LIQUID CHROMATOGRAPHIC METHODS FOR DETERMINATION OF

THE NEW ANTIEPILEPTIC DRUGS: STIRIPENTOL, RETIGABINE,

RUFINAMIDE AND PERAMPANEL.

A project work
submitted in partial fulfillment of requirement of degree of
BACHELOR OF PHARMACY
FINAL YEAR

To the faculty of Science & Technology (Pharmacy)

Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad

By
Mr. Om Jaiswal
(B. Pharm Final Year)

Guided by
Mr. Nanasaheb Dharbale
(M. Pharm)

Department of Pharmaceutical Chemistry

Shri Bhagwan College of Pharmacy
Dr. Y. S Khedkar Marg, Cidco N6,
Aurangabad. 431003
(M.S) India
2021

1
 SHRI BHAGWAN COLLEGE OF PHARMACY
Dr. Y. S Khedkar Marg, Cidco N6, Aurangabad. 431003 (M.S) INDIA
Ph. No. +91 240 2241850 Email: bhagwanpharmacy@gmail.com

CERTIFICATE
This is to certify that the thesis entitled “LIQUID CHROMATOGRAPHY FOR
DETERMINATION OF NEW ANTIEPILEPTIC DRUGS: STIRIPENTOL,
RETIGABINE, RUFINAMIDE AND PERAMPANEL” represents the bonafide work
of Mr. Om Jaiswal submitted in partial fulfillment of the requirement for the degree of
‘Bachelor of Pharmacy’. The work has been carried out in Shri Bhagwan College of
Pharmacy, affiliated to Dr. Babasaheb Ambedkar Marathwada University under the
guidance of Mr. Nanasaheb Dharbale.

Date: Mr. P.V. Mhaske

Place: Aurangabad Principal

2
 SHRI BHAGWAN COLLEGE OF PHARMACY
Dr. Y. S Khedkar Marg, Cidco N6, Aurangabad. 431003 (M.S) INDIA
Ph. No. +91 240 2241850 Email: bhagwanpharmacy@gmail.com

CERTIFICATE
This is to certify that the thesis entitled “LIQUID CHROMATOGRAPHY FOR
DETERMINATION OF NEW ANTIEPILEPTIC DRUGS: STIRIPENTOL,
RETIGABINE, RUFINAMIDE AND PERAMPANEL” represents the bonafide work
of Mr. Om Jaiswal submitted in partial fulfillment of the requirement for the degree of
‘Bachelor of Pharmacy’. The work has been carried out in Department of Science &
Technology of Shri Bhagwan College of Pharmacy, affiliated to Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad under our guidance. All the materials obtained from
other sources have been duly acknowledged in the thesis.

Date: Mr. Nanasaheb Dharbale

Place: Aurangabad Guide

3
 SHRI BHAGWAN COLLEGE OF PHARMACY
Dr. Y. S Khedkar Marg, Cidco N6, Aurangabad. 431003 (M.S) INDIA
Ph. No. +91 240 2241850 Email: bhagwanpharmacy@gmail.com

Declaration
I undersigned, Mr. Om Jaiswal, Aurangabad hereby declare that the thesis entitled
“LIQUID CHROMATOGRAPHY FOR DETERMINATION OF NEW
ANTIEPILEPTIC DRUGS: STIRIPENTOL, RETIGABINE, RUFINAMIDE AND
PERAMPANEL” is based on original research work and has not been submitted
previously for the award of any degree or diploma by me to any other university. The thesis
work was carried and submitted in the fulfillment of the requirement of the Degree of
“Bachelor of Pharmacy”, in the faculty of Science & Technology of Dr. Babasaheb
Ambedkar Marathwada University, Aurangabad, under the guidance of Mr. Nanasaheb
Dharbale. I hereby declare that the information given in the thesis is true as per my efforts
to accomplish this research work.

Date:
Place: Aurangabad Mr. Om S. Jaiswal

4
 Acknowledgement

“Words never express human sentiments. This is only an attempt to express my gratitude
which comes from my heart.”
I express my special thanks to honorable Principal sir, Shri Bhagwan College of
Pharmacy. Aurangabad for providing us excellent infrastructural facilities in campus.
I am honored to express my profound and deep sense of gratitude
towards my guide Mr. NANASAHEB DHARBALE sir of Shri Bhagwan College of
Pharmacy for his creative suggestions, helpful discussion, unfailing advice, constant
encouragement during the Practice school. I consider myself privileged to have worked
under him as he always shared his vast experience so generously and patiently.
I am grateful to my family. For their kind support and motivation which has helped me
to complete this work successfully.

Mr. Om S. Jaiswal

5
Table of Contents
Abstract: ................................................................................................................... 9
Introduction: ........................................................................................................... 10
Classification of epilepsies.................................................................................. 10
Mortality of Epilepsy .......................................................................................... 11
New Antiepieptic Drugs: .................................................................................... 12
Pharmacology ......................................................................................................... 14
Pharmacokinetic properties: ................................................................................... 15
Physicochemical properties and drug stability: ...................................................... 17
Sample preparation: ................................................................................................ 21
LC methods: ........................................................................................................... 22
Chromatographic columns and mobile phases: ...................................................... 25
Detection systems ................................................................................................... 26
Conclusion: ............................................................................................................. 28
References: ............................................................................................................. 29

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Figure Contents

Figure 1 Classification of epilepses .................................................................................. 11

Figure 2 Structure of antiepileptic drugs .......................................................................... 12

7
Table Contents

Table 1: Main pharmacokinetic properties of the new antiepileptic drugs stiripentol

(STP), retigabine (RTG), rufinamide (RFM) and perampanel (PER) in humans. 16

Table 2: Physicochemical properties of stiripentol (STP), retigabine (RTG), rufinamide

(RFM) and perampanel (PER). ......................................................................................... 17
Table 3 Stability of stiripentol (STP), retigabine (RTG), rufinamide (RFM) and perampanel
(PER) in organic solutions and in human and animal biological matrices. ...................... 20

8
Abstract
New antiepileptic drugs perampanel, retigabine, rufinamide and stiripentol have recently
been developed for different types of epilepsy. Innovation in the antiepileptics
armamentarium, a More investigations into their drug properties will be conducted.
Besides, considering their broad anticonvulsant activities, an extension of their therapeutic
indications may be worthy of investigation, especially regarding other seizure types as well
as other central nervous system disorders. Although the various liquid chromatographic
(LC) methods associated with ultraviolet, fluorescence, mass or tandem-mass spectrometry
detection have already been developed for the determination of perampanel, retigabine,
rufinamide and stiripentol, new and more cost-effective methods are yet required.
Therefore, this review summarizes key analytical aspects in relation to the liquid
chromatographic methods designed for the analysis of perampanel, retigabine (and its main
active metabolite), rufinamide and stiripentol in biological samples and pharmaceutical
dosage forms. In addition, the physicochemical properties and stability of targeted
chemicals will also be consistent targeted. Therefore, this review collects, for the first time,
important background information on LC methods developed and used for perampanel,
retigabine, rufinamide and stiripentol, which should be considered a starting point if new
(bio)analytical techniques are aimed to be implemented for these drugs. (1)

9
Introduction
• Epilepsy is a chronic disease of the brain characterized by an enduring (i.e.,
persisting) predisposition to generate seizures, unprovoked by any immediate
central nervous system insult, and by the neurobiologic, cognitive, psychological,
and social consequences of seizure recurrences.(2)
• Epilepsy is a disease of the brain characterized by an enduring predisposition to
generate seizures and by the neurobiologic, cognitive, psychological, and social
consequences of seizure recurrences.
• Epilepsy exists when someone has an epileptic seizure and their brain
“demonstrates a pathologic and enduring tendency to have recurrent seizures”.
• Epilepsy is one of the most common neurological disorders worldwide, it affects
about 1% of 2% of the population.
• Currently, there are more than 20 antiepileptic drugs (AEDs) available for clinical
use, which is often divided into three different generations.
• First-generation AEDs present several drawbacks such as narrow therapeutic
indices, complex pharmacokinetic profiles, saturable metabolism, high plasma
protein binding (PPB), and high potential for drug interactions, and may induce
anticonvulsant hypersensitivity syndrome. (3)
• However, the accumulated experience in its clinical use is enormous, this is the
reason why these AEDs are still the first choice for many epilepsy disorders. To
overcome the above disadvantages and to achieve higher efficacy and better
tolerability, second-generation AEDs were then developed, followed by third
generation AEDs, starting with lacosamide approval in 2008.

Classification of epilepsies
• The epilepsy type classification is broader in scope than is the seizure classification,
and considers the possibility of having multiple seizure types, and incorporates
information about the overall clinical picture, imaging, genetics, laboratory tests,
prognoses and comorbidities.
• Epilepsy types are classified as:

10
 1) Focal
2) Generalized
3) Combined Generalized and Focal
4) Unknown.

Figure 1 Classification of epilepses

Mortality of Epilepsy

• Epilepsy accounts for a significant proportion of the world’s disease burden,

affecting around 50 million people worldwide.
• The estimated proportion of the general population with active epilepsy (i.e.
continuing seizures or with the need for treatment) at a given time is between 4 and
10 per 1000 people.(4)
• Globally, an estimated five million people are diagnosed with epilepsy each year.
In high-income countries, there are estimated to be 49 per 100 000 people
diagnosed with epilepsy each year.
• There were four times geographical variations in the age-standardised incidence
rates of idiopathic epilepsy, with the highest rates observed in Ecuador (70·9 per

11
 100 000 person-years) and Mexico (56·0 per 100 000 person-years) and the lowest
rates in North Korea (17·0 per 100 000 person-years) and China (19·7 per 100 000
person-years). (5)

New Antiepileptic Drugs

• The new class of drugs are improved in their pharmacological profiles, third-
generation AEDs also introduced new chemicals structures that allow them
to act by new mechanisms of action, interact with different therapeutic targets
and modulate different mechanisms of neuronal excitability that were not
covered by first- and second-generation AEDs.
• Therefore, to increase the therapeutic effectiveness, to improve tolerance and
reduce toxicity, the introduction of third-generation AEDs as an integrated
treatment in patients already treated with other types of AEDs are more
common. Examples of AEDs with such characteristics are shown in Fig. 2.

Figure 2 Structure of antiepileptic drugs

• Retigabine (RTG) (also known as ezogabine in the USA), rufinamide (RFM),

stiripentol (STP) and perampanel (PER). RFM and STP are both have been
classified as orphaned drugs since their clinical manifestations specifically
prescribed to treat very low epilepsy syndromes population growth. (6)

12
 • Specifically, RFM is indicated to treat a severe, chronic, and multiple drug-
resistance epileptic encephalopathy mostly characterized by the occurrence of
multiple seizure types so called Lennox-Gastaut syndrome. Also used for the file
treatment of super-refractory tonic-clonic status epilepticus and as comprehensive
treatment of partial seizures in adults and adolescents.
• On the other hand, STP is indicated as a combination therapy with clobazam and
valproic acid treatment not only Dravet syndrome in children, but also in
adolescence and old age. This syndrome, also known as myoclonic epilepsy of
infancy.
• In opposition to STP and RFM, RTG and PER are widely used as complementary
therapies in different types of epileptic seizures. Recently, clinical evidence also
shows that PER is effective in monotherapy for the treatment of focal seizures.
• While the new AEDs present a better security profile, they are associated with
serious and toxic effects, most of them depending on dose. So, whatever the
therapeutic reference range for plasma / serum concentration range of this new
AEDs are currently controversial, undeniably an important role that therapeutic
drug monitoring (TDM) plays a therapeutic guidance because treatment of epilepsy
is mainly prophylactic. Therefore, the use of TDM in these new AEDs is also useful
and, sometimes, it is critical. (7)
• However, to accurately apply TDM, the measurement of RFM, STP, RTG and PER
concentrations in plasma, serum or whole blood must be performed with validated
bioanalytical methods.
• Though, to correlate AEDs concentrations with respective clinical effects, the
concentration of drugs in brain may still be more determinant than that in blood.
• Nevertheless, as the drug quantification in patients' brains is not feasible, it is of
great importance to perform non-clinical pharmacokinetic and pharmacodynamic
assays in order to correlate, as accurately as possible, the plasma drug
concentrations with drug concentrations in brain.
• For that, the development of methods for quantification of these AEDs in several
laboratory animals’ matrices used to perform these studies becomes mandatory.
Considering new AEDs, there is also evidence of significant diversity among

13
 people in their pharmacokinetics, mainly derived from differences in hepatic
metabolism.
• In fact, appropriate pharmacokinetic bonds and their effect on pharmacodynamics
should be in-depth research, especially on new AEDs, such as RTG, RFM, STP and
PER, because the information currently available is scarce.
• Therefore, to obtain this evidence effectively and accurately, drug concentration in
biological samples should also be measure for that reason, bioanalysis analysis is
important. In this case, it is important using fully proven bioanalytical methods for
reliable data.

Pharmacology
• The precise mechanisms by which rufinamide exerts its antiepileptic effect are
unknown. In vitro studies suggest that a principal mechanism of action is the
modulation of activity in sodium channels, particularly prolongation of the inactive
state. (8)
• Rufinamide has no effect on benzodiazepine or GABA receptors or on adenosine
uptake; it also has no significant interactions with glutamate, adrenergic,
tryptophan, histamine, and muscarinic cholinergic receptors.
• Efficacy in all seizure models suggests that rufinamide is likely to be of value in a
broad spectrum of seizure types, although results in animal models may not
translate to humans.
• In vitro studies have shown that perampanel is a selective, noncompetitive
antagonist at the AMPA receptor
• Perampanel protect from tonic–clonic generalized seizures in audiogenic and
maximal electric shock–induced seizure tests, and from absence or myoclonic
seizures in pentylenetetrazole-induced seizure tests.
• In tests the potency of perampanel was higher than carbamazepine and sodium
valproate.
• Retigabine inhibit EA in pathologic brain tissue from the presumed epileptogenic
zone resected in these patients with pharmacoresistant partial epilepsy.

14
 • Retigabine is a potent anticonvulsant against generalized seizures induced by
disturbance of both excitatory and inhibitory transmission. However, RTG/EZG
was unable to prevent clonic seizures induced by bicuculline, or motor seizures
induced by blocking spinal glycinergic transmission using strychnine. (9)
• Stiripentol is particularly potent in combination with CBZ.
• Stiripentol potentiates GABA transmission by elevating the levels of the inhibitory
neurotransmitters in the brain.
• Stiripentol is a positive allosteric modulator of GABA-A receptors in the brain that
enhances the opening duration of the channel by binding to a site different than the
benzodiazepine binding site

Pharmacokinetic properties
• In general, third-generation AEDs have a better pharmacokinetic profile than older
ones. The main pharmacokinetic features of RTG, RFM, STP and PER in humans
summarized in Table 1. With the exception of RTG, which shows a 60% oral
availability, all the other three AEDs have the highest oral availability (85%), with
PER presenting the ideal value of approximately 100%.
• On average, the time to reach maximum plasma concentration (tmax) after oral
administration is shorter for both RTG and PER, than that for STP and RFM. Food
is also responsible for RTG and PER tmax delay up to 2 h, but for both drugs, the
extent of absorption and their bioavailability are not substantially affected.
• In the case of RFM and STP, they are recommended to be administered with food,
because it is demonstrated that food intake not only increases the bioavailability of
RFM, but also protects STP from fast degradation in the acidic environment found
on an empty stomach.
• Actually, STP degradation in acidic conditions was demonstrated during a stability
study that used 0.5 M hydrochloric acid, a similar condition of the stomach
environment.
• As for PPB, there are others differences between the four AEDs (Table 1). STP
and PER exist especially with high PPB values of 99% and 95% respectively, it is
very important to take these values into consideration when detecting plasma

15
 concentrations practiced, especially during TDM studies and therapeutic reference
ranges optimization. (10)
• By analyzing Table1, it is also evident that there are different characteristics in
relation to apparent volume of distribution (Vd) and elimination half-life (t1/ 2el).
RTG shows a larger Vd comparatively with PER and RFM. Regarding RFM and
PER Vd values, it can be expected that both present similar body distribution
patterns. In case of STP, its Vd values were found to be variable, probably as a result
of its non-linear pharmacokinetics, its strong PPB and its complex and extensive
metabolism.

Table 1: Main pharmacokinetic properties of the new antiepileptic drugs stiripentol (STP), retigabine (RTG),
rufinamide (RFM) and perampanel (PER) in humans.

Tss Refere
Drug tmax Vd Active Main
PPB T1/2el nce
Fpo (day (L/kg metabo excretion Linear PK Food effect
(%) (h) (h) range
(%) s) ) lite routes
(mg/L)
No;
Renal (70% systemic
as exposure
Food avoids
metabolites increases
STP 1.5- 4.5- variab degradation in
99 1-2 no ) Feces and 4-22
≥90 3 13 le acidic
(13% e24% clearance
environment
as decreases
unchanged) with higher
doses
Food
N- Renal increases Not
RTG 0.5- acetyl- (85%) Cₘₐₓ, but not establis
80 1-2 8 6-8.7 No hed
60 2 retigabi Feces AUC; tmax
ne (14%) increase up to
2h
No; Cₘₐₓ
and AUC
increase in Food 4-31
RFM 0.7- Renal
26-35 4-6 2 6-12 no a less than increases Cₘₐₓ
≥85 1.1 (84.7%)
dose and AUC
proportiona
l manner
Feces (70% Food
as increases tmax 0.086-1
PER 0.5- 10-
95 105 1.1 no metabolites Yes up to 2 h, but
=100 205 19
) Renal not absorption
(30%) extent

16
 AUC: area under the curve; Cmax: peak plasma drug concentration; Fpo: oral bioavailability; PER:
perampanel; PK: pharmacokinetics; PPB: plasma protein binding; RFM: rufinamide; RTG: retigabine; STP:
stiripentol; t1/2el: elimination half-life; tmax: time to reach maximum plasma concentration; tss: time to reach
steady state; Vd: apparent volume of distribution.

Table 2: Physicochemical properties of stiripentol (STP), retigabine (RTG), rufinamide (RFM) and perampanel
(PER).
Water
MW Log Log D 0.1 M HCl
pKa solubility
Drug MF (g/mol) P (pH 7.4) solubility
(mg/L)
(mg/L)
14.34 (strongest 3.53
STP C14H18O3 234.3 acidic); -3.1 or 2.80 49.2 Instable
(strongest basic) 2.94
13.6 (strongest 2.0
RTG C10H18FN3O2 303.3 acidic); 3.99 or 2.24 10.0 NR
(strongest basic) 2.5
12.69 (strongest
RFM C10H8F2N4O 238.2 acidic); -1.1 0.88 0.42 40.0 63
(strongest basic)
PER C12H15N3O 349.4 3.24 (weak base) 3.7 2.92 5.6 470

Physicochemical properties and drug stability

• For the development of new bioanalytical methods, firstly we should consider the
physicochemical properties of the target analytes.
• In the case of RTG, RFM, STP and PER, it is still the case, the information available
in the books regarding those properties is still young (Table 2).
• Considering their chemical structures, depicted in Fig. 1, the differences between
the chemical groups that assembly these four AEDs are evident. PER is chemically
a 2-(2-oxo-1-phenyl-5-pyridinyl-1,2-dihydropyridin-3-yl) benzonitrile hydrate,
whose structure is responsible for the selective non-competitive antagonism of
AMPA ionotropic glutamate receptors.(11)
• STP (4,4- dimethyl-1-[3,4(methylenedioxy)-phenyl]-1-penten-3-ol) is an aromatic
allylic alcohol with no carbonyl or nitrogenous heterocycle in its structure that
usually is responsible for the antiepileptic properties of the majority of AEDs. A

17
 particular feature of STP is the presence of a chiral center in C-3, resulting in a
racemic mixture of R(+)-STP and S(-)-STP enantiomers, with the R(+)-STP
presenting an anticonvulsant potency approximately 2.4-fold greater than that of
the S(-)-enantiomer.
• RFM (1-[(2,6-difluorophenyl) methyl]-1H-1,2,3-triazole-4-carboxamide) presents
a triazole ring that can be responsible for its activity in different seizure types being,
until now, the first AED with that unique chemical structure.
• On the other hand, RTG (N-(2-amino-4-(4-fluorobenzylamino) phenyl carbamic
acid ethyl ester) is an ester similar to the central analgesic flupirtine but structurally
different from any other AEDs.
• All four AEDs are weak bases, being their water solubility dependent on the pH
values of the dissolution media. Therefore, a higher solubility is expected in acidic
aqueous conditions instead of basic aqueous solutions, being this evident for PER
and RFM, which have a higher solubility in a 0.1 M hydrochloride acid solution
than in water (Table 2).
• PER, RTG and STP present a low water solubility, which is reflected by their high
values of octanol/water partition coefficient (Log P) and distribution coefficient
(Log D) measured in a buffer with a pH value of 7.4 (Table 2).
• Therefore, a higher solubility of PER, RTG and STP in organic solvents is
expected, being this a reasonable explanation to prepare their stock and working
solutions using medium polarity solvents as acetonitrile, methanol and ethanol.
• In opposition, by analyzing RFM Log P and Log D values, this AED presents a
lower lipophilic profile when compared with PER, RTG and STP. This could be
the reason why, in several studies, RFM powder was dissolved in methanol is
mentioned, a most polar solvent than acetonitrile, in a mixture of acetonitrile and
water, or in a mixture of methanol and water or other polar solvents.(12)
• Another key factor to be considered in the development of new chromatographic
methods is the stability of the analytes in biological samples and in stock and
working solutions. Stability is usually related to compounds physicochemical
properties, storage conditions, container systems, and the biological matrix itself.

18
 • The accuracy of assays at each concentration level should be within ±15% of
nominal concentrations. All the revised publications that performed stability studies
adopted this acceptance criterion, and these studies are summarized in Table 3.
• Stability studies under refrigerated storage conditions (e.g., 4°C) are also frequently
performed; STP, RTG, RFM and PER were shown to be stable after at least 24 h in
those conditions.
• Besides, stability after three freeze-thaw cycles (24 h per cycle) and long-term
stability tests performed at freezing temperatures in a time period ranging from 3
weeks to 376 days were also demonstrated for the four compounds.
• All storage conditions (sample type, storage time and temperature) applied in
stability studies of the AEDs here studied are also described in Table 3.
• Contrary to stability determination in biological matrices, only a few studies
determined stability of PER, RTG, RFM and STP in stock and working solutions
(Table 3).
• Stress degradation studies were carried out under acid and basic hydrolysis,
oxidation, thermal and photolytic forced conditions.
• RTG proved to be sensitive to UV light exposure and susceptible to acidic and basic
hydrolysis. Furthermore, like RFM, RTG can also be destroyed under oxidative
conditions, while RFM and STP are degraded by acid hydrolysis.
• In fact, STP instability in acidic conditions has practical implications, being
mandatory to administer this drug orally with meals in order to avoid its acidic
degradation by gastric acid.
• In all cases, none of the degradation products showed to interfere with the retention
time of the analyte.

19
 Table 3 Stability of stiripentol (STP), retigabine (RTG), rufinamide (RFM) and perampanel (PER) in organic
solutions and in human and animal biological matrices.

20
Sample preparation
• Sample preparation procedures are critical and time-consuming steps applied to
biological samples before the analysis of target analytes.(13)
• Sample preparation is essential since the presence of small molecules and
macromolecules, salts and other matrix endogenous compounds may influence the
detection of the analytes under investigation. Furthermore, those interferences can
also be incompatible with the chromatographic system, particularly with
chromatographic columns.
• Before the beginning of sample pretreatment, it is common to add an internal
standard (IS) to the sample. The use of an IS aims to compensate the loss of analytes
during all steps of sample preparation and chromatographic procedures, leading to
an increased accuracy and precision of the method.
• The IS compound should preferentially have a chemical structure and
physicochemical properties comparable to those of target drugs, must not be present
in the original samples and not react or be destroyed by the sample components,
mobile phase or stationary phase of the chromatographic columns.
• Chemical structures of IS that have been used in bioanalytical assays for
quantification of the target AEDs herein considered (i.e. STP, RTG, RFM and
PER).
• A good example of this is the use of flupirtine as IS in the determination of RTG in
human plasma. Regarding this matter, some studies have even used analogue
compounds or labeled isotopes of the original analytes to increase the similarities,
which is preferable to the use of other compounds
• Considering the information summarized, it is evident that conventional sample
preparation approaches (LLE, PP and SPE) continue to be the most applied
techniques in studies involving the quantification of STP, RTG, RFM and PER.
• Thus, gathering all the collected information about the different preparation
procedures used in samples containing STP, RTG, N-acetyl-retigabine, RFM or
PER, the authors suggest that in less complex matrices, simpler methods such as

21
 PP, or even mere dilutions steps and centrifugation/filtration procedures can be
used.

LC methods
• Over the years, several LC methods have been developed to determine the new
AEDs STP, RTG, RFM and PER.

• In the last decade, LC has been the dominant methodology used for analytical and
bioanalytical purposes in laboratories worldwide especially in the pharmaceutical
field.
• Throughout drug development programs, a variety of LC methods are also applied
to guarantee the quality control of pharmaceutical forms, conduct accelerated
stability studies and evaluate the presence of impurities. (14)

Table 5. -Liquid chromatography techniques and analytical conditions for the determination
of stiripentol (STP), retigabine (RTG) and its active metabolite N-acetyl retigabine, rufinamide (RFM)
and perampanel (PER) in different matrices.

22
 Drug Matrix Techn Detectio Internal Elution Column Mobile Flow Temp Retention LLO
ique n Standard Phase rate (℃) time(min Q

23
)

PER Huma HPLC FLD Mirtazapin Isocrati Kinetex PFP 30 mM sodium 0.8 NR 2.08 20
n (Ex/Em: e c (100mm4.6 mm, acetatebuffer ng/m
plasma 290/430 2.6mm) pH 3.7 L
nm)
PER Huma HPLC UV (320 Promethaz Isocrati Two reverse- Water/acetonitr 1.5 50 4.8 25
n nm inehydroc c phase ile(60:40,V/V) ng/m
plasm hlorid C18ChromolithP pH 2.3 L
erformance
STP Huma HPLC UV 3-bromo- NR NR
columns(100 Acetonitrile/wa NR NR NR 0.25
n N- mm4.6 mm) ter(60:40,V/V) mg/l
plasma propylcinn
amide
STP Huma HPLC FLD Xanthone Isocrati Discovery® HS- 25 mM 1.5 50 4.6 0.05
n (Ex/Em: c C18 (150 mm 4.6 phosphate mg/m
Plasm 210/ 400 mm, 3 mm) buffer pH 2.6/ L
a nm)
RTG Huma HPLC UV (240 Flupirtine Isocrati C18 chromolith Water/acetonitr 1.5 50 3.2 25
n nm) maleate c performance (100 ile/ methanol ng/m
plasma mm 4.6 mm) (72:18:10, V/ L
V/V) with 0.1%
of 85%
phosphoric
acid.
 Drug Matrix Tech Detecti Internal Elution Column Mobile Flow Temp Retention LLO
nique on Standard Phase rate (℃) time(min Q

24
)

RTG Huma HPL UV NR Gradien Kromasil C18 20 mM 0.5 21-23 NR NR

n C (220nm t (250 mm 4.6 mm, potassium
plasma ) 5 mm) hydrogen
and phosphate
RTG Huma
urine HPL MS/MS D-10328 Isocrati LiChrospher 60, Acetonitrile/2.2
buffer pH 0.5 NR 2.118 1
n C (m/z (retigabine c RP-Select B (75 5 mM ng/m
plasm 274 / structural mm 4 mm, 5 mm) ammonium L
232) analogue) acetate pH 6.0
RFM Huma HPL MS/MS NR NR NR (55:45,
NR V/V) NR NR NR 40
n C mg/l
RFM plasma
Huma HPL MS/MS Lacosamid Isocrati Zorbax SB-C18 0.1% formic 0.3 40 2.6 40
n C (m/z e c (100 mm 3 mm, acid in water/ mg/m
Plasm 239 / 3.5 mm) methanol L
a 127) (50:50, V/V)
RFM Huma HPL UV NR Isocrati Supelcosil LC18 Acetonitrile/me 1.2 RT NR 50
from
n C (230 c (150 mm 4.6 mm, thanol/ 0.02 M ng/m
0 to
plasma nm) 5 mm) potassium 15 L
min;
hydrogen
2
phosphate from
15 to
(18:8:74,
30
V/V/V) min
Chromatographic columns and mobile phases
• During the development of new HPLC methods, steps like column (stationary
phase) selection and optimization of the mobile phase composition, pH and flow-
rate are critical.(15)
• The choice of these parameters is mostly based on the desirable peak characteristics
(height/area, tailing, shape, symmetry, separation, theoretical plates), run time and
solvent consumption.
• Methods that quantify the herein studied analytes in bulk and pharmaceutical
formulations using HPLC or UHPLC were mostly applied for RTG and RFM [],
but also, in a smaller proportion, for STP. In four of the six studies that quantified
RTG in bulk and/or pharmaceutical formulations, gradient elution programs were
used to pump different mobile phases through the chromatographic system.
• All the other HPLC or UHPLC methods for the determination of RTG, RFM and
STP in bulk and pharmaceutical formulations resorted to isocratic elution, with
chromatographic separations also achieved on reversed phase C18 bonded to silica
columns.
• The only case where a C18 column was not used was in which a chiral stationary
phase based on polysaccharides, linked to a silica matrix, was employed to obtain
the best conditions for STP enantiomers separation.
• Concerning the methods applied to biological samples analysis and bearing in mind
their increased complexity when compared with bulk and pharmaceutical
formulations, buffers as aqueous phase is usually required to obtain selectivity,
accuracy, precision, and sensitivity in shorter run time.
• According to article, the majority of the HPLC or UHPLC assays developed to
determine STP, RTG, RFM and PER in biological matrices applied isocratic elution
procedures.
• In fact, those that applied gradient elution programs either aimed the simultaneous
determination of different AEDs in a short run-time or were focused on
pharmacokinetic studies involving STP, RTG, N-acetyl-retigabine, RFM and PER.

25
 • Regarding the type of stationary phase used for biological matrices analysis, table
clearly shows that the chromatographic separation of STP, RTG, RFM and PER
has mostly been achieved using porous silica C18 reversed-phase columns.
• The use of guard-columns is also highly noticeable in many chromatographic
methods. The aim is mostly to increase column lifetime by protecting them from
quick deactivation and destruction, a situation that is more frequent when dealing
with biological samples.
• In fact, the robustness of HPLC methods is frequently compromised by the column
aging, usually accompanied by an increase of column pressure and an increase of
peak tailing, leading to oscillations in analytes retention time. Therefore, column
aging must be monitored, and the column must be changed particularly when
analytes signals decrease, a situation that is easily observed when low analytes'
concentrations are being measured.

Detection systems
• Different types of detection systems have been used in quantitative HPLC methods,
the most commonly being used the UV/ diode-array detectors (DAD), the
fluorescence detectors and several MS methodologies. UV detectors are the less
expensive detection systems but present poor selectivity as a disadvantage. (16)
• The use of a DAD is of great value by allowing the simultaneous detection of
several compounds at different wavelengths in the same chromatographic run.
• The information summarized in article RFM absorbs UV light at very distinct
wavelength ranges than STP and RTG, contrasting with the similarities between
UV absorbance of both RTG and STP.
• The lower limit of quantification (LLOQ) achieved was much higher than the those
in other methods that used MS or fluorescence detection, demonstrating the lower
sensitivity of UV detectors than that of the others used.
• In fact, since PER have native fluorescence, this detection type has been widely
used for its quantification in biological matrices.

26
 • To apply fluorescence detectors, a specific excitation wavelength must be set to
allow the passage of the analyte to a more energetic state that will further enable
the isolation of the desired emission wavelength. (17)

• In more recent years, MS and MS/MS detection systems have been becoming
widely used in the analysis of pharmaceutical compounds in biological matrices.

• Both systems offer a good selectivity and sensitivity, with MS/MS presenting an
additional selectivity generated by the specific analytes’ signatures as a result of
the precursor to product ions transition.

27
Conclusion
This review gathered important data related to LC methods developed, validated and
applied for determination of STP, RTG, N-acetyl-retigabine, RFM and PER in human
plasma as matrices. These analytical tools were developed not only to be applied in quality
control studies of pharmaceutical formulations but mostly, to support
pharmacodynamic and pharmacokinetic studies intended to investigate drug interactions,
toxicity and therapeutic efficacy of STP, RTG, RFM and PER. This range of applications
was only possible because HPLC techniques present several advantages, compared with
other analytical methodologies, such as good sensitivity, high resolution, versatility,
shorter analysis time per sample, and automation capability. Sample preparation methods,
chromatographic columns, mobile phases, and detectors selection are particularly
important to successfully achieve all those points. In the methodologies herein reviewed,
PP is the predominant technique used to prepare samples containing STP, RTG, N-acetyl-
retigabine, RFM or PER. Regarding the physicochemical properties of the analytes here
addressed, and particularly considering their polarity and hydrophobicity, the majority of
reported HPLC methods used a reversed-phase separation, with the silica-based
C18 columns as stationary phase. Thus, the development of new and more cost-effective
methods for research purposes and clinical application is a continuous challenge.
Therefore, more work needs to be performed in order to quantify new generation
of AEDs such as STP, RTG and its active metabolite N-acetyl-retigabine, RFM and PER
in an easier and faster manner. The use of different types of samples, such as saliva and
urine, as well as the use of simpler and less time-consuming extraction procedures can be
a starting point to expand the knowledge on these new AEDs and make available novel and
improved bioanalytical tools for application in TDM.

28
References
[1] E. Ben-Menachem, Medical management of refractory epilepsy-Practical treatment
with novel antiepileptic drugs, Epilepsia 55 (2014) 3e8.

[2] S.S. Chung, K. Kelly, C. Schusse, New and emerging treatments for epilepsy: review
of clinical studies of lacosamide, eslicarbazepine acetate, ezogabine, rufinamide,
perampanel, and electrical stimulation therapy, J. Epilepsy Res. 1 (2011) 35e46.

[3] E. Burakgazi, J.A. French, Seminar in epileptology treatment of epilepsy in adults,

Epileptic Disord. 18 (2016) 228e239.

[4] S. De Biase, G.L. Gigli, A. Nilo, et al., Pharmacokinetic and pharmacodynamic

considerations for the clinical efficacy of perampanel in focal onset seizures, Expet Opin.
Drug Metabol. Toxicol. 15 (2018) 93e102.

[5] P. LaPenna, L.M. Tormoehlen, The pharmacology and toxicology of thirdgeneration

anticonvulsant drugs, J. Med. Toxicol. 13 (2017) 329e342.

[6] M. Shaju, S. Abraham, Innovations in epilepsy management - an overview, J. Pharm.

Pharmaceut. Sci. 16 (2013) 564e576.

[7] M.D. Krasowski, G.A. McMillin, Advances in anti-epileptic drug testing, Clin. Chim.
Acta 436 (2014) 224e236.

[8] L. Santulli, A. Coppola, S. Balestrini, et al., The challenges of treating epilepsy with 25
antiepileptic drugs, Pharmacol. Res. 107 (2016) 211e219.

[9] L.G.M. van Rooij, M.P.H. van den Broek, C.M.A. Rademaker, et al., Clinical
management of seizures in newborns: diagnosis and treatment, Pediatr, Drugs 15 (2013)
9e18. [10] S. Stefanovic, S.M. Jankovic, M. Novakovic, et al., Pharmacodynamics and
common drug-drug interactions of the third-generation antiepileptic drugs, Expert Opin.
Drug Metab. Toxicol. 14 (2018) 153e159.

[11] M.V. Spanaki, G.L. Barkley, An overview of third-generation antiseizure drugs:

clobazam, lacosamide, rufinamide, and vigabatrin, Neurol. Clin. Pract. 2 (2012) 236e241.

29
[12] M. Mula, Third generation antiepileptic drug monotherapies in adults with epilepsy,
Expert Rev. Neurother. 16 (2016) 1087e1092. [13] J.W. Wheless, B. Vazquez,
Rufinamide: a novel broad-spectrum antiepileptic drug, Epilepsy Current 10 (2010) 1e6.

[14] A.G.B. Thompson, H.R. Cock, Successful treatment of super-refractory tonic status
epilepticus with rufinamide: first clinical report, Seizure 39 (2016) 1e4.

[15] P. Striano, R. McMurray, E. Santamarina, et al., Rufinamide for the treatment of

Lennox-Gastaut syndrome: evidence from clinical trials and clinical practice, Epileptic
Disord. 20 (2018) 13e29.

[16] C. Chiron, M. Helias, A. Kaminska, et al., Do children with Dravet syndrome continue
to benefit from stiripentol for long through adulthood? Epilepsia 59 (2018) 1705e1717.

[17] C. Chiron, Stiripentol for the treatment of seizures associated with Dravet syndrome,
Expert Rev. Neurother. 19 (2019) 301e310.

[18] S. Jacob, A.B. Nair, An updated overview on therapeutic drug monitoring of recent
antiepileptic drugs, Drugs R. D. 16 (2016) 303e316.

[19] K.C. Nickels, E.C. Wirrell, Stiripentol in the management of epilepsy, CNS Drugs 31
(2017) 405e416.

[20] European Medicines Agency, Stiripentol (Diacomit), Summary of product

characteristics. https://www.ema.europa.eu/en/documents/productinformation/diacomit-
epar-product-information_en.pdf. (Accessed 5 October 2020).

[21] E.P. Yıldız, M.U. Ozkan, T.A. Uzunhan, Efficacy of stiripentol and the clinical
outcome in Dravet syndrome, J. Child Neurol. 34 (2019) 33e37.

[22] C.M. Amabile, A. Vasudevan, Ezogabine: a novel antiepileptic for adjunctive

treatment of partial-onset seizures, Pharmacotherapy 33 (2013) 187e194.

[23] European Medicines Agency, Retigabine (Trobalt), Summary of product

characteristics. https://www.ema.europa.eu/en/documents/productinformation/trobalt-
epar-product-information_en.pdf. (Accessed 5 October 2020).

30
[24] O. Takenaka, J. Ferry, K. Saeki, et al., Pharmacokinetic/pharmacodynamic analysis
of adjunctive perampanel in subjects with partial-onset seizures, Acta Neurol. Scand. 137
(2017) 400e408.

[25] P.N. Patsalos, E.P. Spencer, D.J. Berry, Therapeutic drug monitoring of antiepileptic
drugs in epilepsy: a 2018 update, Ther. Drug Monit. 40 (2018) 526e548.

[26] L.A. Rudzinski, N.J. Velez-Ruiz, E.R. Gedzelman, et al., new antiepileptic drugs:
focus on ezogabine, clobazam, and perampanel, J. Invest. Med. 64 (2016) 1087e1101.

[27] A. Gil-Nagel, S. Burd, M. Toledo, et al., A retrospective, multicentre study of

perampanel given as monotherapy in routine clinical care in people with epilepsy, Seizure
54 (2018) 61e66.

[28] T. Tomson, S.I. Johannessen, Therapeutic monitoring of the new antiepileptic drugs,
Eur. J. Clin. Pharmacol. 55 (2000) 697e705.

[29] D. Milosheska, I. Grabnar, T. Vovk, Dried blood spots for monitoring and
individualization of antiepileptic drug treatment, Eur. J. Pharmaceut. Sci. 75 (2015) 25e39.

[30] P.N. Patsalos, D.J. Berry, B.F.D. Bourgeois, et al., Antiepileptic drugs - best practice
guidelines for therapeutic drug monitoring: a position paper by the subcommission on
therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies, Epilepsia 49
(2008) 1239e1276.

[31] S.I. Johannessen, T. Tomson, Pharmacokinetic variability of newer antiepieptic

drugs. When is monitoring needed? Clin. Pharmacokinet. 45 (2006) 1061e1075.

[32] P.N. Patsalos, M. Zugman, C. Lake, et al., Serum protein binding of 25 antiepileptic
drugs in a routine clinical setting: a comparison of free non eprotein-bound concentrations,
Epilepsia 58 (2017) 1234e1243.

31