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  • 1 Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, , Kasr-El-Aini, 11562, Cairo, , Egypt
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Abstract

Due to the wide applicability of separation techniques that rely on the property of differential migration in pharmaceutical formulations analysis, different analytical strategies have been proposed to resolve mixtures of multi-components pharmaceuticals. Three separation methods were developed and validated for the simultaneous determination of Paracetamol (PAR), Pseudoephedrine HCl (PSE) and Chlorpheniramine maleate (CHP). The first method is a thin-layer chromatographic (TLC) separation, followed by densitometric measurement. The separation was carried out on aluminium sheet of silica gel 60 F254 using ethanol:chloroform:ammonia (1:7:0.4, by volume) as the mobile phase. Determination of PAR, PSE and CHP was successfully applied over the concentration ranges of 3–25 µg/band, 0.5–10 µg/band and 0.1–6 µg/band, respectively. The second method is HPLC separation that was achieved on C18 column using the mobile phase acetonitrile:phosphate buffer pH 5 (10:90, v/v) at a flow rate 1 mL min−1. PAR, PSE and CHP were determined by HPLC in concentration ranges of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1, respectively. The third method is a capillary electrophoresis (CE) separation. The electrophoretic separation was achieved using 20 mM phosphate buffer (pH 6.5) at 20 kV. The linearity was reached over concentration ranges of 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1 for PAR, PSE and CHP, respectively. The developed methods were validated with respect to linearity, precision, accuracy and system suitability. The proposed methods were successfully applied for bulk powder and dosage form analysis with RSD of precision <2%. Moreover, statistical comparison with the official methods confirms the methods' validity.

Abstract

Due to the wide applicability of separation techniques that rely on the property of differential migration in pharmaceutical formulations analysis, different analytical strategies have been proposed to resolve mixtures of multi-components pharmaceuticals. Three separation methods were developed and validated for the simultaneous determination of Paracetamol (PAR), Pseudoephedrine HCl (PSE) and Chlorpheniramine maleate (CHP). The first method is a thin-layer chromatographic (TLC) separation, followed by densitometric measurement. The separation was carried out on aluminium sheet of silica gel 60 F254 using ethanol:chloroform:ammonia (1:7:0.4, by volume) as the mobile phase. Determination of PAR, PSE and CHP was successfully applied over the concentration ranges of 3–25 µg/band, 0.5–10 µg/band and 0.1–6 µg/band, respectively. The second method is HPLC separation that was achieved on C18 column using the mobile phase acetonitrile:phosphate buffer pH 5 (10:90, v/v) at a flow rate 1 mL min−1. PAR, PSE and CHP were determined by HPLC in concentration ranges of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1, respectively. The third method is a capillary electrophoresis (CE) separation. The electrophoretic separation was achieved using 20 mM phosphate buffer (pH 6.5) at 20 kV. The linearity was reached over concentration ranges of 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1 for PAR, PSE and CHP, respectively. The developed methods were validated with respect to linearity, precision, accuracy and system suitability. The proposed methods were successfully applied for bulk powder and dosage form analysis with RSD of precision <2%. Moreover, statistical comparison with the official methods confirms the methods' validity.

Introduction

Common cold is a respiratory viral illness that can be managed by relieving symptoms using many combinations of drugs like analgesics, anti-inflammatories, decongestants and antihistamines [1, 2]. One of these combinations is the mixture of Paracetamol (PAR), Pseudoephedrine HCl (PSE) and Chlorpheniramine maleate (CHP).

Paracetamol (PAR), N-(4-hydroxyphenyl) acetamide (Fig. 1a) is a popular analgesic and antipyretic[3] which is used as pain killer and fever treatment in a variety of pain and cough/cold formulations designed for adults and children. It is considered by The World Health Organization to be an essential medicine in a basic health system. It is an official drug in British [3] and United States [4] pharmacopoeias. Pseudoephedrine HCl (PSE), 2-(Methylamino)-1-phenylpropan-1-ol hydrochloride (Fig. 1b), is an adrenoceptor agonist [3] which is used due to its sympathomimetic effect to relieve nasal congestion. It is an official drug in British [3] and United States [4] pharmacopoeias. Chlorpheniramine maleate (CHP), 3-(4-Chlorophenyl)-N,N-dimethyl-3-(pyridin-2-yl)propan-1-amine hydrogen (Z)-butenedioate (Fig. 1c), is an antihistamine [3] and used to control allergy-like symptoms such as sneezing, runny nose and watery eyes. It is an official drug in British [3] and United States [4] pharmacopoeias.

Fig. 1.
Fig. 1.

Chemical structure of (a) Paracetamol (b) Pseudoephedrine HCl (c) Chlorpheniramine maleate

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

Separation techniques that rely on the property of differential migration play a great role in pharmaceutical formulations analysis. Various analytical strategies have been proposed to enhance the assessment of multi-components pharmaceuticals.

To our knowledge, only liquid chromatographic methods were reported for the simultaneous determination of PAR, PSE and CHP in tablets, dog and human plasma [5–10]. However, there are no reported TLC-densitometric or capillary electrophoretic methods. So, it was worthy to develop and validate different accurate methods for simultaneous determination of the PAR, PSE and CHP. The aim of this work is to manipulate the chromatographic and electrophoretic behaviors of the three analytes in three different procedures for their qualitative and quantitative determination in bulk powder and dosage for in pharmaceutical formulations.

Experimental

Apparatus

Thin-layer chromatography (TLC) was performed using a Camag 3S/N 30319 TLC scanner with winCATS software; an ultraviolet (UV) lamp with a short wavelength at 254 nm (Desaga, Wiesloch, Germany), a Camag Linomat autosampler (Muttenz, Switzerland), a Camag microsyringe (100 µL) and; and TLC plates precoated with silica gel 60 F254 10x20 cm, 0.25 mm thickness (Merck, Darmstadt, Germany).

HPLC was performed using an Agilent pump with different flow rates (model 1260 Infinity Series liquid; Agilent, Germany) equipped with a quaternary gradient pumping system and diode array detector and a 20 µL injection loop. A X-Bridge C18 column (250 mm, 4.6 mm, i.d. 5 µm) was used as stationary phase. The samples were injected with a 50 µL Hamilton analytical syringe. Data analysis and system monitoring was accomplished using Chemstation software (Agilent, USA).

CE was performed using an Agilent G 7100A-DAD equipped with an automatic injector and an autosampler, coupled with a photodiode array detector. A bare fused-silica capillaries (Agilent Technologies, Germany) 50 μm id, with effective length of 25 cm was used.

Sonicator (Memmert, Germany). Jenway 3505 pH-meter (Jenway, UK), was employed for pH adjustment.

Materials

  • Paracetamol sample was kindly obtained from Amoun Pharmaceutical Co., the percentage purity was found to be 100.16 ± 0.792 according to official method [4].

  • Pseudoephedrine HCl sample was kindly obtained from Amoun Pharmaceutical Co., the percentage purity was found to be 99.52 ± 1.020 according to official method [3].

  • Chlorpheniramine maleate sample was kindly supplied by Sigma Pharmaceutical Industries, the percentage purity was found to be 99.33 ± 0.670 according to official method [3].

  • Cetal Cold and Flu caplets, Batch No. 1704160 labeled to contain 500 mg PAR, 30 mg PSE and 2 mg CHP, manufactured by EIPICO (10th of Ramadan City, Egypt) were purchased from national market.

Chemicals and reagents

  • All chemicals used throughout this work were of analytical grade, and solvents were of HPLC grade.

  • Methanol, acetonitrile, potassium dihydrogen phosphate, sodium hydroxide and phosphoric acid are from sigma-Aldrich, Germany.

  • Ethanol, chloroform, and ammonia are from Adwic, El-Nasr Pharmaceutical Chemicals Co. Cairo, Egypt.

  • Double distilled deionized water is from Egypt Otsuka Pharmaceutical Co. Cairo, Egypt.

  • Borate and Phosphate buffer (pH 5, 6.5 & 8) were prepared according to British Pharmacopoeia [3].

Solutions

For TLC-densitometric method

  • Stock solutions of PAR, PSE and CHP (1 mg mL−1) were prepared in methanol.

For HPLC method

  • Stock solution of PAR (5 mg mL−1) was prepared in mobile phase (acetonitrile: phosphate buffer pH 5 (10:90, v/v)).

  • Stock solutions of PSE and CHP (1 mg mL−1) were prepared in mobile phase.

  • Working solution of PAR (1 mg mL−1) was prepared from its corresponding stock solution in mobile phase.

  • Working solution of PSE (100 μg mL−1) was prepared from its corresponding stock solution in mobile phase.

  • Working solution of CHP (20 μg mL−1) was prepared from its corresponding stock solution in mobile phase.

For CE method

  • Stock solution of PAR (5 mg mL−1) was prepared in methanol.

  • Stock solutions of PSE and CHP (1 mg mL−1) were prepared in methanol.

  • Working solution of PAR (1 mg mL−1) was prepared from corresponding stock solution in 20 mM phosphate buffer (pH 6.5) as background electrolyte (BGE).

  • Working solutions of PSE and CHP (100 μg mL−1) were prepared from their corresponding stock solutions in 20 mM phosphate buffer (pH 6.5) as background electrolyte (BGE).

Procedures

TLC-densitometric method

Aliquots equivalent to 3–25 µg of PAR, 0.5–10 µg of PSE and 0.1–6 µg of CHP standard stock solutions (1 mg mL−1) were applied to the TLC plates using applicator with 100 µL Camag microsyringe. The plates were developed to a distance of approximately 9.5 cm by the ascending technique with ethanol:chloroform:ammonia (1:7:0.4, by volume) as the mobile phase. The plates were removed, air-dried. The chromatogram was scanned at 208 nm. The calibration curves representing the relationship between the recorded area under the peak and the corresponding concentrations of the drug in micrograms per band were plotted, and the regression equations were computed.

HPLC method

Suitable aliquots were accurately transferred from PAR, PSE and CHP working solutions into a set of 10-mL volumetric flasks and the volumes were completed to the mark with mobile phase to prepare concentrations in the range of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1, respectively. The samples and the mobile phase were filtered through a 0.45 µm millipore membrane filter. The mobile phase was then degassed for about 15 min in an ultrasonic bath prior to use. Then, 20 µL aliquot of each solution was injected into a X-Bridge C18 column (250 × 4.6 mm, i.d. 5 µm) using the mobile phase acetonitrile: phosphate buffer pH 5 (10:90, v/v) at a flow rate 1 mL min−1 with detection at 208 nm. Calibration curves were constructed by plotting the peak area and the corresponding concentrations of each drug and the regression equations were computed. The system suitability parameters, retention time, tailing factor, separation of peaks (resolution), theoretical plate count (N), height equivalent to theoretical plate (HETP) and column retention were studied.

CE method

Suitable aliquots were accurately transferred from PAR, PSE and CHP working solutions into a set of 10-mL volumetric flasks and the volumes were completed to the mark with 20 mM phosphate buffer (pH 6.5) to construct calibrations for PAR, PSE and CHP in the range of 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1, respectively.

A bare fused-silica capillary with 50 μm id, and effective length of 25 cm was used. Each run consisted of preconditioning, injection of the sample and post-conditioning. Preconditioning was implemented by flushing with 0.1 M NaOH, water and BGE for 5 min each. Samples were filtered through a 0.22 µm millipore membrane filter then introduced into the capillary hydrodynamically using a pressure of 50 mbar for 5 s with UV detection at 200 nm and separation voltage of 20 kV. The capillary was then flushed with water for 5 min as post-conditioning. Calibration curves were constructed by relating peak areas with the corresponding concentrations of each component and the regression equations were computed.

Application to pharmaceutical formulation

The film coat was removed using methanol and the contents of ten tablets were accurately weighed and powdered.

For TLC-densitometric method: A weight equivalent to one tablet was transferred into 100-mL volumetric flask, complete to the mark with methanol and then sonicated for 15 min. The solution was filtered into 100-mL volumetric flask.

For HPLC method: A weight equivalent to one tablet was transferred into 250-mL volumetric flask, complete to the mark with mobile phase and then sonicated for 15 min. The solution was filtered into 250-mL volumetric flask.

For CE method: A weight equivalent to one tablet was transferred into 250-mL volumetric flask, complete to the mark with methanol and then sonicated for 15 min. The solution was filtered into 250-mL volumetric flask.

Aliquots from the prepared solutions were transferred to 10-mL volumetric flasks and diluted with the appropriate solvent to prepare tablet solutions. The general procedure previously described for each method was followed to determine the concentration of each drug in the prepared dosage form solutions.

For applying the standard addition technique, known amounts of each standard component were separately added to aliquots of the prepared tablet solution and concentration of the added standard was determined after subtraction.

Results and discussion

Methods development and optimization

TLC – densitometric method

Several trials were conducted to develop the optimum chromatographic conditions for the sufficient separation of the proposed components. First attempts using mixtures of methanol, isopropanol and ethyl acetate in different ratios made the separation not achieved efficiently. Chloroform was then tested with ethanol in different ratios which improved the separation. Addition of acetic acid or formic acid to chloroform-ethanol system worsened the separation. However, separation was enhanced when ammonia was added in different ratios. Finally, optimum separation with good resolution was achieved when ethanol:chloroform:ammonia (1:7:0.4, by volume) was used as a developing system. Different scanning wavelengths were tested; detection at 208 nm was suitable providing good sensitivity for all components. Three well resolved bands of PAR, PSE and CHP were obtained having Rf values of 0.32, 0.43 and 0.81, respectively, Fig. 2.

Fig. 2.
Fig. 2.

TLC chromatogram of 6 µg/band standard PAR (Rf = 0.32), 6 µg/band standard PSE (Rf = 0.43) and 6 µg/band standard CHP (Rf = 0.81) using a mobile phase of ethanol:chloroform:ammonia (1:7:0.4 by volume) and detection at 208 nm

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

A polynomial relationship was found to exist between the area under the peak of the separated bands at the selected wavelength and the corresponding concentration of PAR (3–25 μg/band), PSE (0.5–10 μg/band) and CHP (0.1–6 μg/band), Fig. 3.

Fig. 3.
Fig. 3.

Scanning profile of the TLC chromatogram of (a) PAR (3–25 µg/band), (b) PSE (0.5–10 µg/band) and (c) CHP (0.1–6 µg/band) at 208 nm

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

HPLC method

To optimize the proposed HPLC method, the chromatographic conditions were investigated and tested. Parameters affecting chromatographic separation were studied during method development and optimization to obtain the optimum separation between PAR, PSE and CHP. The initial trials were achieved using X-Bridge C18 column. This column is categorized as fully porous stationary phase which offers pH stability over wide pH range. This feature is important in method development for pharmaceutical compounds and can be used for analysis of weak acidic or basic analytes. X-Bridge C18 column has the advantage of mechanical stability which increases the column efficiency. Different simple systems were tried for chromatographic separation of the proposed components such as methanol/water and acetonitrile/water but poor resolution was obtained. Also the effect of pH was studied by using buffers of different pH values and buffer pH 5 was found to be optimum. Trials revealed that buffer and acetonitrile mixture was appropriate for such separation, so the next factor to optimize was the ratio between acetonitrile and the buffer. Percentage of acetonitrile with different combinations was varied and best resolution was achieved using a mobile phase consisting of acetonitrile: phosphate buffer pH 5 (10:90, v/v). Various mobile phase flow rates (0.8, 1 and 1.2 mL min−1) were tested to obtain the best resolution within a short analysis time. Using 1 mL min−1 flow rate gave the optimum resolution with the shortest analysis time.

Finally, the optimum stationary/mobile phase matching trials for the HPLC system were achieved by using a X-Bridge C18 column (250 × 4.6 mm, i.d. 5 µm) with a mobile phase consisting of acetonitrile: phosphate buffer pH 5 (10:90, v/v) at flow rate of 1 mL min−1, followed by UV detection at 208 nm (Fig. 4). Chromatographic separation allows complete separation of the three components with optimum resolution. Linear relation was obtained between peak areas and the corresponding concentration of the components.

Fig. 4.
Fig. 4.

HPLC chromatogram of 10 μg mL−1 standard CHP (tR = 2.857), 50 μg mL−1 standard PAR (tR = 5.392) and 30 μg mL−1 standard PSE (tR = 7.211) using a X-Bridge C18 column (250X4.6 mm, 5 µm i.d.), mobile phase of acetonitrile:phosphate buffer pH 5 (10:90 V/V) at flow rate of 1 mL min−1 and detection at 208 nm

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

Comparing the proposed HPLC method with the previously published methods, it is found that some methods were developed to determine PAR, PSE and CHP in human or dog plasma [6–7], while other methods were designed to quantify the three mentioned drugs in pharmaceutical formulations [5, 8–10]. The proposed method is more time saving than other methods [5, 8–10] intended to assess PAR, PSE and CHP in pharmaceutical formulations due to the shorter run time. Moreover, the proposed HPLC method has wider concentration ranges for PAR, PSE and CHP determination and found to be the most sensitive method for PAR determination.

CE method

All the experimental conditions and parameters that can affect the electrophoretic separation were investigated and optimized to reach optimum separation between the studied drugs.

First, borate and phosphate buffers were investigated at pH of 5.5, 6.5 and 8. Borate buffer produced bad resolution with slow migration rates. On the other hand, good separation was achieved upon using phosphate buffer. Buffers with pH 5.5 and 8 showed poor peak shape and poor resolution, while buffer with pH 6.5 was the optimum in producing well resolved peaks with good accuracy and precisions of the components to be separated in a short time. According to the ionization and charge/size of the components, the expected order of elution is PSE, CHP and PAR. The basic pKa of PSE and CHP are 9.52 and 9.4, respectively, while PAR is a weakly acidic substance. Both PSE and CHP are ionized at pH <7.5 with positively charged amino group, while PAR remains unionized. Therefore, PSE was eluted first at pH 6.5 due to its smaller size than CHP and PAR was the last eluted component.

The effect of BGE concentration on the separation was investigated in the range from 20 to 50 mM. There was a delay in migration time when the concentration of the buffer increased. Also, an increase in current was observed while increasing buffer molarity which may lead to Joule heating. Finally, 20 mM phosphate buffer (pH 6.5) gave sharp peaks with optimum resolution. Voltages of 15–25 kV were tried and 20 kV permits efficient separation with reasonable current intensity. Detection was tried at different wavelengths and the best sensitivity was obtained at 200 nm, Fig. 5.

Fig. 5.
Fig. 5.

Capillary electrophoresis (CE) electropherogram of 10 μg mL−1 standard PSE (MT = 3.072), 5 μg mL−1 standard CHP (MT = 3.405) and 60 μg mL−1 standard PAR (MT = 3.873) using using 20 mM phosphate buffer pH 6.5 as BGE and 20 kV at 200 nm

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

Moreover, the peak purity of the three compounds was checked through their UV-absorption spectra by using the photodiode array detector proofing that each peak is pure and contains only one substance, Fig. 6.

Fig. 6.
Fig. 6.

Peak purity plot of (a) PSE, (b) CHP and (c) PAR and their corresponding absorption spectra

Citation: Acta Chromatographica 2021; 10.1556/1326.2021.00954

Methods validation

The proposed methods were validated according to ICH guidelines (Table 1). The linearity of the studied components was assessed between the peak area and the related concentrations of PAR, PSE and CHP. Polynomial relationships are found to exist when plotting concentrations of PAR, PSE and CHP against the corresponding peak areas in TLC-densitometric method over concentration ranges of 3–25 µg/band, 0.5–10 µg/band and 0.1–6 µg/band, respectively. While, Linear regressions are obtained for PAR, PSE and CHP, respectively in the range of 5–400 μg mL−1, 2–40 μg mL−1 and 0.5–16 μg mL−1 for HPLC method and 30–250 μg mL−1, 5–50 μg mL−1 and 0.8–20 μg mL−1 for CE method.

Table 1.

Assay and validation parameters for the determination of PAR, PSE and CHP by the proposed methods

ParameterTLCHPLCCEAcceptance criteria
PARPSECHPPARPSECHPPARPSECHP
Range3–25 µg/band0.5–10 µg/band0.1–6 µg/band5–400 μg mL−12–40 μg mL−10.5–16 μg mL−130–250 μg mL−15–50 μg mL−10.8–20 μg mL−1concentration interval over which linearity and accuracy are obtained
SlopeSlope 1* = −0.2832

Slope 2*= 15.898
Slope 1*= −2.957

Slope 2*= 57.152
Slope 1*= −7.7758

Slope 2* = 96.009
11.59430.94344.8110.95640.82821.3521
Intercept308.666.42760.2797.96888.2159.316411.7215.06640.7579
Mean of recovery of calibration99.9499.0499.8899.91100.1999.8299.86100.07100.12100 ± 2%
SD of recovery of calibration1.6371.8021.4810.5851.1200.9440.6600.9130.760SD ≤ 2%
Correlation coefficient (r)0.99960.99991.0001.0000.99990.99990.99991.00001.000≥0.999
Accuracy99.03 ± 0.8099.88 ± 0.56100.39± 0.7099.99 ± 0.79100.44 ± 0.5098.54 ± 0.64100.34 ± 0.6999.51 ± 0.55100.26 ± 0.56100 ± 2%
Repeatability**100.40 ± 0.660100.19 ± 0.82999.96 ± 0.269100.31 ± 0.587100.16 ± 0.47399.58 ± 0.78399.47 ± 0.627101.75 ± 0.92198.11 ± 0.968RSD ≤2%
Intermediate precision**100.76 ± 1.16599.15 ± 1.20499.77 ± 0.879101.54 ± 0.911100.58 ± 0.70599.32 ± 1.11499.37 ± 0.819100.90 ± 1.62298.27 ± 1.131RSD ≤2%
LOD***0.66 µg/band0.04 µg/band0.03 µg/band1.45 μg mL−10.50 μg mL−10.14 μg mL−16.44 μg mL−10.09 μg mL−10.25 μg mL−1
LOQ***2.00 µg/band0.13 µg/band0.09 µg/band4.40 μg mL−11.51 μg mL−10.42 μg mL−119.51 μg mL−10.26 μg mL−10.77 μg mL−1

* Slope 1 and 2 are the coefficients of X 2 and X, respectively. Following a polynomial regression A = ax 2 + bx + c Where, A is the peak area, x is the concentration (µg/band), a and b are coefficients 1 and 2, respectively and c is the intercept.

** Mean values ± relative standard deviations of three concentrations of PAR, PSE and CHP analyzed intra-daily in triplicate (repeatability) and on three successive days (intermediate precision).

*** Limits of detection and quantitation are determined via calculations, LOD = 3.3 × SD/slope, LOQ = 10 × SD/slope.

Accuracy was determined using three concentration levels in three replicates. The accuracy mean percentage recoveries of PAR, PSE and CHP were 99.03 ± 0.80, 99.88 ± 0.56 and 100.39 ± 0.70, respectively using TLC-densitometric method, 99.99 ± 0.79, 100.44 ± 0.50 and 98.54 ± 0.64, respectively using HPLC method and 100.34 ± 0.69, 99.51 ± 0.55 and 100.26 ± 0.56, respectively using CE method. The obtained results proved that the developed methods are accurate and reliable.

Precision was also tested with respect to the intra‐day (repeatability) and inter‐day (intermediate) variations. Intra‐day precision was tested in triplicate within short interval times in the same day while intermediate precision was determined on three successive days. The obtained relative standard deviation values of intra-day and inter-day variations were <2%, indicating good precision. LOD and LOQ were evaluated using the mathematical method and illustrated in Table 1.

An overall system suitability testing was calculated (Table 2). Satisfactory system suitability parameters including resolution (RS), tailing factor (T), capacity factor (k), selectivity factor (α), column efficiency (N), and height equivalent to theoretical plates were obtained for the proposed TLC, HPLC and CE methods.

Table 2.

Parameters required for system suitability tests of TLC–densitometric, HPLC and CE methods

TLCHPLCCEReference values [11–13]
PARPSECHPCHPPARPSEPSECHPPAR
Retention/Migration time (min)2.8575.3927.2113.0723.4063.873
Rf0.320.430.81
Resolution (Rs)1.685.058.344.402.4813.168Rs< 1.5
Tailing factor (T)1.030.950.981.051.101.171.031.081.13T = 0.8–1.2
Capacity factor (k)2.575.748.011 > K> 10
Selectivity factor (α)1.345.052.231.391.111.14α < 1
Column efficiency (N)655.361,183.364,199.044,797.0313,302.6012,307.3414,353.9613,941.6320,694.06≥2,000 (HPLC and CE)
Height equivalent to theoretical plate (cm)0.00390.00290.00150.00520.00190.00200.00170.00180.0012The smaller the value the higher the column efficiency

Application to pharmaceutical formulation

The proposed separation methods were successfully applied for the simultaneous determination of PAR, PSE and CHP in their pharmaceutical formulation, Table 3. The obtained percentage recoveries of PAR, PSE and CHP were 100.43 ± 0.589, 98.43 ± 0.523 and 100.25 ± 1.347, respectively using TLC-densitometric method, 101.54 ± 0.590, 99.57 ± 0.946 and 98.19 ± 0.839, respectively using HPLC method and 97.81 ± 1.049, 99.95 ± 0.900 and 99.46 ± 1.156, respectively using CE method.

Table 3.

Determination of PAR, PSE and CHP in dosage form and application of standard addition technique using the proposed methods

TLCHPLCCE
PARPSECHPPARPSECHPPARPSECHP
Cetal cold and flu *Found10.04 µg/band1.48 µg/band0.20 µg/band101.54 μg mL−117.92 μg mL−11.18 μg mL−1195.61 μg mL−111.99 μg mL−10.80 μg mL−1
% Recovery ±S.D100.43 ± 0.58998.43 ± 0.523100.25 ± 1.347101.54 ± 0.59099.57 ± 0.94698.19 ± 0.83997.81 ± 1.04999.95 ± 0.90099.46 ± 1.156
Recovery of standard added %*98.04 ± 0.33799.39 ± 1.01598.58 ± 0.852100.89 ± 0.56899.49 ± 1.05499.53± 0.90499.97 ± 0.77099.80 ± 0.71699.06 ± 0.481

*Average of three determinations.

The standard addition technique was applied to assess the validity of the proposed methods, Table 3. Good accuracy confirmed that the excipients in pharmaceutical formulation did not interfere in the determination of the drugs. Finally, the results obtained by applying the proposed methods for the determination of PAR, PSE and CHP were statistically compared with the official methods [3–4] and showed no significant difference with respect to accuracy and precision (Table 4).

Table 4.

Statistical comparison for the results obtained by the proposed methods and the official method for the analysis of PAR, PSE and CHP

TLCHPLCCEOfficial method
PARPSECHPPARPSECHPPARPSECHPPAR [4]PSE [3]CHP [3]
Mean99.9499.0499.8899.91100.1999.8299.86100.07100.12100.1699.5299.33
S.D.1.6371.8021.4810.5851.1200.9440.6600.9130.7600.7921.0200.670
Variance2.6783.2482.1930.3421.2550.8910.4360.8340.5780.6271.0400.449
n877777766666
Student's t-test0.329 (2.179)0.604 (2.201)0.883 (2.201)0.624 (2.201)1.125 (2.201)1.068 (2.201)0.722 (2.201)0.984 (2.228)1.903 (2.228)
F value4.27 (4.88)3.12 (4.95)4.89 (4.95)1.83 (4.39)1.21 (4.95)1.98 (4.95)1.44 (4.39)1.25 (5.05)1.29 (5.05)

Figures between parentheses represent the corresponding tabulated values of t and F at P = 0.05.

The proposed TLC – densitometric, HPLC and CE methods succeeded in the resolution and sensitive quantitation of PAR, PSE and CHP. Under the optimized chromatographic conditions, excellent separation of three mentioned drugs was obtained. Proposing different separation methods offers more than one way to quantify the desired components according to the available resources.

The developed TLC – densitometric method has the strength of being simple and accurate. The TLC – densitometric method is economic as it saves cost by using inexpensive apparatus and solvents. In addition, it saves time and effort as multiple samples could be applied to a single plate and analyzed per one development. The proposed HPLC method has the advantage of being accurate, reproducible and more precise than the developed TLC – densitometric with no excessive data manipulation. Furthermore, the HPLC method allows optimum separation of the three components with the highest resolution and speed analysis due to the help of a pump to push the mobile phase through the column. The presented CE method has shorter run time than HPLC method which is time saving with the added advantage of low consumption of solvents and samples. The method can be fully automated. Moreover, it has the advantage of purity checking of the separated components through their UV-absorption spectra and confirming that each peak is pure and contains only one substance.

Conclusion

Three separation methods were developed to determine PAR, PSE and CHP in their ternary mixture and pharmaceutical dosage form. TLC-densitometry, HPLC and capillary electrophoresis were proposed, optimized and validated. These are the first developed TLC-densitometry and capillary electrophoresis methods for simultaneous determination of this combination. Good resolution was obtained between the proposed components. The developed TLC method is economic and highly sensitive. The presented HPLC method is fast and accurate. The presented CE method can be fully automated therefore; it is simple, time and cost saving and reproducible.

The three mentioned methods are characterized by broad applicability, use of minimal solvents volume and short analysis time with no need of data manipulation. Moreover, the proposed methods were validated with respect to linearity, precision, accuracy and system suitability. So, they can be applied for assessment of pharmaceutical formulations containing PAR, PSE and CHP in quality control laboratories.

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  • 1.

    Simasek, M. ; Blandino, D. Treatment of the common cold. Am. Fam. Phys. 2007, 75, 515520.

  • 2.

    Picon, P. D. ; Costa, M. B. ; da Veiga Picon, R. ; Fendt, L. C. C. ; Suksteris, M. L. ; Saccilotto, I. C. ; Dornelles, A. D. ; Schmidt, L. F. C. Symptomatic treatment of the common cold with a fixed-dose combination of paracetamol, chlorphenamine and phenylephrine: a randomized, placebo-controlled trial. BMC Infect. Dis. 2013, 13, 18.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    British Pharmacopoeia. In British Pharmacopoeia, 5th ed.; Stationary Office, Appendix 1D: Buckingham Palace Road, London, UK, 2016.

  • 4.

    The United States of Pharmacopoeia-39/National Formulary-34. United States Pharmacopoeial Convention, Inc.: Rockville, Maryland, USA, 2016.

    • Search Google Scholar
    • Export Citation
  • 5.

    Biemer, T. A. Simultaneous analysis of acetaminophen, pseudoephedrine hydrochloride and chlorpheniramine maleate in a cold tablet using an isocratic, mixed micellar high-performance liquid chromatographic mobile phase. J. Chromatogr. A 1987, 410, 206210.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Liao, Q. ; Xie, Z. ; Pan, B. ; Zhu, C. ; Yao, M. ; Xu, X. ; Wan, J. Lc–Ms–Ms simultaneous determination of paracetamol, pseudoephedrine and chlorpheniramine in human plasma: application to a pharmacokinetic study. Chromatographia 2008, 67, 687694.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Zou, H. ; Gao, S. ; Chen, W. ; Zhong, Y. ; Jiang, X. ; Pei, Y. Simultaneous quantitation of paracetamol, pseudoephedrine and chlorpheniramine in dog plasma by Lc-Ms-Ms. Chromatographia 2008, 68, 251257.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Kalogria, E. ; Koupparis, M. ; Panderi, I. A porous graphitized carbon column Hplc method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation. J. AOAC Int. 2010, 93, 10931101.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Rajurkar, S. Simultaneous determination of chlorpheniramine maleate, paracetamol and pseudoephedrine hydrochloride in pharmaceutical preparations by Hplc. Int. J. Life Sci. Pharma Res. 2011, 1, 94100.

    • Search Google Scholar
    • Export Citation
  • 10.

    Dong, Y.-M. ; Li, N. ; An, Q. ; Lu, N.-W. A novel nonionic micellar liquid chromatographic method for simultaneous determination of pseudoephedrine, paracetamol, and chlorpheniramine in cold compound preparations. J. Liquid Chromatogr. Relat. Tech. 2015, 38, 251258.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Weston, A. ; Brown, P. R. High Performance Liquid Chromatography & Capillary Electrophoresis: Principles and Practices. Elsevier: San Diego, California, USA, 1997; p. 715.

    • Search Google Scholar
    • Export Citation
  • 12.

    Variyar, P. S. ; Chatterjee, S. ; Sharma, A. Fundamentals and theory of Hptlc-based separation. In High-Performance Thin-Layer Chromatography (Hptlc), Springer: 2011; pp. 2739.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Adamovics, J. A. Chromatographic Analysis of Pharmaceuticals, 2nd ed.; Routledge: Madison Avenue, New York, USA, 2017, 74, 1117.

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Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

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  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • Ł. Komsta (Medical University of Lublin, Lublin, Poland)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • J. Sherma (Lafayette College, Easton, PA, USA)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)

 

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

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Impact Factor 1,418
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without
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