772 S. Mennickent et al. J. Sep. Sci.

2007, 30, 772 – 777

Sigrid Mennickent1 Short Communication

Loreto Pino1
Mario Vega2
Carmen Gloria Godoy1
Marta de Diego1
1
Department of Pharmacy,
Faculty of Pharmacy, University
of Concepci
n, Concepci
n,
Chile
2
Department of Bromatology,
Nutrition and Dietetic, Faculty
of Pharmacy, University of
Concepci
n, Concepci
n, Chile
Quantitative determination of haloperidol in tablets
by high performance thin-layer chromatography
A densitometric high performance thin-layer chromatography (HPTLC) method was
developed and validated for the quantitative analysis of haloperidol in tablets. Chro-
matographic separation was achieved on precoated silica gel F 254 HPTLC plates
using a mixture of acetone/chloroform/n-butanol/acetic acid glacial/water
(5:10:10:2.5:2.5 v/v/v/v/v) as the mobile phase. Quantitative analysis was carried out
at a wavelength of 254 nm. The method was linear in the 10–100 ng/lL range, with
a determination coefficient of 0.999. The coefficients of variation for precision were
not higher than 2.35%. The detection limit was 0.89 ng/lL, and the quantification
limit was 2.71 ng/lL. The accuracy ranged from 97.76 to 100.33%, with a CV not
higher than 4.50%. This method was successfully applied to quantify haloperidol in
real pharmaceutical samples, including the comparison with HPLC measurements.
The method was fast, specific, with a good precision and accuracy for the quantita-
tive determination of haloperidol in tablets.
Keywords: Drugs / Haloperidol / HPTLC / Pharmaceutical preparations / Quantitative analysis /
Received: October 4, 2006; revised: November 23, 2006; accepted: December 9, 2006
DOI 10.1002/jssc.200600408

1 Introduction
Haloperidol, a butyrophenone derivative, is used for the
symptomatic management of psychotic disorders. Its
chemical structure is shown in Fig. 1. Drug therapy is
integral to the management of acute psychotic episodes
and accompanying violent behavior in patients with
schizophrenia, and is generally required for a long-term
stabilization to improve symptoms between episodes
and to minimize the risk of recurrent acute episodes.
Haloperidol is often chosen as the agent to terminate
mania, and it is also used for the control of tics and vocal Figure 1. Chemical structure of haloperidol.
utterances of Tourette’s syndrome in children and
adults.
Other uses of haloperidol are for the treatment of delir- It is very important to determine the quantity of halo-
ium, disruptive behavior disorder, and attention deficit peridol in its dosage forms because photolytic degrada-
hyperactivity disorder in children who exhibit excessive tion can be an important limiting factor, resulting in
motor activity accompanying conduct, prevention, and drug loss and potency reduction [1–7].
control of severe nausea and vomiting; and for the treat- Some HPLC [8–10], spectrophotometry [11], and few
ment of alcohol dependence [1–4]. high performance thin-layer chromatography (HPTLC)
[12, 13] methods have been used to determine haloperi-
Correspondence: Professor Sigrid Mennickent, Department of dol in tablets. The official method for the quantitative
Pharmacy, Faculty of Pharmacy, University of Concepci
n, P. O. determination of haloperidol in this dosage form is by
Box 237, Concepci
n, Chile HPLC [6].
E-mail: smennick@udec.cl HPTLC is a technique carried out within a short period
Fax: +56-41-2207086
of time; it requires few mobile phases, and allows the
Abbreviations: HPTLC, high performance thin-layer chromato- analysis of a large number of samples simultaneously.
graphy The ability of HPTLC to analyze many samples in parallel

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J. Sep. Sci. 2007, 30, 772 – 777
has the advantage over other techniques because the sep-
aration of 10 or 20 samples takes the same time as the
separation of one sample. Amounts of the order of nano-
grams (UV) and smaller than picograms (fluorescence)
can be detected.
The other HPTLC methods found in the bibliography
for the quantitative determination of haloperidol in
tablets are either older [12], considering that at the pre-
sent time new spectrodensitometers, new softwares for
the readings, and new application devices of samples are
available for better results; or have validation param-
eters [13] easier to improve. Also, we found another
HPTLC method for the analysis of haloperidol tablets,
but with a different aim of the study [14] as was devel-
oped for an assay for the determination of impurities in
haloperidol pharmaceuticals.
Due to the mentioned aspects of these HPTLC methods
and the costly chemicals and columns used in HPLC and
the importance of time and speed of analysis, the aim of
this study was to develop and validate a simple, rapid,
sensitive, accurate, precise, and economical HPTLC
method for the quantitative determination of haloperi-
dol in tablets.
2 Experimental
2.1 Material
2.1.1 Instrumentation
HPTLC system: Spectrodensitometer Scanner 3 Camag
(Muttenz, Switzerland), equipped with the software
CATS 1.2.3 (Camag). Band application device: ATS auto-
matic (Camag); chromatographic chamber (Camag); and
HPTLC plates precoated with silica gel F 254 (Merck,
Darmstadt, Germany).
2.1.2 Reagents and chemicals
USP standard of haloperidol was obtained from Sigma–
Aldrich (St. Louis, MO). Ethanol, acetone, chloroform, n-
butanol, acetic acid glacial, mehanol, and monobasic
potassium phosphate were from Merck. All of the
reagents were of proanalysis quality. Pharmaceutical pre-
parations of haloperidol were purchased from a local
drug store.
2.2 Methods
2.2.1 Standard solution preparation
A stock solution of haloperidol was prepared by dissolv-
ing an appropriate amount in ethanol to a concentration
of 1 mg/mL. Working solutions of analyte were prepared
by appropriate dilutions of the stock solution with etha-
nol.
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Other Techniques 773
2.2.2 Sample preparation
Pharmaceutical preparations were tablets, nominally
containing 1 mg of haloperidol and some nonspecific
excipients. They were processed as follows: 20 tablets
were weighed and ground into fine powder and an accu-
rately weighed portion equivalent to 10 mg of haloperi-
dol was diluted to 100 mL with ethanol [6]. The solution
was centrifuged and the supernatant was used. Appropri-
ate dilutions were done for validation of the method.
2.2.3 Chromatographic conditions
Chromatography was carried out on silica gel F254
HPTLC plates, previously activated at 1208C for 20 min.
Sample application was done on 4-mm bands using an
automatic ATS device (Camag). For the chromatographic
development, acetone/chloroform/n-butanol/acetic acid
glacial/water (5:10:10:2.5:2.5 by vol.) was used as the
mobile phase. The length of development was 5 cm in
about 12 min. Vertical development chambers were
used.
Densitometry readings were carried out using a scan-
ner 3 Camag spectrodensitometer assisted by a computer
equipped with software WINCATS 1.2.3, and a deuterium
lamp was used as the radiation source. Determination
was performed at a wavelength of 254 nm.
2.2.4 Validation
The parameters validated were linearity, detection limit,
quantification limit, precision, accuracy, selectivity, and
robustness.
Validation was performed in compliance with interna-
tional standards [6, 15–17], using adequate statistics esti-
mates (RSD, Student’s t-test, ANOVA) [15, 18].
2.2.5 HPLC procedure
The pharmaceutical preparation was processed as men-
tioned in Section 2.2.2 [6]. The sample solution was cen-
trifuged and the supernatant was used. This procedure
was carried out in triplicate. Then, 20 lL of each test solu-
tion was injected on an HPLC system Perkin-Elmer series
200 (Norwalk, Ct, USA) with an Applied Biosystems
model 785-A programmable absorbance detector (Foster,
CA, USA) and a Perkin-Elmer Nelson model 1022 data pro-
cessor (Norwalk). The column was a Lichrospher 100 RP-
18, 25 cm64 mm with 5 lm particle size (Merck). The
mobile phase was methanol/monobasic potassium phos-
phate buffer (0.05 M) (60:40 v/v) (pH 4); flow rate was
1 mL/min, and detector wavelength was 254 nm. The buf-
fer was prepared by dissolving 1.36 g of monobasic potas-
sium phosphate (KH2PO4) in water, and diluting with
water to 200 mL.
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774 S. Mennickent et al.
3 Results and discussion
In order to validate an efficient method for the analysis
of haloperidol in tablets, preliminary tests were perform-
ed with the objective to select adequate and optimum
conditions. Parameters, such as detection wavelength,
ideal mobile phase and their proportions, and concentra-
tion of the standard solutions were exhaustively studied.
Several binary or ternary eluents were tested using
different proportions of solvents, such as chloroform,
methanol, isopropanol, acetone, ethyl acetate, acetic
acid glacial, water, n-butanol, ammonia.
3.1 Linearity
The standard curve of haloperidol was linear over the
concentration range of 10–100 ng/lL (10, 30, 50, 80, and
100 ng/lL). Each solution was spotted four times. This
range includes the solution concentration of the tablets
as assayed by USP 2005.
The calibration curve for haloperidol had a correlation
coefficient of 0.999, indicating a linear relationship
between the concentration of haloperidol and peak area
over the range investigated. The slope of the curve was
14.04 and the intercept was 10.84. The analysis of var-
iance (ANOVA) showed an Fc of 14.52 and an F theoretical
(a = 0.05; t = 1.18) of 4.41. Ho (b = 0) was rejected. (b, slope
of the curve).
3.2 LOD and LOQ
The LOD was 0.89 ng/lL and the LOQ was 2.71 ng/lL,
determined for three concentrations (10, 30, and 50 ng/
lL) by using the equations [16, 17]: LOD = 3.3 r/b;
LOQ = 10 r/b, where r is the SD of the response and “b”
corresponds to the slope obtained from the linearity
study of the method.
LOD and LOQ obtained are adequate values for the
detection and quantification of haloperidol in tablets.
3.3 Precision
Repeatability was determined by the measurement of
instrumental and intra-assay precision.
Instrumental precision was measured for ten spots of
the standard at three different concentrations (10, 50,
and 100 ng/lL).
The repeatability or intra-assay precision was studied
by analyzing repeatedly, in the same laboratory and on
the same day, five solutions of three concentrations (10,
50, and 100 ng/lL) from pharmaceutical samples, each of
which was independently prepared and each of them
being applied five times [6, 15–17]. Intermediate preci-
sion (interassay precision) included analyses at the same
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J. Sep. Sci. 2007, 30, 772 – 777
Table 1. Precision of the method
CV (%)
Concentration Instrumental Intra-assay Intermediate
(ng/lL) precisiona) precisionb) precisionc)
10 2.33 1.26 2.35
50 0.73 1.02 2.10
100 0.52 0.64 1.80
a) n = 10; analyzed on the same day.
b) n = 5; analyzed on the same day (for each concentration).
c) n = 9; analyzed on three different days (for each concen-
tration).
three concentrations; each solution was analyzed three
times on the same day for three different days.
Precision analysis studies showed an instrumental pre-
cision between 0.52 and 2.33%; an intra-assay variation
between 0.64 and 1.26% and an inter-assay variation
between 1.80 and 2.35% (Table 1). These values are ade-
quate for the analysis of drugs in concentrations as low
as nanograms [15–17]. All the values for intra-assay preci-
sion were under 2% of CV, and only one mean value for
instrumental precision was above 1%. Precision criteria
for an assay method are that the instrumental precision
will be 1% (CV) and the intra-assay precision will be 2%
(CV) [6, 15–17].
3.4 Accuracy
The accuracy was determined by addition standard,
applying the method to pharmaceuticals preparation to
which known amounts of standard substance corre-
sponding to 50, 100, and 150% of the concentration
expected in the samples were added. The accuracy was
then calculated from the test results as the percentage of
analyte recovered by the assay. The recovery obtained did
not differ from the real value in more than 2.24% and
was independent of the concentration with a CV less
than 4.50% as shown in Table 2. Only one mean value
was above 2% (2.24%) of difference between results of
study and 100% recovery.
3.5 Selectivity
The selectivity was evaluated after induced degradation
of haloperidol by exposing a solution (1 mg/mL) and pow-
der of haloperidol standard to UV light (k = 254 nm) and
by exposing another solution of 1 mg/mL and powder of
haloperidol standard to direct sunlight [6]. Later, a dilu-
tion of these solutions at an estimated concentration of
80 ng/lL was spotted, although the real concentration
was smaller because of the induced degradation of the
haloperidol in this study.
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J. Sep. Sci. 2007, 30, 772 – 777 Other Techniques 775

Table 2. Method accuracy

Added concentration Found concentration Accuracy (%)b) CV (%)c)

(ng/lL) (ng/lL)a)

Intra-assay (n = 9)
50 49.53 € 0.94 99.06 1.90
100 100.33 € 1.45 100.33 1.40
150 152.00 € 0.61 101.33 0.40
Inter-assay (n = 27)
50 48.88 € 2.20 97.76 4.50
100 100.83 € 2.52 100.83 2.50
150 150.00 € 0.80 100.00 0.53

a) Each value is the mean € SD.

b) (Observed concentration/added concentration)6100.
c) CV.

Figure 2. Peak of haloperidol. Solu-

tion concentration: 80 ng/lL.

When the solutions were analyzed, one degradation
product was found. No interferences were found
between haloperidol and its degradation product
(R = 2.07 between both peaks) (Figs. 2 and 3).
Degradation was higher in solutions than in powder
samples and was higher when exposed to direct sunlight
than UV radiation. Concentration of haloperidol was cal-
culated from the equation of the curve of the linearity
study. The degradation percentage was between 52.52
and 75.95%.
The excipients present in the tablets do not interfere
in the analysis. These are eliminated at the centrifuga-
tion step (see sample preparation in Section 2.2.2), as
they are not dissolved by the working solvent.
3.6 Robustness
The robustness was evaluated by an intentional minor
modification in the composition of the mobile phase
used in the proposed method. The tested mobile phase
was acetone/chloroform/n-butanol/acetic acid glacial/
water (10:5:10:2.5:2.5, by vol.) (for optimal mobile phase
see Section 2.2.3). The comparison of the values from the
two mobile phases was based on an analysis of variance

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776 S. Mennickent et al. J. Sep. Sci. 2007, 30, 772 – 777

Figure 3. Selectivity of analytical method. Substance 2: degradation product(s).

(ANOVA) treatment (F calculated: 3.47; F theoretical: 4.76; Table 3. Comparison of the determination of haloperidol in
r = 0.05). This suggests that the proposed method is tablets by the proposed method and by HPLC
robust under slight mobile phase variations.
Parameter HPTLC HPLC

Labeled claim (mg per tablet) 1.00 1.00

3.7 Application of the method
This method was used for the quantitative determina-
tion of haloperidol in tablets (Fig. 4). Table 3 compares
the results of the analysis of haloperidol between the pro-
posed (HPTLC) and official USP (HPLC) method. The
amounts of haloperidol found in tablets are fairly close
to the labeled amounts for both techniques. The results
obtained from the two methods were statistically com-
pared with each other at the 95% confidence level with
the aid of t- and F-tests. No significant difference was
found between both methods with respect to mean
values and SDs since the calculated t- and F-values were
less than the corresponding theoretical ones.
4 Concluding remarks
The results indicate that the HPTLC method is useful for
the determination of haloperidol in tablets. The method
has the required linearity, precision, accuracy, selectiv-
ity, detection, and quantification limits needed for the
quantitative determination of haloperidol in this dosage
form.
Amount founda) (mg per tablet) 0.94 0.93
RSD (%) 2.77 2.58
tcalc.: 0.63; t theoreticalb): 1.89
Fcalc.: 1.17; F theoreticalb): 3.44
a) Each result is the mean of nine experiments (three solu-
tions, three determinations of each of them).
b) a = 0.05.
The method is fast, simple, efficient, and easy to per-
form. HPTLC separation was obtained within 12 min and
the method allows for a large number of samples to be
measured simultaneously with a very good accuracy, sen-
sitivity, and precision which is very important especially
in quality control.
The procedure works without separation steps, only by
centrifugation, because the excipients do not dissolve in
the working solvent.
The method has a lower LOD, a better recovery percen-
tage, and a better precision than other HPTLC methods
for the quantitative determination of haloperidol in
tablets [12, 13]. The proposed method has an accuracy

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J. Sep. Sci. 2007, 30, 772 – 777
Figure 4. Quantitative determination of haloperidol in tablets by HPTLC method. Track 1 and 16: standard 100 ng/lL, Track 2
and 17: standard 80 ng/lL, Track 3 and 18: standard 50 ng/lL, tracks 4–15: samples.
between 97.76 and 100.33%, whereas the method by
Maslanka and Krzek [13] showed a recovery of 93.30% for
haloperidol. The CV for precision of our proposed
method was not higher than 2.35%, with all values for
intra-assay study under 2%, whereas the CV for precision
of the method of Maslanka and Krzek was 3.11% for halo-
peridol determination. Therefore, the proposed method
is a better HPTLC method for the quantitative analysis of
haloperidol in tablets.
The authors acknowledge the Research Council at the University
of Concepci
n (Project DIUC no. 201.074.034-1.0).
5 References
[1] Baldessarini, R. J., Tarazi, F. I., in: Hardman, J., Limbird, L. (Eds.),
Las Bases Farmacol
gicas de la Teraputica, Mc Graw-Hill, Mexico
2003, pp. 493 – 528.
[2] Delgado, J., Remers, W., Wilson and Gisvolds. Textbook of Organic
Medicinal and Pharmaceutical Chemistry, Lippincott-Raeven, Phila-
delphia 1998.
[3] Mc Evoy, G., AHFS Drug Information, American Society of Health-
System Pharmacists, Bethesda 2006.
[4] Sweetman, S., Martindale, Gua Completa de Consulta Farmacotera-
putica, Pharma Editores S. L., Barcelona 2003.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Other Techniques 777
[5] Moffat, A., Osselton, M. D., Widdop, B., Clarke's Analysis of Drugs
and Poisons, Pharmaceutical Press, London 2004.
[6] The United States Pharmacopeia, 29th Edn., U. S. Pharmacopeial
Convection Inc., Rockville, MD 2005.
[7] Yoshioka, S., Stella, V., Stability of Drugs and Dosage Forms, Kluwer
Academic/Plenum Publishers, New York 2000.
[8] Ali, I., Aboul-Enein, H., J. Liq. Chromatogr. Relat. Technol. 2005, 28,
3169 – 3179.
[9] Lea, A. R., Haley, D. M., Daguid, P. R., J. Chromatogr. 1982, 250, 35 –
42.
[10] Trabelsi, H., Bouzouita, K., Safta, F., J. Pharm. Biomed. Anal. 2002,
29, 649 – 657.
[11] Ouanes, S., Kallel, M., Trabelsi, H., Safta, F., Bouzouita, K., J.
Pharm. Biomed. Anal. 1998, 17, 316 – 364.
[12] Sodhi, R. A., Charla, J. L., Sane, R. T., Indian Drugs 1996, 33, 601 –
603.
[13] Maslanka, A., Krzek, J., J. AOAC Inter. 2005, 88, 70 – 79.
[14] Krzek, J., Maslanka, A., Acta Pol. Pharm. 2000, 57, 23 – 26.
[15] Quattrochi, O., Abelaira, S., Laba, R., Introducci
n a la HPLC. Aplica-
ci
n y prctica, Artes Grficas Farro, S. A., Ed., Buenos Aires 1992.
[16] Shabir, G., J. Chromatogr. A 2003, 987, 57 – 66.
[17] FDA Reviewer Guidance Validation of Chromatographic Meth-
ods, CDER, USA 1994.
[18] Daniel, W., Bioestadstica. Base para el Anlisis de las Ciencias de la
Salud, Uteha Noriega Editores, Mexico 1996.
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