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TLC determination of tiapride hydrochloride and its impurities in

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Article  in  Journal of Liquid Chromatography & Related Technologies · June 2012

DOI: 10.1080/10826076.2012.675870

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Journal of Liquid Chromatography & Related Technologies, 35:1336–1345, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2012.675870

TLC DETERMINATION OF TIAPRIDE HYDROCHLORIDE

AND ITS IMPURITIES IN PHARMACEUTICALS

Katarina Ranković, Slavica Filipić, Katarina Nikolić, and Danica Agbaba

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade,
Belgrade, Serbia

& A simple and sensitive HPTLC method for separation and determination of tiapride hydro-
chloride and its degradation products has been developed and evaluated. Analysis of tiapride
hydrochloride and impurities III, VII, and VIII, was performed on silica gel 60 F254 HPTLC plates
using methylene chloride-methanol-concentrated ammonia, 9:1.6:0.1 (v=v) as mobile phase. Detec-
tion was performed at 240 nm. All validation requirements, specificity, linearity (r
0.997), recov-
ery (95.08–100.39%), limit of quantification (0.1–0.2%), and robustness were examined and
fulfilled, and the proposed method can be successfully applied for the quality control analysis of
commercially available tablets and injections.

Keywords dosage forms, HPTLC, impurities, pharmaceutical preparations, quantifi-

cation, tiapride hydrochloride

INTRODUCTION
Tiapride (N-[2-(diethylamino)ethyl]-2-methoxy-5-(methylsulfonyl)-
benzamide) hydrochloride) is a substituted benzamide with the D2=D3
dopamine receptor antagonist activity and it belongs to the atypical antipsy-
chotics with general properties similar to those of sulpiride. It is used in the
treatment of behavioral disorders and dyskinesia, Tourette’s syndrome,
Huntington’s disease, severe persistent pain, and alcohol abuse and
dependence.[1–3]
Different analytical methods have been developed for determination of
tiapride in biological samples and pharmaceutical dosage forms. High per-
formance liquid chromatography (HPLC)[4] and hydrophilic interaction
liquid chromatography-tandem mass spectrometry (HILIC-MS=MS)[5] have
been used for determination of tiapride in human plasma. Concentrations

Address correspondence to Danica Agbaba, Department of Pharmaceutical Chemistry, Faculty

of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia. E-mail: danica@
pharmacy.bg.ac.rs
 Determination of Tiapride Hydrochloride and Impurities 1337

of tiapride in serum and urine in patients with tardive dyskinesia and

Huntington’s disease were measured by the SP-HPLC method, in order
to assess pharmacokinetics of drug in a clinical situation.[6] Capillary
electrophoresis (CE) with cyclodextrin additives,[7] gas chromatography=
ion trap mass spectrometry (GC=MS),[8] gas chromatography with surface
ionization detector,[9] spectrofluorimetry,[10] and column-switching
HPLC[11] were applied for determination of tiapride in the presence of
other drugs. The SPE-HPLC-PDA-FL method for the determination of
tiapride and phase I metabolite was developed, validated, and applied to
xenobiochemical and pharmacokinetic studies in humans and animals.[3]
Tiapride is a very stable substance, but it can be degraded under certain
conditions. Hydrolysis under the forced acidic and basic conditions leads to
the hydrolysis of the amide bond to give 2-metoxy-5-methylsulphonylben-
zoic acid (impurity III) and to the cleavage of the methoxy group (which
is a vinylogous ester and therefore activated to hydrolytic cleavage by the
virtue of being ortho to the ester functionality and para to the sulphone),
to yield N-(-2-diethylaminoethyl)-2-hydroxy-5-methylsulfonylbenzamide
(impurity VII). Also, tiapride can be oxidized under relatively drastic con-
ditions to give N-oxide, that is, N-(2-diethylaminoethyl)-2-methoxy-
5-methylsulphonylbenzamide-N0 -oxide (impurity VIII) (Figure 1).
A limited amount of research was found dealing with the examination
of the tiapride impurities. Capillary zone electrophoresis was applied
for the separation and determination of tiapride hydrochloride and its two
synthetic impurities, 2-metoxy-5-methylsulphonylbenzoic acid and 2-metoxy-
5-methylsulphonylbenzoate.[12] Four different stability-indicating proce-
dures, second derivative (D2), first derivative of the ratio spectra (1DD),
spectrofluorimetry, and high performance column liquid chromatography

FIGURE 1 The chemical structure of tiapride and its degradation products.

1338 K. Rankovic´ et al.

(LC), were employed for determination of tiapride in the presence of its

acid-induced degradation products, 2-methoxy-5-(methylsulfonyl) benzoic
acid and 2-diethylaminoethylamine.[13]
For the tiapride purity testing, European Pharmacopoeia recommends
high-performance liquid chromatography for the quantitative analysis of
2-metoxy-5-methylsulphonylbenzoic acid and methyl 2-metoxy-5-methylsul-
phonylbenzoate, while N,N-diethylethane-1,2-diamine is devoid of the UV
chromophore and it can be semi-quantitatively determined by thin-layer
chromatography and visualization of the chromatogram by spraying with
the ninhydrin solution.[14]
In the literature, no reports are available on the thin-layer chro-
matographic methods for determination of tiapride and its degradation
impurities.
In this paper, we present a simple, rapid, accurate, and precise HPTLC
method for simultaneous determination of tiapride hydrochloride and its
three degradation impurities (III, VII, and VIII), which have to be moni-
tored in pharmaceutical dosage forms according to the manufacturer
requirements of 0.2%.

EXPERIMENTAL
Materials
Tiapride hydrochloride (N-[2-(diethylamino)ethyl]-2-methoxy-5-(methyl-
sulfonyl) benzamide) hydrochloride), 2-methoxy-5-(methylsulfonyl) benzoic
acid (impurity III), N-(2-diethylaminoethyl)-2-hydroxy-5-(methylsulfonyl)
benzamide (impurity VII), and N-(2-diethylaminoethyl)-2-methoxy-5-(methyl-
sulfonyl) benzamide-N-oxide (impurity VIII) were kindly donated by Sanofi-
Synthelabo (France).
Tiapridal tablets and Tiapridal injections were manufactured by Sanofi-
Synthelabo (France). All solvents used were of an analytical grade.

Solutions

Standard Solutions
Stock solutions of standard substances were prepared in the following
way: Tiapride hydrochloride (27.81 mg tiapride hydrochloride, equivalent
to 25 mg tiapride, was dissolved in a 25-mL volumetric flask to obtain
1 mg mL1 solution), and impurities III, VII, and VIII (0.1 mg mL1) were
prepared in chloroform.
 Determination of Tiapride Hydrochloride and Impurities 1339

For the calibration curves, a series of six solutions were prepared from
the stock solutions in the concentration range from 0.3 to 0.7 mg mL1 for
tiapride, and from 0.02 to 0.1 mg mL1 for impurities III, VII, and VIII.

Sample Solutions
Ten tablets containing 100 mg tiapride in the form of tiapride hydro-
chloride were weighted and pulverized. The quantity of the powdered
tablets equivalent to the average mass of one tablet was transferred to the
10-mL volumetric flask and dissolved in 5 mL chloroform by use of an ultra-
sonic bath for 10 min. The solution was then diluted to the volume with the
same solvent and filtered through the 0.45-mm pore size membrane filter
(Millipore). The filtrate with the 10 mg mL1 concentration of tiapride
was applied to the plate for the assay of the impurities.
Likewise, 2 mL tiapride injections equivalent to 100 mg tiapride in the
form of tiapride hydrochloride was dissolved in water, then transferred to
the 10-mL volumetric flask and diluted to volume with methanol. The
obtained solution containing 10 mg mL1 tiapride was used for the assay
of the impurities.
For the tiapride assay, solutions of tablets and injections in the concen-
tration of 0.5 mg mL1 tiapride were used.

Chromatography
HPTLC was performed on the 20 cm  10 cm plates, precoated with sil-
ica gel 60 F254 (Merck, Darmstadt, Germany). The standards and sample
solutions were applied to the plates in the quantities of 1 mL by use of a
Linomat IV automatic TLC applicator (Camag, Muttenz, Switzerland).
Ascending chromatography with methylene chloride-methanol-concen-
trated ammonia 9:1.6:0.1 (v=v) as the mobile phase was performed in
the twin-trough TLC chamber previously saturated for 20 min with the
mobile phase vapor. The developed plates were dried in air and chromato-
grams were recorded by scanning at 240 nm with a Camag Scanner II in the
absorbance=reflectance mode.

RESULTS AND DISCUSSION

Optimization of chromatographic conditions in order to gain a
reproducible method for the separation of tiapride and its degradation
impurities, involves adequate selection of the appropriate stationary and
mobile phase.
Mixture of methylene chloride-methanol-concentrated ammonia in the
ratio 9:1.4:0.2 (v=v) was selected at an initial stage. Satisfactory resolution
1340 K. Rankovic´ et al.

was observed, but with too strong retention of impurity III. Due to the
presence of the carboxylic moiety in the structure of impurity III, high
affinity of this compound to the stationary phase and the low peak
symmetry were the critical factors in the optimization process. To obtain
a satisfactory resolution and retention, and to avoid the peak tailing and
asymmetry, alkalinity of mobile phase was decreased and the volume of
methanol was increased until the optimal conditions were achieved. Final
composition of mobile phase used for validation of the method was
methylene chloride-methanol-concentrated ammonia in the ratio
9:1.6:0.1 (v=v).
The effect of different stationary phases was investigated by use of con-
ventional TLC, HPTLC, and LiChrospher Si 60 F254s plates. The sharpest
zones for all the investigated compounds and the best resolution were
achieved by use of the HPTLC plates.
After recording the UV spectra of tiapride hydrochloride and its impu-
rities in situ, the wavelength maximum of 240 nm was selected for all the
quantitative measurements (Figure 2).
Reproducibility of the separation by use of the selected HPTLC method
was confirmed by the low relative standard deviation of the migration
distances for all examined substances (MD  RSD): 18.6 mm  0.91%,
24.8 mm  1.21%, 31.6 mm  0.44%, and 60.6 mm  1.2% for impurities
III, VII, VIII, and tiapride hydrochloride, respectively. The HPTLC chroma-
tograms illustrating separation of tiapride from the three degradation
impurities are shown in Figure 3.

FIGURE 2 In situ UV spectra of tiapride hydrochloride (1), impurity III (2), impurity VII (3), and
impurity VIII (4).
 Determination of Tiapride Hydrochloride and Impurities 1341

FIGURE 3 Densitograms obtained from standard of impurity III (1); standard of impurity VII (2);
standard of impurity VIII (3); mixture of tiapride and 0.2% impurities III, VII, and VIII (4); mixture
of tiapride and 0.4% impurities III, VII, and VIII (5); sample of Tiapridal tablets (6); and sample of
Tiapridal injections (7).

Once the optimal separation conditions have been established, vali-

dation of the method was performed according to the ICH guidelines.[15,16]
Specificity, linearity, precision, accuracy, limit of detection (LOD), limit of
quantification (LOQ), and robustness were tested during the validation
process.
Specificity of the method was investigated by examining potential interfer-
ences between tiapride hydrochloride and its impurities with the tablet excipi-
ents. The solution of the tablet excipients (placebo) and the solution of
placebo spiked with tiapride hydrochloride and with the examined impurities
were tested. No interfering peaks were observed on the chromatograms of the
placebo solution and the four well separated peaks were observed on the
chromatograms of the solution of placebo spiked with tiapride hydrochloride
and its impurities. Therefore, the specificity of the method was proven.
The linearity was evaluated with the six different concentrations within
the range from 300 to 700 ng per band for tiapride and from 20 to 100 ng
per band for impurities III, VII, and VIII. The calibration curves were con-
structed for the dependences between the peak area and the amount of the
substance applied, and the obtained regression equations are presented in
Table 1. The linear relationships were proven for all the analyzed com-
pounds with the correlation coefficients (r) above 0.997. Also, the residuals
were randomly distributed around the regression line and did not show any
tendency to exponential function.[16]
The repeatability of the method was assessed by chromatographing the
replicate applications (n ¼ 6) of tiapride hydrochloride and impurities III,
1342 K. Rankovic´ et al.

TABLE 1 Linearity Data

Substance Concentration Range (ng band1) Calibration Curve y ¼ ax þ b r

Tiapride 300–700 y ¼ 1.987 x þ 68.900 0.999

Impurity III 20–100 y ¼ 8.441 x þ 11.430 0.999
Impurity VII 20–100 y ¼ 5.379 x þ 10.790 0.997
Impurity VIII 20–100 y ¼ 7.194 x þ 2.608 0.998

VII, and VIII at the three different concentrations. Because the obtained
relative standard deviations for all the concentration levels were below
2%, the precision of the method was confirmed. The important statistical
values are given in Table 2.
Closeness of the measured value to the true value (accuracy) was calcu-
lated as the recovery (%) of the analyte spiked in the placebo mixtures. The
accuracy studies were performed at the three different concentrations of
each impurity. Three solutions at each concentration level (400, 500, and
600 ng band1 for tiapride hydrochloride, and 20, 40, and 60 ng band1
for impurities III, VII, and VIII) were freshly prepared and applied onto
the plates. The recovery values for all the concentrations were determined
by a comparison with the known amount added. The accuracy of the
method was proved, since the calculated percent recovery for every concen-
tration level was in the range (95.08%–100.39%) (Table 3).
The limit of detection (LOD) and the limit of quantification (LOQ)
were determined by fitting the interday, back-calculated standard deviation
of each calibration standard.
The y-intercept was then equal to SD0 (the estimated standard deviation
at the zero concentration). According to the defined LOD and LOQ values
(3SD0 and 10SD0, respectively), the sample mass applied to the plate at

TABLE 2 Precision of the Method

Compound Concentration (ng band1) Found Concentration (ng band1) RSD (%)

Tiapride 400 400.83  5.79a 1.44

500 488.13  4.39 0.90
600 605.67  9.85 1.63
20 20.5  0.37 1.80
Impurity III 40 39.42  0.38 0.96
60 60.18  0.26 0.43
20 20.57  0.41 1.99
Impurity VII 40 38.46  0.76 1.98
60 60.91  0.63 1.03
20 19.75  0.34 1.72
Impurity VIII 40 38.90  0.76 1.95
60 61.22  1.21 1.98
a
SD, Standard deviation. n ¼ 6.
 Determination of Tiapride Hydrochloride and Impurities 1343

TABLE 3 Accuracy of the Method Expressed as Recovery of the Analyte Spiked to Placebo Mixtures

Concentration Found concentration

Compound (ng band1) (ng band1) Recovery (%) RSD (%)

Tiapride 400 401.55 100.39 1.23

500 490.12 98.02 0.47
600 595.48 99.25 0.55
20 19.92 99.59 1.36
Impurity III 40 39.88 99.70 0.95
60 59.29 98.82 0.82
20 19.70 98.51 1.54
Impurity VII 40 39.54 98.85 1.70
60 57.05 95.08 1.16
20 19.85 99.27 2.59
Impurity VIII 40 39.88 99.70 2.12
60 57.29 95.49 1.29

which the analyte can be detected or quantified was established. LODs for
impurities III, VII, and VIII were found equal to 0.004, 0.007, and 0.006 mg
band1 (equivalent to the impurity levels of 0.04, 0.07, and 0.06% for impu-
rities III, VII, and VIII, respectively). LOQs for impurities III, VII, and VIII
were found equal to 0.01, 0.02, and 0.02 mg band1 (equivalent to impurity
levels of 0.1, 0.2, and 0.2% for impurities III, VII, and VIII, respectively).
Robustness is a measure of the capacity of the method to remain unaf-
fected by small but deliberate variation of the method conditions, and it is
an indication of the reliability of the method.[17] As planar chromato-
graphy is an ‘‘open’’ chromatographic method, the environmental con-
ditions can significantly influence its results. Thus, the temperature was
chosen as one of the critical factors that has to be tested. Apart from the
temperature changes, we also examined the influence of the contents of
methanol in mobile phase and the chamber geometry on resolution of
peaks. Temperature between 8 and 30 C, the contents of methanol
10% in mobile phase, the twin-trough and the flat bottom chamber had
no observable effect on the separation, since the resolution (RS) between
the critical peaks was in all cases above 1.6.

TABLE 4 Results of HPTLC Determination of Tiapride Hydrochloride and Its Impurities

Tiapride

Found Impurity Impurity Impurity

Sample Expected (mean  SD) III VII VIII

Tiapridal tablets 100 mg per tablet 97.44  1.7 n.d. n.d. n.d.
Tiapridal injections 100 mg in 2 mL 97.19  1.8 n.d. n.d. n.d.
of solution

n.d., not detected - concentration below the limit of detection of the method.
1344 K. Rankovic´ et al.

After evaluation of the proposed method, its applicability to determi-

nation of the contents of tiapride hydrochloride, and impurities III, VII,
and VIII was examined by analyzing the commercially available tablets
and injections. The high recovery values for tiapride obtained for the
pharmaceutical dosage forms and the low values of the relative standard
deviation obtained for the tiapride tablets and injections, confirm the
accuracy of the method (Table 4). No traces of impurities III, VII, and VIII
were detected in the examined dosage forms (Figure 3).

CONCLUSION
A simple, fast, and economical HPTLC method for the simultaneous
separation and determination of tiapride hydrochloride and its degradati-
on impurities III, VII, and VIII, was developed and validated. Sensitivity of
the method was proved at the nanogram level, and the obtained results
suggest that the proposed HPTLC method can be used as a precise and
reproducible stability indicating method for tiapride hydrochloride in
pharmaceutical dosage forms.

ACKNOWLEDGMENT
This work was supported by the Ministry of Science and Technological
Development of the Republic of Serbia, Contract No. 172033.

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