Vol. 14 | No.

4 |2236-2245| October- December | 2021

ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP
http://www.rasayanjournal.com
http://www.rasayanjournal.co.in

SIMULTANEOUS QUANTIFICATION OF TIAGABINE

AND ITS RELATED SUBSTANCE BY A STABILITY-
INDICATING RP-HPLC METHOD
V. N. V. Kishore1, G.V. Ramana2, R. Venkata Nadh3, and Giri Prasad
Gorumutchu4
1
Department of Chemistry, AG&SGS College, Vuyyuru-521165, India
2
Department of Chemistry, Andhra Loyola College, Vijayawada-520008, A.P., India
3
Industrial Chemical Product Development and Analysis Centre,
GITAM Deemed to be University - Bengaluru Campus, Karnataka-561203, India
4
Department of Chemistry, AG&SGS College, Vuyyuru-521165, India

Corresponding Author: doctornadh@yahoo.co.in
ABSTRACT
The proposed study is the development of the RP-HPLC method for simultaneous quantification of tiagabine (TBN)
and its related substance – A (TBN RS-A). The method was validated for its applicability both in bulk drug and
tablet dosage form. For an isocratic elution, a mobile phase comprising of methanol: 0.1 mM acetate buffer mixture
(65:45 v/v, pH 5.6) was used at 1 ml/min flow rate and ProntoSIL ODS C18 column (250mm × 4.5 mm; 5µm)
column. At 240 nm as max, sharp peaks of TBN RS-A and TBN were observed at 3.7 and 5.2 min respectively. As
per the ICH guidelines, the method was validated. Validation of the method was done as per the guidelines of ICH.
Linear regression for the calibration curve was carried out for concentration ranges of 75-450 and 1-6 µg/mL
respectively for TBN and TBN RS-A. The respective detection limits (LOQ and LOD) of the TBN and RS-A were
found to be (2.014 and 0.032 µg/ml) and (6.103 and 0.098 µg/ml). The method was successfully extended for
stability-indicating studies under different stress conditions.
Keywords: Tiagabine, Related Substance A, HPLC Analysis, Method Validation, Forced Degradation.
RASĀYANJ. Chem., Vol. 14, No.4, 2021

INTRODUCTION
Tiagabine (TBN) is a well-known drug to treat epilepsy. It is effectively used for patients having
refractory focal seizures as an adjunctive therapy. TBN mechanism of action is demonstrated from its
inhibition of the uptake of GABA (γ-aminobutyric acid). In the form of its hydrochloride, the tablet
dosage forms available in the market are 2.5, 5, 10, and 15 mg. In focal seizures, its efficacy was
established with the given divergent mechanism when compared to other antiepileptic drugs. The time
duration of its peak levels is 0.5 to 2.3 hours.1A comparative study on the low and high dosages (6 to 36
mg/day) has revealed that the adverse effects related to CNS are dosage dependent. 2 Similarly, adverse
effects are also affected by the frequency of dosage. This conclusion was drawn based on the tolerability
studies in patients with refractory focal seizures where severe adverse effects were noticed with 2 times
daily dosage of TBN compared to and 3 times as a part of adjunctive therapy.3 Hence, it is advised the
initial dosage level as 4 or 5 mg/day with two to four dosing frequencies per day. The dosage increment is
4 or 5 mg/day on weekly basis. To avoid a sudden rise in plasma concentrations, TBN supplementation
should be done along with feeding. Multiple dosages per day are the major drawback of this drug because
of its tiny half-life. Five to nine hours of the half-life of elimination is observed, however, co-medication
involving inducing of enzymes will further reduce its half-life to two to four hours. Promoted clearance
can be observed in children.4,5
Due to stimulation of its metabolism, tiagabine clearance is improved by antiepileptic drugs, capable of
inducing the enzymes. CYP3A4 cytochrome mediates the oxidative metabolism of TBN which leads to
purging. The use of tiagabine in unclassified epilepsies and generalized epilepsy syndromes should be

Rasayan J. Chem., 14(4), 2236-2245(2021)

http://doi.org/10.31788/RJC.2021.1446412 This work is licensed under a CC BY 4.0 license.
 Vol. 14 | No. 4 |2236-2245| October- December | 2021

avoided, and off-label use in other indications is discouraged. Given prospective induction of seizures as
well as slow-wave action in the electroencephalogram, TBN usage is put off in off-label usage and
unclassified epilepsies. Other adverse effects include CNS, dizziness, mood depression, etc. Given the
prospective improvement in the occurrence of adverse effects, close monitoring of patients is required
who suffer from hepatic function impairment.6 And also better to avoid the usage of TBN in patients with
impaired liver function.
Tiagabine (TBN) belongs to a subclass of piperidinecarboxylic acid derivative and is white to beige
powder. It can be derived from (R)-Nipecotic acid. Its chemical name is “(3R)-1-[4,4-bis(3-
methylthiophen-2-yl)but-3-enyl]piperidine-3-carboxylic acid”. Its hydrochloride empirical formula is
C20H25NO2S2.HCl with a molecular weight of 412.0. The related substance compound A is chemically
known as R-(-)- 1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]- 3-Piperidine ethyl carboxylate hydrochloride
with chemical formula C22H29NO2S2.HCl and molecular weight of 440.06 g/mol. The chemical structures
of TBN and TBN RS –A are presented in Figure 1. 0.91 is the conversion factor to translate the dosage
from its hydrochloride form to free base. It exhibits good solubility inpolar solvents (freely soluble in
water) and is almost insoluble in a non-polar solvent like n-heptane.7

A B
Fig.-1: (A) Chemical Structure of Tiagabine (TBN) and (B) Related Compound
The literature survey revealed various types of analytical methods for the quantification of TBN. Callen et
al and Chorghade et al8,9 synthesized the key degradation products of TBN tablets and from TBN
oxidation. Chollet et al10 monitored the antiepileptic drug – TBN by GC-MS assay method. Rustum et al11
proposed a chiral HPLC method to quantify the S-(+)-enantiomer of TBN in its bulk drug and to separate
its S (+), R (−)-enantiomers as well as two chiral precursors. Hubert‐Roux et al12 identified degradation
products of TBN using LC-MS-MS and UPLC-HRMS. Rajasingam et al13 studied the stress degradation
and proposed an RP-HPLC method to determine TBN in presence of its degradation products. Vaishnav
et al14 proposed a stability-indicating RP-HPLC method for the determination in tablet dosage of TBN.
The concentration of TBN in human plasma was determined by Gustavson and Chu 15 using HPLC with
the electrochemical detector and an HPLC micro method was proposed by Ratnaraj and Patsalos. 16
Similarly, TBN in serum was quantified by using LC-MS/MS17 and LC- diode-array detector.18
Nagalakshmi et al19 determined TBN in Pharmaceutical Formulations by spectrophotometry.
Some of the methods proposed in the literature involve the usage of expensive instruments (like LC-MS-
MS and UPLC-HRMS) and most of the methods didn’t pay attention to impurity profiling. Monitoring
the impurity profile is important to maintain the quality of drugs and patient safety. The intrinsic stability
of the drug substances, as well as drug product, can be understood from the forced degradation studies
(FDS)20,21. Hence, the present study is an effort is to develop a stability-indicating HPLC method for
simultaneous quantification of TBN with its related substance-A. So that the quality in the pharmaceutical
industry can be regulated to release the drug into the global market.
EXPERIMENTAL
Material and Methods
Analytical grade sodium acetate and acetic acid were used in the present study and purchased from Fisher
Scientific, Mumbai. CH3CN and CH3OH (HPLC grade) were procured from Merck chemicals, Mumbai.
Milli-Q water obtained from Ultrapure Water Purification System (Merck Millipore- Germany) was used
in this study. Standard drug Tiagabine hydrochloride and its Impurity A were obtained from Sun
Pharmaceutical Industries Ltd., Mumbai.
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Instrumentation
Analysis was carried on Agilent HPLC (California, United States) equipped with a quaternary pump (G
1311), Thermostatic auto (G 1329A) sampler of sample volume capacity of 0. 1 – 1500 µL, thermostat
column temperature control (COLCOM G1316A) and, UV detector (G 1314). Agilent chem station LC
software was used to analyze the column eluents. U-2900/2910 Double Beam Spectrophotometer, Hitachi
was used to record the UV-Visible spectra. Ultrasonicator (1.5L) was used to sonicate the mobile phase
and to prepare the sample solutions. Digital pH meter (Systronics make) and electronic analytical balance
(Denver make, SI-234) were used.

General Procedures
Preparation of standard solutions: Ten and one mg of standard drug – Tiagabine hydrochlorideand its
impurity – A were weighed accurately and transferred to separate 10 ml volumetric flasks. Sonication was
done to dissolve completely in methanol. A nylon membrane filter (0.45 μm) was used to filter and made
up to the mark with the same solvent. These stock solutions were suitably used in the present method.
Method validation and forced degradation studies: The final optimized conditions are validated as per
existing guidelines (Q2R1, ICH2005)22 to verify the method reliability. All the stress samples were diluted
appropriately to prepare the final concentration containing the drug with proposed method conditions
(TBN of 300 µg/ml) and compared with standard and blank chromatograms.

RESULTS AND DISCUSSION

Method Development
Based on the absorption maxima observed for TBN and RS-A the detector wavelength was set at 240 nm
(Fig.-2) for simultaneous quantification of tiagabine (TBN) and its related substance- A (TBN RS-A).

Fig.-2: The UV-Visible Spectrum of TBN and TBN-RS-A

The isocratic elution with mobile phase with different compositions was optimized along with other
parameters (like column, mobile phase composition, buffers, pH, flow rate, wavelength, etc) until the
good separation of TBN and TBN RS-A peaks along with acceptable system suitability conditions. Few
representative chromatograms of method development are shown in Fig.-3 with an isocratic flow rate of
1.0 ml/min, Zodiac ODS C18 column (250 mm × 4.5 mm; 5µm) (trials - a and b), ProntoSIL ODS C18
column (250 mm × 4.5 mm; 5µm) (trials – c, d, e and optimized condition), Methanol: Acetonitrile
(50:50 and 80:20 v/v for trials - a and b), Methanol: water (50:50 and 60:40 v/v for trials – c and d),
Methanol: 0.1 mM Acetate buffer (60:40 and 65:45 v/v for trials –e and optimized condition) and pH (4.5
for trial-c and 5.6 for trials-c, d, e and optimized conditions).
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(a) (b)

(c) (d)

(e)
Fig.-3: Trial Chromatograms for Method Development
The peaks of TBN and TBN-RS-A were not separated initially (Fig.-3a and b). Both the shape and
resolution of a peak are strongly influenced by the mobile phase pH. Due to the acidic nature of TBN, the
maintenance of pH below and above the analyte’s pKa will lead to the maintenance of analyte in
unionized and ionized forms respectively.23-26 Then, choose the mobile phase with acidic pH, by taking
into consideration of the pKa value, solubility, and polarity of TBN and RS-A. A split in the peak was
observed at pH 4.5 (Fig.-3c), which can be understood from the fact that pKa1 of TBN (w.r.t. carboxylic

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acid)27 is 3.56 and peak splits or shoulders are observed when the mobile phase pH is near to the pKa of
the analyte. pH was raised to 5.6 because the best results are obtained (Fig.-3d) by maintaining the pH of
the buffer by two units minimum away from the required analyte’s pKa.28-29

Fig.-4: Chromatogram of a Standard Solution of TBN and RS-A under the Proposed Condition

However, to convert the broader peaks to sharp peaks, the composition of the mobile phase was changed
to Methanol: 0.1 mM Acetate buffer (60:40 v/v) (Fig.-2e). Fine-tuning was done to achieve optimized
conditions (Table-1) and the obtained standard chromatogram is presented in Fig.-3.
Table-1: Optimized Chromatographic Conditions
S. No. Parameter Optimized Condition
Column ProntoSIL ODS C18 column
1
(250 mm × 4.5 mm; 5µm)
2 Mobile Phase Methanol: 0.1 mM Acetate buffer (65:45 v/v)

3 Mobile phase pH 5.6

4 Mobile phase flow rate (mL/min) 1.0

5 Elution Isocratic
6 Wavelength (nm) 240
7 Sample volume 20 μL

System Suitability and Specificity

Substantiated the performance of the system from the obtained system suitability parameters and the
corresponding tabulated parameters were satisfactory (Table-2). In addition, non-interference of either
diluent or placebo was observed at the retention times of TBN and TBN-RS-A.
Table-2: System Suitability Conditions
Results Observed
Parameter
Tiagabine Impurity A
API Concentration (µg/ml) 300 4
Retention time (min) 5.2 3.7
Peak Area (mV) 498876 76124
Resolution 5.15 --
Theoretical plates 4890 3462
Tailing factor 1.08 0.99
Stability Indicating Studies
A forced degradation study was conducted to understand the stability-indicating nature as well as the
specificity of the current method. No interference or well separation of the peaks corresponding to both
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TBN and its degradation products is evident from the chromatograms of degradation studies under
different stress conditions (Fig.-5). The sensitivity of the TBN towards acidic and alkaline conditions is
established from the high percentage of degradation. Similarly, TBN is less susceptible to oxidation and
thermal degradation conditions (Table-3).

(a) (b)

(c) (d)

(e)
Fig.-4: Chromatograms of Stability Indicating Studies under Stress Conditions a) Acidic, b) Alkali, c) Peroxide, d)
Thermal and e) UV condition
Method Validation
Validated the above-optimized method for simultaneous quantification of tiagabine (TBN) and its related
substance – A (TBN RS-A) as per the existing guidelines (Q2R1, ICH2005). 22
Linearity
Calibration graphs were drawn by plotting the average response of three measurements against the
concentration (75 to 450 µg/ml TBN and 1 to 6 µg/ml TBN-RS-A), where a good correlation coefficient
was observed with R2> 0.999 (Fig.-6).
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Table-3: Results of Forced Degradation Studies

Amount of
% Degradation
Degraded
Parameter Stress Conditions w.r.t. Control
Sample (A)
Sample*(B)
(µg/mL)
Control Sample
No Exposure 302.31 NA
(No Degradation)
1 mL of 0.5 N HCl for 12 hr at
Acid Degradation
room temperature 268.83 10.10
1 mL of 0.5 N NaOH for 12 hr
Base Degradation
at room temperature 271.66 10.06
2 mL of 10% H2O2 for 1 hr at
Oxidation
room temperature 288.14 4.61
Thermal degradation 70 °C for 48 hrs 278.61 7.76
Photolytic degradation (UV) 200-Watt hours/square meter 264.79 9.15
*B=(302.31-A)/302.31*100
120000
Tiagabine Impurity A
800000
100000
700000
Peak Area
600000 80000
Peak Area

500000 y = 18511x + 2142.9

60000 R² = 0.999
400000
y = 1542.9x + 31152
300000 40000
R² = 0.999
200000
20000
100000
0 0
0 100 200 300 400 500 0 1 2 3 4 5 6 7
Concentration in µg/ml Concentration in µg/ml
Fig.-6: Calibration Graphs of TBN and RS-A
Method Precision (M.P.) and Intermediate Precision (I.P.) (Ruggedness)
Six standard samples (containing a mixture of 300 µg mL-1 of TBN and 4 µg mL-1 of TBN-RS-A) were
injected and the average values of system suitability parameters were noted (Table-4). Under similar
conditions, M.P. and I.P. were carried to compare the precision values (Table-5). The precision of the
method is confirmed from the statistical results (% RSD < 1 for both TBN and TBN-RSA). Percentage
RSD values of the M.P. and I.P. (0.189 and 0.231) of the present method are better than values reported in
the literature by Rajasingam et al13 (0.48 and 1.53) and Vaishnav et al14 (0.376 and 0.834).
LOD and LOQ
The lower detection and quantification values (TBN and TBN-RS-A) were found to be (2.014 and 0.032
µg/ml) and (6.103 and 0.098 µg/ml). The method is sensitive as the LOD and LOQ values were found to
be well below the specified limit.
Robustness
Chromatographic method conditions (mobile phase ratio, pH, and wavelength) were intentionally altered
to verify whether the proposed method is robust or not. As the system suitability parameters (RT (Min),
USP resolution, Theoretical plates, and Peak area) are unaffected (Table 6), the robustness of the method
is proved.
Table-4: Comparison of System Suitability Parameters in Precision Experiments
System Suitability M.P. I.P.
Parameter Tiagabine IMP A Tiagabine IMP A
USP resolution 5.14 - 5.14 -
USP tailing factor 1.06 0.96 1.07 0.961
USP plate count 4764 3405 4840 3388
Retention time (min) 5.28 3.65 5.25 3.64
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Peak area 497160 75869 497596 76448

SD of area 937.94 182.41 1147 423
% RSD of area 0.189 0.240 0.231 0.552
* from six standard injectionsat 300 µg mL–1 of Tiagabine and 4 µg mL–1 of Imp-A

Table-5: Comparison of Precision Values

% Assay
S. No. Tiagabine Impurity A
M.P. I.P. M.P. I.P.
1 99.48 99.72 99.59 100.53
2 99.70 99.22 100.03 100.55
3 99.53 99.81 99.40 99.38
4 99.35 99.94 99.21 100.89
5 99.95 99.70 99.61 100.94
6 99.79 99.96 99.57 99.82
Mean 99.73 99.63 100.35 99.57
Std. Dev 0.245 0.201 0.570 0.246
% RSD 0.246 0.202 0.568 0.247
*at 300 µg mL–1 of Tiagabine and 4 µg mL–1 of Imp-A
Table-6: Results of Robustness
% Change
Altered Actual Altered RT USP Theor Peak
in Peak
Parameter Condition Condition (Min) Resolution plates Area
Area
Tiagabine
Control
NA --- 5.27 5.15 4890 498876
Condition ---
Mobile phase 60:40 5.25 5.17 4781 499687 0.16
65:45
ratio (v/v)* 70:30 5.25 5.12 4896 497869 0.20
5.7 5.25 5.19 4769 497970 0.18
pH 5.6
5.5 5.28 5.12 4849 497585 0.26
Wave 243 5.25 5.13 4436 499076 0.04
240
length (nm) 253 5.28 5.18 4749 497425 0.29
Impurity-A
Control
NA --- 3.75 - 3462 76125 ---
Condition
Mobile phase 60:40 3.65 - 3362 76759 0.83
65:45
ratio* 70:30 3.63 - 3440 76588 0.61
5.7 3.62 - 3352 76026 0.13
pH 5.6
5.5 3.68 - 3417 75964 0.21
Wave 243 3.60 - 3475 75877 0.33
240
length (nm) 253 3.70 - 3518 75887 0.31
* Methanol: 0.1M Acetate buffer (v/v)
Accuracy
The accuracy of the proposed method was established from the fact that %RSD values of TBN and TBN-
RS-A were less than 1% different levels of recovery (50%, 100%, and 150%), and the recovered amounts
were found to be closer to the taken values (Table-7).
CONCLUSION
Simultaneous determination of related substance –A along with the drug TBN using simple
chromatographic conditions is demonstrated in the current work. The method is sensitive as the LOD and
LOQ values were found to be well below the specified limit. The degradation impurities do not interfere
with the TBN and its RS, thus the method is stability-indicating. In the current method, evaluated the
factors which remarkably control the resolution of the peaks. The method conditions are successfully

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validated and found the successful application of the method for Tiagabine analysis and stability
indicating studies.
Table-7: Recovery Studies
Amount Recovered
Level of Statistical
(Practical) % Recovery
Recovery Evaluation Statistical Values
(µg mL-1)
(%)
Tiagabine Imp-A Tiagabine Imp-A Tiagabine Imp-A
225 3 97.65 98.91 % Mean 98.37 99.12
50 225 3 98.10 98.96 SD 0.677 0.320
225 3 98.45 99.49 %RSD 0.688 0.323
300 4 99.69 99.83 % Mean 99.45 100.52
100 300 4 99.00 100.89 SD 0.389 0.601
300 4 99.66 100.84 %RSD 0.391 0.598
375 5 99.80 99.83 % Mean 99.98 100.23
150 375 5 100.14 100.55 SD 0.169 0.369
375 5 99.99 100.324 %RSD 0.169 0.368

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