Critical Reviews in Analytical Chemistry

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Stability Indicating Methods for Determination of

Third Generation Antiepileptic Drugs and Their
Related Substances

Jéssica Domingos da Silva, Lucio Mendes Cabral & Valéria Pereira de Sousa

To cite this article: Jéssica Domingos da Silva, Lucio Mendes Cabral & Valéria Pereira de
Sousa (2021): Stability Indicating Methods for Determination of Third Generation Antiepileptic
Drugs and Their Related Substances, Critical Reviews in Analytical Chemistry, DOI:
10.1080/10408347.2021.1890544

To link to this article: https://doi.org/10.1080/10408347.2021.1890544

Published online: 06 Mar 2021.

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CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
https://doi.org/10.1080/10408347.2021.1890544

REVIEW ARTICLE

Stability Indicating Methods for Determination of Third Generation

Antiepileptic Drugs and Their Related Substances
J

essica Domingos da Silva , Lucio Mendes Cabral , and Val
eria Pereira de Sousa
Department of Drugs and Pharmaceutics, Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil

ABSTRACT KEYWORDS
The third generation of antiepileptic drugs that have been approved by international regulatory Antiepileptic drugs; third
agencies between 2007 and 2018 include rufinamide, stiripentol, eslicarbazepine acetate, lacosa- generation; stability
mide, perampanel, brivaracetam and everolimus. As part of demonstrating their safety profile, sta- indicating methods; HPLC;
related substances
bility indicating methods are developed to monitor these drugs and their impurities. In this
context, this review describe some characteristics, impurities and the stability indicating methods
used for the determination of these drugs and the presence of their related substances. Through
a search in official compendia and scientific articles, fifty-six analytical methodologies were identi-
fied up to October 2020. The methodologies were developed using techniques of HPLC, UPLC,
HPTLC, GC and UV/Vis spectrophotometry. A majority of the methods (
70%) employed HPLC-UV.
A number of these antiepileptic drugs were found to have had a small number of studies related
to their stability and for the detection of impurities. The presentation of the current level of
research on third generation antiepileptic drugs highlights the need for new stability and safety
studies that are necessary to develop new pharmaceutical products containing these drugs.

1. Introduction some advantages such as more favorable pharmacokinetics,

Epilepsy is one of the most common chronic neurological
disorders in the world with recurrent crises that affects
approximately 50 million people of all ages.[1] An epileptic
seizure is defined as a transient occurrence of abnormally
excessive or synchronous neuronal activity in the brain that
is visibly apparent.[2] Antiepileptic drugs (AEDs) used to
treat epilepsy aim to decrease the probability of seizures by
acting on the main mechanisms contributing to brain
excitability.[3]
Pharmacological treatment of epilepsy began in the 20th
century with discovery of phenobarbital, which represents
the first generation of AEDs. The second generation com-
prise vigabatrin, oxcarbazepine, lamotrigine, felbamate gaba-
pentin, topiramate, tiagabine, levetiracetam and zonisamide,
these drugs were licensed during the period from 1980 to
2000.[4,5] Currently, there are at least 27 molecules approved
for use as an AED by the Food and Drug Administration
(FDA) and European Medicines Agency (EMA).[6] The
third, and most recent, generation of AEDs is formed by
rufinamide (RUF), stiripentol (STP), lacosamide (LAC), esli-
carbazebine acetate (ESA), perampanel (PER), brivaracetam
(BRV) and everolimus (EVR). These drugs were approved
for treatment of epilepsy between 2007 and 2018 by the
FDA and EMA.[4,5,7] The search for new molecules and for-
mulations that aim to improve efficacy continues, especially
in cases of resistance to therapy, also known as refractory
epilepsy.[8] In addition, third generation AEDs can offer
improved tolerability, lower teratogenic potential and/or
reduced probability to cause enzyme inductions and drug
interactions.[9,10]
The monitoring for impurities and degradation products
is essential for establishing a safety profile for these seven
most recently approved AEDs, since these related substances
may have genotoxic potential and induce adverse effects.[11]
Thresholds of impurities and degradation products are
expressed as total daily intake (TDI) of these related sub-
stances or as a percentage of active pharmaceutical ingredi-
ent (API) determined by maximum daily dose of API. It is
recommended that the exposure be zero or very close to
zero. To meet this recommendation, monitoring must be
performed through highly sensitive analytical methods with
an adequate capacity to detect these related substances.[12–14]
Considering this objective, the purpose of this review is
to describe the most current analytical methodologies that
are being applied to determine the third generation AEDs in
the presence of their related substances. In addition, we pre-
sent an overview of the chemical characteristics of these
drugs and their organic impurities reported in the literature.
In addition, we identify several of the products that can be
formed in forced degradation studies. The analytical meth-
ods described here were compiled from official compendia
and published scientific articles, which were found by data-
base searches as recently as October 2020 in Google Scholar,
Science Direct and PubMed. The search terms include the

CONTACT Valeria Pereira de Sousa valeria@pharma.ufrj.br Department of Drugs and Pharmaceutics, Faculty of Pharmacy, Federal University of Rio de
Janeiro, Av. Carlos Chagas Filho, 373, CCS, Bss, sl15, Rio de Janeiro, RJ 21941-902, Brazil.
ß 2021 Taylor & Francis Group, LLC
2 J. D. DASILVA ET AL.

Table 1. Therapeutics indications, mechanisms of action and dosage forms of third generation AEDs.
AED Approval date Indication Mechanism of Action Dosage forms Reference
RUF 2007 (EMA) Lennox-Gastaut syndrome in patients aged 1 year Modulation of VGSCs, Tablet [15,16]

2008 (FDA) particularly the Oral suspension

Nav1.1 isoform
STP 2007 (EMA) Dravet’s syndrome in a patient aged 2 years in Positive allosteric modulator Capsule Powder for [17,18]

2018 (FDA) association with clobazam and valproate at GABAA receptors oral suspension
[19,20]
LAC 2008 (FDA and EMA) Seizures with partial onset with or without secondary Blocking VGSCs through slow Tablet
generalization in patients aged 4 years inactivation Oral solution
Intravenous injection
[21,22]
ESA 2009 (EMA) Refractory partial-onset seizures, with or without Blocking VGSCs through slow Tablet
2013 (FDA) secondary generalization in adult patients (FDA) inactivation Oral suspension
and children aged  6 years (EMA)
[23,24]
PER 2012 (FDA and EMA) Primary generalized tonic-clonic seizures and focal Non-competitive negative Tablet
epilepsies with or without generalized seizures in allosteric antagonist of Oral suspension
patients aged  12 years AMPA receptors
[25,26]
BRV 2016 (FDA and EMA) Partial onset seizures with or without secondary Binding to SV2A Tablet
generalization in patients aged 4 years Oral solution
Intravenous injection
[27,28]
EVR 2017 (EMA) Tuberous sclerosis complex associated partial-onset Inhibition of the Tablet
2018 (FDA) seizures in patients aged 2 years mTOR pathway Dispersible tablet
AMPA - a-amino-3-hydroxyl-5-methyl-4- isoxazole-propionic acid; GABA- gamma-amino butyric acid; mTOR - mammalian target of rapamycin; SV2A- synaptic ves-
icle protein 2 A; VGSCs – voltage-gated sodium channels.

individual names of each AED combined with “stability
indicating methods”, “forced degradation” and “impurities”.
The intention of the data compiled in this review is to sup-
port researchers and manufacturers in the development of
stability studies and analytical methodologies for determin-
ing the third generation AEDs and their related substances.
2. Mechanisms of action and therapeutic indications
The third generation of AEDs described in this review act in
the reduction of abnormal cerebral hyperexcitability through
five different mechanisms of action as summarized in
Table 1. RUF, LAC and ESA act in the modulation of volt-
age-controlled ion channels; STP increases inhibition of
gamma-amino butyric acid (GABA); BRV modulates synap-
tic release by binding to protein 2 A on synaptic vesicle
(SV2A); PER inhibits synaptic excitation mediated by ino-
tropic glutamate receptors by binding to a-amino-3-
hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptors;
and EVR is considered an agent with a targeted mechanism
to inhibit mTOR protein pathway, which is the mammalian
target of rapamycin.[3,29]
These AEDs also differ in terms of their therapeutic indi-
cation, also summarized in Table 1. RUF, STP and EVR are
considered orphan drugs and indicated for the treatment of
seizures associated with rare diseases, such as Lennox-
Gastaut and Dravet syndromes as well as the tuberous scler-
osis complex.[15–18,27,28] LAC, BRV and ESA are indicated
for the treatment of partial seizures with or without second-
ary generalization in pediatric and adult patients.[19–22,25,26]
PER is indicated for the treatment of generalized and partial
seizures in adult patients only.[23,24]
3. Chemical characteristics
In addition to their different mechanisms of action and
therapeutic indications, third generation AEDs also have dif-
ferent chemical structures, solubility and physical chemical
characteristics, as shown in Table 2. The chemical structure
RUF shows that it is a triazole derivative and PER is a
bipyridine derivative. Neither have a chiral carbon in their
structures.[33,34] STP is a derivative of a-ethylene alcohol
whereby the dosage forms are composed by racemic equi-
molar mixture of the (R) - (þ) - and (S) - (-) - stiripentol
enantiomers.[35,36] LAC is a functionalized amino acid with
a substitution on carbon 2 that originates a chiral center.
The (R) -enantiomer is the pharmacologically active form
with antiepileptic activity.[37,38]
ESA is a prodrug derived from the same family as carba-
mazepine and oxcarbazepine that has in its chemical struc-
ture a dibenzazepine nucleus with the 5-carboxamide
substituent but structurally different in positions 10 and
11.[32] The hydrolysis of this prodrug generates oxcarbaze-
pine and the active metabolite (S) -licarbazepine (eslicarba-
zepine). Oxcarbazepine is metabolized to (R)-licarbazepine
and these two enantiomers are biologically active with the S
form displaying less toxicity and is more efficient at crossing
the blood brain barrier.[39]
BRV is a 2-pyrrolidone derivative of levetiracetam with
two chiral centers, one between the bonding of the butana-
mide portion to the pyrrolidone ring, and the other at pos-
ition 4 of pyrrolidone ring, which permits formation of four
different diastereoisomers. (S, R) -brivaracetam is the form
present in pharmaceutical forms, as it has been rationally
designed to have a greater affinity for binding to SV2A
protein.[40,41]
EVR is a hydroxyethyl derived from rapamycin (siroli-
mus), a macrolide isolated from Streptomyces hygroscopicus.
This drug contains 15 asymmetric carbons and 4 substituted
double bonds that maintain the original rapamycin configur-
ation. EVR is unstable at temperatures above 25  C and is
extremely sensitive to oxidation, which is stabilized with the
antioxidant butylhydroxytoluene (BHT).[42,43]
4. Stability indicating analytical methods
Stability indicating methods are essential for detecting deg-
radation products formed during forced degradation studies
Table 2. Chemical structure and physicochemical characteristics of third generation AEDs.
Molecular Molecular Melting
AED Chemical Structure IUPAC name formula Weight (g/mol) pka Log P range ( C) Solubility References
[15,30,31]
RUF 1- (2,6-difluorobenzyl) -1H-1,2,3-triazole-4-carboxamide C10H8F2N4O 238.19 14.37 ± 0.50 0.65 233  238 Insoluble in water, slightly
soluble in gastric and
intestinal fluids, ethanol
and methanol
[30,31]
STP rac-(E)-1-(benzo [d] [1,3]dioxol-5-yl)-4,4-dimethylpent- C14H18O3 234.29 14.45 ± 0.20 2.95 64 Insoluble in water, soluble in
1-en-3-ol acetone and alcohol,
moderately soluble
in chloroform
[30,31]
LAC (R) -2-acetamido-N-benzyl-3-methoxypropionamide C13H18N2O3 250.29 14.19 ± 0.46 0.728 140–146 Soluble in aqueous medium

[30–32]
ESA (10S) -5-carbamoyl-10,11-dihydro-5H-dibenzo [b, C17H16N2O3 296.32 13.97 ± 0.40 8.8 184–187 Soluble in organic solvents
f]azepin-10-yl acetate and aqueous buffers (pH
1.2  7.4 at 37  C)

[30,31,33]
PER 2- (2-oxo-1-phenyl-5- (pyridin-2-yl) -1,2-dihydropyridin- C23H15N3O 349.38 4.73 ± 0.19 1.36 180 Insoluble in water, with
3-yl) benzonitrile improvement in pH acidic

[25,30]
BRV (2S) -2- [ (40 R) -2-oxo-4-propylpyrrolidin-1- C11H20N2O2 212.29 15.74 ± 0.50 1.04 76–78.7 Soluble in aqueous medium
yl] butanamide and organic solvents

[30,31]
EVR (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S, C53H83NO14 958.22 10.40 ± 0.70 4.18 97–101 Slightly soluble in water,
35R) 1,18-Dihydroxy-12-[(2R)-1-[(1S,3R,4R) 4- (2- soluble in organic solvents
hydroxy-ethoxy)-3-methoxycyclohexyl]propan-2-yl]-
19,30-di-methoxy-15,17,21,23,29,35-hexamethyl-
11,36-dioxa-4-azatricyclo [30.3.1.04,9]hexatriaconta-
16,24,26,28-tetraene-2,3,10,14,20-pentone
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
3
4 J. D. DASILVA ET AL.
Figure 1. Frequency of use for the techniques used in the stability indicating
methods (n ¼ 56) applied to determination of third generation AEDs in the
presence of their related substances. Data based on the review of the scien-
tific literature.
or that may be formed over the shelf life of the product.
These methods must be able to quantify the drug in the
presence of related substances, excipients and matrix com-
ponents.[13,44,45] Analytical methods reported in this review
were developed for the determination of third generation
AEDs in presence of organic impurities present with the
API, in the dosage forms or identified in forced degrad-
ation studies.
Organic impurities can originate from starting materials,
intermediate by-products or degradation, any of which can
arise during the manufacturing and/or storage of the API
and finished product.[13] Seventy-nine organic impurities for
third generation AEDs have been reported in the literature:
3 for RUF, 1 for STP, 26 for LAC, 21 for ESA, 13 for PER,
4 for BRV and 11 for EVR. Chemical structures, nomencla-
tures, formulas and molecular weights of these related sub-
stances are detailed in Supplementary data (Appendix 1).
Liquid chromatography, either HPLC (High Performance
Liquid Chromatography) or UPLC (Ultra Performance
Liquid Chromatography), were the most used techniques for
the development of the indicative stability methods for third
generation AEDs as shown in Figure 1. These techniques
allow for the separation of several components quickly at
high resolution and sensitivity.[46] Other techniques
employed were HPTLC (High performance thin layer chro-
matography), GC (Gas chromatography) and UV/Vis spec-
trophotometry. In most methodologies, detection was
performed by UV due to the presence of chromophoric
groups in the structures of the AEDs (Figure 1).
Employment of photodiode array detectors (DAD) delivers
sensitivity, compatibility with most solvents and coverage
over a wide linear range of concentrations with an absorp-
tion profile proportional to concentration that follows Beer’s
law.[47] The stationary phases most often used were the
reverse phase (columns C18 and C8), but the separation of
enantiomeric impurities employed direct methods that
included chiral separation columns (CSP) derived from pol-
ysaccharides. Among the AEDs reported in this review,
RUF, LAC and EVR have official methods described in US
Pharmacopeia (USP 43) for determining these drugs and
their related substances in API and pharmaceutical forms[48]
and European Pharmacopeia (Eur. Ph. 10.0th)[49]. These
methodologies are detailed in Table 3 as well as non-
compendial methods for the determination of these third
generation AEDs in presence of impurities
and degradation products.
4.1. Rufinamide
Several methodologies have been found for the determin-
ation of RUF in the presence of its three related substances
(compounds 1-3; Appendix 1). USP 43 describes two meth-
ods, one for the API and the other for tablets, for the deter-
mination of RUF and two of its related substances
(compounds 1 and 2; Appendix 1). The two methodologies
employ reverse phase HPLC (column C18), the minimum
differences between these methods is the composition of
mobile phase (80% of potassium phosphate buffer in tablet
method instead of 83% of water in API method) and detec-
tion (210 nm for tablet method and 220 nm for API
method). The resolutions recommend by USP 43 between
RUF and compound 1 (Appendix 1) for tablet and API
methods cannot be less than 1.5 and 2.5, respectively.[48]
Most of the methods for RUF found in the literature also
used HPLC and the most applied parameters were C18 and
C8 stationary phases (lenghts with a UV detection in the
range of 210 to 243 nm. The mobile phases consisted of
aqueous buffers and solvents such as acetonitrile, methanol
and tetrahydrofuran. Shorter RUF retention times of 2.4
from 4.0 min were observed in methods that used aceto-
nitrile in the mobile phase.[50–53,93] In contrast, the use of
methanol as an organic modifier increases RUF retention in
stationary phase with an elution between 5 to 8 min, since
this solvent has lower eluotropic strength than aceto-
nitrile[54–56]. Hassib et al.,[54] method shower greater sensi-
tivity when compared to other methodologies with isocratic
elution, however it was applied only for a separation of RUF
and compound 1. The method of Mahamudi et al.[51],
obtained adequate separation between RUF and compounds
1 and 2, but the authors did not present the LOD values,
not allowing to assess the sensitivity of the method.
Parashar et al.[94] and Ranjith et al.[58] were the only works
in which separated RUF from all three related substances
using gradient mode. Although these methods showed good
sensitivity, the disadvantage was a an increase in the reten-
tion time of RUF and compounds 1,2 and 3 (19.98 to
35.31 min) as well as the total run time of 90 min.
Two methods used UPLC[59] and HTPLC[60] as the chroma-
tographic separation technique for determination of RUF in the
presence of its related substances in the API and when formu-
lated into tablets. The method developed for UPLC was advan-
tageous, because the separation of RUF and all three related
substances has been achieved with an analysis time of 17 min
and a resolution greater than 2. Composition of the mobile
phase for this methodology was similar to that recommended
by USP 43 for tablets (Table 3) and only differed by the pH of
the buffer.[59] HPTLC has proven to be an alternative technique
for the development of a stability indicating method. Using a
mobile phase composed of chloroform, methanol and glacial
acetic acid (9:1:0.1, v/v/v), it was shown that RUF could be sep-
arated from its degradation product (compound 3; Appendix
1)[62]. Another alternative for determining RUF in the presence
of degradation product was through the use of first derivative
ratio spectrophotometry. In this methodology, RUF was deter-
mined by measuring the peak amplitude at 269.5 nm.[54]
Table 3. Stability indicating methods by chromatography for the determination of third generation AEDs in presence of their related substances.
Method/ Retention
AED Matrix Detection Stationary phase/ Temperature (  C) Mobile phase/Flow rate/Injection volume time Run Time LOD/LOQ References
[48]
RUF API HPLC-UV C18 (125 x 4.6 mm, 5 lm) at RT MeOH: tetrahydrofuran: water (12:5:83, v/v). FR: – – –
220 nm 1.0 ml.min-1; IV: 20 mL
[48]
Tablet HPLC-UV C18 (125 x 4.6 mm, 5 lm) at RT MeOH: tetrahydrofuran: 2.7g/L of potassium dihydrogen – – –
210 nm phosphate in water (15:5:80, v/v). FR: 1.0 ml.min-1; IV:
25 mL
[50]
API, tablet HPLC-UV C18 (250 x 4.6 mm, 5 mm) at 25  C 10 mM tetra butyl ammonium hydrogen sulfate buffer 3.53 10.0 0.29 /0.87 mg.mL-1
215 nm pH 3.37: ACN (50: 50, v/v). FR: 1.0 ml.min-1; IV: 20 mL
[51]
API HPLC-UV Xbrigde C18 (250 mm  4.6 mm, 5 lm) Water: ACN (50: 50, v/v). FR: 1.0 ml.min-1 – – –
215 nm at RT
[52]
API, tablet HPLC-UV C18 (250 x 4.6 mm, 5 mm) at 25  C ACN: water (60:40, v/v). FR: 0.8 ml.min-1; IV: 20 mL 3.89 10.0 0.24/0.73 mg.mL-1
215 nm
[53]
API, tablet HPLC-UV Phenomenex Luna C18 (250 [ 4.6 mm, 5 ACN: water (60: 40, v/v). FR: 1.0 ml.min-1; IV: 20 mL 3.047 7.0 0.89/2.69 mg.mL-1
210 nm mm) at 25  C
[54]
API, tablet HPLC-UV Kromasil C8 (250mm x 4.6 mm,5 mm) ACN: water (50: 50, v/v). FR: 1.0 ml.min-1; IV: 20 mL 4.008 – 0.778/2.356 mg.mL-1
210 nm
[55]
API, tablet HPLC-UV C18 (250 x 4.6 mm, 5 mm) at 25  C MeOH: water (52:48, v/v). FR: 1.0 ml.min-1;IV: 20 mL 5.55 10.0 0.0028/0.0086 mg.mL-1
210 nm
R [56]
API, tablet HPLC-UV PhenomenexV Hyperclone BDS C18 (250 x MeOH: 0.1 M octane sulfonic acid, 0.1 M, potassium – – 0.156/0.311 mg.mL-1
210 nm 4.6 mm, 5 mm) at 35  C dihydrogen phosphate buffer: water pH 6.5
(30:10:5:55, v/v/v/v). FR: 1.0 ml.min-1;IV: 20 mL
[57]
API HPLC-UV Inertsil ODS 3V C18 (250 mm  4.6 mm, 5 0.1% ortophosphoric acid in water and MeOH: ACN: 20.03 90.0 0.003% / 0.01%
220 nm mm) at 30  C tetrahydrofuran (90:70:30, v/v/v); gradient elution. FR:
1.0 ml.min-1; IV: 50 mL
[58]
API HPLC-UV Inertsil ODS-3V (250  4.6 mm, 5 mm) 0.1% triethylamine in water pH 2.2 and MeOH: 19.89 90.0 0.0046 / 0.014 mg.mL-1
215 nm at 35  C tetrahydrofuran (98:2, v/v); gradient elution. FR:
1.0 ml.min-1; IV: 50 mL
[59]
API, tablet HPLC-UV Acquity TM UPLC BEH C18 (150 mm  0.02 M potassium dihydrogen orthophosphate buffer pH 4.16 17.0 0.01% /0.03%
210 nm 2.1 mm, 1.7 mm) at 40  C 4.5: MeOH: tetrahydrofuran (80:15:5, v/v). FR:
0.3 ml.min-1; IV: 2 mL
[60]
API, tablet HPTLC Precoated silica gel aluminum plate Chloroform: MeOH : glacial acetic acid (9:1:0.1, v/v/v) – – 196.59/595.74 ng.spot-1
210 nm 60 F254
[61]
STP API, capsule HPLC-UV Symmetry C18 (75  4.6 mm, 3.5 mm) ACN: 50 mM potassium dihydrogen phosphate buffer pH 1.80 6.0 0.081/0.242 mg.mL-1
262.5 nm at 25  C 4.1 (60 : 40, v/v).FR: 1.0 ml.min-1; IV: 20 mL
[49]
LAC API HPLC-UV Amylose derivative (250  4.6 mm, 10 mm) Water: 2-propanol: heptane (3:100:900, v/v/v). FR: 25.0 42.5 –
215 nm at RT 1.0 ml.min-1; IV: 20 mL
[49]
API HPLC-UV C8 (150  4.6 mm, 3.5 mm) at RT 0.1% solution of trifluoroacetic acid and trifluoroacetic 12.0 21.0 –
258 nm acid: ACN: MeOH (0.3:500:500, v/v/v); gradient elution.
FR: 1.2 ml.min-1; IV: 20 mL
[49]
Tablet, infusion HPLC-UV C8 (150  4.6 mm, 5 mm) at 35  C Methanesulfonic acid: ACN: water (0.75:130:870, v/v/v). 6.0 15.0 –
215 nm FR: 2.0 ml.min-1; IV: 5 mL
[49]
Oral solution HPLC-UV C18 (250  4.6 mm, 5 mm) at 30  C Trifluoroacetic acid: ACN: water (0.56:100:900, v/v/v) and 27.0 33.0 –
215 nm trifluoroacetic acid: ACN (0.5:1000, v/v); gradient
elution. FR: 1.5 ml.min-1; IV: 5 mL
[62]
API HPLC-UV Lichrosphere C18 (250  4.6 mm, 5 mm) 0.1 % trimethylamine in water pH 3.0: MeOH (70:30, v/ 10.7 15.0 0.036/ 0.108 mg.mL-1
215 nm at 25  C v). FR: 1.0 ml.min-1; IV: 25 mL
[63]
API HPLC-UV Hypersil BDS C18 (250  4.6 mm, 5 mm) 0.01M monobasic potassium phosphate pH 4.0: ACN 3.6 10.0 2.8/8.9 mg.mL-1
215 nm at 25  C (30:70, v/v). FR: 1.0 ml.min-1; IV: 20 mL
[64]
API, tablet HPLC-UV Hypersil C18 (150  4.6 mm, 5 mm) at RT ACN: water (20:80, v/v). FR: 1.0 ml.min-1;IV: 20 mL 8.9 – 2/ 5 mg.mL-1
258 nm
[65]
API HPLC-UV Develosil ODS HG-5 (150  4.6 mm, 5 mm) 0.01M sodium dihydrogen phosphate monohydrate 16.58 60.0 0.011/ 0.036 mg.mL-1
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY

210 nm at 40  C buffer pH 3.0 and ACN; gradient elution. FR:

1.0 ml.min-1; IV: 30 mL
(continued)
5
Table 3. Continued. 6
Method/ Retention
AED Matrix Detection Stationary phase/ Temperature (  C) Mobile phase/Flow rate/Injection volume time Run Time LOD/LOQ References
[66]
API, tablet HPLC-UV Apollo Grace C18 (250  4.6 mm, 5 mm) 0.01M sodium dihydrogen ortho phosphate buffer pH 5.6 10.0 –
215 nm at RT 3.5: ACN (70:30, v/v). FR: 1.0 ml.min-1; IV: 20 mL
[67]
API HPLC-UV Kromasil C18 (250  4.6 mm, 5 mm) at RT 0.01 M potassium dihydrogen orthophosphate buffer pH 14.64 22.0 –
220 nm 3.0 and ACN; gradient elution. FR: 1.0 ml.min-1;
IV: 20 mL
[68]
API HPLC-UV Kinetex C18 (150  4.6 mm, 5 mm) at 25  C 0.02 M potassium dihydrogen phosphate buffer pH 4.5 13.3 26.0 0.08/ 0.2 mg.mL-1
J. D. DASILVA ET AL.

210 nm and MeOH; gradient elution. FR: 1.0 ml.min-1; IV:10 mL

[69]
Oral solution HPLC-UV Waters Symmetry C8, (250  4.6 mm, 5 mm) 0.012 M sodium dihydrogen phosphate anhydrous buffer 25.67 85.0 –/0.05%
210 nm at 30  C pH 2.0 and water: ACN (20:80); gradient elution. FR:
1.2 ml.min-1; IV: 10 mL
[70]
API, tablet HPLC-UV Regis Pack (250  4.6 mm, 5 mm) at 25  C 100 % Ethanol. FR: 1.0 ml.min-1; IV: 10 mL 7.06 30.0 0.0429/ 0.1286 mg.mL-1
210 nm
[71]
API HPLC-UV Chiralcel OD-H, (250  4.6 mm, 5 mm) Mixture of n- hexane–ethanol (74:6, v/v) in isopropyl 12.93 25.0 –
210 nm at 25  C alcohol–trifluoroacetic acid (72:6:0.08, v/v). FR:
0.5 ml.min-1; IV: 10 mL
[72]
API, tablet HPLC-UV Chiralpak-IC (250  4.6 mm, 5 mm) at 27  C N-hexane: ethanol (85:15, v/v). FR: 1.0 ml.min-1; IV: 10 mL 11.5 25.0 –
210 nm
[67]
API HPLC-MS Kromasil C18 (250  4.6 mm, 5 mm) at RT 0.01 M ammonium acetate buffer pH 3.0 and ACN; – – –
gradient elution. FR: 1.0 ml.min-1; IV: 10 mL
[68]
API HPLC-MS Kinetex C18 (150  4.6 mm, 5 mm) at 25  C 0.02 M ammonium formate buffer pH 4.5 and MeOH; – – –
gradient elution
[73]
API HPLC-MS Agilent Zorbax SB C18 (150  4.6 mm, 0.02 M ammonium acetate buffer with 0.1% formic acid 10.0 25.0 –
5 lm) at 30  C and ACN; gradient elution. IV: 5 mL
[74]
API UPLC-UV Acquity HSS C18 (2.1  100 mm, 1.8 mm) 0.01 M sodium dihydrogen phosphate monohydrate 2.1 5.0 –
210 nm at 45  C buffer pH 2.0: ACN (85:15, v/v). FR: 0.7 ml.min-1;
IV: 1 mL
[75]
API, tablet HPTLC Precoated silica gel aluminum Toluene: ethyl acetate: MeOH: triethylamine (7:2:1:0.1 v/ – – 893.65/2708.03 ng.spot-1
210 nm plate 60 F254 v/v/v)
[76]
API, tablet GC-FID Capilar column HP-5 (30 m x 0.320 mm) Helio gas. FR: 2.0 ml.min-1. IV: 1 mL (split mode, 5.7 7.0 5.87/19.57 mg.mL-1
260  C 100  C for 1 min to 250  C (40  C/min) ratio 50:1)
[77]
ESA API HPLC-UV Chiralpak IC-3 (150  4.6 mm, 3 mm) Dichloromethane: ethanol (90:10, v/v). FR: 1.0 ml.min-1; 6.51 – Enantiomer: 0.3/
240 nm at 25  C IV: 20 mL 0.9 mg.mL-1
[77]
API HPLC-UV Chiralpak IC-3 (150  4.6 mm, 3 mm) 100% ACN. FR: 0.5 ml.min-1; IV: 10 mL 11.84 – Enantiomer: 0.3/
220 nm at 25  C 1.0 mg.mL-1
[78,79]
API HPLC-UV C8 (250  4.6 mm, 5 mm) at 35  C 0.01 M potassium dihydrogen phosphate buffer pH 5.0: 14.35 60.0 0.020 / 0.060 mg.mL-1
215 nm ACN (95:5 v/v) and ACN: water (80:20 v/v); gradient
elution. FR: 1.0 ml.min-1; IV: 10 mL
[80]
API HPLC-UV Zorbax SB C18 (150  4.6 mm, 3.5 mm)a at 0.01 M potassium dihydrogen phosphate buffer: MeOH 7.0 20.0 0.05/0.15 mg.mL-1
210 nm 27  C at room temperature (80:20, v/v) and ACN: MeOH: water (75:5:20, v/v);
gradient elution. FR: 1.0 ml.min-1; IV: 10 mL
[81]
API HPLC-UV Inertsil ODS 3 V (150  4.6 mm, 5 mm) at 0.1% orthophosphoric acid in water: MeOH: ACN 4.2 10.0 2.40/7.29 mg.mL-1
210 nm room temperature (500:250:250, v/v). FR: 1.5 ml.min-1; IV: 20 mL
[78,79]
API HPLC-MS RP8 (250  4.6 mm, 5 mm) at 35  C 0.01 M ammonium bicarbonate buffer and ACN (95:5, v/ – – –
v) and ACN: water (80:20, v/v); gradient elution. FR:
1.0 ml.min-1; IV: 10 mL
[82]
API UPLC-UV Acquity BEH C18 (150  2.1 mm, 1.7 mm) Mobile phase A and phase B (50:50) 2.6 3.5 0.38/ 1.15 mg.mL-1
215 nm at 30  C (A) 0.01 M potassium dihydrogen orthophosphate and
ACN (90:10, v/v), (B) ACN: water: MeOH (75:5:25, v/v/v).
FR: 0.5 ml.min-1; IV: 2 mL
[82]
API HPTLC Precoated silica gel aluminum Toluene: MeOH: acetone (60:20:20, v/v/v) – – 893.65/2708.03 ng.spot-1
254 nm plate 60 F254
[83]
PER API, tablet HPLC-UV ODS partisil (250  4.6 mm, 5 lm) at room ACN: 0.1% trimethylamine in water pH 2.5 (60:40 v/v). 3.0 7.0 0.32/ 0.98 mg.mL-1
227 nm temperature FR: 1.5 ml.min-1; IV: 20 mL
 [84]
API HPLC-UV Shimadzu Wondasil C18 (250 mm x 0.01M potassium dihydrogen phosphate buffer and ACN; 24.88 40.0 0.19/4.78 mg.mL-1
220 nm 4.6 mm, 5 mm) at room temperature gradient elution. FR: 1.0 ml.min-1; IV: 20 mL
[85]
API HPLC- MS Dikma Diamonsil 0.01 M ammonium acetate buffer and ACN; – 40.0 –
C18 (250  4.6 mm, 5 mm) at 30  C gradient elution
[85]
API, tablet UPLC-UV Phenomenex C8 (150  4.6 mm, 3.5 mm) Acetic acid: ACN: water (0.1:50: 50, v/v/v). FR: 1.0 ml.min- 10.41 15.0 –
1
240 nm at 25  C ; IV: 20 mL
[86]
API UPLC-UV- MS Acquity BEH C18 (100  2.1 mm, 1.7 mm) 0.1 % formic acid in water: ACN (60:40, v/v). FR: 3.1 6.0 0.03/ 0.06 mg.mL-1
290 nm at 40  C 0.4 ml.min-1
[87]
BRV API HPLC-UV C18 (250  4.6 mm, 5 mm) at room MeOH: water: ACN (30:10:60 v/v). FR: 1.0 ml.min-1; 7.9 – 0.1/ 0.4 mg.mL-1
242 nm temperature IV: 20 mL
[88]
API, tablet HPLC-UV ODS 3 V (150  4.6 mm, 5 mm) at 30  C 0.1% trifluoroacetic acid in water: ACN (60:40 v/v). FR: 3.1 – 0.047/ 0.132 mg.mL-1
210 nm 1.0 ml.min-1; IV: 10 mL
[89]
API UPLC- UV Acquity BEH C18 (100  2.1 mm, 1.7 mm) 0.1 % trifluroroacetic acid in water and water: ACN (20: 3.1 10.0 0.05/0.018 mg.mL-1
230 nm at 35  C 80 v/v); gradient elution. FR: 0.3 ml.min-1; IV: 1 mL
[90]
API UPLC-UV -MS Chiral PAK IG-U (100  3.0 mm, 1.6 lm) 0.01 M ammonium bicarbonate:ACN (65:35, v/v). FR: 9.4 15.0 0.3/0.8 mg.mL-1
215 nm at 40  C 0.3 ml.min-1; IV: 5 mL
[49]
EVR API HPLC-UV C18 (150  2.1 mm, 2.7 mm) at 60  C Formic acid: MeOH: 0.7 g/L solution of ammonium 16.0 28.0 –
278 nm format: ACN (0.05:100:450:450 v/v/v/v) and formic
acid: MeOH: 0.96 g/L solution of ammonium formate:
ACN; gradient elution. FR: 0.9 ml.min-1; IV: 3.5 ml;
autosampler at 6  C
[49]
API HPLC-UV Base – deactivated C18 (150  2.1 mm, ACN: 0.27 g/L solution of potassium dihydrogen 15.0 43.0 –
210 nm 2.7 mm) at 50  C phosphate (40:60 v/v) and ACN; gradient elution. FR:
1.1 ml.min-1; IV: 10 ml
[91]
API, tablet HPLC-UV Hypersil BDS C18 (100  4.6 mm, 5 lm) Ammonium acetate buffer: ACN (50:50 v/v) pH 6.5. FR: 3.1 6.0 0.036/ 0.109 mg.mL-1
280 nm at 30  C 1.0 ml.min-1; IV: 10 mL
[92]
Tablet HPLC-UV Zorbax SB C18 (250  4.6 mm, 5 mm) Ammonia solution and formic acid and MeOH: ACN 38.3 100.0 –
280 nm at 50  C (30:70, v/v); gradient elution. FR: 1.0 ml.min-1;
IV: 50 mL
ACN – acetonitrile; API- active pharmaceutical ingredient; FID – Flame ionization detector; FR- flow rate; GC-Gas chromatography; HPLC – High performance liquid chromatography; HPTLC- High performance thin layer
chromatography; IV – injection volume; LOD- limit of detection; LOQ – limit of quantification; MeOH – methanol; MS- mass spectrometry; RT – room temperature.
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
7
8 J. D. DASILVA ET AL.
In forced degradation studies, RUF proved to be more
susceptible to oxidation and hydrolysis by both acid and
alkaline conditions. No degradation was observed under
thermal and photolytic conditions. One product (compound
3; Appendix 1) was formed in acidic and alkaline hydrolysis
(1 N HCl and 1 N NaOH).[58] Another study demonstrated
that this product could be methylated by methanol (co-solv-
ent) present in acid hydrolysis, forming a pseudo degrad-
ation product (compound 2; Appendix 1)[52].
4.2. Stiripentol
Only one HPLC-UV method was reported in the literature
to determine STP in the presence of its degradation product
(compound 4; Appendix 1)[63]. The chromatographic condi-
tions employed a column C18 along with a mobile phase
composed of phosphate buffer and acetonitrile in the iso-
cratic mode, which enabled the elution of STP at 1.8 min
and the degradation product at 4.25 min that gave a com-
plete analysis time of 6 min. This methodology showed good
sensitivity (0.081 mg.mL1) and advantageous for separated
STP and its degradation product. In this work, the authors
observed that STP was highly susceptible to acid hydrolysis
with an almost total decomposition in 0.5 N HCl. In con-
trast, it proved to be stable under alkaline, oxidative, photo-
lytic and thermal conditions.[61]
4.3. Lacosamide
Different methodologies were found in the literature for the
determination of LAC in the presence of its 26 related sub-
stances (compounds 5-30; Appendix 1). Four HPLC-UV
methods described in Eur. Ph 10.0th are presented in
Table 3 for the determination of LAC and 11 impurities
(compounds 5-15; Appendix 1) in the API, infusion, oral
solution and tablet.[49] One of these methodologies is for the
determination of an enantiomeric purity in the API that
specifies the use of a chiral separation column derived from
amylose. This determination is important as the R enantio-
meric form is responsible for the pharmacological action of
LAC (see Section 3). Methodologies for determining LAC
and impurities in the API, tablets and infusion recommend
the use of a C8 column, whereas for oral solution, the rec-
ommendation is to employ a C18 column. All three method-
ologies incorporate acetonitrile as an organic modifier. The
chromatographic conditions employed in these methods dir-
ectly impact in the retention time of LAC and its related
substances, to guarantee the resolution recommended by
official monograph. For the tablet and infusion method, the
resolution proposed between compounds 8 and 9 (Appendix
1) is 1.5. For these same related substances in the oral solu-
tion method, the resolution recommended is at least 3.0. In
the API methodology the suggested resolutions between
LAC and compounds 11 and 7 (Appendix 1) are at least 2.0
and 4.5, respectively.[49]
Two reviews[95,96] describe some stability indicating
methods for determination of LAC and its related substan-
ces published through 2016, but did not include any
pharmacopeial methods. The chromatographic conditions
most often used in non-compendial HPLC methods
described in these reviews and published after 2016 were:
reverse stationary phase (C18 and C8) with a temperature
range of 25 to 45  C, a mobile phase composed of either
sodium or potassium phosphate buffers, an acid pH and
organic modifiers as acetonitrile or methanol with a detec-
tion at 210, 215, 220 and 258 nm.[62–69,95,96] The method of
Ramisetti et al.[68] demonstrated to be sensitive, the LOD
value (0.08 mg.mL1) was not the lowest among all the
methods described in the present review, but it proved to be
the most comprehensive for separating LAC and six degrad-
ation products (compounds 8.14, 15,19, 25-27, Appendix 1),
with run time of 26 min, selectivity and precision.
The chiral separation of LAC from its (S)-enantiomer
(compound 7; Appendix 1) was performed by HPLC using
chiral separation columns. In the chiral method that used
RegisPack column, based on tris-(3,5-dimethylphenyl carba-
moyl) of amylose and a mobile phase composed of 100%
ethanol, retention time of LAC and its enantiomer was
5.36 min and 7.02 min, respectively.[70] Shorter retention times
were observed with this methodology when compared to
methods that used the cellulose-derived columns of Chiracel
OD-H, based on tris- (3, 5-dimethylphenyl carbamate) of cel-
lulose and Chiralpak-IC, based on tris- (3, 5-dichlorophenyl
carbamate) of cellulose. Even with their longer retention
times, these methods proved to be enantioselective.[71,72]
Mass spectrometry (MS) coupled to HPLC was another
technique used both for determination of LAC and to support
the identification of degradation products (compounds 8, 14-
19, 25-30; Appendix 1). The methodologies developed used
volatile buffers such as ammonium acetate and formate with
either acetonitrile or methanol as an organic solvent in combin-
ation with a C18 column. Ionization was performed by electro-
spray (ESI) in both positive and negative modes. High
resolution spectrometers (HRMS), such as Ion trap and Time-
of-flight (TOF) were used for determination of mass and frag-
mentation pattern of LAC and degradation products.[67,68,73]
Other non-HPLC techniques, such as UPLC, HPTLC and
GC, were also reported to separate LAC from its related
substances. The parameters used in the UPLC-UV technique
were similar to some of those described for HPLC methods
such as using a C18 column, potassium phosphate buffer
and methanol as a mobile phase in an isocratic mode. This
methodology show to be an alternative to HPLC methods,
with total analysis time of 6 min and resolution greater than
2 between peaks of LAC and 4 impurities (compounds
11,16,19 and 20; Appendix 1)[78]. The HPTLC methodology
proved to be selective for the determination of LAC using a
mobile phase composed of toluene, ethyl acetate, methanol
and triethylamine (7:2:1:0.1, v/v/v/v) that allowed for the
separation of LAC from 3 of its degradation products (com-
pounds 8, 11 and 24; Appendix 1)[79]. Another technique
used to determine LAC in forced degradation studies was
gas chromatography with flame ionization detection (GC-
FID). In this method, the mobile phase was composed of
helium gas and a temperature gradient was applied to the
column that showed an elution of LAC at approximately

5.7 min without any interference from peaks formed in deg-
radation conditions.[76]
Degradation products (compounds 8,11,14-17,24-30;
Appendix 1) of LAC were formed by oxidation (30% H2O2
at 100  C for 0.5 h), acidic and alkaline hydrolysis in differ-
ent concentrations of stressors (0.1 M and 2 M HCl; 0.05 M,
1 M and 2 M NaOH), reaction times and temperature.
Under thermal and photolytic conditions, LAC showed
insignificant degradation.[62,67,68,76]
4.4. Eslicarbazepine acetate
Analytical methods described in the literature for determin-
ing ESA in the presence of its 21 related substances (com-
pounds 31-51; Appendix 1) widely employed HPLC[77–81].
Two reported on the use of UPLC[82] and HPTLC.[97] Two
different HPLC methodologies were developed in same
study for the separation of ESA and its (R)-enantiomer
(compound 31; Appendix 1) in API.[77] In these methods,
chiral separation was performed through a Chiralpak-IC-3
column that is based on tris-(3,5-dichlorophenyl carbamate)
of cellulose. One used a normal phase containing dichloro-
methane and ethanol, and the other used an organic polar
solvent, 100% acetonitrile. In the normal phase method, the
retention times were 4.24 min for the (R)-enantiomer and
6.77 min for ESA with a resolution of 6. In the polar organic
method, the resolution was 3 with longer retention times of
10.18 and 11.77 min for (R)-enantiomer and ESA, respect-
ively.[77] Control of the enantiomeric purity ensures that
during hydrolysis there is a greater conversion of the pro-
drug into the active metabolite (S)-licarbazepine and not the
formation of carbamazepine-10, 11-epoxide, which is the
metabolite responsible for adverse effects.[39]
In the Mohamed et al (2016) review are described three
stability indicating methods.[78,80,81] The first method
described uses column C8 and was developed for the separ-
ation of ESA in face of potential bulk impurities and deg-
radation products (compounds 32-46, Appendix 1)[82]. This
method proved to be more sensitive (LOD 0.02 mg.mL1)
than the other two methods described in the review, how-
ever it presented longer run time (60 min) to obtain
adequate resolution between the chromatographic peaks.
The other two methods used column C18 as a stationary
phase, with shorter retention times for ESA, when compared
to the first methodology. In addition, the methods proved to
be selective in determining ESA in the presence of its related
substances (compounds 33-35, 39, 41, 48-50, Appendix 1).
Thomas et al. (2012, 2014)[78,79] used the same HRMS
(HPLC-Ion trap) methodology at two works as support tool
for the identification of unknown impurities and degrad-
ation products. In this methodology, unlike the HPLC-UV
method, ammonium bicarbonate buffer was used as the
mobile phase since it is volatile and compatible with ioniza-
tion by ESI and APCI (atmospheric pressure
ionization).[78,79,98]
An analytical method developed for UPLC used chroma-
tographic conditions similar to the methodologies for HPLC
in the separation of ESA from degradation product
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 9
(compound 51; Appendix 1). This methodology enabled
rapid analyzes with an elution of ESA and product in 2.6
and 1.8 min, respectively.[82] An HPTLC methodology, using
toluene, methanol and acetone (60: 20:20, v/v/v) as the
mobile phase, also proved to be selective for the analysis of
samples from forced degradation studies without interfer-
ence in the elution of ESA.[97]
Two degradation products (compound 33 and 51;
Appendix 1) of ESA described in the literature were formed
by oxidation (reflux with 30% H2O2 for 24 h), neutral
hydrolysis (60  C for 48 h) and, most notably, by acidic
(0.5 N HCl for 1 h and 2 N HCl for 15 min, both in room
temperature) and alkaline hydrolysis (0.001 N NaOH for
15 min and 0.015 N NaOH for 2 min, both in room
temperature).[78,82]
4.5. Perampanel
Analytical methodologies for the separation of PER and 13
of its related substances (compounds 52-64; Appendix 1)
were performed by HPLC-UV and UPLC-UV.[83–86] The
chromatographic conditions employed columns of C18 and
C8, a mobile phase composed by phosphate buffer, acidified
water and acetonitrile in isocratic and gradient mode, and a
detection in the UV range of 220 to 290 nm. Saida et al.[86]
UPLC – UV methodology is the most sensitive, with shorter
retention times for PER and its degradation products (com-
pounds 52 and 53, Appendix 1). However, Xia et al.[83]
methodology even with longer retention time and run time,
it is the most advantageous for separating PER from thirteen
related substances (compounds 52-64, Appendix 1).
HPLC-MS was used again to support in the identification
of degradation products. In one study, the determination of
the mass of degradation products (compounds 52, 53, 62-64;
Appendix 1) was performed using an HPLC-MS (triple
quadrupole) methodology with ionization by ESI in positive
mode. The coupling to MS differed extensively from the
method for HPLC with a UV detection in relation to the
composition of mobile phase, time and gradient proportion,
as well as column temperature.[84]
PER was shown to be stable under thermal and photo-
lytic conditions although degradation products were formed
under oxidation as well as acid and alkaline hydrolysis with
different concentrations of stressors (0.5 M and 1 M HCl;
1 M NaOH, H2O2 at 3% and 30%), temperatures (60 and
100  C) and reaction times[84,86]
4.6. Brivaracetam
BRV has four related substances with structures and nomen-
clatures described in the literature, three diasteroisomers
(compounds 65-67; Appendix 1) and one degradation prod-
uct (compound 68; Appendix 1). Analytical methods devel-
oped for BRV determination in presence of these substances
were performed by HPLC and UPLC coupled to DAD/UV
detectors.[87–90] In the HPLC-UV methods, a longer reten-
tion time of 7.9 min was observed for BRV when methanol
was used in mobile phase.[79] BRV retention time was
10 J. D. DASILVA ET AL.
reduced to 3.1 min when the mobile phase was composed of
acetonitrile and acidified water together with a column tem-
perature that was maintained at 30  C.[88] In addition, sensi-
tivity was improved with value of LOD 2-fold lower than
previous method.
In the UPLC-UV method, a C18 column was also used
with a gradient elution of mobile phase that resulted in a
BRV retention time of 3.1 min and that provided an
adequate separation from its impurities with resolution
greater than 2.[89] Chiral separation of BRV and its three
diastereomers was achieved by UPLC-UV using a Chiral
PAK IG-U column (tris (3, 5-chloromethylphenyl carba-
mate) of amylose), ammonium bicarbonate buffer and aceto-
nitrile as the mobile phase in the isocratic mode. With these
chromatographic conditions, a complete analysis was
obtained in 15 min with an elution of BRV at 9.4 min and
separation of the stereoisomers with a resolution greater
than 1.5. This chiral methodology was also used with an
UPLC-MS (Ion trap) with ionization by ESI to support in
the identification of diasteroisomer (R,R)-brivaracetam
(compound 66; Appendix 1) in alkaline hydrolysis.[90]
In forced degradation studies of BRV, the formation of dia-
stereomer (R,R)-brivaracetam (compound 66; Appendix 1) was
observed after alkaline hydrolysis in 1 N NaOH for 4 h.
Another degradation product (compound 68; Appendix 1) was
also formed in alkaline hydrolysis in 1 N NaOH, but with 24 h
of reaction. In both studies, BRV was directly exposed to deg-
radation conditions without a co-solvent.[88,90] These studies
indicated that BRV is stable under conditions of acidic, ther-
mal, photolytic and oxidative degradation.[90]
4.7. Everolimus
EVR has 14 related substances described in the literature, of
which, 11 have known chemical structures (compounds 69-
79; Appendix 1). Among the analytical methods used for
determining EVR in presence of these impurities, two are
found in Eur. Ph. 10.0th and are applied for the analysis of
the API.[49] The first methodology is for separation of EVR
and sirolimus (compound 69; Appendix 1), that as discuss
in Section 3 is the compound from which EVR is derived.
The second method was developed to determine EVR in the
presence of 7 impurities, 4 with known structures (com-
pounds 71-74; Appendix 1) and 3 unknown structures. The
two methodologies are reverse phase (column C18) with an
elution in a gradient mode. Only for a second method, Eur.
Ph. 10.0th recommend resolutions at least 1.5 between EVR
and compound 70 and 72 (Appendix 1) peaks.
Another two methods found in the literature for the deter-
mination of EVR in the presence of its related substances are
also by reverse phase HPLC-UV.[91,92] The method with an iso-
cratic elution, showed good sensitivity (LOD 0.036 mg.mL1)
and the EVR retention time was 3.1 min.[90] In other method, a
Quality by Design (QbD) approach by Design of Experiments
(DOE) was applied to determine EVR in presence of 8 impur-
ities (compounds 69-70, 75-79; Appendix 1). One of criteria for
optimizing the method was the resolution between EVR and
sirolimus (compound 69; Appendix 1) to achieve a value of
2.6. However, the overall analysis time was greater than
100 min.[92] EVR showed little degradation under the stress
conditions used in these two studies and without the formation
of degradation products.[91,92]
5. Conclusion
This review covered the stability indicating methods for the
separation of third generation AEDs from their impurities
and degradation products. The frequency of the techniques
used in the methods for obtaining fast and sensitive analyses
were: HPLC (79%), UPLC (12%), HPTLC (5%), GC (2%)
and UV/Vis spectrophotometry (2%). Mass spectrometry
was employed mainly for the identification of impurities
and degradation products that provided support information
in the structural elucidation of these compounds. USP 43
and Ph. Eur. 10th describe methods for separating RUF,
LAC and EVR from their related substances in dosage forms
and API. The official methods, as well as most of the meth-
odologies found in scientific articles, suggest the use of
liquid chromatography, UV detection and reverse phase col-
umns (C18 and C8). Chiral separation of LAC, ESA and
BRV enantiomers was performed using direct methods with
chiral separation columns. In general, the AEDs reported in
this review showed susceptibility to degradation under oxi-
dative condition as well as to acid and alkaline hydrolysis.
Forced degradation studies along with the use of known
impurities, allowed for the demonstration of analytical
methodologies selectivity. Some of these AEDs, having been
only approved during the last decade, have few studies
focused on their stability and the identification of their
impurities/degradation products. Importantly, this review
shows that further studies are clearly needed to contribute
to the establishment of more complete safety profiles of
these third-generation antiepileptic drugs.
Acknowledgments
The authors acknowledge CAPES (Bras
ılia, Brazil), CNPq (Bras
ılia,
Brazil) and FAPERJ (Rio de Janeiro, Brazil). We are grateful to
William Provence Jr for English review.
Disclosure statement
The authors declare that there are no conflicts of interest.
Funding
This article was funded by Conselho Nacional de Desenvolvimento
Cient
ıfico e Tecnol

ogico;Coordenaç~ao de Aperfeiçoamento de Pessoal
de N
ıvel Superior;Fundaç~ao Carlos Chagas Filho de Amparo a Pesquisa
do Estado do Rio de Janeiro.
ORCID
J
essica Domingos da Silva http://orcid.org/0000-0001-8728-1906
Lucio Mendes Cabral http://orcid.org/0000-0002-4550-5729
Val
eria Pereira de Sousa http://orcid.org/0000-0003-1589-0846

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