Accepted Manuscript

Title: “Ghost peaks” of ezetimibe: Solution degradation

products of ezetimibe in acetonitrile induced by alkaline
impurities from glass HPLC vials

Author: Jianyang Jin Zhiying Wang Jinsheng Lin Wenquan

Zhu Chengzhen Gu Min Li

PII: S0731-7085(16)31151-7
DOI: http://dx.doi.org/doi:10.1016/j.jpba.2017.02.059
Reference: PBA 11135

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 16-11-2016

Revised date: 28-1-2017
Accepted date: 1-2-2017

Please cite this article as: J. Jin, Z. Wang, J. Lin, W. Zhu, C. Gu, M. Li, “Ghost peaks”
of ezetimibe: solution degradation products of ezetimibe in acetonitrile induced by
alkaline impurities from glass HPLC vials, Journal of Pharmaceutical and Biomedical
Analysis (2017), http://dx.doi.org/10.1016/j.jpba.2017.02.059

This is a PDF file of an unedited manuscript that has been accepted for publication.
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 “Ghost peaks” of ezetimibe: solution degradation products of ezetimibe in
acetonitrile induced by alkaline impurities from glass HPLC vials

Jianyang Jin 1, Zhiying Wang 2, Jinsheng Lin 1, Wenquan Zhu 1,

Chengzhen Gu 1, and Min Li 1,3,*

1
Center of Excellence for Modern Analytical Technologies (CEMAT)
Huahai Pharmaceutical Co., Ltd, Xunqiao, Linhai, Zhejiang, P.R. China 317024
2
Waters Corp., Shanghai

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Huahai US, Inc. 2001 Eastpark Blvd, Cranbury, NJ 08512, USA

* Corresponding author: minli88@yahoo.com

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Key words: Ezetimibe; base-catalyzed; degradation; LC-MS; HPLC vial

Abstract
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Degradation of ezetimibe solution in pure acetonitrile occurs when these are stored in
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glass HPLC vials. The occurrence of the two main degradation peaks and one minor
peak was unpredictable at the time of each sample preparation and over time, it
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appeared that approximately 15% of the sample solutions in glass HPLC vials show
the degradation peaks. Once the degradation peaks occurred in a particular vial,
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typically within 24 hours, their quantity keeps growing until reaching a total yield of
about 4 to 5%. Through a comprehensive investigation it is determined that the
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solution degradation is caused by a base-catalyzed process, during which ezetimibe

undergoes 1) dimerization to form two dimeric impurities, which have not been
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reported in the literature, and 2) to a less degree, isomerization to produce an isomeric

impurity that has been reported before.

Page 1 of 17
1. Introduction
Ezetimibe (1), [(3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-
(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one], is a
first-in-class cholesterol-lowering therapeutic agent developed by the former
Schering-Plough Corp. (which was merged with Merck Co., Inc. in 2009). It works by
inhibiting cholesterol absorption in the small intestine. It has a substituted

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four-membered β-lactam ring in the center of the molecule. Several forced
degradation studies were reported during various analytical method development

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programs prior to 2011; these studies were summarized by Lestari et al. [1].

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Nonetheless, the degradants in these studies were either not characterized, in vast
majority cases, or improperly characterized. In 2011, Gajjar and Shah reported the
isolation and structure characterization of a major degradant of ezetimibe formed
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under alkaline condition [2]. Nevertheless, the proposed structure was soon found to
be incorrect by Barhate and Mohanraj [3], but they did not provide an alternative
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structure. The correct structure of this degradant (2, Scheme 1) was soon reported by
Filip et al. [4] and Sánta et al. [5]; it turned out that 2 is formed via intramolecular
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isomerization. Furthermore, Sánta et al. found that the correct structure of this
degradant was separately determined by Swamy et al. several years earlier through
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single crystallography and NMR [6], and this degradant was generated under strong
acidic condition in the latter study. Batova et al. performed a forced degradation study
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of ezetimibe in alkaline pH range [7]; the major degradation product was determined
to be 2 in pH 7 – 12.5. The formation mechanism of 2 is unusual: the hydroxyl group
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attacks the phenolic benzylic position, rather than the carbonyl group, rendering the
amide as the leaving group. At pH higher than 12.5, hydrolysis of the amide bond
became the major degradation pathway (Pathway b, Scheme 1), resulting in the
formation of the amino acid 3. The latter pathway is common for a typical β-lactam
[8]. Further stress under the strong alkaline condition (pH > 12.5) led to the formation
of the cyclic acid 4.

Since ezetimibe is essentially insoluble in water, analytical methods of the drug

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Page 2 of 17
substance with reversed phase column chromatography typically utilize pure or mixed
organic solutions with little or no aqueous component in the sample diluent. During
our method development for ezetimibe, we initially chose pure acetonitrile as the
sample diluent. An odd phenomenon was observed: two unpredictable degradation
peaks occurred in about 15% of the sample solutions, when they were stored in glass
HPLC vials. When the two peaks occurred in a particular vial, typically within 24

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hours, their quantity keeps growing until about 4 to 5%. Interestingly, the sample
solutions stored in volumetric flasks never displayed the two peaks above the

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detection limit. It seemed that something leaked out of the surface of those

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odd-behaving, minority glass HPLC vials and went into the acetonitrile solution,
which caused degradation of ezetimibe. The aim of this study was to investigate the
root cause of the formation of the two degradants utilizing liquid
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chromatography-high resolution tandem mass spectrometry (LC-MS/MS).
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2. Experimental
2.1 Materials
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Ezetimibe and ezetimibe isomeric impurity (2) were manufactured by Huahai

Pharmaceutical Co., Ltd. Acetonitrile of HPLC grade was purchased from Merck.
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Ammonium acetate and triethylamine of HPLC grade were purchased from

Sigma-Aldrich and DAMAS-BETA, respectively. Acetic acid of AR grade was
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product of Hangzhou Chemical Reagent. Borosilicate glass HPLC vials, deactivated

borosilicate glass HPLC vials, polypropylene HPLC vials, vial caps (12x32 mm), and
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PTFE/Silicone pre-slit septa were products of Waters Corp. Borosilicate glass HPLC
vials were also procured from Agilent.

2.2 HPLC analysis

HPLC separation was carried out on Waters 2695 HPLC system equipped with a
Kromasol 100-5 C18 column (250×4.6 mm, 5 µm) using a mobile phase system
consisting of A, water, and B, acetonitrile. The analyses were performed at 30ºC with
a flow rate of 1.0 mL/min and a gradient program varied according to the following
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program: 0 min (50% B), 5 min (50% B), 15 min (80% B), 25 min (80% B), 30 min
(50% B) and 35 min (50% B). HPLC/UV chromatograms were collected at 230 nm
using Waters 2996 variable wavelength UV detector.

2.3 LC-PDA-MS and MS/MS analyses

High resolution LC–PDA–MS and MS/MS analyses were performed on a Waters

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Xevo G2-S Q-Tof mass spectrometer interfaced to a Waters ACQUITY UPLCTM
system equipped with a PDA detector.

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The UPLC separation on the Waters system was carried out on a BEH C18 (100 x 2.1
mm, 1.7 µm) column using a mobile phase system consisting of A, water, and B,
acetonitrile. The analyses were performed at 30ºC with a flow rate of 0.45 mL/min
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and a gradient program: 0 min (50% B), 2 min (50% B), 6 min (80% B), 8 min (50%
B)，and 10 min (50% B). UV spectra were collected from 190 to 400 nm from the
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PDA detector. The Waters Q-TOF mass spectrometer was operated at positive
electrospray mode in vast majority cases with the following source parameters: cone
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gas 10 L/hr, desolvation gas 800 L/hr, desolvation temperature 400 ℃, source
temperature 110℃, and capillary voltage 0.8 kV. The acquisition mode was MSE and
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acquisition range was m/z 100 – 1200.

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2.4 Base-catalyzed forced degradation of ezetimibe in acetonitrile versus stabilization

of ezetimibe solutions with weak acid and neutral buffer
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For the base-catalyzed forced degradation study, 0.1% of triethylamine was added
into a stock solution of ezetimibe in acetonitrile and then it was divided into nine
borosilicate glass HPLC vials, respectively. The nine solutions were immediately
analyzed by HPLC with the method described in Section 2.2 Afterward, the nine
solutions were allowed to stand at room temperature for 3 days, and analyzed by
HPLC again.

For the stabilization of ezetimibe solution with weak acid, 0.1% acetic acid was added
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into a stock solution of ezetimibe in acetonitrile and then it was divided into five
borosilicate glass HPLC vials. For the stabilization of ezetimibe solution with neutral
buffer, a stock solution of ezetimibe in acetonitrile containing 0.1% ammonium
acetate was prepared and then it was divided into five borosilicate glass HPLC vials.
The two sets of the solutions were allowed to stand at room temperature for 3 days
and then analyzed by HPLC (Section 2.2).

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3. Results and discussion

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3.1 Random occurrence of “ghost peaks” from ezetimibe solution in pure acetonitrile

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As mentioned in the Introduction, our initial method for ezetimibe utilized pure
acetonitrile as the sample diluent. In the course of investigating these solutions two
degradation peaks eluting after ezetimibe were observed (Fig. 1) in a seemingly
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unpredictable, random fashion. Over a period between 24 hours and up to a few days,
approximately 15% of the sample solutions in glass HPLC vials would eventually
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display the two peaks. Several different brands of glass HPLC vials from various
vendors were tried but the phenomenon remained more or less the same. Plastic
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HPLC vials were also tried; the frequency of the “ghost peaks” occurrence was much
lower than that from the glass vials, but the problem remained unresolved. Once the
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“ghost peaks” occurred in a particular vial, typically within 24 hours, they would keep
growing until reaching a combined yield of up to 4 to 5%. Interestingly, the sample
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solutions stored in volumetric flasks never displayed the two “ghost peaks” above the
detection limit.
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3.2 LC-PDA-MS and MS/MS analyses

An ezetimibe solution in pure acetonitrile that displayed the two “ghost peaks” was
analyzed by LC-PDA-high resolution MS and MS/MS. The two “ghost peaks” eluted
at 3.92 and 4.15 min (Fig. 2), and showed m/z values at 819.3063 and 819.3055
(Refer to Supplementary materials), respectively, indicating they are isomers toward
each other. Their accurate m/z values of 819.3063 and 819.3055 match a formula of
C48H43N2O6F4 (theoretical m/z: 819.3052), corresponding to the protonated ion of an
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“M+M = 2M” type dimer of ezetimibe [9]. The MS spectra of both dimers also
showed sodiated ions at m/z 841. In order to make sure that both the m/z 819 and m/z
841 ions are not gas phase dimers, the sample solution was also analyzed under ESI
negative mode; [M–H]- ions at m/z 817 from both the 3.92 and 4.15 min peaks were
observed (Refer to Supplementary materials), confirming the two degradation
products are indeed covalent dimers of ezetimibe. Both protonated ions of the dimers

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(m/z 819) had extensive in-source fragmentation, with m/z 392 and m/z 410 as the
major fragments, suggesting the dimers are not very stable. MS/MS fragmentation of

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both m/z 819 ions produces similar, but differentiable fragmentation patterns (or MS2

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fingerprints) (Fig. 3), suggesting that they are isomers with similar structures [10].
The UV spectra of the 3.92 and 4.15 min peaks are essentially identical (Fig. 4),
indicating that both species have the same or similar chromophore.
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During the course of the investigation, it was noticed that a small solution degradation
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peak at 12.27 min (Fig. 1) was also formed along with the two dimeric degradants.
The 12.27 min peak corresponds to the 2.28 min peak in the LC-MS analysis as
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shown in Fig. 2 and it displays an accurate mass that is the same as ezetimibe (results
not shown), indicating that this is an isomeric degradant of ezetimibe. Upon a search
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in the literature, it turned out that this isomeric degradant was a major degradation
product that was formed when ezetimibe was subjected to either strong acidic [6] or
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alkaline conditions [2-5, 7]. The structure of this degradant had been a subject of
controversy [2,3]; nevertheless, its correct structure (2, Scheme 1) was finally firmly
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established after several groups published their findings [4,5, 7]. As illustrated in
Scheme 1, degradant 2 is formed via a rearrangement mechanism, in which the
hydroxyl group attacks the phenolic benzylic position, resulting in the opening of the
β-lactam and concurrent formation of a six-membered tetrahydropyran ring. This
rearrangement is unusual, as one would expect that the hydroxyl group should attack
the carbonyl of the β-lactam ring (because the β-lactam ring of a penicillin antibiotic
typically opens up through the cleavage of the amide bond, when the β-lactam is
attacked by a nucleophile) [8]. In the present case of ezetimibe rearrangement, the
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Page 6 of 17
hydroxyl group attacks the phenolic benzylic position, rather than the carbonyl group,
rendering the amide as the leaving group.

3.3 Forced degradation study under alkaline condition to consistently reproduce the
“ghost peaks”
Small quantities of alkaline impurities may leach out of the surface of a glass vial.

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The basicity of these impurities can be significantly enhanced in an aprotic solvent
[11-12], such as acetonitrile, to such a level that may enable them to catalyze the

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solution degradation of ezetimibe. To test this hypothesis, we added 0.1% of

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triethylamine into a stock solution of ezetimibe in acetonitrile and then divided it into
nine glass vials, respectively. The nine solutions were immediately analyzed by HPLC
sequentially and the first solution analyzed produced Dimer 1 and Dimer 2 in 0.42%
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and 0.96% yields, respectively. In the same solution, the isomeric degradant 2 was
also seen at a level of 0.13%. By the time the 9th solution was analyzed (~6 hours
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later), Dimer 1, Dimer 2 and Degradant 2 were observed at 0.98%, 2.49%, and 0.35%,
respectively. Furthermore, after standing at room temperature for 3 days, the average
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levels of these three degradants were found to be 1.42%, 3.93%, and 2.48%,
respectively (Table 1). On the other hand, of the five control solutions of ezetimibe in
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pure acetonitrile with nothing else added, only one solution underwent meaningful
degradation with the yields of Dimer 1, Dimer 2, and Degradant 2 at 0.13%, 0.26, and
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0%, respectively, during the same 3 day period.

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3.4 Stabilization of ezetimibe solution in acetonitrile with weak acid or neutral buffer
To further confirm the hypothesis of base-catalyzed degradation and to provide a
practical way of stabilizing the ezetimibe solution for the HPLC method, we figured
that addition of a weak acid or neutral buffer may prevent the base-catalyzed
degradation. Hence, to one set of five ezetimibe solutions in acetonitrile, 0.1% acetic
acid was added into each solution; to another set of five ezetimibe solutions in
acetonitrile, 0.1% ammonium acetate was added into each solution. After standing at
room temperature for 3 days, none of the solutions underwent degradation. We also
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noticed that the current USP method for ezetimibe drug substance also utilizes 0.1%
acetic acid in acetonitrile as the sample diluent. Therefore, we can speculate that
similar degradation problem might have been encountered during the development of
that method. On the other hand, the random solution degradation of ezetimibe in
acetonitrile remained with the use of either polypropylene or deactivated borosilicate
glass vials.

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3.5 The nature of the two main “ghost peaks” and proposed degradation pathway of

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ezetimibe under alkaline conditions in aprotic solvent

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Based upon the LC-PDA-MS and MS/MS results presented above as well as the
degradation chemistry of ezetimibe reported in the literature, there is no doubt that the
two main “ghost peaks” are ezetimibe dimers, which were solution degradants of
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ezetimibe formed under alkaline conditions in aprotic solvent. The exact structures of
the two dimers are currently not known without further structure elucidation.
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Nevertheless, we may speculate that they could be formed via the nucleophilic attack
to the first ezetimibe molecule by either the benzilic hydroxyl (to give Dimer 1) or
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phenolic hydroxyl group (to give Dimer 2) from the second ezetimibe molecule
(Scheme 1, Pathway c). The site of nucleophilic attack on the first ezetimibe molecule
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could be the 4-hydroxylbenzylic carbon or the carbonyl of the β-lactam ring. During
the period of this manuscript being reviewed, we became aware of a manuscript,
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which was recently published in this journal, describing a very similar phenomenon of
solution degradation caused by alkaline impurities leaching out of glass HPLC vials
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[13].

4. Conclusions
Through a comprehensive investigation, we have determined that the root cause for
the unpredictable occurrence of the two “ghost peaks” is due to base-catalyzed
degradation of ezetimibe in acetonitrile, an aprotic solvent. The major degradation
products are two dimeric impurities, which have not been reported in the literature. A
minor degradation product is found to be an isomer of ezetimibe, whose formation
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Page 8 of 17
mechanism and structure has been reported in the literature. The exact structures of
the two dimers have not been determined at this point and it is beyond the scope of
our current HPLC method development effort.

References
1. M.L.A.D. Lestari, F. Ardiana, G. Indrayanto, Chapter 3 – Ezetimibe, Prof. Drug

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Subs. Excip. Rel. Meth. 36 (2011) 103 – 149.
2. A.K. Gajjar, V.D. Shah, Isolation and structure elucidation of major alkaline

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degradant of ezetimibe, J. Pharm. Biomed. Anal. 55 (2011) 225–229.

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3. C.R. Barhate, K. Mohanraj, What is the degradation product of ezetimibe? J.
Pharm. Biomed. Anal. 55 (2011) 1237–1238.
4. K. Filip, K. Bankowski, K. Sidoryk, J. Zagrodzka, M. Laszcz, K. Trzcinska, A.
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Szyprowska, P. Cmoch , W. Maruszak, Physicochemical characterization of
ezetimibe and its impurities, J. Mol. Struct. 991 (2011) 162–170.
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5. Z. Sánta, J. Kóti, K. Szőke, K. Vukics, C. Szántay Jr. Structure of the major
degradant of ezetimibe, J. Pharm. Biomed. Anal. 58 (2012) 125–129.
ed

6. G.Y.S.K. Swamy, K. Ravikumar, L.K. Wadhwa, R. Saxena, S. Singh, (2R*, 3R*,

6S*)-N, 6-Bis (4-fluorophenyl)-2-(4-hydroxyphenyl)-3, 4, 5,
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6-tetrahydro-2H-pyran-3-carboxamide, Acta Cryst. E 61 (2005) o3608–o3610.

7. J. Batova, A. Imramovsky, J. Hajicek, L. Hejtmankova, J. Hanusek, Kinetics and
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mechanism of the base-catalyzed rearrangement and hydrolysis of ezetimibe, J.

Pharm. Sci., 103 (2014) 2240-2247.
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8. A. Llinas, B. Vilanova, J. Frau, F. Munoz, J. Donoso, M.I. Page, Chemical

reactivity of penicillins and cephalosporins. Intramolecular involvement of the
acyl-amido side chain, J. Org. Chem. 63 (1998) 9052-9060.
9. M. Li, Organic Chemistry of Drug Degradation, Section 4.7 Dimerization/
Oligomerization, pp 139-144, Royal Society of Chemistry, London, UK, 2012.
10. M. Li, X. Wang, B. Chen, M. Lin, A.V. Buevich, T.-M. Chan, A.M. Rustum, Use
of liquid chromatography/tandem mass spectrometric molecular fingerprinting for
the rapid structural identification of pharmaceutical impurities. Rapid Commun.
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 Mass Spectrom. 23 (2009) 3533 – 3542.
11. B.A. Trofimov,; A.M. Valsil’tsov,; S.V. Amosova, Studied the basicity of alkali
metal hydroxides in a dipolar aprotic solvent DMSO, Russ. Chem. Bull. 35 (1986)
682–686.
12. M. Li, B. Chen, S. Monteiro, A.M. Rustum, Mechanism of base-catalyzed
autooxidation of corticosteroids containing 20-keto-21-hydroxyl side chain.

t
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Tetrahedron Lett. 50 (2009) 4575–4581.
13. R. Arvary, I. Mangion, The importance of vial composition in HPLC analysis: An

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unusual case of phosphorous pseudorotation. J. Pharm. Biomed. Anal. 134 (2017)

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237-242.

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Fig. 1. UV chromatograms at 230 nm of ezetimibe solutions in pure acetonitrile in

glass vials using the method described in Section 2.2. Upper: an example of normal
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behaving chromatogram; lower: an example of chromatogram with “ghost peaks”.
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Page 11 of 17
 t
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Fig. 2. LC-MS analysis of the ezetimibe solution in pure acetonitrile in glass vial that

displayed “ghost peaks” using the method outlined in Section 2.3; the original HPLC
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analysis of the solution is presented in Fig. 1 (lower). Upper: UV chromatogram at

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230 nm; lower, total ion chromatogram (TIC).

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Page 12 of 17
 t
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Fig. 3. Upper: MS2 spectrum of the protonated ion of 3.92 min peak (m/z 819); lower:
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MS2 spectrum of the protonated ion of 4.15 min peak (m/z 819). Both spectra are

normalized in Y-axis against the most abundant fragment peak, m/z 392.
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Page 13 of 17
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Fig. 4. UV spectra of Ezetimibe and 3 solution degradation peaks at 2.28 min, 3.92
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min, and 4.15 min (from Fig. 2, upper).

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Page 14 of 17
 F OH

a: H
ay N
hw tion
Pat meriza ition, ons, ity
Iso c ond oluti kalin O
idic us s th al
g ac queo t wi F
tron .5 a ven
In s 7 - 12 tic sol Ezetimibe isomeric degradant (2)
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p n ap r o (Reported previously and
or i observed in current study as a minor degradant)

OH OH
OH OH F
OH
Pathway b:
O
Hydrolysis

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pH > 12.5 F

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N O OH
F O OH
O
3 F 4
F
Path
In a way
Ezetimibe (1) c:
(cur protic s

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stud nt w
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lkali
nity Ezetimibe Dimer 1 + Ezetimibe Dimer 2

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Scheme 1. Degradation chemistry of ezetimibe based on the literature [7] as well the

current study.
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Page 15 of 17
Table 1. Formation of ezetimibe degradants under the condition of
base-catalyzed forced degradation (Experimental Set 1)1 versus one of the five
control runs (Experimental Set 2)2
Vial No./Exp Set Dimer 1 Dimer 2 Isomeric Degradant (2)

1/Exp Set one3 0.42% 0.96% 0.13%

9/Exp Set one4 0.98% 2.49% 0.35%

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1-9/Exp Set one5 1.42% 3.93% 2.48%

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1/Exp Set two6 0.13% 0.26% 0%

Notes: All the analyses were carried out with the HPLC method as described in

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Section 2.2. 1The forced degradation study with base (0.1% triethylamine); 2The
control runs are five replicate ezetimibe solutions in acetonitrile that were allowed to
stand at room temperature for 3 days. 3The first of the nine vials analyzed sequentially;
4
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The last of the nine vials analyzed sequentially (~6 hours later). 5The results shown
are the average values of the nine vials that stood at room temperature for 3 days;
6
The results shown are from one of the five control runs. The remaining four control
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runs did not show any degradants above the detection limit.
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Highlights

Resolved the “ghost peaks” phenomenon in HPLC analysis of Ezetimibe.

Elucidated the mechanism of base-catalyzed degradation of Ezetimibe in solution

of acetonitrile.

The “ghost peaks” are products of base-catalyzed dimerization caused by traces

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of basic materials eluted from HPLC vials.

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