Major degradation product identified in several pharmaceutical formulations against the common cold

Article abstract of DOI:10.1021/ac0611263

Different pharmaceutical preparations against the common cold contain acetaminophen, phenylephrine hydrochloride, and chlorpheniramine maleate. A degradation product had been discovered in these preparations after short- and long-term stability studies. T

Full text of DOI:10.1021/ac0611263

Anal. Chem. 2006, 78, 7891-7895
Correspondence
Major Degradation Product Identified in Several
Pharmaceutical Formulations against the Common
Cold
J. Wong, L. Wiseman, S. Al-Mamoon, T. Cooper, L.-K. Zhang, and T.-M. Chan*
Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, Schering-Plough HealthCare
Products, 3030 Jackson Avenue, Memphis, Tennessee 38151
from those reported in the literature.2 Another possible compound
that has the same molecular formula was then synthesized, and
its NMR spectra matched those reported in the literature. We
report that the correct structure of the degradant should be 2-[2-
hydroxy-2-(3-hydroxyphenyl)ethyl]methylaminosuccinic acid (com-
pound III, Figure 1).
Different pharmaceutical preparations against the com-
mon cold contain acetaminophen, phenylephrine hydro-
chloride, and chlorpheniramine maleate. A degradation
product had been discovered in these preparations after
short- and long-term stability studies. This degradation
product was isolated and found to be an adduct of
phenylephrine and maleic acid. An account of the isolation
and characterization of this compound was published.
Our interest in this area led us to synthesize the com-
pound, and we found that the synthesized compound does
not have the same spectroscopic properties described in
the original paper. Our subsequent work identified the
structure of the degradation product as a “Michael addi-
tion” product of phenylephrine and maleic acid.
EXPERIMENTAL SECTION
Materials. Phenylephrine HCl was from Boehringer Ingelheim
(Ingelheim, Germany). Excipient blends were from Schering-
Plough Healthcare Products (Memphis, TN). Acetonitrile and
water were Optima grade from Fisher Scientific (Pittsburgh, PA).
Sodium perchlorate, (99%) was from Acros Organics/Fisher
Scientific (Pittsburgh, PA). Perchloric acid (70% ACS grade) was
from Acros Organics/Fisher Scientific (Pittsburgh, PA).
Synthesis. Compounds II and III were synthesized in Scher-
ing-Plough Research Institute. The compounds were synthesized
from I in reactions shown in Figure 1. Details of the syntheses
will be published in the future.
High-Performance Liquid Chromatography (HPLC). The
HPLC system consisted of an Agilent Technologies (Palo Alto,
CA) 1100 quaternary pump with a photodiode array detector set
at 215, 265, and 275 nm. HPLC conditions were very similar to
those described in the paper by Marin and co-workers2 (method
1). The analytical column was a Discovery HS PEG column 4.6 ×
150 mm (5 µm) (Supelco, Bellefonte, PA) with the column
temperature set to 35 °C. The flow rate was 1.0 mL/min and
injection volume 10 µL. The mobile phase was sodium phosphate
buffer 20 mM at pH 7.0/actonitrile 90:10 (v/v).
For better resolution, another HPLC method (method 2) was
used. The analytical column was Zorbax SB-CN 4.6 × 250 mm (5
µm) (Agilent) with the column temperature set to 25 °C. The flow
rate was 1.0 mL/min and injection volume 10 µL. The mobile
phase consisted of (A) 0.1% (v/v) perchloric acid with 100 mM
sodium perchlorate and (B) acetonitrile. Mobile phases were
filtered through 0.45-µm membrane and degassed before use. A
4-min initial isocratic (94:6 A/B) run was followed by a 16-min
gradient with increasing percent B to elute the peaks (10:90 A/B),
followed by regeneration of the starting conditions.
Phenylephrine is a decongestant1 used in commercial drug
products as a replacement for pseudoephedrine and is being
evaluated by various drug manufacturers or has already been
marketed. Its most common form is the hydrochloride salt. The
combination of phenylephrine with other active ingredients,
namely, chlorpheniramine maleate and acetaminophen, has
been reported to produce a small amount of a degradant with a
molecular mass of 283. This degradant has been reported2 as
being the reaction product of phenylephrine and maleic acid
and was assigned a structure of 3-hydroxy-N-[2-hydroxy-2-(3-
hydroxyphenyl)ethyl]-N-methylsuccinamic acid (compound II,
Figure 1).
During pharmaceutical development3 in which phenylephrine
HCl and dexbrompheniramine maleate were combined one at a
time with various common tablet excipients, we observed a
degradant with the same molecular mass of 283, similar HPLC
profile (RRT), and UV spectrum. Compound II (3-hydroxy-N-[2-
hydroxy-2-(3-hydroxyphenyl)ethyl]-N-methylsuccinamic acid) was
synthesized and found to have NMR spectra that are different
* To whom correspondence should be addressed. E-mail: tze-ming.chan@
spcorp.com.
(1) 21 CFR Part 341.
(2) Marin, A.; Espada, A.; Vidal, P.; Barbas, C. Anal. Chem. 2005, 77, 471-
477.
(3) International Conference on Harmonization (ICH) Q8: Guidance for
Industry. Pharmaceutical Development.
10.1021/ac0611263 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/27/2006
Analytical Chemistry, Vol. 78, No. 22, November 15, 2006 7891

Figure 1.
Figure 2. (1) Standard solution, (2) solution of stressed active and excipient, (3) solution of stressed active and excipient with III added, and
(4) solution of stressed active and excipient with II added.
7892 Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

Figure 3.
Figure 4.
Excipient Compatibility/Stability Study. Two sets of active/
excipient mix generally resulted in higher amounts of degradation
products. Among them was one with molecular mass of 283
(referred as the unknown in the rest of this paper). The amount
of this particular degradation product produced over all active/
excipient sets made was 1.5-11.7%, by area, relative to phenyl-
ephrine HCl standard response.
Analysis. Using the HPLC conditions in the paper of Marin
and co-workers (method 1), the unknown degradation product
has a RRT of 0.75 (phenylephrine, RRT ) 1.0). Compound III
has the same RRT, while II has a RRT of 0.79. For better
resolution, a different column (method 2) was used. Figure 2
presents an overlay of four chromatograms as analyzed by method
2. HPLC analysis of the stressed active/excipient mix pro-
duced an unknown peak at RRT 0.90 (phenylephrine RRT, 1.0).
Spiking the solution with a solution of III resulted in an increased
response of the peak (chromatogram 3, Figure 2). For com-
parison, a solution of stressed active/excipient was spiked with a
solution of II (chromatogram 4, Figure 2). Spiking the stressed
active/excipient solution with III matches the retention time
of the unknown peak and the response is enhanced. Spiking
excipient mixes were made. One set consisted of heating at 60
°C for 1 month. The second set consisted of the identical
components as the first except that ∼10% (w/w) water was added
followed by heating to 60 °C for 1 month. Both sets of mixes
contained the actives phenylephrine HCl and dexbromphe-
niramine maleate along with a single tablet excipient. Both sets
of active/excipient mixes (with and without water) produced
multiple degradants, and samples were analyzed by HPLC.
HPLC Sample Preparation. The concentration of phenyl-
ephrine HCl in standard and sample solutions was ∼0.5 mg/mL
in diluent. The dilute used was water/acetonitrile 80:20 (v/v).
Excipient mixes were added quantitatively into volumetric flasks
with the aid of some acetonitrile followed by dilution with diluent
to a phenylephrine concentration of 0.5 mg/mL. Filtration,
centrifugation, or both were performed as necessary to obtain a
clear sample solution for analysis.
RESULTS
Both sets of active/excipient mixes (with and without water)
produced multiple degradants. The addition of water to the active/
Analytical Chemistry, Vol. 78, No. 22, November 15, 2006 7893

Table 1. Assignments of the Proton and Carbon NMR Resonances for Compounds II and IIIa
compound II compound III
13C δ (ppm)
1H δ (ppm)
13C δ (ppm)
1H δ (ppm)
1
172.55, 172.3, 172.2
39.2, 39.25, 39.3
64.8, 65.0, 65.2, 65.4
172.15, 172.1, 171.9
56.2, 56.4, 56.5
70.5, 70.7, 70.75, 70.9
144.75, 145.0, 145.3, 145.4
112.7, 112.8, 112.9
157.2, 157.25, 157.3, 157.4
114.0, 114.1, 114.2
129.0, 129.1, 129.2
116.5, 116.55, 116.6
34.1, 34.3, 36.4, 36.5
171.3/174,4, 174.7
32.5, 33.1
66.0, 66.8
171.3/174.4, 174.7
62.1, 62.6
69.0, 69.6
143.8, 143.9
113.9, 114.0
159.1
2
2.33 2.54, 2.33 2.60, 2.39, 2.67
4.60, 4.69, 4.75
2.90 3.09, 2.92 3.10
4.27, 4.31
3
4
1′
2′
3′
4′
5′
6′
7′
8′
1′′
3.16 3.49, 3.32, 3.22 3.70, 3.43
4.62, 4.64, 4.71, 4.73
3.21 3.42, 3.24 3.46
5.04
6.76, 6.79, 6.81
6.88
6.62, 6.64
116.4
6.72
7.09, 7.11, 7.12
6.73, 6.77, 6.78
2.78, 2.85, 2.96, 3.00
130.9
118.1, 118.2
39.1, 39.5
7.11
6.88
2.95, 2.99
a NMR spectra were obtained on a Varian Inova 500-MHz spectrometer at 25 °C. Spectra of II were obtained in DMSO-d6, and those of III were
obtained in methanol-d4.
techniques such as HSQC, HSQCTOCSY, and HMBC. The proton
and carbon assignments are summarized in Table 1.
The NMR spectra of II are more complicated; each diastere-
omer in the sample also exist as two rotomers due to the restricted
rotation around the NC4 amide bond. Therefore, there are four
sets of peaks in the NMR spectra due to the four rotomers. In
the HMBC spectrum, correlation was observed between the
N-methyl proton (H1′′) and the methylene carbon C1′, and
between H1′′ and C4 (172 ppm). No HMBC correlation was
observed between H1′′ and C3 as expected since they are
separated by four bonds.
In the HMBC spectrum of III, correlation was observed
between the N-methyl proton (H1′′) and the methylene carbon
C1′, and between H1′′ and C3, which is the same as observed by
Marin and co-workers.2 No HMBC correlation was observed
between H1′′ and the carbonyl carbons (160-170 ppm) as
expected. NMR spectra (proton, carbon, HSQC, and HMBC
spectra) of III obtained in D2O were nearly identical to those
reported by Marin and co-workers.2 The proton NMR spectrum
Figure 5. Proton NMR spectrum of III, obtained on a Varian Inova
shown in Figure 5 was nearly identical to the one reported by
500-MHz spectrometer. Spectrum was obtained in D2O. Since the
Marin and co-workers.2
resonances were sensitive to the pH of the solution, a small amount
of NaOD was added to the D2O solution until the spectrum was nearly
identical to that reported by Marin and co-workers.
CONCLUSIONS
the solution with II does not match the retention time of the
unknown peak. The UV spectra of the unknown peak and of
III are given in Figure 3. An overlay of the spectra is presented
Two new compounds were synthesized and fully characterized
by MS and NMR. These compounds are potential candidates for
a degradation product in pharmaceutical formulation against the
in Figure 4.
common cold containing phenylephrine and maleates (chlorphe-
Characterization of Compounds II and III. The compounds
niramine maleate or dexbrompheniramine maleate). In the
were characterized by mass spectrometry and NMR spectroscopy.
literature,2 this degradation product was assigned the structure
of 3-hydroxy-N-[2-hydroxy-2-(3-hydroxyphenyl)ethyl]-N-methylsuc-
In their mass spectrum, both compounds give a M + H+ ion of
284, and the high-resolution mass spectrometry confirms a
cinamic acid. The syntheses of these two compounds allow us to
molecular formula of C13H17NO6 for both compounds.
conclude that this degradation product was misidentified, and its
Structures of both compounds were determined by 2D NMR.
structure should be 2-[2-hydroxy-2-(3-hydroxyphenyl)ethyl]-
methylaminosuccinic acid. It is a “Michael addition” product
between phenylephrine and maleic acid, which should be readily
formed under fairly mild conditions such as the stressed condi-
tions used for the excipient compatibility and stability studies.
Since the structures contain an additional chiral center, both
synthetic samples are a mixture of two diastereomers, and two
sets of peaks were observed in NMR. All proton and carbon
resonances in the spectra were assigned, and the connectivities
between proton and carbon nuclei were confirmed by 2D NMR
7894 Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

ACKNOWLEDGMENT
Received for review June 21, 2006. Accepted September
11, 2006.
We thank Dr. B. Pramanik, Dr. P. McNamara, and Dr. M.
Senior for their helpful discussions, Dr. Mohammed Kabir for pro-
viding formulation samples, and Ms H. Yun for library research.
AC0611263
Analytical Chemistry, Vol. 78, No. 22, November 15, 2006 7895