Slow, spontaneous degradation of lansoprazole, omeprazole and pantoprazole tablets: Isolation and structural characterization of the toxic antioxidants 3H-benzimidazole-2-thiones

Article abstract of DOI:10.1691/ph.2013.2144

The spontaneous degradation of lansoprazole, omeprazole and pantoprazole tablets upon long-term and forced storage conditions was determined by high performance liquid chromatography (HPLC). The more abundant products could be isolated by liquid chromatography and their molecular weights determined by Mass Spectrometry (MS). Their structures, established according to their spectroscopic data, were compared to those of either the literature or of authentic samples. Thus lansoprazole led mainly to a mixture of 3H-benzimidazole-2-thione (2a) and 3H-benzimidazole-2-one (2c), omeprazole mainly to a mixture of 5-methoxy-3H-benzimidazole-2-thione (1a) and 2-hydroxymethyl-3, 5-dimethyl- 4-methoxypyridine (1b), and pantoprazole, to 5-difluoromethoxy-3H-benzimidazole-2-thione (3a) and 2-hydroxymethyl-3, 4-dimethoxypyridine (3b). Although some of the degradation products had already been observed under different conditions, the detection of benzimidazole-2- thiones is unprecedented and their involvement as possible physiological, yet toxic antioxidants must be emphasized. Plausible, unified mechanisms for the formation of the different degradation products observed herein and in previous papers from the literature are suggested.

Full text of DOI:10.1691/ph.2013.2144

ORIGINAL ARTICLES
1
Department of Chemistry , Faculty of Science, University of Aleppo, Syria; Institut Parisien de Chimie Moléculaire UMR
2
CNRS 7201 , Université Pierre et Marie Curie, Paris, France
Slow, spontaneous degradation of lansoprazole, omeprazole and
pantoprazole tablets: isolation and structural characterization of the toxic
antioxidants 3H-benzimidazole-2-thiones
1
1
2
2
2
A. Rajab , M. Touma , H. Rudler , C. Afonso , M. Seuleiman
Received July 27, 2012, accepted January 25, 2013
Dr. Mannan Seuleiman, Institut Parisien de Chemie Moléculaire - Case Courrier 42. 4, place Jussieu, 75252 Paris
cedex 05- France
mannan.seuleiman@upmc.fr
Pharmazie 68: 749–754 (2013)
doi: 10.1691/ph.2013.2144
The spontaneous degradation of lansoprazole, omeprazole and pantoprazole tablets upon long-term
and forced storage conditions was determined by high performance liquid chromatography (HPLC).
The more abundant products could be isolated by liquid chromatography and their molecular weights
determined by Mass Spectrometry (MS). Their structures, established according to their spectroscopic
data, were compared to those of either the literature or of authentic samples. Thus lansoprazole led
mainly to a mixture of 3H-benzimidazole-2-thione (2a) and 3H-benzimidazole-2-one (2c), omeprazole
mainly to a mixture of 5-methoxy-3H-benzimidazole-2-thione (1a) and 2-hydroxymethyl-3, 5-dimethyl-
4
2
-methoxypyridine (1b), and pantoprazole, to 5-difluoromethoxy-3H-benzimidazole-2-thione (3a) and
-hydroxymethyl-3, 4-dimethoxypyridine (3b). Although some of the degradation products had already
been observed under different conditions, the detection of benzimidazole-2-thiones is unprecedented and
their involvement as possible physiological, yet toxic antioxidants must be emphasized. Plausible, unified
mechanisms for the formation of the different degradation products observed herein and in previous papers
from the literature are suggested.
+
+
1
. Introduction
(H , K )-ATPase inhibitory activity in stimulated gastric glands,
but low reactivity (high chemical stability) at neutral pH.
In vitro investigations on the mechanism of action of these com-
pounds have demonstrated that they are indeed pro-drugs which
are transformed under physiological conditions into cyclic sulfe-
namides the pharmacologically active intermediates (Krüger
et al. 1990; Hogg 1990).
As a result of their inherent instability, it was mandatory to
determine the presence of impurities in the final pharmaceu-
tical ingredients and to establish their structure. Although the
degradation of lansoprazole, omeprazole and pantoprazole had
already been described under various conditions, the exact struc-
ture of the newly formed degradation products had up to now
not been determined (EL-Sherif et al. 2006; USP 2004). Several
papers devoted to the analytical data on these drugs and describ-
ing, on the one hand, the possible presence of process-related
impurities, originating directly from the batches, appeared in
the literature (Reddy et al. 2007; Ph. Eur 7.6 2013). On the
other hand omeprazole has recently been observed in surface
waters together with some degradation products, eg the toxic
2-hydroxymethylpyridines (Apollo Scientific Ltd, Safety Data
Sheet 2005), resulting, according to these authors, from its inter-
action with water and acids (Della Greca et al. 2006).
Lansoprazole, omeprazole and pantoprazole are [(pyridyl-
methyl) sulfinyl] benzimidazoles, so-called proton-pump
inhibitors, known for their high activity as gastric (H , K )-
ATPase inhibitors. [(Pyridylmethyl) sulfinyl] benzimidazoles
possess long-lasting acid antisecretory properties associated
with anti-ulcer and anti-gastroesophageal reflux activities
+
+
(Brandstrom et al. 1989; Lindberg et al. 1986; Sachs et al 1995;
Krüger et al 1990; Rackür et al 1985; Hogg 1990). They are also
known for their effectiveness in the Zollinger-Ellison syndrome
(Del Valle et al. 2003). More recently, their antioxidant proper-
ties have also been put forth in efforts to explain their role in the
gastro-protection towards radical species (Kohler et al. 2010;
Biswas et al. 2003; Becker et al. 2006; Natale et al. 2004; Koch
et al. 2004; Lapenna et al. 1996; Backlund et al. 1999). Yet, no
clear-cut structure-related arguments in favour of theses proper-
ties have been given, their antioxidant properties being possibly
either inherent to the drugs themselves or to some of their degra-
dation products: Lansoprazole, Omeprazole like benzimidazole
and 2-mercaptobenzimidazole interact with the cytochrome P-
4
50 system in the liver and inhibit several liver-monooxygenase
activities. These compounds appeared to be rather unstable with
respect to external factors such as acids (Stroyer et al. 2006).
Efforts have been made to improve their stability by modifying
for example the substituents in the pyridine and in the benz-
imidazole moieties so as to obtain compounds with both high
However, to the best of our knowledge, no quantitative data
on the direct, per se, slow, long term transformation of these
compounds, as raw material or in the form of enteric-coated
tablets can be found in the literature. Therefore we carried out
Pharmazie 68 (2013)
749

ORIGINAL ARTICLES
H
N
H
N
H
N
O
S
N
S
+
O
O
CF3
N
N
H
N
H
CH3
2
(Lansoprazole)
2a
2c
Scheme 1: Degradation products of lansoprazole 2.
◦
stress studies on these blister-protected pharmaceuticals in order
to establish their inherent stability (according to ICH guidelines)
and to identify their possible degradation products.
Under long-term regular conditions (25 C, 60% humidity), the
enteric-protected tablets of lansoprazole underwent a slow trans-
formation, already detectable by HPLC after a short period of
time, giving seven compounds, the most abundant degradation
product having the lowest retention time. Under accelerated con-
The aim of this paper is first to confirm that indeed a slow
degradation of a series of proton-pump inhibitors, lansoprazole,
omeprazole, and pantoprazole takes place over time, both under
◦
ditions (40 C, 75% humidity), such tablets led much faster to
◦
regular (25 C, 60% room humidity (RH)), and under accel-
a mixture of the same products. The decrease of the amount
of lansoprazole versus time, determined by HPLC is shown in
(Fig. 1).
The more abundant degradation products were isolated via suc-
cessive column and thin- layer chromatography as described
above, and their purity checked again by HPLC, giving two well-
separated peaks, with retention times of 0.46 and 0.92 minutes.
The first product 2c, (m/z = 135), a white solid with m.p. >
◦
erated storage conditions (40 C, 75% RH); second to isolate
and determine the structure of the most abundant degradation
products. As it turned out, the results described herein differ
mainly from previous observations of the literature in that, in
all the cases examined, 3H-benzimidazole-2-thiones 1a, 2a, 3a,
among others, were detected as degradation products. The pres-
ence in the proton-pump inhibitors of even tiny amounts of
these compounds, the antioxidant properties of which are well
established, might possibly account for their observed acid-
independent protective effect during various gastric mucosal
damage conditions. Reversely, the known adverse effects of
these degradation products (potent thyroid toxicity) should be
addressed and care should be taken in order to limit this degrada-
tion process (Gaorski et al. 1991; Kawasaki et al. 1998; Sakemi
et al. 2002).
◦
1
300 C, exhibited a very simple H NMR spectrum in DMSO,
showing two series of signals, at respectively δ10.69 ppm
attributable to NH protons, and at δ6.94, attributable to aromatic
protons.
The 13C NMR spectrum, disclosed four signals at δ 155.89,
129.88, 120.75, and 108.73 ppm. All these data, together with
the elemental analysis, agreed with structure 2c, thus with the
formation of 3H-benzimidazole-2-one.
To the more retained product, isolated as a high-melting
solid, was given structure 2a. The elemental analysis con-
firmed the presence of sulphur and agreed with the formula
2
. Investigations and results
C7H6N2S (Table 1) as did the electro- spray mass spectrum
Samples of the various commercially available products were
placed in a climatic chamber (Angelantini, model CH1500)
+
(
M Na = 173.014).
The 13C NMR spectrum of 2a was very close to the spectrum of
◦
under regular storage conditions (25 C, 60% RH, for three
2
c, with signals at δ168.33, 132.66, 122.57, and 109.95 ppm.
◦
years) or under accelerated conditions (40 C, 75% RH, for eight
1
The H NMR spectrum disclosed the expected signals at δ12.52
NH) and at δ 7.11 ppm. Altogether, these data matched with the
structure 2a, (Scheme 1) (Sutter et al. 1999).
months). The chemical analyses of successive samples were per-
formed respectively either after each 6 months or monthly, using
HPLC.
(
2
.2. Degradation products of omeprazole
2
.1. Degradation products of lansoprazole
In the case of Omeprazole tablets, the purity of the starting
tablets was again determined by HPLC.
In the case of lansoprazole tablets, the purity of the starting
tablets was determined by HPLC: only lansoprazole (2) could
be detected.
Underlong-termregularconditions, theenteric-protectedtablets
of omeprazole led to seven compounds, the most abundant hav-
ing the lowest retention time. At higher temperature, such tablets
led much faster to a mixture of the same products. The decrease
of the amount of Omeprazole versus time, determined by HPLC
is shown in (Fig. 2). The more abundant degradation products
(retention times 0.54 and 0.94 minutes) were isolated as for
lansoprazole.
The ESI mass spectrum as well as the elemental analysis of the
first product, which clearly indicated the presence of sulphur
agreed with those of 5-methoxy-3H-benzimidazole-2-thione
(
1a), a result confirmed by the NMR data of the isolated com-
1
pound. Thus, the H NMR spectrum depicted signals at δ 12.42
(
m, 2H), 7.08 (m, 1H), 6.72 (m, 2H) and 3.80 (s, 3H) ppm. The
C NMR spectrum showed signals at δ 168.01, 156.14, 133.44,
1
3
1
26.75, 110.42, 109.92, 94.86 and 55.83 ppm.
◦
All these data together with the melting point, 255 C, were
Fig. 1: Decrease of the amount of lansoprazole under long-term regular conditions
◦
◦
(
25 C, 60% RH) and under accelerated conditions (40 C, 75% RH).
in agreement with literature values for 1a. Structure 1b was
7
50
Pharmazie 68 (2013)

ORIGINAL ARTICLES
carbons, at δ 170.31, 147.42, 133.82, and 130.90 ppm, and at δ
Accelerated conditions
Long term conditions
Omeprazole
117.78 (t, J = 74 Hz), 115.22, 111.74 and 101.30 ppm for four
tertiary carbons, among which figures the signal for the carbon
of the CHF2 group, giving a typical triplet with a large coupling
constant (J = 257Hz).
1
1
02,00
00,00
9
9
9
9
9
8
8
8
8,00
6,00
4,00
2,00
0,00
8,00
6,00
4,00
All these data matched thus with a structure such as 3a, the
known 2-difluoro-2-benzimidazole-thione. To the second prod-
uct (Rt1.56 mn), isolated as a white solid, m.p. 85 C, was given
structure 3b on the following grounds. Its mass spectrum agreed
with the molecular formula C8H11NO3.
◦
0
5
10
15
20
25
30
35
40
The proton NMR spectrum disclosed typical signals for two
adjacent protons of a substituted pyridine, at δ 8.17 (d, J = 5 Hz)
and 7.05 (d, J = 5 Hz) ppm, for two protons of a CH2OH group,
at δ 4.52 ppm, as a singlet, and for the protons of two methoxy
groups at δ 3.87 and 3.75 ppm.
Time (month)
Fig. 2: Decrease of the amount of omeprazole under long term regular conditions
◦ ◦
25 C, 60% RH) and under accelerated conditions (40 C, 75% RH).
(
assigned to the second, more retained product on the follow-
ing grounds: the ESI mass spectrum was in accordance with
such a structure. The elemental analysis confirmed the molec-
13
The C NMR spectrum disclosed signals at δ 158.31, 152.75,
144.79, 141.91 and 106.74 ppm for the five aromatic carbons,
together with signals at δ 60.69, δ 59.49 and δ 55.50 ppm for
the carbons of the two methoxy groups and the carbon of the
hydroxymethyl group.
1
ular formula together with the presence of nitrogen. The H
NMR spectrum disclosed a series of expected signals at δ 7.87
6
a
d
(
(
1H, H ), 5.07 (1H, OH), 4.41 (2H, H ), 3.50 (3H, H ), 2.00
c
d
13
6H, H and H ). The C NMR spectrum, showing up signals
3. Discussion
4
2
6
5
at δ 163.74 (C ), 156.56 (C ), 147.39 (C ), 124.83 (C ), 122.57
C ), 61.67 (C ), 9.20 (C ) ppm confirmed these assignments.
All these data together with the melting point, (55-60 C), were
again in agreement with those of the literature for 1b. Scheme 2
features the degradation of omeprazole.
3
b
d
(
The in vivo and in vitro transformations of [(pyridylmethyl)
sulfinyl] benzimidazoles has been the matter of numerous stud-
ies. Early reports clearly demonstrated that under physiological
highly acidic conditions, these substrates underwent fast and
deep transformations, giving inter alia the real active principle
originating from a sulfenic acid.
In contrast, experiments conducted at higher pH indicated that
the drugs were quite stable. The presence of impurities in the
laboratory batches, or even in the finished drugs eg starting mate-
rials or their derivatives such as 1a, 2a, 2c and 3a (Ph.Eu.7.5)
and side-reaction products, has also been disclosed (Reddy et al.
◦
2
.3. Degradation products of pantoprazole
In the case of pantoprazole tablets, the purity of the starting
tablets was again determined by HPLC: only pantoprazole (3)
could be detected.
As for the previous compounds, time-dependent degradation of
pantoprazole was observed, the degradation being again faster
at higher temperature and leading to a more complex mixture of
products (presence of four extra products), most of them being
however the same as those obtained under long-term, regular
conditions. The decrease of the amount of Pantoprazole versus
time, determined by HPLC is shown in Fig. 3.
Two of the more abundant products could again be separated
and isolated and their structures fully established (Scheme 3).
A first product 3a (Rt 0.52 mn) obtained as a solid, m.p. 290 C,
contained again sulphur according to the elemental analysis
Table 1) and agreed with the molecular formula C8H6F2N2OS.
2007). Finally, the abiotic transformations of these substrates
in an aquatic environment has been outlined, confirming that
water, in the absence of any other reagent, was able to induce
the degradation of omeprazole and lansoprazole into the benz-
imidazolones 2a and more surprisingly, into benzimidazoles 5
and hydroxymethylpyridines 1b.
Moreover, uptonow, thestriking formationofbenzimidazole-2-
thiones1a, 2aand3afromthedrugs1-3, besidesbenzimidazole-
2-ones 2c, and to a lesser extend, of hydroxymethylpyridines 1b
and 3b in all the cases examined herein, had never been observed
before.
Since no acid is present in the commercially available tablets,
it seems clear that the transformations observed under non-
physiological conditions must be different from a mechanistic
pointofview. Ithasbeenshownthatinthefinalproduct, obtained
on an industrial scale, water is always present; therefore only
water would possibly be able to interact either with the start-
ing substrates or with their degradation products (Della Greca
◦
(
1
The H NMR spectrum disclosed signals at δ 12.69 ppm (2 NH),
at δ 7.17 ppm, as a triplet J = 74 Hz, typical, according to the
literature for the CHF2 proton observed for example for panto-
prazole, at δ 7.20 (d, 1H, J = 2 Hz) and δ = 6.89 (m, 2H) ppm for
the aromatic protons. The 13C NMR spectrum exhibited a series
of signals which, according to a DEPT sequence, are quaternary
2006). According to the structure of 1b and 3b, the main degra-
dation products observed herein, it is clear that the sulfur-carbon
bond, which links the sulfur atom to the methylpyridine ring, has
been cleaved, and that a hydration reaction took place.
Evidence for the existence of a week carbon-sulfur bond in lan-
soprazole came from the examination of its MS/MS spectrum
Pantoprazole
1
1
02,00
00,00
9
9
9
9
9
8
8
8
8
8,00
6,00
4,00
2,00
0,00
8,00
6,00
4,00
2,00
Accelerated condition
Long term condition
(Fig. 4). It is indeed known that fragmentation of a molecule
is virtually absent in electrospray mass spectrometry, but that
it can be easily induced by MS/MS technique which employs
collision-induced dissociation to fragment a precursor ion.
0
10
20
30
40
Time (month)
3
.1. ESI - MS analysis of lansoprazole
Fig. 3: Decrease (%) of pantoprazole in the tablets with time, under long-term
conditions (25 oC, 60% RH) and under accelerated conditions (40 oC, 75%
In methanol solvent, the negative ion spectra from 10 μM solu-
RH).
tion of lansoprazole revealed a prominent molecular anion for
Pharmazie 68 (2013)
751

ORIGINAL ARTICLES
or 3b upon a hydration step and to thiooxiranes 8. 8 could
evolve via three routes I, II and III. Extrusion of either oxygen
via I), or sulfur (via II), or SO (via III) would lead respec-
(
tively to the observed benzimidazole-2-thiones 1a, 2a or 3a,
the benzimidazole-2-ones 2c and to the benzimidazoles 5, the
formation of which had indeed been observed by Della Greca
and coworkers (Sutter et al 1999). Taken together, the slow for-
mation of all of the observed products might be explained, on
a formal point of view, by the involvement and evolution of
pseudo-carbenoidspecies(Gaorskietal(1991)resultingfroman
intramolecular self-degradation of the [(pyridylmethyl) sulfinyl]
benzimidazoles 1-3.
Fig. 4: (a) ESI spectrum of lansoprazole recorded in the negative ion mode and (b)
−
CID spectrum of the [M-H] ion m/z 368.
These slow degradation reactions, though interesting on a chem-
ical point of view, yet raise the question of the health impact
of the formed products. Although it has been established that
the acute toxicity of 2-mercaptobenzimidazole (eg tyrotoxicity
in rats), a rubber-stabilizer and corrosion-inhibitor antioxidant
the singly charged molecule at m/z 368.0. The molecular mass of
the uncharged lansoprazole is 369.076, and the signal at m/z 368
+
–
represents the deprotonated Lansoprazole. In addition, [M Cl]
anion is detected at m/z 404 and the non-covalent deprotonated
–
(Kawasaki et al 1998; Sakemi et al 2002; Xu et al 1995) is rather
dimer [2M-H] is detected at m/z 737.
low, its potent toxicity has been demonstrated. Moreover, it is a
fat-soluble compound, which might also act as an endocrine dis-
rupter. Since it has been recommended that the contamination
level by such compounds should be reduced as far as possible,
care should thus be taken to limit their amount in the drugs
administered over long period of times. This might be achieved
in the case of the proton-pump inhibitors by a strict observation
of the stocking conditions.
3
.2. MS/MS of lansoprazole
The Collision Induced Dissociation (CID) spectrum of lansopra-
zole deprotonated molecule showed a single, prominent product
ion at m/z 164 (plus a weak fragment at m/z 149). The calcu-
lated m/z values of the proposed structures for these ions were
in acceptable agreement with the measured values.
Cleavage of the carbon-sulfur bond which links the benzim-
idazole ring to the pyridine ring took therefore place. This
fragmentation process results in the formation of A and B, A
being a stable radical anion. (Scheme 4). Omeprazole and pan-
toprazole behaved similarly. Having established the position of
the weakest bond in lansoprazole, one can speculate about a
mechanism accounting for the slow transformation of lansopra-
zole, omeprazole and pantoprazole under long term, non-acidic
conditions (Scheme 5). These drugs contain two nucleophilic
centers able to react with the electrophilc carbon - nitrogen dou-
ble bond: the nitrogen atom of the pyridine and the oxygen atom
of the sulfoxide. In the first case, under acidic conditions, such
an interaction leads to the physiological active species of the
drugs. The second case, (Scheme 5, 1-3 → 7) might be related,
at least in its first steps, to the Moffatt oxidation of alcohols
into ketones (Moffatt 1971), which involves indeed the interac-
tion of a sulfoxide, dimethylsulfoxide, with a carbon-nitrogen
double bond of an imide, dicyclohexylcarbodiimide. Applied to
the case of lansoprazole, such a concerted, intramolecular nucle-
ophilic addition, followed by a deprotonation reaction involving
the oxygen atom, the carbon-nitrogen double bond and a hydro-
gen atom of the methylene group in 1-3, might lead to 7. The
first step is likely to be a reversible process with the equilibrium
lying far on the side of the starting drugs 1-3. We hypothesized
therefore that the evolution of such a key-intermediate 7 might
lead to all of the observed products (Scheme 5).
3
.4. Conclusion
A complex, slow transformation of lansoprazole, omeprazole
and pantoprazole tablets under normal and accelerated condi-
tions has been observed: it takes place even at room temperature
and is accelerated at higher temperature, lansoprazole show-
ing the highest stability. Among the products isolated by liquid
chromatography and characterized by spectroscopic methods,
the three corresponding benzimidazole-2-thiones 1a, 2a and 3a
could be identified as products of spontaneous decomposition
for the first time. The mechanism of formation of these degra-
dation products is probably not related to the mechanism of
formation of the physiological active intermediates, but is due
to a slow intramolecular degradation process.
4
. Experimental
4.1. Samples and chemicals
The standard samples of lansoprazole, omeprazole, and pantoprazole, were
obtained from Sl Drugs and Pharmaceuticals. Their purity was established
as 99.93, 99.97 and 99.91 % respectively. The investigated commercially
available sample of Omepral (40 mg capsules) were obtained from Asia
Pharmaceutical Industries (Aleppo, Syria). Commercially available Duo-
gaste (30 mg capsules) and Progast tablets containing 45.104 mg of sodium
pantoprazole (equivalent to 40 mg of pantoprazole), were obtained from
Kaspar-Chabani Pharma (Aleppo, Syria) in the form of enteric-coated
tablets.
A multistep cleavage of the carbon-sulfur bond in 7 might lead
via the carbenoid species 9 to the hydroxymethylpyridines 1b
Triethylamine, potassium hydroxide, potassium dihydrogen phosphate,
dipotassium hydrogen phosphate, silica gel 60 (0.063–0.200 mm) and silica
CH3
CH3
H
H
O
S
N
N
N
N
S
+
OCH3
HOH C
OCH3
N
N
H
2
H CO
3
H CO
3
CH3
CH3
1
(Omeprazole)
1a
1b
Scheme 2: Degradation products of omeprazole 1.
7
52
Pharmazie 68 (2013)

ORIGINAL ARTICLES
gel60F254 (0.063–0.200mm)wereobtainedfromMerck. Dichloromethane,
isopropanol, and acetonitrile were obtained from LAB-SCAN (Ireland).
was calculated by measuring the areas of the samples with respect to the
areas of the standard.
The degradation products of the various samples were isolated by
using LC. The column was filled with
a suspension of silica
4
.2. Chromatography methods
gel in methanol. The following mobile phase, obtained by mixing:
dichloromethane/isopropanol/triethylamine (80/20/0.5) was then used for
the separation of the different degradation products from the starting sam-
ples. The samples were prepared in the following way: the product obtained
after storage either under normal or accelerated conditions in the form
of blister-protected tablets was grinded and the powder was thoroughly
extracted with methanol with vigorous shaking; the suspension was then
filtered, and the filtrate submitted to LC. The various new compounds were
separated according to their polarity by using the prepared mobile phase.
Samples of the collected fractions were checked by TLC. A final purification
of the isolated products was carried out by using preparative thin layer chro-
matography and acetonitrile as the eluant. Finally the purity of the isolated
A Liquid Column Chromatography (LC) gradient method was developed for
the separation and the determination of the purity of the isolated degradation
products. An Agilent (model 1100, USA) HPLC apparatus was used. The
buffer solution used for the preparation of the mobile phase was prepared
from KH2PO4 (3.00 g) and K2HPO4 dissolved in water (1000 ml). The
pH was adjusted to 7.4 with KOH (1N). The mobile phase was prepared
by mixing 1000 ml of the previously prepared buffer with acetonitrile (500
ml). The pH of the solution was adjusted to 7.4 using KOH (1N). Three
samples of the pharmaceutical standard drugs (0.1 mg/ml) were prepared
together with three samples of the degraded samples. The samples were
injected in the HPLC column and the percentage of the active substance
H
H
O
S
N
N
N
N
S
+
OCH3
N
N
H
HOH C
OCH3
F2HC-O
F2HC-O
2
OCH3
OCH3
3b
3
(Pantoprazole)
3a
Scheme 3: Degradation products of pantoprazole 3.
-
N
-
N
O
S
N
N
O
S●
+
●
O
CF3
H C
O
CF3
2
N
N
CH3
CH3
A
B
Scheme 4: MS/MS sulfur carbone bond cleavage in lansoprazole.
Scheme 5: Suggested mechanism.
Pharmazie 68 (2013)
753

ORIGINAL ARTICLES
compounds was determined by HPLC, by using a DAD UV detector. The
formations of omeprazole \& its analogues. Acta Chem Scandinav 43:
newanalytical method hasbeen validated forLansoprazole, Omeprazole and
Pantoprazole by accuracy, precision, range, linearity, specificity/selectivity,
limit of detection (LOD) and limit of quantitative (LOQ) tests.
536–548.
Della Greca M, Iesce MR, Previtera L, Rubino M, Temussi F, Brigante M
(2006) Degradation of lansoprazole and omeprazole in aquatic environ-
ment. Chemosphere 63: 1087–1093.
Del Valle J, Scheiman JM (2003) Zollinger-Ellison syndrome. In: Yamada
T (ed) Textbook of Gastroenterology Vol. 1, Lippincott Williams and
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EL-Sherif ZA, Mohamed AO, EL-Bardicy MG, EL-Tarras MF (2006).
Reverse-phase high performance liquid chromatography method for the
determination of lansoprazole, omeprazole and pantoprazole sodium
sesquihydrate in presence of their acid-induced degradation products.
Chem Pharm Bull 54: 814–818.
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prazole: investigation of key parameters that influence the water content
in final API. Organic Proc Res Devel 14: 229–233.
The selectivity of the HPLC method was illustrated in their chromatograms,
where complete separation of lansoprazole, omeprazole and pantoprazole
from their degradation products were noticed with sharp peak and clear base-
line separation. Linear correlation was obtained between the peaks areas and
the corresponding concentrations for lansoprazole, omeprazole and panto-
prazole in the range of 2- 3 mg/100 mL. The regression equations were
calculated. The accuracy of the proposed method was checked by analysing
differentconcentrationsofthedrugsinpurepowderedform. Theresultsindi-
cate that the accuracy of the proposed method is not affected by the presence
of excipients. The proposed HPLC method was successfully applied for the
determination of these drugs in their formulations (blister protected tablets
and powders).
4
.3. NMR spectroscopy
Gaorski CL, Aranyi C, Vana S, Rajendran N, Abdo K, Levine SL (1991)
Prechronic inhalation toxicity studies of 2-mercaptoimidazole (2-MBI)
in F344/N rats. Fund Appl Tox 16: 161–171.
Hogg DR (1990) In: Patai S (ed) The Chemistry of Sulphenic Acids and
Esters.Wiley, New York, pp 362–402 ICH (2006).
The H and 13C NMR spectra and DEPT (Distortion Enhancement by Polar-
1
ization Transfer) experiments for the different isolated compounds were
carried out at 100 and 400 MHz on Brucker AVANCE spectrometers either
1
in DMSO-D6 or in methanol-D3. The H chemical shift values were reported
on the δ scale in ppm, relative to TMS, and the chemical shift values were
reported relative to CDCl3 (δ = 77.0 ppm) and DMSO-D6 (δ = 30.50 ppm)
as internal standards, respectively.
Kawasaki Y, Umemura T, Saito M, Momma J, Sekiguchi H, Matsumoto
M, Sakemi K, Isama K, Inoue T, Kurokawa Y, Tsuda M (1998) Toxi-
city of a rubber antioxidant, 2-mercaptobenzimidazole by repeated oral
administration in rats. J Tox Sci 23: 53–68.
Koch TR, Petro A, Darrabie M, Opara EC (2004) Effects of the H,K-ATPase
inhibitor, esomeprazole magnesium, on gut total antioxidant capacity in
mice. J Nutr Biochem 15: 522–526.
Kohler JE, Blass AL, Liu J, Tai K, Soybel DI (2010) Antioxidant pre-
treatment prevents omeprazole-induced toxicity in an in vitro model of
infectious gastritis. Free Rad Biol Med 49: 786–791.
4
.4. Mass spectrometry
The analysis was performed on a SHIMADZU model LC/MS-2010 2V
mass spectrometer. High-resolution positive and negative ESI mass spec-
tra were acquired with a recent ultra-high resolution mass spectrometer, the
hybrid linear ion trap LTQ-Orbitrap-XL (Thermo Fisher Scientific, Les Ulis,
France). The analyses were performed by flow-injection analysis using a LC
pump. Each direct introduction analysis was carried out by injecting 20 ␮L
sample within a flow rate of 100 ␮L/min of a mobile phase consisting of
methanol (around 100 ng of sample were injected). The electrospray voltage
was set to 4.5 kV, the capillary voltage and the tube lens offset were set to 20
V and 70 V, respectively. The sheath and auxiliary gas flows (both nitrogen)
were optimized at 40 and 15 (arbitrary units) and the drying gas temperature
Krüger U, Senn-Bilfinger J, Sturm E, Figala V, Klemm K, Kohl B, Rainer
G, Schaefer H, Blake TJ, Darkin DW, Ife RJ, Leach CA, Mitchell RC,
+
+
Pepper ES, Salter CJ, Viney NJ (1990) (H -K )-ATPase inhibiting 2-
(2-pyridyl)sulfinyl)benzimidazoles. Evidence for the involvment of a
(
sulfenic acid in their reactions J Org Chem 55: 4163–4168.
Lapenna D, de Gioia S, Ciofani G, Festi D, Cuccurullo F (1996) Antioxidant
properties of omeprazole. FEBS Lett 382(1–2): 189–192.
Lindberg P, Nordberg P, Alminger T, Brandstrom A (1986) The mechanism
of action of the gastric acid secretion inhibitor omeprazole. J Med Chem
29: 1327–1329.
◦
was set to 250 C. The mass resolving power (full width at half maximum)
was set at 6.10 FWHM at m/z 400. Tandem mass spectrometry was carried
out with an electrospray-quadrupole ion trap mass spectrometer (Esquire
4
3000, Bruker, Bremen, Germany) operated in the negative ion mode. Sam-
ples (10 μL in MeOH) were directly infused using a syringe pump with a
flow rate of 120 μL/min. Sequential MS experiments have been carried out
with a Low Mass Cut Off (LMCO) corresponding to 28% of the m/z of the
precursor ion (qz = 0,25).
Moffatt JG (1971) Sulfoxide-carbodiimide reactions. X. Mechanism of the
oxidation reaction. J Org Chem 36:1909–1912.
n
Natale G, Lazzeri G, Lubrano V, Colucci R, Vassalle C, Fornai M, Biandizzi
C, Del Tacca M (2004) Mechanism of gastroprotection by lansopra-
zole pretreatment against experimentally induced injury in rats: role of
mucosaloxidativedamageandsulfhydrylcompounds. ToxicolApplPhar-
macol 195: 62–72.
Rackür G, Bickel M, Fehlhaber HW, Herling A, Hitzel V, Lang HJ, Rösner
M, Weyer R (1985) 2-((2-Pyridyl) sulfinyl benzimidazoles: acid sensi-
tive suicide inhibitors of the proton transport system in the parietal cell.
Biochem Biophys Res Comm 128: 477–484.
Acknowledgements: The authors wish to thank the Department of Chem-
istry - Faculty of Science - University of Aleppo - (Aleppo - Syria), K and
C. Pharma (Aleppo - Syria) and Université Pierre et Marie Curie.
Reddy GM, Bhaskar BV, Reddy PP, Ashok S, Sudhakar P, Babu JM, Vyas
K, Mukkanti K (2007) Structural identification of potential impurities of
Pantoprazole sodium. J Pharm Biomed Anal 45: 201–210.
Sachs G, Shin JM, Briving C, Wallmark B, Hersey S (1995) The pharma-
cology of the gastric acid pump: The H ,K ATPase. Ann Rev Pharmacol
Toxicol 35: 277–305.
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