Direct monitoring of drug degradation by easy ambient sonic-spray ionization mass spectrometry: The case of enalapril

Article abstract of DOI:10.1002/jms.2014

Using enalapril maleate as a test case, the ability of ambient mass spectrometry, namely, via easy ambient sonic-spray ionization mass spectrometry (EASI-MS), to perform direct monitoring of drug degradation has been tested. Two manufacturing processes were investigated (direct compression and wet granulation), and the formation of degradation products was measured via both EASI-MS and high-performance liquid chromatography with ultraviolet detection for a total period of 18 months. Both techniques provide comparable results, which indicate that direct analysis by ambient mass spectrometric techniques presents a viable alternative for drug degradation monitoring with superior simplicity, throughput, and reliability (no sample manipulation), and comparable quantitative results. In terms of qualitative monitoring, the full mass spectra with intact species provided by EASI-MS allow for comprehensive monitoring of known and unknown (or unexpected) degradation products. Copyright

Full text of DOI:10.1002/jms.2014

Research Article
Received: 18 July 2011
Revised: 19 October 2011
Accepted: 1 November 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.2014
Direct monitoring of drug degradation by easy
ambient sonic-spray ionization mass
spectrometry: the case of enalapril
Phellipe H. Amaral,a Raquel Fernandes,b Marcos N. Eberlinb* and
Nelci F. Höehra*
Using enalapril maleate as a test case, the ability of ambient mass spectrometry, namely, via easy ambient sonic-spray
ionization mass spectrometry (EASI-MS), to perform direct monitoring of drug degradation has been tested. Two manufacturing
processes were investigated (direct compression and wet granulation), and the formation of degradation products was
measured via both EASI-MS and high-performance liquid chromatography with ultraviolet detection for a total period of
18 months. Both techniques provide comparable results, which indicate that direct analysis by ambient mass spectrometric
techniques presents a viable alternative for drug degradation monitoring with superior simplicity, throughput, and reli-
ability (no sample manipulation), and comparable quantitative results. In terms of qualitative monitoring, the full mass
spectra with intact species provided by EASI-MS allow for comprehensive monitoring of known and unknown (or unex-
pected) degradation products. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: ambient mass spectrometry; sonic-spray ionization; drugs; drug degradation products
Enalapril maleate, a drug that acts as an angiotensin-converting
enzyme inhibitor, constitutes one of the major drugs used to
INTRODUCTION
The stability of a pharmaceutical formulation is a major factor
affecting the quality of drug products and their efficacy.[1] Drug
stability is mainly affected by the exposure of the product to
environmental conditions such as temperature, humidity, and light.
Its chemical composition and the physical-chemical properties of
excipients and active ingredients and their relative quantities in
the formulation as well as the manufacturing process and conditions
of storage and transportation are also major factors influencing drug
stability. Metabolites created in the human body are also crucial for
overall efficacy and safety of a drug. In modern pharmaceutical drug
discovery and development, it is of crucial importance to identify
unknown compounds arising from impurities or degradation with
the highest possible confidence because of their potential pharma-
cological effects on humans.[2,3]
treat essential and renovascular hypertension and congestive
heart failure,[10] was used in commercial formulations as a test case.
Two manufacturing processes were also compared.
EXPERIMENTAL
Chemicals
Formic acid, methanol, acetonitrile, and phosphoric acid
were purchased from Sigma-Aldrich (São Paulo, Brazil) and used
without further purification. Deionized water was obtained from
a MilliQ (Millipore, Billerica, MA, USA) purification unit.
Mass spectrometry
Recently, a set of techniques known collectively as ambient
ionization mass spectrometric techniques[4] have been devel-
oped and applied with success to the direct analysis of drug
formulations.[5] We have also introduced an ambient ionization
technique, namely, easy ambient sonic-spray ionization (EASI),[6, 7]
and tested it for direct drug analysis with superior simplicity
and signal-to-noise ratios.[8] EASI produces a bipolar stream of
charged droplets and is also attractive for being inherently free
from electrical or discharge interferences and of greater simplic-
ity because it requires no voltage and temperature or irradiation
assistance and may incorporate Venturi pumping.[9] Herein we
tested whether ambient ionization mass spectrometry, as
exemplified by EASI, applied direct to the drug formulation with-
out any sample preparation or pre-separation, would provide
reliable monitoring of drug degradation as compared with the
more conventional monitoring performed via high-performance
liquid chromatography–ultraviolet (HPLC-UV) using drug extracts.
The EASI-MS experiments were performed in a mass spectrometer
(LCMS-2010EV-Shimadzu Corp., Japan) equipped with a home-
made EASI source described in detail elsewhere.[8] To produce
*
*
Correspondence to: Marcos N. Eberlin, ThoMSon Mass Spectrometry Labora-
tory, Institute of Chemistry, University of Campinas- UNICAMP, 13084-971,
Campinas SP, Brazil. E-mail: eberlin@iqm.unicamp.br
Nelci F. Höehr, Faculty of Medical Sciences, Department of Clinical Pathology,
University of Campinas- UNICAMP, 13084-971, Campinas SP, Brazil. Email:
nelci@fcm.unicamp.br
a Faculty of Medical Sciences, Department of Clinical Pathology, University of
Campinas- UNICAMP, Campinas SP, Brazil
b ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas- UNICAMP, 13084-971, Campinas SP, Brazil
J. Mass. Spectrom. (2011), 1269–1273
Copyright © 2011 John Wiley & Sons, Ltd.

P. H. Amaral et al.
the sonic spray, an acidic solution of methanol (0.1 v%) at 20
ml minÀ1 and N2 (100 psi) was used. Experiments were performed
in the positive ion mode.
RESULTS AND DISCUSSION
Drug degradation was followed for a total period of 18 months,
and analysis was performed every 3 months. Figure 2 shows the
EASI(+)-MS for a fresh enalapril tablet (20 mg of enalapril maleate
in a 200 mg Æ 5% total weight) as well as representative spectra
for a tablet after 12 and 18 months during natural aging at
controlled conditions (climatic chamber at 40 ꢀC and 75% relative
humidity). For the fresh tablet (Figure 2a), the active drug,
namely, the protonated enalapril molecule [M + H]+ of m/z 377
as well as its sodiated molecule [M + Na]+ of m/z 399 are
the two predominant ions. However, two known degradation
products of enalapril (Scheme 1),[11] namely, enalaprilat as the
[M + H]+ ion of m/z 349 and DKP as [M + Na]+ of m/z 381 are also
detected for the fresh formulation, indicating degradation by
hydrolysis and dehydration during manufacturing by the wet
granulation process. Lactose, the major excipient, is also detected
as [M + Na ]+ of m/z 365, which is also beneficial for the monitor-
ing of formulation and its stability. The other excipients (sodium
bicarbonate, amide, silicon dioxide, magnesium stearate, and iron
oxide) are not detected by EASI(+)-MS.
After a year inside a climate chamber at 40 ꢀC and 75% relative
humidity, as expected, the abundance of the enalapril ions of m/z
377 and 399 decreases, with the subsequent increase of the
relative abundances of the ions of m/z 381 and 349, whereas that
from the lactose excipient (m/z 365) remained quite unaltered.
After 18 months (Figure 2), however, degradation is quite severe,
and the enalaprin ions of m/z 377 and 399 are much reduced
in abundance whereas those for elaprilat (m/z 349) and DKP
(m/z 381) are now dominant. Interesting, EASI-MS is also able at
this point to detected further degradation products via the ions
of m/z 252 and 331. Scheme 2 depicts two possible structures
for these products of 251 and 330 Da.
High-performance liquid chromatography
The HPLC-UV is equipped with a 215-nm detector and a 4.6-mm Â
25-cm column that contains 5-mm packing L7. The column tem-
perature was maintained at 50 ꢀC, and the flow rate was of 2 ml
minÀ1. The injection volume was 50 ml. The buffer solution was
prepared as follows: 1.38 g of monobasic sodium phosphate
was dissolved in approximately 800 ml of water, and the pH
was adjusted to 2.2. with phosphoric acid, followed by dilution
with water to 1000 ml, and mixing. The mobile phase was a
mixture of the buffer solution and acetonitrile (75:25). Ten tablets
were used to prepare approximately 0.2 mg mlÀ1 aqueous
solution of enalapril maleate. The buffer solution was added in
a 2 : 1 ratio, and the mixture was sonicated for 15 min and shaken
by mechanical means for 30 min, then diluted with the buffer
solution to volume, shaken again, and sonicated for additional
15 min. The resulting mixture was filtered (0.45-mm filter). HPLC
quantitation of enalaprilat and diketopiperazine (DKP) were
performed using U. S. Pharmacopia (USP) standards (Figure 1).
Figure 3 shows the EASI-MS data for degradation monitoring
for tablets manufactured by the direct compression process, via
which the formulation is not subjected to high temperature or
humidity. As compared with Figure 2, very similar spectra were
obtained, but with some substantial differences in terms of
Figure 1. Schematic illustration of the EASI-MS monitoring of drug
degradation.
Figure 2. EASI-MS for enalapril manufactured by the wet granulation process at different periods of stability in a climatic chamber at 40 ꢀC and 75%
relative humidity.
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J. Mass. Spectrom. (2011), 1269–1273

Drug degradation by EASI-MS
O
N
OH
humidity), the relative abundances of the enalapril ions decrease
and those of the degradation products enalaprilat (m/z 349) and
DKP (m/z 381) increase, but to lesser extents than those observed
for the tablets manufactured by the wet granulation process. Note
that the further degradation products detected via the ions of m/z
331 and 252 in Figure 2 (18 months) are not seen in Figure 3
(18 months).
O
H
N
O
O
Enalapril
(376 Da)
O
Figure 4 shows a typical HPLC-UV chromatogram for an extract
from a pool of 10 tablets of enalapril manufactured via the wet
granulation process after 12 months of prolonged degradation.
Note the peak for enalapril at 3.8 min and those for its widely
known degradation products enalaprilat at 1.9 min and DKP at
11.7 min. The peak at approximately 1 min is due to maleic acid.
Using USP standards and by constructing the corresponding
calibration curves, quantitation was performed, and the results
are summarized in Figure 5.
O
C
N
N
H
N
OH
N
O
O
O
O
HO
O
Enalaprilat
(348 Da)
DKP
(358 Da)
Scheme 1. Structures of Enalapril and two of its known degradation
products.
Figure 5 compares the degradation profiles obtained by
both the HPLC-UV and the EASI-MS monitoring. Note that for
HPLC-UV, quantitation was performed via classical procedures
using USP standards for the degradation products. For EASI-MS,
semi-quantitation is performed via direct comparison of relative
abundances assuming equal ionization efficiencies. Although
there is a small quantitative difference, qualitatively both profiles
are quite similar indicating the usefulness of EASI-MS for direct
monitoring of the degradation profile of this drug.
O
OH
N
H
N
N
O
O
O
HO
O
HO
330 Da
251 Da
Figure 6 shows EASI-MS data for tablets manufactured using
the wet granulation process and exposed to accelerated and
severe degradation conditions, that is, (i) high acidic media as
simulated by dropping a drop of an aqueous 1-N HCl solution
directly on the top of the tablet and letting it dry at ambient
conditions and (ii) high temperature as simulated by exposing
the tablet to 100 ꢀC for 4 h. As compared with the prolonged
degradation (Figures 2 and 3), EASI(+)-MS is able to detect
two additional degradation products via the ions of m/z 313
and 303. Although secure characterization of these degradation
products would require a more extensive structural investigation,
Scheme 2 depicts two possible structures corresponding to
[M–H2O–EtOH] of 312 Da and [M–CO–EtOH] of 312 Da.
O
N
O
N
N
N
O
C
O
HO
O
302 Da
312 Da
Scheme 2. Putative structures for the Enalapril degradation products
detected by EASI-MS.
degradation extent. For the fresh sample, only the enalapril
(m/z 377 and 399) and lactose (m/z 365) ions are detected with
no signs of degradation. After 18 months of prolongated exposition
to humidity and heat (climate chamber at 40 ꢀC and 75% relative
Figure 3. EASI-MS for enalapril manufactured by the direct granulation process at different periods of stability in a climatic chamber at 40 ꢀC and 75%
relative humidity.
J. Mass. Spectrom. (2011), 1269–1273
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P. H. Amaral et al.
Figure 4. HPLC-UV chromatogram for a methanolic extract from a poll of 10 tablets of enalapril maleate produced by the wet granulation process after
12 months of prolonged degradation of a climatic chamber at 40 ꢀC and 75% relative humidity.
Figure 5. Comparison of degradation profiles obtained by HPLC-UV (upper panel) and EASI-MS (lower panel) monitoring.
spectrometry to directly monitor drug degradation, the general-
ity and robustness of the approach should be ideally tested with
CONCLUSIONS
a comprehensive set of chemicals from different classes of drugs
such as antibiotics, steroidal hormones, and vitamins. For most
commercial drugs, however, a simple preliminary test could
be easily performed by analyzing an extensively degraded
tablet and by evaluating the ionization efficiencies of known
degradation products. Because of ion suppression effects, it is
also predictable that EASI-MS should work best for polar drugs
forming polar degradation products. Fortunately this seems to
be a common feature of many commercial drugs.
Using enalapril maleate as a test case, the ability of EASI-MS to
monitor drug degradation directly from intact tablets has been
demonstrated. As compared with classical HPLC-UV monitoring,
EASI-MS has been shown to be able to provide similar qualitative
and quantitative results with much superior speed and simplicity
and with no need of sample preparation and pre-separation,
and without inherent risks of contamination and artifacts
associated with these procedures. Although the test case evalu-
ated herein points to the viability of ambient ionization mass
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J. Mass. Spectrom. (2011), 1269–1273

Drug degradation by EASI-MS
Figure 6. EASI-MS for enalapril manufactured by the wet granulation process and submitted to accelerated and drastic acid (concentrated HCl) and
thermal (100 ꢀC for 4 h) degradation.
2005, 77, 2297–2302. c) J. J. Perez, G. A. Harris, J. E. Chipuk,
J. S. Brodbelt, M. D. Green, C. Y. Hampton, F. M. Fernandez.
Acknowledgements
Analyst 2010, 135, 712–719. d) J. S. Sampson, A. M. Hawkridge,
D. C. Muddiman. J. Am. Soc. Mass Spectrom. 2008, 19, 1527–1534. e)
Y. Y. Liu, Z. Q. Lin, S. C. Zhang, C. D. Yang, X. R. Zhang. Anal. Bional.
Chem. 2009, 395, 591–599.
The authors thank the Brazilian science foundations FAPESP,
CNPq, and CAPES for financial assistance.
[6] A. Hirabayashi, M. Sakairi, H. Koizumi. Sonic Spray Ionization Method
for Atmospheric Pressure Ionization Mass Spectrometry. Anal. Chem.
1994, 66, 4557–4559.
[7] R. Haddad, R. Sparrapan, T. Kotiaho, M. N. Eberlin. Easy Ambient
Sonic-Spray Ionization-Membrane Interface Mass Spectrometry
for Direct Analysis of Solution Constituents. Anal. Chem. 2007,
80, 898.
REFERENCES
[1] M. A. M. Shehata, M. A. El Sayed, M. F. El Tarras, M. G. El-Bardicy.
Stability indicating methods for determination of vincamine,
J. Pharm. Biomed. Anal. 2005, 38, 72–78.
[2] M. M. Al-Omari, Abdelah, A. A. Badwan, A. M. Y. Jaber. Effect of
the drug-matrix on the stability of enalapril maleate in tablet
formulations. J. Pharm. Biomed. Anal. 2001, 25, 893–902.
[8] R. Haddad, R. Sparrapan, M. N. Eberlin. Desorption sonic spray
ionization for (high) voltage-free ambient mass spectrometry. Rapid
Commun. Mass Spectrom. 2006, 20, 2901–2905.
[3] ICH Harmonised Tripartite Guidelines, Impurities in New Drug
Substances Q3A(R1), International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for
human use. 7 Feb, 2002.
[4] For recent reviews see: a) G. A. Harris, A. S. Galhena, F. M. Fernandez.
Ambient Sampling/Ionization Mass Spectrometry: Applications and
Current Trends. Anal. Chem. 2011, 83, 4508. b) R. M. Alberici,
R. C. Simas, G. B. Sanvido, W. Romão, P. M. Lalli, M. Benassi, I. B. S. Cunha,
M. N. Eberlin. Anal. Bioanal. Chem. 2010, 398, 265–294. c) D. R. Ifa,
C. P. Wu, Z. Ouyang, R. G. Cooks. Analyst 2010, 135, 669–681. d) H. Chen,
G. Gamez, R. Zenobi. J. Am. Soc. Mass Spectrom. 2009, 20, 1947–1963.
[5] a) Z. Takáts, J. M. Wiseman, B. Gologan, R. G. Cooks, Science 2004,
306, 471–473. b) R. B. Cody, J. A. Laramee, H. D. Durst. Anal. Chem.
[9] V. G. Santos, T. Regiani, F. F. G. Dias, W. Romão, C. F. Klitzke, F. Coelho,
M. N. Eberlin. Venturi Easy Ambient Sonic-Spray Ionization. Anal.
Chem. 2011, 83, 1375–1380.
[10] J. Biollaz, J. L. Schelling, B. Jacot Des Combes, D. B. Brunner,
G. Desponds, H. R. Brunner, E. H. Ulm, M. Hichens, H. J. Gomez. Enalapril
maleate and lysine analogue (MK-521) in normal volunteers;
relationship between plasma drug levels and the rennin angiotensin
system. Br. J. Clin. Pharmacol. 1982, 14, 363–368.
[11] S. Pérez, P. Eichhorn, D. Barcelo. Structural characterization of
photodegradation products of enalapril and its metabolite
enalaprilat obtained under simulated environmental conditions by
hybrid quadrupole-linear ion trap-MS and quadrupole-time-of-
flight-MS. Anal. Chem. 2007, 79, 8293–8300.
J. Mass. Spectrom. (2011), 1269–1273
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