Rapid Validated Thin-Layer Chromatography–Densitometry for the Simultaneous Determination of Three Co-formulated Drugs Used for Common Cold Treatment

Abstract

A rapid validated thin-layer chromatography (TLC)-densito-metric method has been developed for the simultaneous determination of 3 co-formulated drugs used for common cold and cough treatment. The studied drugs are paracetamol, pseudoephedrine, and chlorpheniramine maleate. The separation was achieved using silica gel 60 F 254 plates and the developing system of metha-nol-toluene-acetic acid (44:16:1, v/v). Densitometry scanning was performed at 254 nm. The method was validated as per the International Conference on Harmonization (ICH) guidelines and was successfully applied for the analysis of pharmaceutical preparation containing the cited ternary mixture without interference from excipients. There is no previously published TLC-densitometric method for the determination of the previously mentioned ternary mixture. The suggested method is rapid and of low cost, so it can be used for quality control analysis.

Full text

Journal of Pla nar Chromatography 29 (2016) 3 127
Summary
A rapid validated thin-layer chromatography (TLC)‒densito-
metric method has been developed for the simultaneous determ i-
nation of 3 co-formulated drugs used for common cold and cough
treatment. The studied drugs are paracetamol, pseudoephedrine,
and chlor pheniramine maleate. The separation was achieved us-
ing silica gel 60 F254 plates and the developing system of metha-
nol‒toluene‒acetic acid (44:16:1, v/ v). Densitometry scanning was
performed at 254 nm. The method was val idated as per the Inter-
national Conference on Harmonization (ICH) guidelines and was
successfully applied for the analysis of pharmaceutical preparation
containing the cited ternary mixture without interference from
excipients. There is no previously published TLC–densitometric
method for the determination of the previously mentioned ternary
mixture. The suggested method is rapid and of low cost, so it can be
used for quality control analysis.
1 Introduction
Paracetamol (PARA), pseudoephedrine (PSE), and chlorphe-
niramine maleate (CPM) have been co-formulated and widely
used for symptomatic treatment of common cold [1, 2]. Par-
acetamol, N-(4-hydroxyphenyl)acetamide, is an analgesic and
antipyretic agent. Pseudoephedrine, 2-methylamino-1-phenyl-
propan-1-ol, is used as a nasal mucosal vasoconstrictor and
M.M. Fouad and C.M. El-Maraghy, Analy tical Che mistry Depa rtment, Facult y
of Pharmacy, October Un iversity for Modern Sciences and Arts (MSA), Egypt;
and M.M. Fouad, Analytical C hemist ry Depart ment, Faculty of Phar macy,
Al-Azhar University, Egypt.
*E-mail: Christine_elmaraghy@hotmail.com; cmagued@msa.eun.eg
decongestant [3]. Chlorpheniramine maleate, 2-pyridinepro-
panamine-(4-chlorophenyl)-N,N-dimethyl-2-butenedioate, is a
reversible competitive inhibitor of the interaction of histamine
with H1 receptors [3]. The structures of these compounds are
shown in Figure 1. The literature review revealed their simul-
         
plasma, including spectrophotometry for the determination
of PSE, CPM, and dextromethorphan (DX) [4], chemometric
methods for the determination of PARA, CPM, and guaiphen-
esin [5], high-performance liquid chromatography (HPLC) for
the determination of PARA, CPM, and PSE in pharmaceuticals
[6–9], and HPLC–mass spectrometry (MS) for their analysis
        -
edge, there was no reported thin-layer chromatography (TLC)–
densitometric method for the simultaneous determination of
PARA, PSE, and CPM. The TLC method has the advantages
over the HPLC method of being more cost- and time-saving
and not requiring pH adjustment of the mobile phase or tedious
 
conduct a comparative study between TLC– densitometry and
the previously published HPLC method for the simultaneous

pharmaceutical preparation.
Rapid Validated Thin-Layer Chromatography–Densitometry
for the Simultaneous Determination of Three Co-formulated
Drugs Used for Common Cold Treatment
Manal M. Fouad and Christine M. El-Maraghy*
Key Words:
Paracetamol
Pseudoephedrine
Chlorpheniramine maleate
Thin-layer chromatography–densitometry
Common cold
Journal of Pla nar Chromatography 32 (2019) 2, 127–131 DOI: 10.155 6/10 06 .2019.3 2. 2.8
09 33- 4173 © Akadémiai Kiadó, Budapest
Figure 1
The chemical structures of paracetamol, pseudoephedrine hydro-
chloride, and chlorpheniramine maleate.

TLC–Densitometric Determination of Three Co-formulated Drugs Used for Common Cold
128 Journal of Planar Chromatography 32 (2019) 2
2 Experimental
2.1 Instruments
A CAMAG TLC scanner (CAMAG, Muttenz, Switzerland)
operated with winCATS software version 3.15, a Linomat
         
(Hamilton, Bonaduz, Switzerland), and pre-coated silica gel
254

2.2 Reagents and Chemicals
Paracetamol (El Nasr Company, Cairo, Egypt), chlorphe-
niramine maleate (ADWIC Pharmaceutical Company, Egypt),
and pseudoephedrine hydrochloride (Delta Pharma Company,
         
respectively, as stated by the supplier were used.
Methanol (Riedel-de Haën, Sigma-Aldrich, Darmstadt, Ger-
   
glacial acetic acid (ADWIC) were used.
2.3 Pharmaceutical Preparation
Cetal Cold&Flu®      


2.4 Preparation of Standard Solutions
 for
PARA and 1 mg mL for PSE and CPM were prepared using
       
freshly prepared by dilution with methanol to obtain solutions
having concentrations of 1 mg mL  
for PSE and CPM.
2.5 Procedures
2.5.1 Chromatographic Conditions
-
  254 aluminum sheets. The samples were applied to
    

from each other and 2 cm from the bottom edge. A mixture of
methanol, toluene, and acetic acid (44:16:1, v/v) was selected
as the mobile phase. Densitometry scanning was performed at
254 nm. The chromatographic chamber was pre-saturated with


2.5.2 Construction of Calibration Curves
-
ing standard solutions of each drug separately, equivalent to

        
plates, and the previous conditions were applied. The relative
         -
ing concentration, from which the regression equations were
calculated.
2.5.3 Assay of Laboratory-Prepared Mixtures
  


each drug in the laboratory-prepared mixtures were calculated
and processed as described. The concentration of the 3 drugs
was calculated using the corresponding calculated regression
equation.
2.5.4 Application to Pharmaceutical Preparation
Ten Cetal Cold&Flu® tablets were accurately weighted and
 
            -
          
      
            
-

 
 PSE. The general pro-
cedure described above was followed, and the concentrations of
PARA, CPM, and PSE were calculated using the corresponding
regression equation. The validity of the results was assessed by
      
concentrations of the 3 pure drugs to the same pharmaceuti-
cal preparation and proceeding as the previously mentioned
procedure.
3 Results and Discussion
         
method for the simultaneous determination of 3 co-formulated
-
         
was no reported TLC–densitometric method for the simultane-
ous determination of PARA, PSE, and CPM. TLC–densitom-
 
instr ument and the solvents used), and rapid, when compared
to HPLC.
Figure 2
TLC densitogram of PARA (60 μg per spot), CPM (20 μg per spot),
and PSE (20 μg per spot), using methanol –tolu ene–acetic acid
(44:16:1, v/v) as the developing system measured at 254 nm.

TLC–Densitometric Determination of Three Co-formulated Drugs Used for Common Cold
Journal of Pla nar Chromatography 32 (2019) 2 129
3.1 Method Optimization
The chromatographic conditions were optimized by spotting
-
tems in order to achieve the best separation. Initially, a system
  v/v) was used, but PARA was
very near to the solvent front. Increasing the polarity of the
developing system by increasing the methanol ratio was done
in order to move PARA away from the solvent front. Acetic acid

error in the retardation factor (RF) calculation. Complete sepa-
 
acetic acid in a ratio of 44:16:1 (v/v). The average RF values of

Figures 2‒5.
3.2 Method Validation
The Inter national Conference on Harmonization (ICH) guide-
lines [13] for method validation were followed.
3.2.1 Linearity
Calibration graphs were found to be linear in the range of


3.2.2 Accuracy
The accuracy of the proposed chromatographic method was

standard solution of PARA, CPM, and PSE in triplicate. Their
concentrations were calculated using the corresponding regres-
sion equation, and then the mean percentage recovery and
the standard deviation (%RSD) were calculated, as shown in
Table 1.
3.2.3 Precision
          
drugs were analyzed on the same day to determine the intra-
 
each concentration were analyzed for 3 separate days by using
the developed chromatographic method and calculating the rel-
ative standard deviation (Table 1).
3.2.4 Specicity
 
     
PARA, CPM, and PSE. Satisfactor y results were obtained as
shown in Table 2.
3.2.5 Robustness
The robustness was investigated by analysis of samples under
small changes in the mobile phase ratio to 42:18:1 (v/v), by
changing the mobile phase volume and the time of the mobile
Figure 4
TLC densitogram of PARA (RF = 0.81 ± 0.02) in the concentration
range of 50– 600 μg per spot.
Figure 5
TLC densitogram of CPM (RF = 0.1 ± 0.01) in the concentration range
1–30 μg per spot.
Figure 3
TLC densitogram of PSE (RF = 0.71 ± 0.02) in the concentration
range of 10–35 μg per spot.

TLC–Densitometric Determination of Three Co-formulated Drugs Used for Common Cold
130 Jo urnal of Planar Chromatography 32 (2019) 2
          
the chromatographic resolution. The results indicated that the

small deliberate variations (Table 3).
Tab le 1
Regression parameters for the determination of PARA, CPM, and
PSE using TLC–densitometry.
Parameters
TLC–densitometry
PA R A CPM PSE
Linearity range
]    
Slope 212.75 1771.9 6 93.14
Intercept 1587. 2 3515 
SE of the slope  95.615    
SE of the intercept 191.83    
Standard deviation of
residuals   76.67   
r)  
]
]
19.7
48.6


 

Accur acy
%RSD)  
Precision
(%RSD)
Inter-day 1.54 1.23 1.67
Intra-day 1.89 1.75 
The inter-day and int ra-day precision values of the samples: PARA


Tab le 2
Results obtained for the analysis of laboratory-prepared mixtures,
by the proposed methods, for the determination of PARA, CPM, and
PSE.
Conc.
( PAR – C PM – PS E)
%Recoverya)
PAR CPM PSE
 99.67 9 8.17  
     
       
     
b) 98.54 99.77 98.77
   
a)Average of three determinations
b)The ratio of Cetal Cold&Flu® pharmaceutical preparation
Tab le 3
Robustness of the proposed TLC–densitometric method for the de-
termination of PARA, CPM, and PSE.
Parameter
PA R A CPM PSE
RF
Mobile phase composition
[methanol–toluene –acetic acid]
(42:18:1, v/v)
v/v)






Mobile phase volume
   
Duration of saturation
   
Tab le 4
System suitability parameters of the develope d TLC– densitometric
method.
Parameter
TLC–densitometry
PA R A CPM PSE
RF   
Tailing factor (T)  
Selectivity factor (α)7.66 1.15
Resolut ion (Rs) 2.2 2.45 –
Tab le 5
Quantitative d etermination of PARA, CPM, and PSE in the phar-
maceutical preparation and application of standard addition tech-
nique.
Pharmaceutical
Preparation laimed Added
 %Recoverya)
Cetal Cold&Flu® tablets

Each labeled to contain

PARA, 2 mg CPM,

PA R A







99.72
 
CPM


3
4
5



 
PSE


1
2
3
98.54
99. 31
99.75
 
a)Average of 3 determinations. The values between parentheses repre-
sent the %Recovery and SD of analysis of pharmaceutical preparation

TLC–Densitometric Determination of Three Co-formulated Drugs Used for Common Cold
Journal of Pla nar Chromatography 32 (2019) 2 131
3.3 System Suitability
       
        
Method performance data including retardation factor (RF),
tailing factor (T), and resolution (Rs) are listed in Table 4.
3.4 Analysis of Pharmaceutical Preparation
The proposed method was applied for the determination of
PARA, CPM, and PSE in Cetal Cold&Flu® tablets. The results
were satisfactor y and in good agreement within the labeled
amount. The interference of excipients in the pharmaceutical
preparation was studied using a standard addition technique.
According to the obtained results, good accuracy and preci-
sion were observed (Table 5). Consequently, the excipients
in the phar maceutical formulations did not interfere in the
simultaneous analysis of the 3 studied drugs in pharmaceutical
preparation.
3.5 Statistical Analysis
The results obtained by applying the proposed chromatographic
method were statistically compared to the reference HPLC
method [8]. The calculated t- and F-values were less than the
 

respect to accuracy and precision, as presented in Table 6.
4 Conclusion
-
titative analysis of PARA, PSE, and CPM in their laborato-
ry-prepared mixtures and in pharmaceutical formulations using
TLC–densitometry. The results showed that the developed
TLC–densitometry had the advantages of being simpler than
the HPLC, as it used simple mobile phase with no pH adjust-
ment, sensitive, and economic, as it saves cost (inexpensive
           
applied onto a single plate per one development). The developed
method can be used in routine quality control testing, allowing
qualitative and quantitative determination with high accuracy
and precision.
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



Tab le 6
Statistical comparison bet ween th e results obtained by the proposed methods and reference method for the determination of PARA, CPM,
and PSE in pure powder form.
Parameter
TLC–densitometry Reference methodb)
PA R A CPM PSE PA R A CPM PSE
Mean 99.43  99.54 99. 21 98.99 9 9.21
SD 1.17 1.55 1.23  1.53 
 1.36  1.51  2.34 1.14
N 555 555
Student’s t-testa)   
F-testa)   
a)The values between parenthesis are the theoretical values of t- and F-test at P
b)The reference HPLC method using a C18              , 2 m L
