tungstrate,
molybdenum trioxide) (7). As a first attempt, cetirizine
hydrochloride was successfully oxidized with 30% aqueous
hydrogen peroxide. Reaction mixture was purified by
preparative TLC as described in the literature (2) and
analyzed by HPLC/UV. The retention time of the
compound isolated from TLC plates corresponded to the
retention time of oxidative peak of interest (Rt=24 min by
HPLC) formed during forced degradation.
Additionally, cetirizine was selectively oxidized with
sodium percarbonate with higher selectivity and yield of
oxidation product than with conventional hydrogen peroxide.
The kinetics of oxidation were monitored by HPLC. Results
showed that in the presence of sodium percarbonate,
conversion of cetirizine approached 50% during 2 h at
80°C. The 1H NMR spectrum of isolated cetirizine
oxidation product was compared to NMR of cetirizine
hydrochloride (Fig. 3).
Fig. 6 Auto-oxidation process.
RESULTS
Preliminary LC/MS Results
The preliminary LC/MS and LC/MS/MS results from the
study of the stressed solution of cetirizine (MW 388.89) in
PEG-containing formulation showed the monochlorinated
cluster of peaks at m/z 405/407 (M+16) and a base peak at
m/z 201. The intense ion at m/z 201 was assumed to be
formed due to the fission of >C-N< bond between
aromatic and aliphatic fragment of molecular ion. The
value of m/z 405 for piperazine containing aliphatic
fragment can be attributed to the monohydroxylated
product or N-oxide, suggesting that oxidation occurs in
the aliphatic portion of cetirizine. There was no evidence of a
m/z fragment of 217 (oxidation on the aromatic fragment)
leading to a possible structure as shown in Fig. 2.
The number of protons in the aromatic region of the
1H NMR of the degradation product was similar to that of
cetirizine, indicating formation of N-oxide, rather than
monohydroxylation. The N-oxide effect can be observed
by the difference in the chemical shifts of the proximal
protons adjacent to the N-oxide carbons (Fig. 3). The
deshielding influence of the N-oxide function, generated
Selective Oxidation of Cetirizine and Identification
of Oxidation Product
In order to model the degradation process of cetirizine in a
PEG-containing formulation, several oxidative methods for
-
HO
HO
H
O
H
-
O
O
.
.
N
+
N
N
N
N
N
O
O
H O
O
O
O
O
2
+
OH
OH
OH
Cl
Cl
Cl
Fig. 7 Proposed mechanism for the oxidation of cetirizine to form N-oxide.

Mechanism of Cetirizine Degradation
1323
on the nitrogen bearing the ethoxyacetic acid chain,
causes a down-field shift of the piperazine (Δδ=0.2) and
ethoxy (Δδ=0.5) protons.
located on the nitrogen bearing the ethoxyacetic acid
chain.
This site of oxidation in the molecule was proposed
based on the correlation of results of degradation in pH
range from 3 to 10 with ionization scheme of cetirizine
This result agreed with the LC/MS data and was
verified by spiking the cetirizine degradation sample
with cetirizine N-oxide standard. The chromatogram
of stressed solution of cetirizine spiked with cetirizine
N-oxide (Fig. 4c) showed retention times 19 and 25 min
for cetirizine and cetirizine-oxidation products, respec-
tively. Chromatograms of two standards—cetirizine (a)
and cetirizine N-oxide (b)—are shown in Fig. 4. Based on
these results, the cetirizine oxidation process could be
explained by N-oxidation of piperazine nitrogen by
hydroperoxides formed from oxidative decomposition of
polyoxyethylene chains.
1
and H NMR data. The ionization scheme of cetirizine
is shown in Fig. 8. The previous studies (8) reported three
macro-pKa values for cetirizine (1.52, 2.92 and 8.27),
the lowest of which was attributed to the nitrogen
nearest to the aromatic rings and pKa 2.9—to the
carboxylic acid. The minimum of the oxidation rate at
pH of 6.5 could be explained by the protection effect of
protons on tertiary amine (9). According to the literature
(8,10), zwitterionic form HX is predominant between pH
3 to 8 (Fig. 8).
We assume that between these values of pH the
À
Á
Effect of pH on Oxidation of Cetirizine in PEG
formulation
protonation of superoxide radical Oꢀ2 ꢁ around pH∼6
À
ꢁÁ
(11) Oꢀ2 ꢁ þ Hþ ! HOO may result in the elimination of
the charged Oꢀ2 ꢁ species and therefore decrease the
oxidation rate of the protonated tertiary amine. This can
happen because the neutral HOO˙ as an oxidizer would
not have the Coloumb attraction as a driving force. As a
result, one could obtain another minimum in the oxidation
rate at pH below 6.
The formation of cetirizine N-oxide in PEG was investigated
in a pH range from 3 to 10. Cetirizine, formulated in PEG,
was subjected to the forced degradation at different temper-
atures (30, 60 and 80°C). The aliquots of the product were
withdrawn at different times and analyzed by HPLC/UV.
The results obtained at 80°C are presented in Fig. 5.
Cetirizine proved to be significantly more stable at pH
4.5 and 6.5 during 48 days exposure at 80°C. Only
0.34–5.7 and 1.5–7.6% of cetirizine N-oxide was formed
at pH 4.5 and 6.5, respectively. N-oxide formation increased
at pH>7 and higher N-oxide formation was observed
between pH 4.5 and 6.5.
The peroxide formation in PEG-containing formulations
can be inhibited by including an anti-oxidant, such as
butylated hydroxytoluene (BHT) and hydroxyl anisole
(BHA). The effect of anti-oxidant (BHA) on the rate of
cetirizine oxidation under stressed conditions was evaluated
in our previous work (12). The data indicated that the
addition of BHA reduced the rate of oxidation of cetirizine.
The results suggested that the degradation of cetirizine in
polyethylene glycol arose from the reaction between the
drug and reactive peroxide intermediates, formed through
oxidation of PEG.
DISCUSSION
Mechanism of Cetirizine Oxidation
The polyoxyethylene-based excipients contain some resid-
ual peroxides and could potentially affect the stability of
oxidation-sensitive pharmaceuticals. The presence of
hydroperoxides and peroxyl radical formed in cetirizine
soft gelatin capsule matrix could be the main reason for
cetirizine oxidation.
The radical-driven process of cetirizine auto-oxidation
predominantly consists of the reactions of initiation, chain
propagation and termination. A well-established auto-
oxidation process is shown in Fig. 6, where R in our case
is –(OCH2CH)O–. The autoxidation starts with decompo-
sition of alkyl polyoxyethylene chain (RH) and subsequent
peroxides formation.
CONCLUSION
The results of degradation of cetirizine in PEG-containing
formulation indicated that cetirizine is susceptible to
oxidation. Degradation of cetirizine in polyethylene
glycol arose from the reaction between the drug and
reactive peroxide intermediates, formed through oxida-
tion of PEG. Cetirizine degradation product was synthe-
sized by the selective oxidation of the sterically less
hindered piperazine nitrogen. The qualitative analyses
1
by HPLC, LC/MS/MS along with H NMR were used
to identify the oxidation product as cetirizine N-oxide.
The mechanism of oxidation was proposed. N- oxidation
was found to be the major pathway for cetirizine
transformation.
The formation of N-oxide progresses according to
Fig. 7, producing cetirizine N-oxide with the oxygen atom

1324
Dyakonov et al.
6. Donbrow M, Azaz E, Pillersdorf A. Autoxidation of polysorbates.
J Pharm Sci. 1978;67(12):1676–81.
REFERENCES
7. Shimizu S, Watanabe N, Kuranishi H. US 5, 668281 (1997).
8. Pagliara A, Testa B, Carrupt PA, Jolliet P, Morin C, Morin D et
al. Molecular properties and pharmacokinetic behaviour of
cetirizine, a zwitterionic H1-receptor antagonist. J Med Chem.
1998;41:853–63.
9. Freed AL, Strohmeyer HE, Mahjour M, Sadineni V, Reid DL,
Kingsmill CA. pH control of nucleophilic/electrophilic oxidation.
Int J Pharm. 2008;357:180–8.
10. Tam KY, Quere L. Multiwavelenght spectrophotometric resolu-
tion of the micro- equilibria of cetirizine. Anal Sci. 2001;17:1203–
8.
11. Salvador A, Sousa J, Pinto RE. Hydroperoxyl Superoxide and pH
Gradients in the Mitochondrial Matrix: A Theoretical Assessment.
Free Radic Biol Med. 2001;31(10):1208–15.
1. Jones JW, Francis JJ. Softgels: consumer perceptions and market
impact relative to other oral dosage forms. Adv ther. 2000;17:5.
2. Makhija SN, Vavia PR. Stability indicating HPTLC method for the
simultaneous determination of pseudoephedrine and cetirizine in
pharmaceutical formulations. J Pharm Biomed Anal. 2001;25:663–7.
3. Benedetti MS, Plisnier M, Kaise J, Maier L, Baltes E, Arendt C
et al. Absorption, distribution, metabolism and excretion of [14C]
levocetirizine, the R enantiomer of cetirizine, in healthy volunteers.
Eur J Clin Pharmacol. 2001;57:571–82.
4. V. Kumar, DS, Kalonia. Removal of Peroxides in Polyethylene
Glycols by Vacuum Drying: Implications in the stability of
Biotech and Parmaceutical Formulations. AAPS PharSciTech. 7(3)
2006; Article 62. DOI; 10 1208 /pt070362.
5. Hamburger R, Azaz E, Donbrow M. Autoxidation of polyox-
yethylenic nonionic surfactants and of polyethylene glycols. Pharm
Acta Helv. 1975;50(1–2):10–7.
12. Nasri H, Dyakonov T, Toops D. Compatibility of shell ingre-
dients with hydrophilic actives in gelatin-based dosage forms,
presented at AAPS, Atlanta (2008).