Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction

Article abstract of DOI:10.1002/jms.3351

Ketorolac, a nonsteroidal anti-inflammatory drug, was subjected to forced degradation studies as per International Conference on Harmonization guidelines. A simple, rapid, precise, and accurate high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to degrade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of ketorolac tromethamine and its degradation products on a Grace Smart C-18 (250-mm-×-4.6-mm i.d., 5-μm) column using 20-mM ammonium formate (pH-=-3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0-ml/min. A total of nine degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M-+-H]⁺ ions of ketorolac and its degradation products. In silico toxicity of the drug and degradation products was investigated by using topkat and derek softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization guidelines. Copyright

Full text of DOI:10.1002/jms.3351

Research article
Received: 20 January 2014
Revised: 17 February 2014
Accepted: 18 February 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3351
Characterization of forced degradation
products of ketorolac tromethamine using
LC/ESI/Q/TOF/MS/MS and in silico toxicity
prediction
Pradipbhai D. Kalariya,a B. Raju,b Roshan M. Borkar,b Deepak Namdev,a
S. Gananadhamu,a Prajwal P. Nandekar,c Abhay T. Sangamwarc
and R. Srinivasa,b*
Ketorolac, a nonsteroidal anti-inflammatory drug, was subjected to forced degradation studies as per International Conference on
Harmonization guidelines. A simple, rapid, precise, and accurate high-performance liquid chromatography combined with
electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed
for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to de-
grade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas
the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of
ketorolac tromethamine and its degradation products on a Grace Smart C-18 (250mm× 4.6 mm i.d., 5 μm) column using 20mM
ammonium formate (pH = 3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0 ml/min. A total of nine
degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of
degradation products have been proposed on the basis of a comparison of the fragmentation of the [M+ H]+ ions of ketorolac
and its degradation products. In silico toxicity of the drug and degradation products was investigated by using TOPKAT and DEREK
softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International
Conference on Harmonization guidelines. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: ketorolac tromethamine; anti-inflammatory; forced degradation; LC/ESI/MS/MS; accurate mass measurements; degradation
products; in silico toxicity
formed under various stress conditions by LC/MS/MS in combina-
Introduction
tion with accurate mass measurements.[7–10]
Ketorolac tromethamine (KETO) [( ) 5-benzoyl-2,3 dihydro-1H-
pyrrolizine-1-carboxylic acid, 2-amino-2-(hydroxymethyl)-1,3-
propanediol] is a nonsteroidal anti-inflammatory drug with an
analgesic efficacy, 800 times more potent than aspirin. It has
both anti-inflammatory and analgesic activity.[1] KETO is mainly
used in the treatment of postsurgical ocular pain and allergic
conjunctivitis.[2] It is associated with a high risk of serious gas-
trointestinal toxicity, peptic ulceration, and acute renal failure.[3]
Concerns about the severe side effects of KETO led to restriction
in its dosage and maximum duration of use for tablets and
intravenous or intramuscular formulation.
A few analytical and bioanalytical methods have been reported in
the literature for the determination of KETO alone or in combination
with other drugs. It includes determination of KETO in biological sam-
ples by using LC and LC/MS methods[11–21] as well as LC/MS/MS
method,[22] in vivo metabolites identification,[23] spectrophotometric
estimation of KETO by different methods,[24] determination of KETO
by HPTLC,[25] determination of KETO and its impurities by capillary
electrochromatography,[26] assay of KETO in pharmaceutical matri-
ces using differential pulse polarography,[27] derivative adsorptive
Stress stability is an integral part of the drug development pro-
cess and explains several factors that affect the expiration dating
of drug products, including the chemical and physical stability
during the preclinical formulation stages, process development,
packaging development, and postmarketing life. As per the regu-
latory guidelines,[4,5] it is essential to establish the stability indi-
cating assay method that gives the idea of inherent stability of
the drug. Characterization of degradants is useful to establish
the mechanism of formation of degradation product (DP) of the
drug and the explanation of side effects of drugs.[6] Recently,
we have reported the structural characterization of DPs of drugs
*
Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, CSIR-
Indian Institute of Chemical Technology, Hyderabad, 500 007, AP, India. E-mail:
srini@iict.res.in; sragampeta@yahoo.co.in
a Department of Pharmaceutical Analysis, National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad, 500037, AP, India
b National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Tech-
nology, Hyderabad, 500 007, AP, India
c
Department of Pharmacoinformatics, National Institute of Pharmaceutical Ed-
ucation and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), Punjab, 160
062, India
J. Mass Spectrom. 2014, 49, 380–391
Copyright © 2014 John Wiley & Sons, Ltd.

Degradation study of ketorolac tromethamine
chronopotentiometry,[28] and high-performance liquid chromatog-
raphy (HPLC) methods.[29–31]
(w/w) hydrogen peroxide were purchased from Merck (Mumbai,
India). HPLC grade water was prepared by filtrating through a
Millipore Milli-Q- plus system (Millipore, Milford, MA, USA).
The hydrolytic degradation behavior of KETO was studied under
acidic and alkaline conditions by Salaris et al.[32] One acid hydrolysis
product was identified that is listed in British Pharmacopoeia as
impurity H, chemically known as methyl (1RS)-5-benzoyl-2,3-
dihydro-1H-pyrrolizine-1-carboxylate (Scheme 1, structure K-8). A
drug substance monograph of KETO in British Pharmacopoeia’09
listed ten impurities.[33] However, the listed impurities are not
classified into DPs and process impurities.
There is no systematic study on the degradation behavior of
ketorolac including developing a stability indicating method, val-
idation of the method, and characterization of all the DPs formed
under ICH recommended stress conditions using LC/ESI/MS/MS.
Even the roles of other stress conditions (e.g. oxidation, photolysis,
and thermal) on the chemical stability of KETO are important.
Hence, the purpose of the present study was to develop a stability
indicating method for KETO and to characterize using LC/ESI/MS/
MS all the DPs formed under the International Conference on
Harmonization (ICH) recommended stress conditions. The results
of the present study may also be helpful for the assessment of the
quality of store products that have expired or on the edge of getting
expired. By considering this information additionally, in silico toxicity
and carcinogenicity of all the proposed degradants was predicted
using TOPKAT (toxicity prediction by computer-assisted technology)
and DEREK (deductive estimate of risk from existing knowledge)
softwares. Some of the DPs may possess potential for genotoxicity,
which create an additional safety concern leading to significant risk
for carcinogenicity or other toxic effects.
Instrumentation and software
The HPLC analysis was performed on Shimadzu separation module
consisting of a binary pump (LC-20 AD); photodiode array detector
(SPD-M20A) and degasser (DGU-20A3). The output signal was mon-
itored and processed using LC-Solution software. All pH measure-
ments were carried out on a glass electrode containing pH meter,
pH tutor (Eutech Instruments), and weighing was carried out on a
Sartorius balance (CPA225D, Germany).
For LC/MS analysis, an Agilent 1200 series HPLC instrument
(Agilent Technologies, USA) attached to a quadrupole time-of-
flight (Q-TOF) mass spectrometer (Q-TOF LC/MS 6510 series clas-
sic G6510A, Agilent Technologies, USA) equipped with an
electrospray ionization (ESI) source. The data acquisition was un-
der the control of Mass Hunter workstation software. The typical
operating source conditions for MS scan of KETO in positive ESI
mode were optimized as follows: the fragmentor voltage was
set at 70 V, the capillary at 3500 V, the skimmer at 60 V, and nitro-
gen was used as the drying (320 °C,10 l/min) and nebulizing
(45 psi) gas. For collision-induced dissociation experiments, keep-
ing MS1 static, the precursor ion of interest was selected using
the quadrupole analyzer, and the product ions were analyzed
using the TOF analyzer. Ultrahigh pure nitrogen gas was used
as collision gas. All the spectra were recorded under identical
experimental conditions and were an average of 20–25 scans.
Photolytic studies were carried out in a photostability chamber
(Osworld OPSH-G-16-GMP series, Osworld Scientific Equipments
Pvt. Ltd. India) set at 40.0 5.0 °C/75.0% relative humidity (RH)
3.0% RH and equipped with an illumination bank on inside
top, consisting of a combination of two black light ultraviolet
lamps and four white fluorescent lamps in accordance with two
option of the ICH guideline Q1B. Assessment of in silico toxicity
was carried out by using TOPKAT (Discovery Studio 2.5, Accelrys,
Inc., San Diego, CA, USA) and DEREK (Nexus v 2.0, Lhasa Ltd.,
Leeds, UK) software.
Experimental
Materials and reagents
Pure ketorolac was obtained as a gift from Symed Labs Hyderabad,
India. HPLC grade acetonitrile was purchased from S. D. Fine
Chemicals (Mumbai, India). All analytical grade reagents: ammonium
formate, formic acid, sodium hydroxide, hydrochloric acid, and 6%
Scheme 1. Proposed structures of protonated degradation products of ketorolac tromethamine formed under various stress conditions.
J. Mass Spectrom. 2014, 49, 380–391
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P. D. Kalariya et al.
Forced degradation studies
Fig. 1. A total of nine DPs were identified and characterized by
using LC/ESI/MS/MS experiments and accurate mass measure-
ments. The proposed structures of DPs and their elemental com-
positions are given in Scheme 1 and Table 2.
Forced degradation of KETO was carried out on the bulk drug
(KETO) as per ICH guidelines Q1A (R2). KETO was subjected to
stress hydrolytic degradation study by refluxing 24 h in acidic
(1.0 N HCl), 24 h in alkaline (1.0 N NaOH), and 72 h in neutral
(H2O) conditions. The optimized oxidative, photolytic, and ther-
mal stress conditions are given in Table 1. The drug solutions
were prepared in 1.0 mg/ml concentration for all stress samples.
Hydrolysis
Initially, KETO was refluxed in 0.5 N HCl and 0.5 N NaOH at 80 °C
for 24 h. Drug was found to be stable in these conditions. While
three DPs (K-1 to K-3) were formed in 1.0 N HCl at 80 °C for
24 h (Fig. 1(A)), only one DP (K-3) was formed in 1.0 N NaOH at
80 °C for 24 h (Fig. 1(B)). In neutral hydrolysis, one DP, K-3, was
Sample preparation
All the stressed samples (hydrolytic, oxidative, thermal, and photo-
lytic stress) were neutralized and diluted with mobile phase and
filtered through 0.22 μm membrane filter before LC/MS analysis.
Results and discussion
Chromatographic conditions
Chromatographic conditions were optimized using a Grace smart
C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase com-
posed of ammonium formate buffer (A) (pH 3.2; 20 mM, pH
adjusted by formic acid) and acetonitrile (B) in a gradient mode.
A linear flow gradient program was set as follows: 0/20, 5/20, 7/
30, 9/45, 18/55, 22/45, and 28/20 (Tmin/%B) where all the DPs of
KETO eluted at adequate retention times (Rts) with good resolu-
tion and symmetrical peak shapes. The column temperature, flow
rate, injection volume, and detector wavelength were at 30 °C,
1.0 ml/min, 20.0 μl, and 280 nm, respectively. For LC/MS analysis,
conditions such as nebulizing gas flow, drying gas temperature,
capillary voltage, drying gas flow, skimmer voltage, and spray
voltage were optimized once more to obtain maximum ioniza-
tion of KETO and all the DPs. The method was validated with
respect to the parameters given in ICH guidelines Q1A (R2).[34]
Degradation behavior of KETO
The degradation behavior of KETO was studied using LC/MS un-
der various forced degradation conditions. Sufficient degradation
was observed in all conditions except in solid photolysis where
drug was found to be stable. The overlay of LC/ESI/MS total ion
chromatograms of all stress degradation samples are given in
Figure 1. LC/ESI/MS/TIC of (A) acidic, (B) basic, (C) neutral, (D) oxidative,
(E) photo neutral, (F) photo acidic, (G) photo basic, (H) photo solid, and (I)
thermal degradation products.
Table 1. Stress conditions for optimum degradation of ketorolac tromethamine (KETO)
Stress conditions
Concentration of stressors
Exposure
Duration
Percent degradation of KETO
condition (°C)
Hydrolysis
Acid
1.0 N HCl
1.0 N NaOH
H2O
80
24 h
18.5
6.2
Base
80
80
24 h
Neutral
Oxidation
72 h
5.5
6.0% H2O2
Room temperature
15 days
30.5
Photolysis
Acid
0.1 N HCl
0.1 N NaOH
H2O
40, 75% RH
40, 75% RH
40, 75% RH
40, 75% RH
70
15 days
15 days
15.2
14.8
11.9
0.0
Base
Neutral
Solid
—
Thermal
—
5.8
RH, relative humidity.
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Degradation study of ketorolac tromethamine
Table 2. Elemental composition of ketorolac tromethamine (KETO) and degradation products
Degradation product
Retention
time (min)
Molecular
Calculated m/z
Observed m/z
Error (ppm)
MS/MS fragment ions
formula [M + H]+
KETO
K-1
K-2
K-3
K-4
K-5
K-6
K-7
K-8
K-9
13.8
5.1
C15H14NO3+
256.0968
228.1018
210.0914
212.1080
244.0967
272.0913
178.0490
238.0863
270.1135
107.0496
256.0962
228.1014
210.0911
212.1082
244.0961
272.0911
178.0488
238.0861
270.1131
107.0495
2.64
1.75
1.43
À0.94
2.46
0.74
1.12
0.84
1.48
0.93
210, 178, 132, 105, 77
200, 184, 150, 105, 77
192, 132, 105, 77
194, 184, 134, 105
226, 216, 198, 170, 158
254, 194, 105
—
C
C
C
C
C
14H14NO2+
14H12NO+
14H14NO+
14H14NO3+
15H14NO4+
13.1
19.3
4.1
6.2
12.3
15.5
7.2
C9H8NO3+
C
C
15H12NO2+
16H16NO3+
—
210, 105
15.0
C7H7O+
77
observed by refluxing the drug in water at 80 °C for 3 days (Fig. 1
(C)) (Schemes 2–4).
three DPs, K-5, K-6, and K-7, were formed during oxidation (Fig. 1
(D), Scheme 5).
Oxidation
Photolytic degradation
Oxidative degradation was performed in 6.0% H2O2 for 15 days.
The drug degraded significantly after keeping the solution for
15 days in the dark room at an ambient temperature. A total of
On exposing the neutral solution of the drug at 1.2 million lux
hours and 200 W h/m2 for 15 days, three DPs (K-1, K-6, and K-9)
were formed (Fig. 1(E)). In acidic drug solution, four DPs (K-4, K-
Scheme 2. Proposed fragmentation pathway of protonated ketorolac tromethamine (KETO) and its degradation products (K-1, K-2, K-3, K-5, K-8, and K-9).
J. Mass Spectrom. 2014, 49, 380–391
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P. D. Kalariya et al.
Scheme 3. Proposed fragmentation pathway of degradant K-4 (m/z 244).
Scheme 4. A probable mechanism of the formation of K-1, K-2, and K-3 under hydrolytic conditions.
6, K-8, and K-9) were formed (Fig. 1(F)); whereas in alkaline drug
solution, three DPs (K-1, K-6, and K-9) formed (Fig. 1(G)). Solid
sample showed no formation of the DPs (Fig. 1(H)).
m/z 178 and m/z 132 are characteristic for 2,3-dihydro-1H-
pyrrolizine skeleton in KETO. The elemental compositions of all
these ions have been confirmed by accurate mass measurements
(Tables 2 and 3).
Thermal degradation
MS/MS of DPs
Thermal degradation sample showed formation of one DP, K-3
(Fig. 1(I)).
Online LC/ESI/MS/MS experiments were performed to characterize
all the DPs (K-1 to K-9) formed under various stress conditions.
Most plausible structures have been proposed for all the DPs
based on the m/z values of their [M+ H]+ ions and the MS/MS data
in combination with elemental compositions derived from accu-
rate mass measurements, as discussed in the succeeding texts.
The ESI/MS/MS spectrum of [M + H]+ ion (m/z 228) of K-1,
eluting at an Rt of 5.1 min (Fig. 2, Table 2), shows structure
indicative fragment ions that are explained in Scheme 2. The
molecular mass of K-1 matches with impurity A, listed in British
LC/ESI/MS/MS study of KETO and its DPs
MS/MS of KETO
The MS/MS spectrum of protonated KETO (Rt = 13.8 min;
[m/z 256]) displays product ions at m/z 210 (loss of HCOOH),
m/z 178 (loss C6H6 from m/z 256), m/z 132 (loss C6H6 from
m/z 210), m/z 105 (PhCO+), and m/z 77 (C6H+5) (Fig. 2)
(Scheme 2).[23] It can be noted that m/z 105 is diagnostic for
the presence of benzoyl group in KETO. The product ions at
Pharmacopoeia.[33]
A
mass difference of 27.9949 u (units)
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Degradation study of ketorolac tromethamine
Scheme 5. Probable mechanisms of formation of degradation products under oxidation and photolytic conditions.
Figure 2. ESI/MS/MS spectrum of [M + H]+ ions of ketorolac tromethamine (KETO) (m/z 206) at 20 eV, K-1 (m/z 228) at 20 eV, K-2 (m/z 210) at 15 eV, K-3
(m/z 212) at 15 eV, and ESI/MS/MS spectrum of [M + H]+ ions of K-4 (m/z 244) at 20 eV.
J. Mass Spectrom. 2014, 49, 380–391
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P. D. Kalariya et al.
Table 3. High resolution mass spectrometry (HRMS) data of product
ions of protonated ketorolac tromethamine (KETO) and its degrada-
tion products
impurity I listed in British Pharmacopoeia.[33] The product ions
observed in the MS/MS spectrum (Fig. 2) are consistent with
the structure (2,3-dihydro-1H-pyrrolizin-5-yl)(phenyl)methanone
(Scheme 2). A probable mechanism for the formation of K-3 is
shown in Scheme 4. The elemental compositions of product ions
have been confirmed by accurate mass measurements (Table 3).
Figure 2 shows the ESI/MS/MS spectrum of [M + H]+ ion
(m/z 244) of K-4 (Rt = 4.1min) (Table 2). The spectrum displays
high abundance [M+ H]+ ion and its product ions at m/z 226
(loss of H2O from m/z 244), m/z 216 (loss of CO from m/z 244),
m/z 198 (loss of CO from m/z 226), m/z 170 (loss of CO from
m/z 198), and m/z 158 (loss of C2H2O2 from m/z 216) (Fig. 2).
Proposed fragmentation pathway for K-4 is given in Scheme 3,
which has been supported by accurate mass measurements.
On the basis of these information, K-4 was identified as
(1,2-dihydroxy-2,3-dihydro-1H-pyrrolizin-5-yl)(phenyl)methanone.
A probable mechanism for the formation of K-4 under pho-
tolytic condition is shown in Scheme 5.
KETO and
degradation
product
Molecular
formula
Observed
m/z
Calculated
Error
(ppm)
m/z
[M + H]+
KETO
C15H14NO3+
256.0962
210.0943
178.0522
132.0467
105.0334
77.0388
256.0959
210.0949
178.0527
132.0461
105.0330
77.0391
À1.17
2.86
C
14H12NO+
C9H8NO3+
C8H7NO+
C7H5O+
C6H5+
2.81
À4.54
À3.81
3.89
K-1
C
C
C
14H14NO2+
13H14NO+
13H14N+
228.1014
200.0714
184.1099
150.0557
210.0911
192.0735
212.1082
194.1895
134.0595
244.0961
226.0859
216.1027
198.0915
170.0961
158.0952
272.0911
254.0813
194.0441
270.1131
107.0495
228.1018
200.0710
184.1101
150.0560
210.0914
192.0737
212.108
1.75
À2.00
1.09
C8H8NO2+
2.00
K-2
K-3
C
C
C
C
14H12NO+
14H10N+
1.43
1.04
The degradant K-5 at m/z 272 [M + H]+ was eluted at 6.2 min,
suggesting the addition of an oxygen to KETO (Fig. 3; Tables 2
and 3). The formation of a diagnostic ion at m/z 254 by the loss
of H2O confirms that K-5 is a hydroxylated DP. The presence of
an abundant ion at m/z 105 and absence of m/z 121 (hydroxy
benzoylation) rule out the hydroxylation in the aromatic ring.
The elemental compositions of K-5 and its product ions have been
confirmed by accurate mass measurements (Tables 2 and 3). All
these data are highly compatible with the proposed structure,
5-benzoyl-2-hydroxy-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid
(Scheme 2). A probable mechanism for the formation of K-5 under
oxidation may involve an addition of OH radical generated from
hydrogen peroxide to KETO (Scheme 5).
The degradant K-6 at m/z 178 was formed under oxidation,
and a photolytic stress condition was eluted at 12.3 min
(Table 2). The mass difference between the protonated drug
(m/z 256) and K-6 (m/z 178) is 78 u, which suggests the loss of
benzene moiety from KETO (Scheme 5). The mass difference
between protonated drug (m/z 256) and K-7 (Rt = 15.5 min) is
18 u indicating that the latter is formed by loss of water mole-
cule. The elemental compositions of [M + H]+ ions of K-6 and
K-7 have been confirmed by accurate mass measurements
(Tables 2 and 3). On the basis of these information, K-6 and
K-7 were identified as 5-formyl-3H-pyrrolizine-1-carboxylic acid
and 5-benzoyl-3H-pyrrolizine-1-carbaldehyde, respectively. Be-
cause of lower abundance of these ions, MS/MS experiments
could not be performed.
14H14NO+
14H12N+
À0.94
2.06
194.1899
134.0591
244.0967
226.0854
216.1023
198.0916
170.0965
158.0958
272.0913
254.0817
194.0443
270.1135
107.0496
C8H8NO+
À2.98
2.46
K-4
C
C
C
C
C
C
C
C
14H14NO3+
14H12NO2+
13H14NO2+
13H12NO+
12H12N+
À2.21
À1.85
0.50
2.35
11H12N+
3.80
K-5
15H14NO4+
15H12NO3+
0.74
1.57
C9H8NO4+
16H15NO3+
C7H7O+
1.03
K-8
K-9
C
1.48
0.93
between K-1 and KETO suggests that K-1 is formed by the loss of
carbonyl group and the presence of an abundant peak at m/z 105
in the MS/MS spectrum confirms that the CO comes from the
carboxy group. The elemental compositions of [M + H]+ ion of
KETO and its product ions have been confirmed by accurate
mass measurements (Table 2). All these data are highly compatible
with the proposed structure, (1-hydroxy-2,3-dihydro-1H-pyrrolizin-
5-yl)(phenyl)methanone proposed for K-1. The formation of K-1
in hydrolytic stress conditions can be explained by decarboxylation
of KETO followed by an addition of ―OH group from water
(Scheme 4). A probable mechanism involving the decarboxylation,
addition of oxygen, removal of singlet oxygen, and followed by
addition of hydrogen radical for the formation of K-1 under
photolysis is illustrated in scheme 5.[35]
The degradant K-8 at m/z 270 ([M + H]+) was eluted at 7.2 min
(Table 2). Molecular mass of K-8 matched with the impurity H
listed in British Pharmacopoeia.[33] The increase of m/z value by
14 u indicates that K-8 is a methylated DP (Scheme 1). Formation
of the diagnostic product ion at m/z 210 from the protonated
molecule by the loss of CH3OH and CO confirms the methylation
of COOH (Fig. 3, Scheme 2).[35] The elemental compositions of
K-8 and its product ions have been confirmed by accurate mass
measurements (Tables 2 and 3). A probable mechanism for the
formation of K-8 is shown in Scheme 5.
A difference of 46 u between [M + H]+ ion of KETO and that of
K-2 (m/z 210) indicates that the latter is formed by loss of HCOOH
from KETO, which has been supported by accurate mass measure-
ments (Table 3). The ESI/MS/MS spectrum of [M+ H]+ ion of K-2
(m/z 210, Rt=13.1 min) displays product ions that are also com-
patible with the structure, phenyl(3H-pyrrolizin-5-yl)methanone
(Fig. 2, Table 2, Scheme 2). It can be noted that protonated K-2 is
also formed as a product ion of protonated KETO in its MS/MS
spectrum. A probable mechanism for the formation of K-2 under
acidic hydrolytic condition is depicted in Scheme 4.
The ESI-MS/MS spectrum of [M + H]+ ion of K-9 (m/z 107,
Rt = 15.0 min) displays product ions that are compatible with
the structure benzaldehyde (Fig. 3, Scheme 2, Table 2). The
elemental compositions of K-9 and its product ions have been
confirmed by accurate mass measurements (Tables 2 and 3). A
probable mechanism for the formation of K-9 under photolytic
condition is shown in Scheme 5.
The [M + H]+ ion of K-3 (Rt = 19.3 min) at m/z 212 (Fig 1, Table 2)
and its elemental composition indicate that it is formed by loss of
carbon dioxide (44 u) from KETO that is found to match with the
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 380–391

Degradation study of ketorolac tromethamine
Figure 3. K-5 (m/z 272) at 20 eV, K-6 (m/z 178) at 15.0 eV, K-7 (m/z 238) at 15 eV, ESI-MS/MS spectrum of [M + H]+ ions of K-8 (m/z 270) at 20 eV, and K-9
(m/z 107) at 15 eV.
Table 4.3 shows qualitative information on skin sensitization
for KETO and its DPs, obtained using DEREK software. This rules-
In silico toxicity prediction
The potential toxicity of KETO and its DPs were assessed by using
TOPKAT and DEREK software.[36] TOPKAT calculations were based on
quantitative structure–toxicity relationship model using statistical
methods such as linear regression of structural descriptors. Toxic-
ity value generated through TOPKAT depends upon the LOG P,
shape index, molecular weight, and symmetry of molecules.
Probability values from 0.0 to 0.30 are considered low probabili-
ties and are likely to produce a negative response in an experi-
mental assay, whereas probability values greater than 0.70 are
considered high and are likely to produce a positive response
in an experimental assay. Probabilities greater than 0.30 but less
than 0.70 are considered indeterminate. Tables 4.1 and 4.2 show
the results of TOPKAT-predicted toxicity and carcinogenicity for KETO
and its DPs. The toxicity of DPs were assessed and compared with
KETO in different animal models. The probabilities of carcinogenic-
ity of KETO in all the models are very less (<0.7) compared with
DPs. All the DPs, except K-1 and K-4, showed higher carcinogenic-
ity potential in different models. For example, NTP Carcinogenicity
Call (male mouse) (v3.2) model indicates higher probability of
carcinogenicity for K-2, K-3, K-7, and K-8 as compared with KETO.
NTP Carcinogenicity Call (female mouse) (v3.2) showed higher
probability of carcinogenicity for K-6, K-7, and K-9 because of
the presence of aldehyde group. Moreover, it is scientifically
proven that aldehyde functional group may have high potential
of genotoxicity and carcinogenicity.[37]
based software derived from the collective knowledge of toxicol-
ogists from academia, industry, and government. The toxicity
predictions are the results of two processes. The program first
checks whether any alerts in the knowledge base match
toxicophores in the query structure. The reasoning engine then
assesses the likelihood of a structure being toxic. There are nine
levels of confidence: certain, probable, plausible, equivocal,
doubted, improbably, impossible, open, and contradicted. DEREK
can be integrated with Lhasa’s Meteor software, which makes
predictions of fate, thereby providing predictions of toxicity for
both parent compounds and their degradants. The presence of
α,β-unsaturated aldehyde structure alert in K-7 may lead to its in-
teraction with skin proteins via a Michael addition mechanism[38]
and therefore likely to cause skin sensitization.
Method validation
Specificity and linearity
Specificity of the method was determined by evaluating the peak
purity of all DPs using photodiode array detector and also con-
firmed by subjecting all the stress samples to LC-MS analysis.
The mass detector showed excellent purity of all DPs and the
drug. Calibration curve was plotted by analysis of working stan-
dard solutions of KETO at six different concentrations in the
J. Mass Spectrom. 2014, 49, 380–391
Copyright © 2014 John Wiley & Sons, Ltd.
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P. D. Kalariya et al.
Table 4.1. Probability values of different toxicity models of the drug and its degradation products (K-1 to K-4) by TOPKAT analyses
Model
KETO
K-1
K-2
K-3
K-4
NTP Carcinogenicity Call (Male Rat) (v3.2)
NTP Carcinogenicity Call (Female Rat) (v3.2)
NTP Carcinogenicity Call (Male Mouse) (v3.2)
NTP Carcinogenicity Call (Female Mouse) (v3.2)
FDA Carcinogenicity Male Rat Non vsCarc (v3.1)
FDA Carcinogenicity Male Rat Single vsMult (v3.1)
FDA Carcinogenicity Female Rat Non vsCarc (v3.1)
FDA Carcinogenicity Female Rat Single vsMult (v3.1)
FDA Carcinogenicity Male Mouse Non vsCarc (v3.1)
FDA Carcinogenicity Male Mouse Single vsMult (v3.1)
FDA Carcinogenicity Female Mouse Non vsCarc (v3.1)
FDA Carcinogenicity Female Mouse Single vsMult (v3.1)
Weight of Evidence Carcinogenicity Call (v5.1)
Ames Mutagenicity (v3.1)a
0.002
0.003
0.454
0.004
0.001
0.000
0.000
0.000
0.004
0.024
0.002
0.013
0.002
0.000
0.000
677.7
11.8
0.000
0.000
0.637
0.000
0.000
0.000
0.000
0.001
0.001
0.000
0.000
0.008
0.051
0.000
0.000
0.000
0.000
1.000
0.000
0.003
0.000
0.000
0.000
0.000
0.221
0.000
0.999
1.000
0.000
0.000
0.002
0.000
1.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.043
0.383
0.000
0.001
0.000
0.000
0.002
0.000
0.000
0.000
0.002
0.060
0.000
0.000
0.000
0.000
0.033
0.999
0.003
Developmental Toxicity Potential (DTP) (v3.1)
Rat Oral LD50 (v3.1) (mg/kg)b
259.6
1700
24.2
1000
Rat Maximum Tolerated Dose – Feed/Water (v6.1) (mg/kg)
Rat Inhalational LC50 (v6.1) (g/m3/H)c
Chronic LOAEL (v3.1) (mg/kg)d
213.2
7.7
144.1
2.1
0.007
7.3
0.002
0.58
8.7
4.4
10.9
9.2
8.6
13.7
Skin Irritation (v6.1)
0.999
1.000
1.000
0.993
0.000
1.000
0.000
0.313
0.276
1.000
1.000
1.000
0.002
0.000
0.000
0.000
0.000
0.138
0.84
Skin Sensitization NEG v SENS (v6.1)e
0.002
0.139
0.000
0.082
0.387
1.000
1.000
1.000
0.000
0.000
0.000
0.000
0.046
0.000
1.000
0.000
0.000
0.678
1.000
Skin Sensitization MLD/MOD v SEV (v6.1)
Ocular Irritancy SEV/MOD vs MLD/NON (v5.1)f
Ocular Irritancy SEV vs MOD (v5.1)
Ocular Irritancy MLD vs NON (v5.1)
Aerobic Biodegradability (v6.1)
Daphnia EC50 (v3.1) (mg/l)g
728.1
550.1
NTP, National Toxicilogy Program; FDA, US Food and Drug Administration.
aAmes mutagenicity test is a biological assay to measure the mutagenic potential of chemical compounds.
bRat oral median lethal dose (LD50) in mg/kg.
cRat inhalational median lethal concentration LC50 in g/m3/H.
dChronic lowest observed adverse effect level (LOAEL).
eFor a chemical, a computed probability less than 0.3 indicates a noncarcinogen (NEG) v sensitive (SENS)
fSeverity specific submodels: NON (none), MLD (mild), MOD (moderate), and SEV (sever).
gDaphnia median effective concentration (EC50) in mg/l.
range of 5.0–50.0 ng/ml. Each solution was injected and
measured in triplicate (n = 3). Standard calibration curves were
plotted by taking peak area on the y-axis and nominal concen-
trations of drug on the x-axis. Limits of detection and limits of
quantification were calculated on the basis of signal-to-noise
ratio that was determined by comparing measured signals from
samples with known low concentrations of analyte with those
of blank samples. For limits of detection and limits of quanti-
fication, signal-to-noise ratio is 3 : 1 and 10 : 1, respectively.
Results obtained from the regression analysis of data are given
in Table 5.
Precision
Precision of the developed method was measured in terms of intra-
day precision (repeatability) and interday precision (reproducibility).
Method repeatability was determined from the results of five sepa-
rate samples prepared at different concentrations. Each sample was
injected in triplicate (n = 3), and peak areas obtained were used to
calculate means and %RSD values. The method reproducibility
was evaluated on four different days, by preparing and analyzing
in triplicate five separate sample solutions at the same concentra-
tion of intraday solutions. Table 7 depicts %RSD for intraday and
interday precision of method for KETO, and results (%RSD< 2.0%)
show the method is precise in terms of intraday and interday preci-
sion (Table 9).
Accuracy
Accuracy was determined by applying the standard addition
method to synthetic mixture of drug product placebo compo-
nent to which known quantities of KETO had been spiked. Each
solution was injected in triplicate analysis, and the percentage
recovery was calculated. The percentage recovery range and
%RSD values were found to be 99.0–101.0 and <2.0%, respec-
tively (Table 6).
Robustness
Robustness measures reliability of an analytical method with
respect to deliberate variations of the method parameters. Robust-
ness of method was evaluated by changing the column tem-
perature (30 °C 5.0°C), flow rate (1.0 0.1ml/min), and pH of the
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Degradation study of ketorolac tromethamine
Table 4.2. Probability values of different toxicity models of degradation products (K-5 to K-9) by TOPKAT analyses
Model
K-5
K-6
K-7
K-8
K-9
NTP Carcinogenicity Call (Male Rat) (v3.2)
NTP Carcinogenicity Call (Female Rat) (v3.2)
NTP Carcinogenicity Call (Male Mouse) (v3.2)
NTP Carcinogenicity Call (Female Mouse) (v3.2)
FDA Carcinogenicity Male Rat Non vsCarc (v3.1)
FDA Carcinogenicity Male Rat Single vsMult (v3.1)
FDA Carcinogenicity Female Rat Non vsCarc (v3.1)
FDA Carcinogenicity Female Rat Single vsMult (v3.1)
FDA Carcinogenicity Male Mouse Non vsCarc (v3.1)
FDA Carcinogenicity Male Mouse Single vsMult (v3.1)
FDA Carcinogenicity Female Mouse Non vsCarc (v3.1)
FDA Carcinogenicity Female Mouse Single vsMult (v3.1)
Weight of Evidence Carcinogenicity Call (v5.1)
Ames Mutagenicity (v3.1)
0.008
0.013
0.001
0.000
0.000
0.000
0.992
0.005
0.002
0.000
0.000
0.097
0.005
0.000
0.000
0.000
0.997
0.166
0.999
0.037
0.000
0.000
0.772
0.010
0.000
0.000
0.421
0.002
0.000
0.029
0.000
0.000
1.000
1.000
0.000
0.000
0.000
1.000
0.001
0.001
0.000
0.007
0.000
0.034
0.000
0.092
0.000
0.718
0.002
0.001
0.000
0.000
0.000
0.000
0.187
0.000
0.000
0.006
0.000
0.000
0.030
0.000
0.000
1.000
0.943
0.626
0.997
1.000
0.393
0.783
0.000
0.030
0.238
0.000
0.012
Developmental Toxicity Potential (DTP) (v3.1)
Rat Oral LD 50 (v3.1) (mg/kg)
3100
64
0.023
1200
683.7
544.9
1300
280.2
Rat Maximum Tolerated Dose – Feed/Water (v6.1) (mg/kg)
Rat Inhalational LC 50 (v6.1) (g/m3/H)
19.2
6.9
47.2
10.0
30.2
100
10
4.5
Chronic LOAEL (v3.1) (mg/kg)
3.1
20.7
8.0
170.0
Skin Irritation (v6.1)
1.000
1.000
1.000
0.009
0.000
1.000
0.075
1.000
1.000
1.000
1.000
1.000
0.000
0.989
0.044
0.000
0.014
Skin Sensitization NEG v SENS (v6.1)
1.000
0.999
1.000
0.000
1.000
1.000
2.7
1.000
1.000
0.000
0.003
1.000
1.000
0.209
0.961
0.993
0.966
0.800
0.061
1.000
Skin Sensitization MLD/MOD v SEV (v6.1)
Ocular Irritancy SEV/MOD vs MLD/NON (v5.1)
Ocular Irritancy SEV vs MOD (v5.1)
Ocular Irritancy MLD vs NON (v5.1)
Aerobic Biodegradability (v6.1)
Daphnia EC50 (v3.1) (mg/l)
445.1
15.9
12.9
Table 4.3. Qualitative toxicity prediction of the ketorolac
tromethamine (KETO) and its degradation products (K-1 to K-9) by
DEREK analyses
Table 5. Parameters of linear regression equations for ketorolac
tromethamine
Parameters
Value
KETO and its degradation
products
Skin sensitization
Calibration range (ng/ml)
Correlation coefficient (r)
Slope
5.0–50.0
0.9999
Structural alert
O
12496
Intercept
À661.9
62.26
1889.21
0.52
R
H
SD of slope
SD of intercept
LOD (ng/ml)
R = any group (except OH)
The skin sensitization of α,β-unsaturated
aldehyde and precursors that interact
with skin proteins via a Michael
addition mechanism
LOQ (ng/ml)
1.48
Comments
SD, standard deviation; LOD, limits of detection; LOQ, limits of
quantification.
KETO, K-1 to K-6, K-8,
NA
K-9
K-7
√
Table 6. Accuracy study of ketorolac tromethamine using standard
additions method (n = 3)
Spiked
concentration
(ng/ml)
Calculated spiked
concentration
(ng/ml) SD; %RSD
Percent
recovery
mobile phase (3.2 0.3) at three different concentrations (5, 30,
and 50 ng/ml). Each sample was injected in triplicate (n = 3),
and peak areas obtained were used to calculate means and %
RSD values. The %RSD for the column temperature, flow rate,
and the pH of the mobile phase was <1%. There were no signif-
icant changes in assay value of the drug that showed the
robustness of the method.
10.0
30.0
50.0
10.10 0.19; 1.91
29.81 0.49; 1.65
50.32 0.65; 1.29
101.0
99.4
100.6
J. Mass Spectrom. 2014, 49, 380–391
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jms

P. D. Kalariya et al.
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(ng/ml)
Intraday precision
Interday precision
Measured concentration Measured concentration
(ng/ml) SD; %RSD
(ng/ml) SD; %RSD
5.0
4.98 0.09; 1.87
10.03 0.10; 1.00
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Conclusion
A selective validated stability indicating LC/MS/MS method
was developed to study the degradation behavior of KETO un-
der hydrolysis (acid, base, and neutral), oxidation, photolysis,
and thermal stress conditions and determined the inherent
stability of the drug. The drug was found to degrade in all
the stress conditions except when the solid was exposed to
photolytic conditions. A total of nine unknown DPs were iden-
tified and characterized using online LC/ESI/MS/MS experi-
ments combined with accurate mass measurements. The
proposed structures of the DPs have been rationalized by ap-
propriate mechanisms. This study may be useful in future in-
vestigation on characterization of process-related impurity.
Further, in silico toxicities were predicted for all DPs using
TOPKAT and DEREK softwares. NTP Carcinogenicity Call (female
mouse) (v3.2) showed higher probability of carcinogenicity for
K-6, K-7, and K-9 because of the presence of aldehyde group.
DEREK software shows structure alert for K-7 that is likely to
cause skin sensitization.
Acknowledgements
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bad and Dr Ahmed Kamal, Project Director, NIPER, Hyderabad
for facilities and their cooperation. P. K and D. N is thankful to
the Ministry of Pharmaceuticals, New Delhi, for providing Junior
Research Fellowship. B. R and R. B are thankful to CSIR (AARF)
and DST, New Delhi, for awarding senior research fellowship.
mers and paracetamol in human plasma: application to
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