The Simultaneous Determination

of Phenylephrine Hydrochloride,
Paracetamol, Chlorpheniramine Maleate
and Dextromethorphan Hydrobromide
in Pharmaceutical Preparations 2007, 66, S93–S96

İ. M. Palabıyık, F. Onur&

Department of Analytical Chemistry, Faculty of Pharmacy, University of Ankara, 06100 Tandoğan, Ankara, Turkey;
E-Mail: [email protected]

Received: 29 January 2007 / Revised: 3 May 2007 / Accepted: 7 May 2007

Online publication: 19 July 2007

sugar-coated tablets also have dyes and

coating materials.
Abstract The aim of this study was to develop
a rapid, accurate and selective LC
A new rapid and sensitive method has been developed and validated for the simultaneous method for the simultaneous determina-
determination of phenylephrine hydrochloride, paracetamol, chlorpheniramine maleate and tion of PHEN, DEX, CHL and PAR in
dextromethorphan hydrobromide in pharmaceutical preparations. The separation was their mixture and in sugar-coated tablets.
achieved on a C18 column using a gradient mobile phase of acetonitrile–sodium perchlorate The method was then subjected to vali-
(pH 3, 0.01 M) at a flow rate of 1.4 mL min)1. Detection was at 204 nm. Pseudoephedrine dation. The validation characteristics
hydrochloride was selected as internal standard. The recovery of the drugs ranged from 97.8 to evaluated were the selectivity, precision,
100.9%. Central composite design was used during validation to calculate method robustness linearity, LOQ and LOD values and the
and the percentage of sodium perchlorate, temperature and flow rate were investigated as robustness. In robustness, we used
factors. The method was found to be applicable for the determination of the four compounds in Response Surface Methodology (RSM), a
sugar-coated tablets. collection of statistical and mathematical
techniques for developing, improving
and optimizing processes and interpret-
ing the relationships between responses
Keywords (performance measure or quality char-
acteristic) and factor effects [14]. An
Column liquid chromatography
important aspect of RSM is design of
Dextromethorphan hydrobromide
the experiment. The aim of experimental
Phenylephrine hydrochloride
design is to minimize the number of
Paracetamol
Chlorpheniramine maleate experiments required, reduction of anal-
ysis time and selection of the points
where the response should be inter-
preted. We used Central Composite
Designs in these studies.
Introduction A variety of methods exist in the
literature for the determination of some
A quaternary mixture of phenylephrine of these compounds [2–12] but none Experimental
hydrochloride (PHEN), chlorphenir- describe the determination of all four
amine (CHL), dextromethorphan compounds simultaneously. Materials and Reagents
hydrobromide (DEX) and paracetamol Determination of the ingredients in a
(PAR) is widely used in diseases accom- pharmaceutical preparation becomes Dextromethorphan hydrobromide, phen-
panied by cough, pain and fever such as more difficult as the number of compo- ylephrine hydrochloride, paracetamol,
the common cold and other viral infec- nents in the mixture increases. In this chlorpheniramine maleate and pseudo-
tions as an analgesic, antipyretic, decon- study, there are four active ingredients ephedrine hydrochloride were kindly
gestant, antihistamine and antitussive [1]. and excipients, which might interfere donated by Ilsan Ilaç ve Hammaddeleri
with the analysis of the active ingredients. San. A.Ş., Istanbul, Turkey and were used
Application of Separation Techniques in Turkey In addition to the normal excipients, without further purification.

Full Short Communication Chromatographia Supplement Vol. 66, 2007 S93

DOI: 10.1365/s10337-007-0324-5

2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
 and the volume was completed to 25 mL
with the same solvent. This mixture was
injected into the chromatographic system.

Commercial Pharmaceutical
Preparation

WINTUS (10 mg dextrometorphan

hydrobromide, 5 mg phenylephrine
hydrochloride, 250 mg paracetamol and
2 mg chlorpheniramine maleate) sugar-
coated tablets from Sandoz Ilac San. Tic.
Asß., Turkey (batch no: 9054878) were
assayed.

Results and Discussion

Method Development
and Optimization

Fig. 1. a Typical chromatogram of the solution of 1 = 32 lg mL)1 PHEN, 2 = 32 lg mL)1 The pH and concentration of sodium
PAR, 3 = 100 lg mL)1 Pseudoephedrine hydrochloride, 4 = 36 lg mL)1 CHL and 5 = perchlorate in mobile phase were opti-
36 lg mL)1 DEX in acetonitrile–sodium perchlorate (pH 3, 0.01 M) (25:75,v/v). b Chromatogram mized for the separation on an ACE
of the sugar-coated tablet selected (WINTUS)
C18 column and pH 3.0 and 0.01 M were
selected. The solvent content in mobile
Acetonitrile (HPLC grade), NaClO4 PAR (2,000 lg mL)1) were prepared in
phase was also studied on the same col-
and HClO4 were provided by Merck, acetonitrile–sodium perchlorate (pH 3,
umn. The optimal mobile phase for good
Darmstadt, Germany. Water for prepa- 0.01 M) (25:75, v/v). A solution of
separation in a gradient elution program
ration of solutions was produced in-house 2,000 lg mL)1 pseudoephedrine hydro-
was found to be acetonitrile–sodium
by a PurelabUHQ water purification chloride internal standard (IS) was also
perchlorate solution (5:95, v/v) initially
system (Elba). The mobile phase and the prepared in acetonitrile–sodium perchlo-
then a linear gradient up to 60% aceto-
solutions for injection were degassed in an rate (pH 3, 0.01 M) (25:75, v/v) as stock
nitrile. The flow rate, column tempera-
ultrasonic bath and were filtered through solution for the HPLC method.
ture and injection volumes were also
a 0.45 lm nylon membrane before use. For preparation of sodium perchlo-
optimized to be 1.4 mL min)1, 35 C and
rate solution, 1.405 g of sodium per-
20 lL, respectively. Pseudoephedrine
chlorate was dissolved in 975 mL distilled
Apparatus hydrochloride, timolol maleate, dorzol-
water and the pH of the resulting solution
amid, ephedrine hydrochloride and
was adjusted to 3.0 with HClO4 and then
An Agilent Technologies HP 1100 chro- phenylephrine hydrochloride were tested
the volume was made up to 1,000 mL
matographic system equipped with a G13 for use as internal standard. Pseudo-
with distilled water.
79A degasser, G1311A quaternary pump, ephedrine hydrochloride could be
61313A injector and G1315B DAD successfully separated from the other
detector was used in conjunction with an compounds so it was used as IS in
ACE C18 column (250 · 4.6 mm, 5 lm Sample Solution this study. The detection wavelength was
particle size). The chromatograms were selected as 204 nm.
recorded and the peaks were quantified The contents of 20 sugar-coated tablets The conditions finally adopted were
using an automatic integrator. The mo- were accurately weighed and powdered an ACE C18 column (250 · 4.6 mm,
bile phase consisted of (A) acetonitrile separately in a mortar. A weight equiva- 5 lm), an intial mobile phase of acetoni-
and (B) sodium perchlorate solution (pH lent to one tablet was dissolved in 50 mL trile-sodium perchlorate (5:95, v/v)
3, 0.01 M). The initial ratio of mobile of acetonitrile–sodium perchlorate (pH 3, initially then programmed linearly up to
phase components (A/B) was 5:95. The 0.01 M) (25:75, v/v) and diluted to 60% of acetonitrile in 8 min at a flow rate
flow rate was set at 1.4 mL min)1 with volume with the same solvent. After of 1.4 mL min)1, 35 C column temper-
20 ll as injection volume and the detec- 30 min. of mechanical shaking and ature and 20 lL injection volume.
tion wavelength was 204 nm. 15 min. of standing in the dark, the solu- Under these chromatographic condi-
tions were filtered through a 0.45 lm tions, PHEN, PAR, pseudoephedrine
Millipore filter in a 50 mL volumetric hydrochloride (IS), CHL and DEX peaks
Solutions flask separately. Then 1 mL of this solu- were well resolved and their retention
tion and 0.5 mL of pseudoephedrine times were found to be 3.91, 4.19, 4.89,
Solutions of DEX (200 lg mL)1), PHEN hydrochloride (IS) from the stock solution 6.41 and 7.06 min, respectively. A typical
(200 lg mL)1), CHL (200 lg mL)1) and were put into a 25 mL volumetric flask chromatogram of the drugs and internal

S94 Chromatographia Supplement Vol. 66, 2007 Full Short Communication

standard is illustrated in Fig. 1. System Table 1. System suitability tests results of PHEN, CHL, PAR and DEX
suitability tests including retention time,
Parameters PHEN CHL PAR DEX
retention factor, separation factor, reso-
lution, asymmetry, and theoretical plates Retention factor 4.23 8.45 4.61 7.58
were carried out and the results are Separation factor 1.08 1.11 1.08 1.11
shown in Table 1. Resolution 2.78 5.2 2.78 5.2
Asymmetry 0.92 0.91 0.98 0.90
Theoretical plates 24485.99 65761.48 28089.76 79772.35

Linearity
Table 2. Statistical data in the method
The calibration curves were established
with ten different concentrations in the Parameters PHEN CHL PAR DEX
range of 0.48–52 lg mL)1 for PHEN,
Linearity range 0.48–52 0.48–44 4–240 0.4–19
0.48–44 lg mL)1 for CHL, 4–240 lg (lg mL)1)
mL)1 for PAR and 0.4–19 lg mL)1 for Slope of the 1.5574 1.6717 1.7023 0.5602
DEX. Each solution was injected three calibration curve ± ± 0.0008 ± 0.0039 ± 0.0005 ± 0.0035
standard error
times and the areas of peaks, as measured
Intercept of the 0.1018 )0.2533 4.2065 0.0827
at 204 nm, were integrated. The ratios of calibration curve ± ± 0.0125 ± 0.0402 ± 0.0502 ± 0.0212
the peaks areas of investigated substances standard error
to that of IS were calculated for each r 0.9999 0.9999 0.9997 0.9997
Mean recovery (%) 99.83 99.10 99.80 99.90
injection. Regression equations were RSD (%) 0.39 0.39 0.23 0.79
established by plotting the ratio of peak
areas to the concentration of each sub- RSD Relative standard deviation
stance. The linearity was evaluated by
linear regression analysis, which was cal-
culated by least square regression. The Table 3. Assay results of commercial preparation (WINTUS  sugar-coated tablet) (Labelled
results are shown in Table 2. claim = 10 mg DEX, 5 mg PHEN, 250 mg PAR and 2 mg CHL/sugar-coated tablet)

PHEN (mg) PAR (mg) CHL (mg) DEX (mg)

mean ± SD* mean ± SD mean ± SD mean ± SD
Sensitivity
4.95 ± 0.04 259.8 ± 1.51 2.00 ± 0.03 10.27 ± 0.05
The calculation method of LOQ (limit of SD standard deviation
quantitation) is based on the standard
deviation (SD) of the response and the
slope (m) of the calibration curve including 10 replicates, was carried out amol is quite high in the formulation, a
according to the formula: LOQ = 10 on three successive days with synthetic bigger peak was observed for PAR when
(SD/m) [13]. LOD (limit of detection) can mixtures of PHEN, CHL, PAR and compared with the other active ingredi-
be calculated based on the SD of the re- DEX. Calculated Snedecor F values ents. Assay results obtained by the
sponse and the slope (m) of the calibra- (£ 0.061) below the tabulated levels were method are shown in Table 3.
tion curve according to the formula: obtained in all cases (F = 4.21, n1 = 2,
LOD = 3.3 (SD/m) [13]. LOQ values n2 = 27) so there were no significant
were calculated as 0.08 lg mL)1 for differences among the results obtained in Robustness
PHEN, 0.24 lg mL)1 for CHL, 0.29 lg the determination of each drug in the
mL)1 for PAR and 0.38 lg mL)1 for presence of the others on three different As defined by the International Confer-
DEX. LOD values were calculated as days. ence on Harmonisation, the robustness of
0.03 lg mL)1 for PHEN, 0.08 lg mL)1 an analytical procedure is a measure of its
for CHL, 0.10 lg mL)1 for PAR and capacity to remain unaffected by small
0.13 lg mL)1 for DEX. Application variations in method parameters and
provides an indication of its reliability
No interfering peak was observed in the during normal usage [13]. For studying
Accuracy and Precision chromatogram of the commercial sugar- the simultaneous variation of the factors
coated tablet formulations under the on response, some designs of experiment
Accuracy was studied using three differ- conditions described. Excipients (lactose, techniques were suggested in robustness
ent solutions containing 12, 24 and 36 lg starch, avicel, povidon, sodium dodecyl- testing including multivariate applica-
mL)1 of PHEN, 1.6, 24 and 36 lg mL)1 sulfate, aerosil and magnesium stearate) tions. Response Surface Methodology
of CHL, 130, 144 and 192 lg mL)1 of and colorant (tartrazine) in the commer- (RSM) is a collection of statistical and
PAR and 4.8, 8.0 and 9.6 lg mL)1 of cial preparation analysed did not inter- mathematical techniques useful for
DEX. Mean recoveries and relative fere in the quantitation of PHEN, PAR, developing, improving and optimizing
standard deviations for the method are CHL and DEX. A chromatogram of the processes and interpreting the relation-
shown in Table 2. The precision, deter- sugar-coated tablets is illustrated in ships between response and factor effects
mined by means of one-way ANOVA Fig. 1b. Because the amount of paracet- [14]. An important aspect of RSM is de-

Full Short Communication Chromatographia Supplement Vol. 66, 2007 S95

sign of the experiment. Composite the other compounds in all of the com- and ternary mixtures in pharmaceutical
Designs are very efficient for a limited binations of these levels. The equation for formulations [2–10, 12]. The method was
number of factors (<5 to 6) [15] and Y was as follows found selective for PHEN, CHL, PAR
includes a combination of a full fractional and DEX in the presence of common
design and an additional design, often a Y ¼ 2:8775 þ 0:1810 x1 þ 0:0502 x2 excipients, dyes and coating materials in
star design. If the centers of both designs þ 0:1191 x3
0:1317 x21 the sugar-coated tablet formulation
coincide they are called central composite analysed. All the statistical values were
designs. For the number of runs, r,

0:0610 x22
0:0663 x23 within the acceptable limits. The low

0:005 x1 x2 þ 0:0075 x2 x3 RSD values indicate good precision and
r ¼ 2k
p þ 2k þ no : high recovery values indicate good
þ 0:0175 x1 x3
accuracy of the method. The method can
[16] where k = number of factors,
also be used for monitoring stability. The
p = number for reduction of the full
developed method has been proved sen-
design, no = number of experiments in Variation of percentage of sodium per- sitive, accurate and precise for the rou-
the center of the design. chlorate and flow rate while the temper- tine analysis of these drugs.
In this equation, 2k)p is the number of ature is maintained constant at 35 C
experiments in the full or fractional de- showed that an increase in the percentage
sign; 2k is the number of experiments in of sodium perchlorate resulted in an in-
the star designs; no is the number of crease of the resolution between PHEN References
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The level is ()1) and (+1) for 2k)p, ± a had no important effect on the response.
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Pharmacy (2000) 20th edn. University of
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k X
k
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X
k
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S96 Chromatographia Supplement Vol. 66, 2007 Full Short Communication