Development and validation of a reversed-phase HPLC method for the determination of pindolol and clopamide in tablets

Abstract

A high-performance liquid chromatographic method was developed for the simultaneous determination of pindolol and clopamide in pharmaceutical dosage forms. The use of a β-cyclodextrin bonded-phase column results in satisfactory separation of both of the compounds. The mobile phase consisted of a mixture of 1.0% w/v triethylamine acetate buffer (pH = 5.5) and methanol (90:10, v/v), pumped at a flow rate 0.8 mL/min. The UV detector was operated at 245 nm. Calibration graphs are linear (r better than 0.99997, n = 5), in concentration range 1.0-3.0 μg/mL for pindolol and 0.5-1.5 mg/mL for clopamide. The intra- and interday R.S.D. values were less than 2.97% (n = 5), while the relative percentage error Er was less than 2.0% (n = 5). Detection limits were 0.12 and 0.16 mg/mL for pindolol and clopamide, respectively. The method was applied in the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Full text

DEVELOPMENT AND VALIDATION OF A
REVERSED-PHASE HPLC METHOD FOR
THE DETERMINATION OF PINDOLOL
AND CLOPAMIDE IN TABLETS
P. Papadopoulos, M. Parissi-Poulou, and I. Panderi*
University of Athens, School of Pharmacy,
Division of Pharmaceutical Chemistry, Panepistimiopolis,
157 71, Athens, Greece
ABSTRACT
A high-performance liquid chromatographic method was
developed for the simultaneous determination of pindolol and
clopamide in pharmaceutical dosage forms. The use of a b-
cyclodextrin bonded-phase column results in satisfactory separa-
tion of both of the compounds. The mobile phase consisted of a
mixture of 1.0% w=v triethylamine acetate buffer (pH ¼5.5) and
methanol (90:10, v=v), pumped at a flow rate 0.8 mL=min. The
UV detector was operated at 245 nm.
Calibration graphs are linear (r better than 0.99997,
n¼5), in concentration range 1.0–3.0 mg=mL for pindolol and
0.5–1.5 mg=mL for clopamide. The intra- and interday R.S.D.
values were less than 2.97% (n¼5), while the relative percen-
tage error E
r
was less than 2.0% (n ¼5). Detection limits were
J. LIQ. CHROM. & REL. TECHNOL., 25(1), 125–136 (2002)
125
Copyright #2002 by Marcel Dekker, Inc. www.dekker.com
*Corresponding author. E-mail: ipanderi@pharm.uoa.gr
D20

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0.12 and 0.16 mg=mL for pindolol and clopamide, respectively.
The method was applied in the quality control of commercial
tablets and content uniformity test and proved to be suitable for
rapid and reliable quality control.
INTRODUCTION
Pindolol (( )-4-(2-hydroxy-3-isopropyl-amino-propoxy)-indole), is a non-
selective b-adrenergic antagonist with intrinsic sympathomimetic activity (1) and
a 5-HT
1A=1B
receptor antagonist. (2) The racemate mixture of this compound is
used for the clinical treatment of angina pectoris and hypertension. (3,4) Its
combination with clopamide (4-chloro-N-(2,6-dimethyl-piperidino)-3-sulpha-
moylbenzamide), a diuretic that reduces the reabsorption of electrolytes from
renal tubules, (5) increases the antihypertensive effects.
Among the methods that have been reported in literature for the
determination of pindolol are potentiometric titration, (6) and UV spectro-
photometry, (7) thin-layer chromatography, (8) gas-chromatography mass
spectroscopy (9) and liquid-chromatography mass spectroscopy. (10,11) Various
high-performance liquid chromatographic methods have also been described for
the determination of pindolol in pharmaceutical formulations (12,13) in the form
of its organic salts (14) and in biological fluids (15,16) including enantioselective
bioanalytical determinations. (17–20) The quantitation of clopamide has been
carried out using various analytical techniques such as high-performance liquid
chromatography, (21–23) derivative spectrophotometry, (24) and gas chromato-
graphy-mass spectroscopy. (25)
A few methods for the simultaneous determination of pindolol and
clopamide have been published. These include derivative spectrophotometry
(26,27) and high-performance liquid chromatography. (28) In the latter method,
chromatographic separation was carried out under isocratic conditions on a
reversed-phase C-18 column, thus, pindolol was early-eluted.
As the combination of these two compounds in antihypertensive therapy
has become popular, we thought that it would be of particular interest to develop
and validate a simple, selective, and reliable HPLC method for their simultaneous
determination. In the present study the applicability of a b-cyclodextrin bonded-
phase column to the HPLC analysis of pindolol and clopamide was evaluated, in
view of the need of selective methods for their determination in pharmaceuticals.
Cyclodextrin columns effect numerous chemical separations by selectively
including a wide variety of organic and inorganic guest molecules in the
cyclodextrin cavity. (29,30) The proposed method is applicable, as well, for
routine analysis and complies well with the validation requirements in the
pharmaceutical industry.
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EXPERIMENTAL
Apparatus
Chromatography was performed on a Waters Model 501 pump, and a
Rheodyne Model 7125 injector with a 20-mL loop. Detection was performed with
a Waters Model 486 UV-Vis detector with a 8-mLflow cell. Integration of the
chromatograms was made with a Hewlett-Packard Model HP-3394A integrator. A
pH meter Metrohm, Model 654 Herisau was used for all pH measurements.
Materials
Solvents were of HPLC grade and were purchased from Lab-Scan Science
Ltd., Ireland. Triethylamine acetate (pro analysi), and glacial acetic acid
(analytical reagent grade) were purchased from Aldrich Ltd. Water was deionised
and further purified by means of a Milli-Q Plus Water Purification System,
Millipore Ltd. Pindolol and clopamide of pharmaceutical purity grade were
kindly provided by Novartis Pharma, while sulfamerazine of pharmaceutical
purity grade was kindly provided by Minerva Hellas. All substances were used
without any further purification. Viskaldix tablets are products of Novartis
Pharma; each tablet was labelled to contain 10.0 mg of pindolol and 5.0 mg of
clopamide.
Methods
Chromatographic separations were performed on a b-cyclodextrin column
(25064.6 mm i.d.), which was obtained from Advanced Separation Technologies
Inc., (Whinappy, NJ, USA) under the commercial name Cyclobond-I. When not
in use the column was stored in 100% methanol.
The mobile phase, 1.0% w=v triethylamine acetate buffer (pH ¼5.5) and
methanol (90:10, v=v), was filtered through a 0.45 mm Millipore filter and
degassed, under vacuum, prior to use. The mobile phase was pumped at a flow
rate 0.8 mL=min. All chromatographic experiments were carried out at room
temperature. The column eluate was monitored at 245 nm, a suitable wavelength
obtained from the PND and CLP UV-spectra.
Stock Standard Solutions
Stock standard solutions of pindolol (PND), 1.0 mg=mL, clopamide (CLP),
1.0 mg=mL, and sulfamerazine, (SLF), 0.5 mg=mL, were prepared by dissolving
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appropriate amounts of the compounds in methanol. These solutions were stored
in the dark under refrigeration at 4C and were found to be stable for several
weeks. A series of mixed standard solutions were prepared by the appropriate
dilution of the above mentioned stock standard solution in mobile phase to reach
concentration ranges of 1.00–3.00 mg=mL and 0.50–1.00 mg=mL for PND and
CLP, respectively. In each sample 1.20 mg=mL of the internal standard SLF was
added. Standard solutions were found to be stable during the analysis time.
Assay Sample Preparation
Twenty tablets were weighed and finely pulverised. An appropriate portion
of this powder, equivalent to 10.0 mg of PND and 5.0 mg of CLP was placed in a
50-mL volumetric flask with 40 mL of methanol. The solution was sonicated for
5 min and diluted to volume with methanol. A portion of this solution was
centrifuged at 4000 rev=min (2890 g) for 15 min. A 5-mL aliquot was transferred
to a 50-mL volumetric flask and diluted to volume with mobile phase.
Consequently, a 500-mL aliquot of this solution was further diluted to 10 mL
mobile phase containing 1.20 mg=mL of the internal standard, SLF; 20 mL sample
was injected into the HPLC system. Peak area ratios of each compound to that of
the internal standard were then measured for the determinations. The same
procedure was followed for the content uniformity test, using one tablet per
sample.
Calibration Procedure
Two calibration curves were constructed by assaying the above mentioned
mixed standard solutions of PND and CLP in mobile phase. The concentration
range covered was 1.00–3.00 mg=mL for PND and 0.50–1.50 mg=mL for CLP.
Triplicate 20-mL injections were made of each solution and the peak area ratio of
each drug to that of the internal standard was plotted against the corresponding
concentration to obtain the calibration graph.
The over-all precision and accuracy of the chromatographic assay was
evaluated by analyzing three series of mixed standard solutions of PND and CLP,
at concentrations of 1.00, 2.00, and 3.00 mg=mL for PND and 0.50, 1.00, and
1.50 mg=mL for CLP. In each sample 1.20 mg=mL of the internal standard SLF
was added. The precision of each method was based on the calculation of the
relative standard deviation (% R.S.D.). An indication of the accuracy was based
on the relative percentage error of the samples (E
r
%).
In order to assess the specificity of the proposed HPLC method, the effect
of the excipients used in the formulation of tablets was evaluated using the
128 PAPADOPOULOS, PARISSI-POULOU, AND PANDERI

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standard addition method. (31) Thus, five equal amounts of tablets equivalent to
10.00 mg of PND and 5.00 mg of CLP, were spiked with different amounts of
reference standards of PND and CLP. The samples were analyzed as mentioned in
the assay procedure, while in each sample, 1.20 mg=mL of the internal standard,
SLF, was added. Peak area ratios of each drug to that of the internal standard were
measured for the determination of both compounds.
RESULTS AND DISCUSSION
Chromatographic Characteristics
Chromatographic separations using CD-bonded phases, are mainly the
result of variations in the stability of inclusion complexes of the analytes with the
cyclodextrin molecules. (32) Thus, a thorough investigation was conducted in
order to choose the optimum conditions for the chromatographic separation of the
analytes on a Cyclobond-I column.
The effect of composition and pH of the mobile phase on the retention time
of PND, CLP, and SLF (internal standard), were investigated. Results of the effect
of methanol concentration in the mobile phase are presented in Figure 1. An
Figure 1. Effect of methanol concentration on the log k0of pindolol, r, clopamide, s,
and sulfamerazine, 6. Column, Cyclobond I (25064.60 mm i.d.); eluent, 1.0% w=v
triethylamine acetate buffer (pH ¼5.5) and methanol; flow rate, 0.8 mL=min; detection
wavelength 245 nm.
DETERMINATION OF PINDOLOL AND CLOPAMIDE 129

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increase in the percentage of methanol decreases the interaction between the
analysed components and the b-cyclodextrin cavity, and results in lower degrees
of retention. The optimum methanol concentration was found to be (10% v=v).
The effect of pH on the retention time of the analytes was also investigated by
changing the pH values of the aqueous component of the mobile phase from 4.5
to 7.0 using triethylamine acetate buffer (1.0% w=v). For all experimental pH
values, the drugs are eluted in order of PND, CLP, and SLF. It is known that the
non-polar and less ionic molecules form more stable inclusion complexes than the
polar and ionic molecules. (33) Consequently, non-polar and uncharged
compounds are, in principle, more strongly retained by cyclodextrin than the
polar and ionic molecules.
Pindolol is a basic substance (pKa 8.8) and owing to protonization the
retention is lower for lower pH values. On the contrary, at a pH value of 4.5
clopamide (weak acid, pKa 7.0) and sulfamerazine (weak acid, pKa 8.0) are non-
ionized, thereby, forming more stable inclusion complexes. Consequently, an
increase in the pH from 4.5 to 7.0 decreases the stability of CLP-b-CD and SLF-
b-CD complexes, and thus, decreases the retention times (Figure 2). Therefore, at
the highest pH value a loss of resolution between CLP and PND is observed. A
pH value of 5.0 was chosen for the optimum separation of the compounds.
The specificity of the HPLC method is illustrated in Figure 3, where
complete separation of the compounds was observed. PND was eluted at
4.66 min, CLP appeared at 5.84 min, while the internal standard, SLF, was eluted
at 6.80 min.
Figure 2. Effect of mobile phase pH on the log k0of pindolol, r, and sulfamerazine, 6.
Column, Cyclobond I (25064.60 mm i.d.); eluent, 1.0% w=v triethylamine acetate buffer
and methanol (90:10, v=v); flow rate, 0.8 mL=min; detection wavelength 245 nm.
130 PAPADOPOULOS, PARISSI-POULOU, AND PANDERI

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Linearity and Reproducibility
Calibration graphs were constructed at six concentration levels in the range
1.0 to 3.00 mg=mL for PND and 0.50 to 1.50 mg=mL for CLP, and three
independent determinations were performed at each concentration (n ¼3). Linear
relationships were obtained between the peak area ratio of each compound and
the corresponding concentration, as shown by the equations presented in Table 1.
The correlation coefficient (r) and the standard error of the estimate (S.E.) of the
calibration lines are also given, along with the S.D. of the slopes and intercepts.
Figure 3. Representative chromatogram of a mixture of pindolol clopamide and
sulfamerazine at retention times 4.66, 5.84, and 6.80 min, respectively. The chromatogram
was obtained from the analysis of tablets and the chromatographic conditions were:
reversed-phase HPLC on a Cyclobond-I column (25064.60 mm i.d.); mobile phase, 1.0%
w=v triethylamine acetate buffer (pH ¼5.5) and methanol (90:10, v=v); flow rate,
0.8 mL=min; detection wavelength 245 nm.
DETERMINATION OF PINDOLOL AND CLOPAMIDE 131

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In order to further evaluate the linearity of the proposed method, five
calibration equations were constructed over a period of four weeks. The average
regression equation for PND:
SPND ¼0:730ð0:014ÞCPND þ0:030ð0:08Þ
and for CLP:
SCLP ¼0:832ð0:009ÞCCLP þ0:080ð0:54Þ
where S is ratio of the peak area amplitude of each compound to that of the
internal standard, and C is the appropriate concentration expressed in mg=mL.
The slopes of the calibration equations of PND and CLP had R.S.D. values of
1.94 and 1.08, respectively, while the correlation coefficient invariably exceeded
0.9998.
Intra-day data for the precision and accuracy of the method given in Table
2, indicate R.S.D.% ¼1.32–2.0 and E
r
¼1.0–1.0 for PND and R.S.D.% ¼
1.01–1.99 and E
r
¼1.0–2.0 for CLP. Moreover, the inter-day R.S.D.% values
(Table 2) for the determination of PND and CLP ranged from 2.02 to 2.97 and
1.99 to 2.04, respectively.
The limit of detection attained as defined by IUPAC,(34) LOD
(k ¼3)
¼
k6S
a
=b (where b is the slope of the calibration graph and S
a
is the standard
deviation of the blank signal) was found to be 0.12 and 0.16 mg=mL for PND and
CLP, respectively. The limits of quantitation LOQ were also attained according to
the IUPAC definition, LOD
(k ¼10)
¼k6S
a
=b, and were found to be 0.42 and
0.48 mg=mL for PND and CLP, respectively.
The statistical evaluation of the HPLC method revealed its good linearity
and reproducibility, and led us to the conclusion that it could have been used for
the reliable determination of CLP and PND in tablets.
Tab le 1. Analytical Data of the Calibration Graphs for the Determination of Pindolol and
Clopamide by High-Performance Liquid Chromatography
Linearity Range (mg=mL) Calibration Equation
a
r
b
PND CLP
1.00–3.00 0.50–1.00 S
PND
¼0.720(0.003)6C
PND
þ0.010(0.03) 0.99997
1.00–3.00 0.50–1.00 S
CLP
¼0.830(0.002)6C
CLP
þ0.020(0.04) 0.99998
a
Ratio of the peak area amplitude of each compound to that of the internal standard, S,
versus concentration of each compound, C, in mg=mL; six standards.
b
Correlation coefficient.
132 PAPADOPOULOS, PARISSI-POULOU, AND PANDERI

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Assay of Pharmaceutical Formulations
The proposed method was evaluated in the assay of commercially available
tablets containing a mixture of PND and CLP in the proportion 2.0:1.0. Ten
replicate determinations were carried out on an accurately weighed amount of the
pulverised tablets equivalent to 10.00mg of PND and 5.00 mg of CLP. The results
obtained gave a mean of 9.89 0.22 with a R.S.D. of 2.22 for PND, and a mean
of 4.990.03 with a R.S.D. of 0.60 for CLP.
The method proved to be suitable for the content uniformity test, where a
great number of assays on individual tablets are required. Commercially available
tablets, containing mixtures of PND and CLP in proportion 2.0:1.0, were
analysed using the proposed methodology and the results are given in Table 3.
Recoveries achieved were in accordance with the actual content of PND and CLP
in tablets.
Tabl e 2. Precision and Accuracy of Within- and Between-Run Analysis for the
Determination of Pindolol and Clopamide by High-Performance Liquid Chromatography
Nominal Concentration
(mg=mL)
Assayed Concentration of
Pindolol (mg=mL)
Assayed Concentration of
Clopamide (mg=mL)
Pindolol Clopamide Means.d. RSD%
a
E
r
(%)
b
Means.d. RSD%
a
E
r
(%)
b
Intra-day (n ¼5)
1.0 0.5 0.990.02 2.02 1.0 0.510.01 1.96 2.0
2.0 1.0 1.990.03 1.50 0.5 0.990.01 1.01 1.0
3.0 1.5 3.030.04 1.32 1.0 1.51 0.03 1.99 0.7
Inter-day (n ¼5)
1.0 0.5 1.010.03 2.97 1.0 0.49 0.01 2.04 2.0
2.0 1.0 1.980.04 2.02 1.0 1.020.02 2.04 2.0
3.0 1.5 3.050.07 2.30 1.7 1.52 0.05 1.99 1.3
a
Percentage relative standard deviation.
b
Relative percentage error.
Tabl e 3. Determination of Clopamide and Pindolol in Commercial Formulations by
High-Performance Liquid Chromatography
Sample Pindolol; Found mg=Tablet Clopamide; Found mg=Tablet
Means.d. (n ¼10) Recovery (%)
a
Means.d. (n ¼10) Recovery (%)
a
Viskaldix 9.940.03 99.4 4.95 0.05 99.0
a
Mean and standard deviation for ten determinations; percentage recovery from the label
claim amount.
DETERMINATION OF PINDOLOL AND CLOPAMIDE 133

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Recovery studies were also performed, by analyzing spiking sample
powders with appropriate amounts of the reference standard of both compounds.
Two calibration curves were then constructed by plotting the amount of the drug
found (mg) versus the amount of the drug added (mg) for each one of the two
compounds. The following linear regression equations were obtained through
regression analysis of data:
Cf
PND ¼0:990ð0:017ÞCa
PND 9:93ð0:17Þ;r¼0:9996
Cf
CLP ¼1:018ð0:011ÞCa
CLP þ4:98ð0:09Þ;r¼0:9998
where Cf
PND and Cf
CLP are the amounts (mg) found for PND and CLP,
respectively, while Ca
PND and Ca
CLP are the amounts (mg) added for PND and CLP,
respectively; r is the correlation coefficient of the calibration equation. The y-axis
intercept of the above mentioned linear regression equations indicate the amount
(mg) of the drug found in the powdered tablets, while the percentage recoveries
were calculated as: % recovery ¼slope6100. The results presented in Table 4
indicate that there is no interference from the excipients used in the formulation
of the tablets.
The proposed method was evaluated by comparison with a zero-crossing,
first-order derivative spectrophotometric method developed in our laboratory. (27)
Commercially available tablets containing mixtures of 10.00 mg of PND and
5.00 mg of CLP were analysed by derivative spectrophotometry and the proposed
HPLC method. The results obtained by both methods are demonstrated in
Table 5.
Tabl e 4. Recoveries of Pindolol and Clopamide in Spiked Commercial Samples
Drug Amount Added (mg) Amount Found (mg) m
a
Recovery
b
Pindolol 5.0 14.85 0.990 99.0
8.0 17.93
10.0 19.75
12.0 21.96
14.0 23.70
Clopamide 2.0 7.09 1.018 101.8
5.0 7.09
7.0 12.10
9.0 14.06
12.0 17.29
a
m is the slope of the linear regression analysis of the amount found versus the amount
added.
b
Recovery (%) ¼m6100.
134 PAPADOPOULOS, PARISSI-POULOU, AND PANDERI

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In conclusion, the proposed high-performance liquid chromatographic
method was evaluated for linearity, reliability, and specificity, and proven to be
convenient and effective for the analysis of clopamide and pindolol in
commercial formulations. Moreover, the proposed method offers a short
analytical run time of 8.00 min and achieved good resolution between PND,
CLP, and the internal standard, SLF. The method was successfully applied to the
determination of clopamide and pindolol mixture in tablets.
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136 PAPADOPOULOS, PARISSI-POULOU, AND PANDERI

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