Tlc methods for the determination of paracetamol, pseudoephedrine and

brompheniramine in Comtrex tablets

ABSTRACT
In this mini review, I focus on the uses of Tlc methods for the determination of paracetamol, pseudoephedrine and brompheniramine in
Comtrex tablets. In general, Comtrex ® tablet is a drug that is used to temporarily treat symptoms caused by the flu, common cold, allergies, or
other breathing illnesses and this tablet is made up of paracetamol, pseudoephedrine, and brompheniramine. This research has overcome the
challenging dosage form ratio (250:15:1) and proposed a chromatographic method was used to analyze the ternary combination showing
different devices, solvents, and sensitivity ranges. For the simultaneous determination of the three drugs, a chromatographic method was
developed and validated, namely thin layer chromatography (TLC). Thin layer chromatography ( TLC) is a fast , sensitive and inexpensive
method used to identify the number of components in a mixture, to test the compound's identity and purity, to track the progress of a reaction,
and to analyze the fractions obtained from column chromatography. With respect to the TLC process, aluminum TLC plates pre-coated with
silica gel 60F254 have been used, methanol: water: ammonia (9:1:0.1, v / v / v) was being used as a mobile phase and the plates were scanned
at 254 nm under GC instrument.

KEYWORDS (MAX 6)

HIGHLIGHTS (BULLET POINT)

INTRODUCTION
Medication is a necessity in human life to help reduce pain and relieve. Nowadays, the need for reliable and secure counter-
medication raises everyday causing drug manufacturers and pharmaceutical companies to incorporate more components in
their types of dosage. the symptoms. Hence, the pharmacists in the world need a fast and accurate method that helps them to
produce and analyze the components or ingredients in medicine. The main drug that has been analyzed in this research is
Comtrex® Maximum Strength tablets where this type of tablets are composed of paracetamol (PAR), pseudoephedrine
hydrochloride (PSE) and brompheniramine maleate (BRM).
There are various active ingredients used in Comtrex® Maximum Strength tablets. For instance, paracetamol (PAR),
pseudoephedrine hydrochloride (PSE) and brompheniramine maleate (BRM) are the most common ingredients to make this
tablet. Generally, the function of paracetamol (also known as N-(4-hydroxyphenyl) acetamide) is as the fever reducer and pain
reliever. In Malaysia, Paracetamol has been recognized by various brands available in the market and the most famous one is
Panadol. PAR can also be given specifically for managing unbearable pain when combined with non-steroidal anti-inflammatory
medications or opioid analgesics. Literature survey showed that PAR was measured by titrimetry, spectrophotometry,
spectrofluorimetry, high performance thin layer chromatography (HPTLC), GC – MS, HPLC – UV, HPLC – MS / MS and micellar
electrokinetic capillary chromatography (MECC) in its single form or in combination with certain medicinal items.
The other compound found in this tablet was PSE [(+)-threo-a-[1-methylamino) ethyl] benzyl alcohol] where it
functions as a decongestant in the nasal passages which shrinks blood vessels to avoid dilated blood vessels that results in
nasal congestion or stuffy nose. Besides, PSE is a sympathomimetic amine that works directly on adrenergic receptors. The
methods found in the literature for PSE quantitation are titrimetry, spectrophotometry, HPTLC,GC , Micellar electrokinetic
chromatography (MEKC), HPLC–UV and capillary electrophoresis (CE).
The third drug which is BRM is an antihistamine used to treat signs of allergies including sneezing , coughing and watery eyes.
Since BRM is a newly released drug, there are only two methods that have been used in the BRM literature along with
phenylephrine and the blood plasma.
According to this research, the determination of cough and cold pharmaceutical preparations is typically difficult,
since these preparations consist of complex formulae containing various active ingredients and a wide variety of additives.
Another challenging part of this research is the separation of these ingredients in the pharmaceutical dosage forms is typically
complicated due to similarities of their physical and chemical properties. Therefore, the researchers aim to carry out various
sensitive, reliable and precise chromatographic method (TLC) for separating and quantifying PAR, PSE and BRM in their
combined dosage type specifically as far as their knowledge was concerned.

REVIEW (TLC)

Apparatus and software

In this experiment, the researchers have determined the apparatus that will be used for the TLC-densitometric
system. Firstly, a CAMAG TLC Scanner 3 S/N 130,319 with CATS software and Linomat 5 autosampler were used to perform the
tlc measurements. Besides, the main and important apparatus in tlc experiment which is TLC aluminum sheets pre-coated with
silica gel 60 F254 in the size of 20 × 20 cm as well as a CAMAG micro syringe of 100 μL were used while conducting the research.

Chemicals and reagents

Before starting the experiment, the researcher has obtained the pure samples of PAR , PSE and BRM from
GlaxoSmithKline located in Cairo, Egypt. The researcher has been informed that the purities of these samples were found to be
at 99.40 ± 0.778, 100.11 ± 0.427 and 99.12 ± 0.699 for PAR, PSE and BRM respectively, according to recorded analytical
methods. Solvent is one of the important things in analytical technique thus methanol (HPLC) was selected to use in the TLC
method which has been received from Fischer Scientific UK Ltd located in Loughborough, UK.
According to the label attached, these Comtrex Maximum Strength coated tablets contained 30 mg of PSE, 500 mg of
PAR and 2 mg BRM with a batch number of A514875. The making of these samples is not as worrying as it being registered
under license from Novartis Consumer Health and it was ordered from the local market. About the preparation of the standard
solution for the tlc method, the researchers made a separation on the standard solutions that consists of (5.0 mg/mL) PAR, (5.0
mg/mL) PSE and (4.0 mg/mL) BRM. By using the same solvent for the dilution process, the researchers have prepared standard
solutions with different concentrations which are (2.5 mg/mL) of PAR, (2.5 mg/mL) of PSE and (2.0 mg/mL) of BRM.

Chromatographic conditions for TLC-densitometric method

In this research, the same chromatographic condition was applied to all trials. The samples were applied in bands
defined as 10 μL / band to the TLC sheets using a 100-μL syringe .The size of tlc plate used was 20 × 20 cm with width of each
band was 6 mm, the bands were about 1 cm apart, and 1 cm away from the bottom of the plate. The researchers used
methanol:water:ammonia (9:1:0.1, v/v/v) as the developing system that being placed in the chromatographic tank for 1 hour at
room temperature. After that, they performed the linear ascending separation to a distance of approximately 8 cm from the
lower edge of the TLC plate. The plates were therefore air-dried, and scanned at 254 nm using a GC instrument. By using the
CAMAG TLC Scanner 3, the detection was conducted in the absorbance mode using a deuterium lamp as the source of radiation
while preserving the slit length at 3 mm × 0.45 mm and the speed of scanning at 20 mm/s.

.
Procedure
For the procedures used by the researchers to conduct the experiment, it began with transferring the aliquots of PAR,
PSE and BRM from their standard solutions into 10 ml volumetric flasks. Then, they marked up the solution with water in order
to produce concentrations of 250.0–2400.0 µg/mL for PAR, 100.0–2250.0 µg/mL for PSE and 20.0–1500 µg/mL for BRM. Next,
by using the micro syringe, about 10 µL of the solution from each concentration were spotted on the labelled tlc plates that
were already developed using methanol:water:ammonia (9:1:0.1, v/v/v). Immediately, they applied all the chromatographic
conditions on the tlc plate. In this context, the densitograms were recorded at 254 nm. The calibration curves describing the
peak area ratio (10 , 15, and 1.2 μg / band of PAR, PSE, and BRM, respectively) were used as an objective standard against the
equivalent concentration of the three compounds, and the regression equations were computed. The linearity ranges for PAR
are 2.5–24.0μg / band, for PSE 1.0–22.5μg / band and for BRM 0.2–15.0μg / band.

Results
Method development
According to the method development for tlc, the researchers decided to try on different developing systems to make
sure that they produce a good separation of the compounds and maximize the proposed method. First and foremost, they
began with applying chloroform:acetone:ammonia (8:2:0.1, v/v/v). Unfortunately, none of the three compounds were
separate. Same goes to the second trial, where they used methanol:chloroform (8:2, v/v) and PAR was extracted but after
seeking various ratios of the two solvents, PSE and BRM were not resolved. Then, the researchers tried to change water with
chloroform and used different ratios of methanol:water and it resulted in separating the PAR and BRM but PSE was tailed.
Methanol: ammonia ratio (10:1, v / v) has been effective in separating PSE and BRM, however PAR appears with the front
solvent.
 Eventually, the researchers managed to get the best results with a ratio of methanol: water: ammonia (9:1:0.1, v / v /
v) as the mobile phase and it came out with sharp symmetrical peaks thus provided the highest resolution. Therefore, they
adjusted and choosed band width of 6 mm to minimize band diffusion. In order to measure the spectra of the three drugs, they
attempted to use wavelengths of 210, 230, 254 and 265 nm where the drugs have high absorbance values and they observed
that at 254 nm, the drugs gave the sharp and symmetrical peaks with minimal level of noise.

After making some calculations, the researchers managed to get the retention factor value, Rf of the drugs which are
0.24 for PSE, 0.32 for BRM and 0.81 for PAR. Based on the tlc chromatogram shown in Fig.2, from which it was clear that each
of the three drugs could be determined without any intervention from the other and the movement of the substances can be
obviously seen.

Fig.2
Method Validation
LINEAR
A linear relation has been observed between each drug's peak area ratios and their equivalent concentrations.
Normally, external standard was used for the TLC and HPLC methods, while internal standard was used for UPLC-MS/MS
methods. The linearity ranges varied between the methods, and the linearity range for the TLC method for PAR, PSE and BRM is
5.0–100.0 μg / mL, 30.0–200.0 μg / mL, and 30.0–200.0 μg / mL , respectively. It can be concluded that the linear relation of the
calibration curves were verified by the values of the correlation coefficients that were reaching unity.

ACCURACY
 In this research, the drugs or the concentrations were performed three times and still using the same procedures as
the first trial in order to ensure that the average results are more reliable than a single trial. Hence, to check the accuracy of
certain results, it can be measured through the percentage recoveries or mean as well as standard deviation.

PRECISION

On the same day, three concentrations of the medications were tested in triplicates to determine intraday precision.
Nevertheless, on three consecutive days the interday precision was achieved by analyzing the same three concentrations of the
drugs in triplicates. The precision was measured as relative standard deviations (RSD per cent). From the findings it was obvious
that the RSD percent for all concentrations did not exceed 2 percent, showing that tlc methods could be regarded as accurate
methods.

ROBUSTNESS
The robustness of the method has been confirmed by the consistency of the drugs' peak area ratios with the intended slight
changes made. In the tlc method, there was A small change in the percentage of methanol ± 2 per cent . In all cases, however,
minor changes were observed in retention times or retention factors, the peak areas remained typically constant and proving
the robustness of the tlc method

CONCLUSION
This research is performed for the simultaneous determination of PAR, PSE and BRM in their pharmaceutical dosage form by
applying a basic chromatographic method which was tlc method. This research actually analyzed the three chromatographic
methods on three different compounds, but to follow the instruction of this assessment, only one chromatographic method
was chosen. Overall, the duration of the tlc method is considered to be fast but slower than the others proposed methods with
a run time ranging from 1 min for UPLC-MS / MS to 4 min for HPLC. Therefore, tlc method also helps in reducing solvent
consumption, which is a privilege from an economic and eco-friendly perspective.Based on the obtained results, TLC benefits
include rapid analysis time, as many samples can be analyzed simultaneously or in other words it reduces analytical time and
thus cost-effective. Besides, the TLC method used low solvent use per sample and results in high degree of accuracy and
precision for instrumental TLC, and it shows sensitivity in the range of nanograms or picograms.

ACKNOWLEDGEMENT
First of all, I would like to thank Allah SWT as I finally managed to finish my final assessment for analytical chemistry. This
assessment has been done with all affordance and despite all the problems happening, I was able to solve it and finished this
assessment on time.
A big thank I addressed to lecturers of analytical chemistry SCH 1014, Dr Wan Hazman and Dr Saiful ‘Arifin for all the guidance,
knowledge, teach and encouragement in finishing this assessment. Not to forget also my family and friends who have always
supported and understand me throughout the period of completing this assessment. Last but not least, I hope that this
assessment can contribute to giving my friends and I the best results for this first year degree.

REFERENCES

TLC, Paracetamol, Brompheniramine maleate, Pseudoephedrine

BRM: brompheniramine maleate

GC: gas chromatography
HPLC–UV: high performance liquid chromatography-ultraviolet detection
MECC: micellar electrokinetic capillary chromatography
PAR: paracetamol
PSE: pseudoephedrine hydrochloride
TLC: thin layer chromatography
UPLC-MS/MS: ultra-performance liquid chromatography-tandem mass spectrometry
 paracetamol, b pseudoephedrine HCl and c brompheniramine maleate