Proceedings of ICoAPS, 2018

Determination of chlorpheniramine maleate in the presence of tartrazine

in tablets using high performance thin layer chromatography

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F. Annuryanti, A. Darmawati, R. Primaharinastiti, M. Kusoiri
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya, Indonesia

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*Corresponding author: A. Darmawati (asri-d@ff.unair.ac.id)

ABSTRACT: Tartrazine is a colouring substance suspected to interfere with the determination of

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chlorpheniramine maleate (CPM) in tablet analyzed using USP39/NF34 method. The purpose of this study,
therefore, was to separate tartrazine using High Performance Thin Layer Chromatography (HPTLC), and five
microliters of samples with standard solution were spotted on the HPTLC plate of silica gel 60 F254.
Furthermore, the result showed the ability of a mixture of isopropanol and NH4OH (24:6 v/v) as a mobile

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phase to separate tartrazine and maleic acid from chlorpheniramine, with Rs value of > 1.5. Meanwhile, the
HPTLC validation method used were as follows: linearity of CPM in the range concentration of 400-5000
ppm was 0.998, while the accuracy and precision was 99.93 % and 3.50%, respectively. In addition, drug
determination in the registered tablets was conducted using the proposed technique, which showed that
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concentrations in samples met the specifications as stated in USP39/NF34 monography.
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Keywords: chlorpheniramine maleate, tartrazine, HPTLC

1. INTRODUCTION

Chlorpheniramine maleate (CPM) is a first generation of antihistamine (AH1) that is widely used for allergic
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disorders, including rhinitis, pruritis and urticaria. In addition, it is commercially available either as a single or
multi component dosage form, and this compound is usually combined with antitussive, analgesic-antipyretic,
and sympathomimetic decongestant agents (ISO, 2016). Furthermore, almost all single tablet formulations are
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yellow in color and the frequently used pigmentation agent is Tartrazine (C.I No.19140 or FD&C No.5).
Moreover, their molecular structures, including that of CPM and Sodium Tartrazine are shown in Figure 1 (FI
V, 2014; FAO, 2016).
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Figure 1. Chemical structures of Chlorpheniramine Maleate (A) and Tartrazine (B).

Different forms of CPM tablets require different determination methods, and the Compendia
recommendation is UV spectrophotometry, which is proceeded by a long process of sample extraction (FI V,
2014; USP 39, 2014). In addition, this assay procedure in bulk materials involves the use of water free
titration (FI V, 2014), or High Performance Liquid Chromatography (HPLC) (USP 39, 2014), while
impurities in the bulk material, including CPM-B and CPM-C, are detected using HPLC (USP 39, 2014) or

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 Proceedings of ICoAPS, 2018
Gas Chromatography (GC) (FI V, 2014). Furthermore, tartrazine, which is the added coloring substance, was
suspected to interfere with the accuracy of determining the amount of drug in the tablet, while using the
compendia method, due to the similarity between the solubility properties of both compounds. In addition,
HPLC, which is an expensive method, was developed for its assessment in a mixtures that contains other
substances, including Codeine, Acetaminophen, Phenylephrine and Aminophylline (Kommana & Basappa,

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2013; Rasal et al. 2014; Ali et al. 2015). This study is, therefore, aimed at developing a High Performance
Thin Layer Chromatograph (HPTLC) for the determination of CPM in the presence of tartrazine contained
within the tablet, hence, the optimization of this method was conducted to obtain a good mobile phase capable
of an appropriate separation between both components, as well as other substances in the tablet formulation.
Subsequently, the HPTLC procedure for the quantitative analysis was validated through the observation of

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selectivity, linearity, accuracy and precision parameters, and the result were expected to be used in product
development processes, especially in method improvement for stability study.

2. MATERIALS AND METHODS

2.1 Materials

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Standard chlorpheniramine maleate (CPM) was procured from BPOM-RI, and dried at 105oC for 3 hour prior
to use. In addition, the tartrazine standard was bought from Fluka, and three commercially registered CPM
tablets (AD, AL, ZE) served as samples. Furthermore, Methanol, ethyl acetate, isopropanol and ammonium
hydroxide were of analytical grade (Merck), and the Matrix tablets consisted of avicel, lactose, amylum,

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magnesium stearate and talc, which were all of pharmaceutical grade. Therefore, HPTLC plates of silica gel
60 GF254 (Merck) was employed as the stationary phase, while the 5 µL capillary pipettes used were products
of Merck. e
2.2 Preparation of CPM standard working solution
Stock solution of CPM was prepared by dissolving an accurate weight of 60 mg CPM standard in a mixture of
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methanol and water (1:1) present in a 10 ml volumetric flask. This mixture was subsequently diluted to obtain
CPM concentrations within the range of 1000-5000 ppm, using the same solvent.

2.3 Preparation of tartrazine solution

Stock solution of tartrazine was made by accurately weighing 50 mg of tartrazine, which was then dissolved
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in a mixture of methanol and water (1:1) present in a 10 ml volumetric flask. This mixture was subsequently
diluted using the same solvent to obtain specific concentrations.
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2.4 Preparation of matrix tablet mixtures

The matrix tablet composition was as follows: avicel (4.500 gram), lactose (4.500 gram), starch (0.750 gram),
magnesium stearate (0.300 gram) and talc (0.250 gram) (Ali et al. 2011). In addition, each CPM tablet was
assumed to possess an equivalent to 150 mg matrix powder, and 4 mg of CPM, thus, all components were
mixed homogenously in a mortar.
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2.5 Preparation of artificial tablet solution

Approximately 900 mg of the matrix tablet was placed in a 10 ml volumetric flask, 4.0 ml of CPM and 80 µl
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of tartrazine stock solutions were then added. Furthermore, a solvent mixture of methanol and water (1:1) was
subsequently incorporated into the volumetric flask, with sufficient quantity that made up two third of the
total volume. This unit was, therefore, agitated in an ultrasonic bath for 15 minutes prior to the agitation on a
vortex apparatus for the following 5 minutes. Finally, the suspension was added up to 10.0 ml with the same
solvent, and subsequently transferred into the covering tube for centrifugation at 3000 rpm for 15 minutes. In
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addition, the supernatant was filtered using Whatman 40 filter paper, followed by spotting of the sample
solution on HPTLC plate with the 5 µl-capillary pipette. This procedure was replicated three times, and also
repeated by adding 2.0 ml and 0.8 ml of CPM stock solution.

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 Proceedings of ICoAPS, 2018
2.6 Preparation of tablet sample solution
Twenty units of each registered CPM tablets were weighed and pulverized homogenously, then a portion that
was equivalent to 24 mg of the active drug was estimated accurately. This was further processed in
accordance with the preparation of the artificial tablet solution described above, without adding the standard
CPM, which were subsequently repeated for the other tablets.

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2.7 Method validation
2.7.1 Optimization of mobile phase
The standard stock solution of CPM and tartrazine were pipetted in order to make a solution that contains

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2000 and 400 ppm, respectively. Therefore, the mobile phase optimization was conducted by spotting five
microliters of each solution onto the HPTLC plate, using various solvent mixtures as mobile phase, and both
chromatograms were analyzed with Shimadzu dual wavelength Chromato Scanner CS-930. Thus, the mobile
phase was selected based on the retardation factor of CPM (0.2 – 0.8), and the resolution (Rs) of CPM and
Tartrazine spots (Rs ≥ 1.5).

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2.7.2 Selectivity
The method selectivity was indicated through the comparison of Rs parameters among CPM and other closer
spots of the sample solutions, while the similarity of Rf value and spectra profile between CPM sample and
standard.

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2.7.3 Linearity
The linearity was tested using the standard solution in the concentration range of 25-200% of the sample.
Therefore, a linear regression was obtained by plotting concentration (X) toward densitometry signal (Y), and
the linearity parameter was applied in coefficient determination (R 2) values that were more than 0.99.
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2.7.4 Accuracy and precision
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The accuracy parameter was stated as the recovery value of the standard CPM that was added quantitatively
to the matrix tablet, and the total concentration added was within the range of 500-2400 ppm, while precision
was evaluated based on the percent coefficient variation (% CV) of CPM recoveries.

2.7.5 Instrument precision

Instrument precision was assessed by scanning six spots of the drug solution at the same concentration, as
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well as the standards into HPTLC plate, which were then eluted with the selected mobile phase. In addition,
the coefficient of variation (CV) of the area ought be no more than 5%.

2.7.6 Determination of CPM in samples

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Three types of registered CPM tablets were analyzed using the proposed HPTLC method in order to identify
their concentrations and compare with the values in the compendial.

3. RESULTS
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3.1 Validation method

3.1.1 Selectivity
Various mixtures of mobile phase were tested to establish the most optimum in the separation of CPM from
other substances, and two mixtures were identified with Rs > 1.5. Therefore, the resolution value of 1.5 means
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the overlapped area between two symmetric peaks was < 0.3% (Spangenberg et al. 2011), hence, the
enhanced propensity of accurately scanning both spots for quantitative analysis. The selective mobile phases
include a mixture of (a) isopropanol and NH4OH, at a ratio of 24:6, and (b) ethyl acetate-methanol-NH4OH, at
a ratio of 15:4:4, respectively and the HPTLC chromatograms are showed at Figure 2.
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3467488
 Proceedings of ICoAPS, 2018

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Figure 2. HPTLC chromatograms of Chlorpheniramine (CP), tartrazine, and maleic acid analyzed using mobile phase type (A) a

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mixture of isopropanol and NH4OH (24:6); and (B) a mixture of ethyl acetate: methanol: NH4OH (15:4:4).

The chromatogram in Figure 3 indicates the CPM in tablet samples (with the codes AD, AL, ZE) to
possess identical spectra with the standard, which means the absence of substances overlapping with the
specific spot. In addition, the resolution between CPM and Tartrazine, which was the closest, using a selected

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mobile phase was 5.7.
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Figure 3. Spectra of CPM standard overlaid with CPM in tablet samples spots (A), and CPM sample with Tartrazine and Maleic
acid in tablet sample spots (B).

3.1.2 Linearity
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Linearity was measuring by spotting the CPM standard working solutions in the concentration range of 400-
5000 ppm onto HPTLC plate. Therefore, all spots were obtained using 5 µl of sample solution, subsequently
scanned at the maximum wavelength of 262 nm, and the results of linearity test are listed in Table 1.

Table 1. The result of CPM linearity test.

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Parameter Mobile phase

(a) (b)
Range of CPM concentrations (499.0-4990) ppm (499.0-4990) ppm
Spotted volume 5 µL 5 µL
Slope (B) 22.653 16.776
Intercept 7429.1 3170.8
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R2 0.9982 0.9992
CV (Vx0) 4.90 % 3.23 %

3.1.3 Accuracy and precision

Accuracy was determined by using the analytical procedures above on artificial CPM tablet powder, standard
CPM, and tartrazine, and the results are shown in Table 2. The range concentration of the drug was within the

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 Proceedings of ICoAPS, 2018
range of 25-100% of the artificial tablet as claimed by the packages, in order to accommodate for the
possibility of applying the assessment process in the development of an analytical method for stability study.

Table 2. Accuracy and precision of CPM standard analyzed using HPTLC method.
Mobile phase (a) Mobile phase (b)
CPM in synthetic tablet powder

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CPM standard Recovery CPM standard Recovery
(%)
(ppm) (%) (ppm) (%)
100 % 2488 103.50 2660 97.96
2660 101.14 2488 100.55
2744 102.06 2744 98.40
50% 1244 101.70 1330 100.34

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1330 101.54 1244 103.17
1372 99.66 1372 103.88
25% 548.8 91.97 532.0 106.33
636.3 100.84 548.8 92.92
497.6 96.93 497.6 97.24
Average 99.93 100.09
CV 3.50 4.03

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3.1.4 Determination of CPM in registered CPM tablets
Three different of registered CPM tablet samples were analyzed by HPTLC method using mobile phase type
A (a mixture of isopropanol : NH4OH at a ratio of (24:6)). The result was presented in Table 3.

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Table 3. Determination of CPM in registered tablet using mobile phase type A.
Sample’s code
AL AD ZE
Weight/tablet (mg) 180.0 + 2.0 150.1+ 3.2 160.0+2.1
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CPM claimed/tablet (mg) 4 4 4
Percentage (%) of CPM claimed:
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Replicate 1 109.6 103.7 107.7
Replicate 2 102.2 110.5 107.6
Replicate 3 103.7 99.42 104.5
Average(%) of CPM claimed 105.1 104.5 106.6
CV (%) 3.71 5.32 1.73
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4. DISCUSSION
4.1 Selectivity
Figure 2 shows two types of mobile phase mixture (A) and (B), which are capable of being used in the
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separation of CPM from other components, with a retardation factor (Rf) of 0.69 and 0.80, respectively.
Furthermore, it was also established that the type A was able to elute CPM and other substances in the tablet
samples, while type B only eluted the active drug, leaving behind other compounds in the starting point,
which comparably occurred an hour earlier. Based on Rf and Rs values, the mobile phase type A was selected
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for the determination of CPM in the tablet, and it is also applicable in the development of analytical methods
for identifying degraded CPM or impurities in a stability study. Contemporarily, there are no data on
established analytical techniques for studying the drug stability in the presence of tartrazine, in tablet using
HPTLC, as most publications are based on determining impurities and related substance in the bulk material
set, using HPLC (USP 39, 2014) or GC-FID (FI V, 2014).
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4.2 Linearity
Linearity of CPM standard working solution that was eluted using mobile phase type A and B is seen in Table
1. Both were observed to show good correlation with the correspondence signal (R 2), which was more than
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0.99, thus, no significant differences were observed for linearity determination of CPM standards.

4.3 Accuracy and precision

According AOAC, the expected recovery and precision of 1% analyte concentration is (97-103) % and 2,7%,
respectively (AOAC, 2012), and the CPM concentration in artificial tablet powder were observed to occur in

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 Proceedings of ICoAPS, 2018
the range of (0.7-2.7)%. Hence, it is concluded that the recovery of (99-100) % of the drug, as stated in Table
2 met the acceptance criteria, while the precision value was slightly higher than specifications in AOAC.
Statistical analysis by independent sample T test program in IBM- SPSS 22 showed the mean drug
recovery, determined by HPTLC using mobile phase A, was not substancially different from the values
obtained with B. Thus, the two tail significant value (P value) was 0.929 (> 0.05).

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4.4 Instrument precision
Precision of the instrument was good, indicated by the CV value of six spots of 1996 ppm and 532 ppm of
CPM standard solution, which were 2.1% and 5.0 %, respectively (data not shown).

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4.5 Determination of CPM in registered CPM tablets
The concentration of three registered CPM tablets were compared with three different compendial methods,
including (1) Indonesia Pharmacope requirement for CPM in the tablet is between 93.0 to 107.0%; (2) this is
90.0-110.0 %, according to USP 39 (2014); and (3) 92.5-107.5%, as stated in British Pharmacope. In
conclusion, the average concentration in all registered formulations met the criteria stated, although some of

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the replication determinations were beyond the upper limit of the Indonesia Pharmacope (FI V, 2014) and
British Pharmacope requirement (British Pharmacope, 2016).

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5. CONCLUSION

The proposed method is expected to be cheaper and more efficient for the determination of CPM with the
presence of tartrazine. However, there is need to optimize the process of preparing the sample, in order to
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ensure that the drug is extracted completely, without significant evaporation of the solvent.
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ACKNOWLEDGEMENT

This study was funded by the Directorate of Research and Community Services, Ministry of Research,
Technology and Higher Education of the Republic of Indonesia.
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REFERENCES

Ali, A., Ahmed, M., Mahmud, T., Qadir, M.A., Nadeem, K. & Saleem, A. 2015. Stability-indicating high-performance liquid
chromatography method for simultaneous determination of aminophylline and chlorpheniramine maleate in pharmaceutical
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formulations. Indian Journal of Pharmaceutical Sciences 77(5): 515–521.

Ali, H., Shoaib, M.H. & Bushra, R. 2011. Formulation development of chlorpheniramine maleate tablet by direct compression.
Jordan Journal of Pharmaceutical Sciences 4(1): 1–8.
AOAC International. 2012. Appendix F: Guidelines for standard method performance requirement. In: AOAC Official Methods of
Analysis: 1-17. Rockville: AOAC International.
British Pharmacope. 2016. Chlorpheniramine maleate. In: British Pharmacopoeia: 1-528. London: The Stationary office.
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FAO. 2016. Tartrazine: chemical and tehcnical assesment. In: 82nd JECFA - Chemical and Technical Assessment (CTA): 1-10.
Rome: Food and Agriculture Organization of the United Nations.
FI V. 2014. Klorfeniramin maleat. In: Farmakope Indonesia (5th eds): 688–690. Jakarta: DepKes RI.
ISO. 2016. Informasi Spesialite Obat Indonesia. Jakarta: PT ISFI Penerbitan.
Kommana, R & Basappa, P. 2013. Validated stability indicating rp-hplc method for simultaneous estimation of codein phosphate
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and chlorpheniramine maleate from their combined liquid dosage form. Chromatography Research International 2013: 1–7.
Rasal, K.S., Suryan, A.L. & Dhaneshwar, S.R. 2014. Simultaneous quantitation and validation of chlorpheniramine maleate,
phenylpropanolamine hydrochloride and paracetamol by RP-HPLC in bulk drug and formulation. International Journal of
Pharmaceutical Sciences and Research 5(12): 5411–5419.
Spangenberg, B., Poole, C.F. & Weins, C. 2011. Quantitative thin-layer chromatography. Berlin: Springer.
USP 39/NF 34. 2014. Chlorpheniramine maleate tablets. In: United State Pharmacopoeia 39/National Formulary 34: 312-316.
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Rockville: The United state Pharmacopoeial Convention.

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3467488