Separation and Assay of Three Anti-Cough Drugs Pseudoephedrine, Dextromethorphan and Chlorpheniramine in Pharmaceutical Forms by using single RP-HPLC Method

 

Yaser Bitar^*

Pharmaceutical Chemistry and Pharmaceutical Quality Control Department - Faculty of Pharmacy

University of Aleppo - Syria.

 

ABSTRACT:

Objective: The objective of this study was to develop and validate a single HPLC method, in order to separate and assay three anti-cough drugs pseudoephedrine, dextromethorphan and chlorpheniramine in pharmaceutical forms. This method was practical additional choice in quality control laboratories. Methods: The chromatographic conditions comprised of a classical C₈-type stationary phase (250 × 4.6 mm, 5μ ), with a mobile phase consisting of 8.57gr/l ammonium dihydrogen phosphate, Acetonitrile, triethylamine and tetrahydrofuran (66:30:2:2 v/v %) respectively, and apparent pH of 3.5 was adjusted with hydrochloric acid. The flow rate was 1ml/min; column temperature set at 30ºC; injection volume was 20µl and the detection wavelengths was at 264 nm, Results: The method was validated for linearity with correlation coefficients very close to one, accuracy with mean recovery values between 98.0-102.0%, precision with relative standard deviations of the calculated concentrations less than 2.0%, specificity in the presence of degradation products and robustness in the case of little change of some chromatographic conditions. Then it was used successfully to separate a mixture of them and to assay these drugs in syrup pharmaceutical form purchased from Syria. This analyzed pharmaceutical form contains three active ingredient within 100±2 % of stated concentration, within the limits specified by British and USP Pharmacopeia. Conclusion: The results presented in this paper showed that the developed method was simple and practical for the separation and determination of the three anti-cough drugs in syrup pharmaceutical form.

 

 

INTRODUCTION:

Medications against the common cough come in different forms (tablets, capsules, syrups, etc.) and usually contain a complex mixture of nitrogenous compounds as active ingredients. These are usually present in varying and very different proportions, have diverse properties inherent to their formulation and desired action, and often possess some similar physical and chemical properties, which turn difficult their separation¹.

 

 

 

The syrup preparation represents complex formulation containing several active ingredients and a broad spectrum of excipients such as flavoring agents, saccharose or aspartame, acidulants, natural or artificial colorings and flavoring agents, dyes sweeteners and preservatives^2-3. These compounds are contained in the pharmaceutical form in very different proportions and present chemical forms of very different nature⁴.

 

Pseudoephedrine hydrochloride (PSD) (Fig. 1), chemically known as (1S, 2S)-2-(methylamino)-1-phenylpropan-1-ol hydrochloride⁵, is a direct-acting and indirect-acting sympathomimetic drug. It is given orally for the symptomatic relief of nasal congestion. It is commonly combined with other ingredients in preparations intended for the relief of cough and cold symptoms⁶.

Dextromethorphan hydrobromide (DXM) (Fig. 1), chemically known as (3-methoxy-17-methylmorphinan hydro bromide monohydrate)⁵, is an opioid like drug acts centrally. It elevates the threshold for coughing, without inhibiting ciliary activity. Dextromethorphan hydrobromide rapidly absorbed from the gastrointestinal tract and converted into lower active metabolite (dextrorphan). The duration of action after oral administration is approximately three to eight hours for Dextromethorphan hydrobromide⁷.

 

Chlorphenamine maleate (CHP) (Fig. 1), chemically known as (3RS)-3-(4-Chlorophenyl)-N, N-dimethyl-3-(pyridin-2-yl)propan-1-amine hydrogen (Z)- butenedioate⁵, is a first-generation alkylamine antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis and urticaria. Its sedative effects are relatively weak compared to other first-generation antihistamines.

 

The combination of antihistamine such as chlorpheniramine maleate and pseudoephedrine hydrochloride and / or dextromethorphan hydrobromide is used to overcome the allergic effects and reduce or relieve cough-cold symptoms⁸.

 

This combination has a long standing tradition in clinical use as a syrup and tablet formulations. Hence, a lot of different analytical method have been described with regard to the determination and separation of PSD, CHP, and/or DXM in pharmaceutical formulations, in combinations of two^9-10 or three^11-12 of these drugs, or with other active principles^9-18.

 

A huge number of chromatographic methods on the separation and determination of these drugs and their combinations focusing on ion-pair high performance liquid chromatography (IPC) have been reported^19-25. However, IPC methods especially in drugs combinations tend to time-consuming due to long equilibration periods and they are often not very robust^26-28. In the other site, RP-HPLC is a wide using in the field of pharmaceutical analysis^29-38. Thus, the aim of this work was to develop and validate a HPLC method without using ion-pair reagents, for the assay of three combination drugs Pseudoephedrine, Dextromethorphan and Chlorpheniramine in its syrups and tablets as bulk and finish pharmaceutical forms.

 

MATERIALS AND METHODS:

Drugs and Chemicals:

The working standards are obtained from Asia Pharmaceutical Industries, Aleppo-Syria, which is the supplier for this research. Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide with Purity (99.89 - 99.31 – 99.54%) respectively, are used without extra purification. other chemicals are used as a HPLC grade which are: Acetonitrile (LiChrosolv, MERCK), Ammonium di-hydrogen phosphate (MERCK), triethylamine (Surechem Products LTD. ScP), tetrahydrofuran (Rectapur, PROLABO), hydrochloric acid (Surechem Products LTD. SCP). Distilled water for HPLC. Pharmaceutical formulation as a syrup (Tusil, batch Nr. 3 MFG Date: 2018 EXP Date: 2021) was obtained from Shifa Pharmaceutical Industries, Aleppo-Syria. All samples, as received, were stored in the dark at ambient temperature and humidity. They were all analyzed within expiry dates.

 

Instrumentation:

The HPLC instrument was of SHIMADZU, Japan (Prominece Liquid Chromatography) with a diode array detector SPD-M20A (Japan), column oven CTO-20A, autosampler CIL-20A, binary pump LC-20A (Japan), Degaser DGU-20A3 and LabSolutions LCsolution Version 1.25 software. The spectrophotometer was Jasco V-530. Analytical balance was of Satorius for weighting. pH meter is made by Metrohm and the ultrasonic bath is made by Clifton. A micro pipette was from ISOLAB, Filters 0.45µm.

 

Standard preparation:

The standard solution was prepared by accurately weighting (150, 10, 75mg) of Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide respectively and dissolving them in the diluent (HCl 0.12mol/l) with dilution to (10ml). After that (1 ml) of this solution was diluted to (10ml) by the same diluent. The final concentration obtained is (1.5, 0.1, 0.75mg/ml) for Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide respectively, which is the standard concentration.

 

Method development and optimization the chromatographic conditions:

Selection of wave length:

The three drugs were prepared in the diluent separately at the standard concentration and scanned in UV-Visible spectrophotometer. A good absorbance noticed at 246 nm for all drugs.

 

Column selection:

Since Dextromethorphan hydrobromide has a strong affinity to hydrophobic phases, the C18 column was deleted. We need a good separation in a short time of analysis, so the available C8 column is Chrometisll 60-5-C8 SH 5µ (250 × 4.6 mm) was selected to start with.

 

Mobile Phase Selection:

Many trails were made to optimize the best mobile phase as different rations of 8.57 gr/l ammonium dihydrogen phosphate, which is selected as a buffer, and acetonitrile were applied.

·       Buffer: Acetonitrile (75:25) pH 3: good resolution with long time for dextromethorphan elution.

·       Buffer: Acetonitrile (50:50) pH 3: good resolution with shorter time for dextromethorphan elution, but it stilled long.

·       Buffer: Acetonitrile (25:75) pH 3: bad resolution with short time for dextromethorphan elution.

Acetonitrile was important to the dextromethorphan elution, but the highly concentrations of acetonitrile resulted in a bad resolution for the drugs. The proportion (25:75) for the acetonitrile and the buffer respectively, was the best since It gives an acceptable resolution but with tailing problems and a slow elution for dextromethorphan.

 

Triethylamine was added to fix the Pseudoephedrine tailing, and a hard work with many trails was done to avoid dextromethorphan tailing problem and its slow elution.

 

The best results were by using THF, which took an important role to avoid dextromethorphan tailing with a fast and accepted elution without interferences with other peaks.

·       Buffer: Acetonitrile: Triethylamine:

THF (71:25:2:2) pH 3: good resolution with long time for dextromethorphan elution. Without tailing.

·       Buffer: Acetonitrile: Triethylamine:

THF (66:30:2:2) pH 3: perfect resolution with short time for dextromethorphan elution. Without tailing.

·       Buffer: Acetonitrile: Triethylamine:

THF (61:35:2:2) pH 3. accepted resolution with short time for dextromethorphan elution. Without tailing.

·       Buffer: Acetonitrile: Triethylamine:

THF (56:40:2:2) pH 3: unaccepted resolution with short time for dextromethorphan elution. Without tailing.

 

The final optimized mobile phase was: (8.57 gr/l) Ammonium di-hydrogen phosphate, Acetonitrile, triethylamine and tetrahydrofuran (66:30:2:2) respectively, with pH 3.5 adjusted by hydro chloride acid.

 

 

 

It was prepared by accurately weighting 8.57 gr of Ammonium di-hydrogen phosphate and dissolving it in 1000ml of distilled water for HPLC. 66 volumes of the buffer added to 30 volumes of acetonitrile and a mixture degassed. Then 2 volumes of triethylamine and tetrahydrofuran were added and adjusted the final mixture by HCl to pH 3.5. At last, the mobile phase was filtered by 0.45 filter.

 

RESULTS AND DISCUSSION:

Selection of detective wavelength:

The Figure 2 shows that Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide have a good absorbance at around 264±2 nm, so detection at 264 nm was selected for the method development purpose.

 

HPLC analysis:

The chromatographic conditions were optimized after performing many trails to achieve the best results to develop a new validated method for separation and assay of Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide. The method was developed and optimized for achieving peak separation with rather short retention times and appropriate peak symmetry. The EMR Chrometisll 60-5-C8 SH 5µ (250 × 4.6 mm) analytical column was used and the final mobile phase was consisted of a mixture of (8.57 gr/l) Ammonium di-hydrogen phosphate, Acetonitrile, triethylamine and tetrahydrofuran (66:30:2:2) respectively, with pH 3.5 adjusted by hydro chloride acid with a flow rate of 1ml/min, temperature 30º C, injection volume 20 µl and detection wavelength at 264 nm. The tailing factor was 1 for Pseudoephedrine hydrochloride, 1.08 for Chlorpheniramine maleate and 1.13 for Dextromethorphan hydrobromide with a good resolution and without interferences between the peaks. Figure 3 shows the standard chromatogram.

 

Analytical method validation:

The validation method was carried out according to ICH guidelines [39] and the United States Pharmacopeia 38[40] recommended test conditions.

 

Linearity and range:

Over a range of 80 to 120 percent, five standard solutions were prepared and injected three times in HPLC. the concentrations were (1.25, 1.375, 1.5, 1.625, 1.75 mg/ml) for Pseudoephedrine hydrochloride, (0.08, 0.09, 0.1, 0.11, 0.12 mg/ml) for Chlorpheniramine maleate and (0.6, 0.675, 0.75, 0.825, 0.9 mg/ml) for Dextromethorphan hydrobromide.

 

 

Figure 4, Figure 5 and Figure 6 show the linearity for each component with a correlation coefficient ≥ 0.998. The resulting correlation coefficients allowing estimating the quality of the curves were 0.9981, 0.9999 and 0.9993 respectively, these values of R² greater than 0.998 indicates a satisfactory linearity. The range 80 to 120 percent was carried out to study the linearity, precision and accuracy.

 

Table 1: Accuracy of pseudoephedrine HCL.

 

Table 2: Accuracy of chlorpheneramine maleate.

 

Table 3: Accuracy of dextromethorphan HBr.

 

Accuracy:

Concentrations of 80, 90, 100, 110, 120 per cent have been used to study the accuracy for each component. (Table 1, Table 2, Table 3). The accuracy was confirmed because the reported results show that the mean recovery of Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide by this method is between 98 – 120% and RSD% is not more than 2%.

 

 

Precision (Repeatability):

Standards of the concentration 100 % for each component were injected six times in HPLC. The results were illustrated in table 4 which show the calculated standard deviation and relative standard deviation of the peak area. The RSD % of repeatability for pseudoephedrine hydrochloride 0.749 %, chlorpheniramine maleate 0.320 % and dextromethorphan hydrobromide 0.380 % is not more than 1 % which means that the repeatability precision of the method is confirmed.

Table 4: Repeatability of HPLC method.

 

Table 5: Intermediate precision of HPLC method.

Intermediate Precision
Analysis done by	No. of injection	Pseudoephedrine hydrochloride		Chlorpheniramine maleate		Dextromethorphan hydrobromide
		Conc. mg/ml	area	Conc. mg/ml	Area	Conc. mg/ml	area
First Analyst (Day1)	1	1.5	4344908	0.1	6333326	0.75	7017864
	2	1.5	4334545	0.1	6301102	0.75	6976564
	3	1.5	4360684	0.1	6327478	0.75	7020956
	4	1.5	4346550	0.1	6306328	0.75	6983537
	5	1.5	4425364	0.1	6355304	0.75	7047176
	6	1.5	4356077	0.1	6311850	0.75	6995487
Second Analyst (Day2)	1	1.5	4467631	0.1	6306943	0.75	6806943
	2	1.5	4481388	0.1	6365209	0.75	6865209
	3	1.5	4462844	0.1	6321409	0.75	6821409
	4	1.5	4460330	0.1	6323906	0.75	6823906
	5	1.5	4507954	0.1	6347643	0.75	6847643
	6	1.5	4484600	0.1	6306418	0.75	6806418
AVR			4419406.250		6325576.333		6917759.333
SD			65625.473		21100.187		96149.635
RSD%			1.485		0.334		1.390

 

For the intermediate precision, the procedure was repeated by another analyst in another day using the same chromatographic conditions and as noticed from table 5, the intermediate precision meets the accepting criteria which the RSD% is not more than 2 % for the total of all calculated area for the twelve injections. RSD of 1.485 %, 0.334 % and 1.390 % were calculated for pseudoephedrine, chlorpheniramine and dextromethorphan respectively. These results indicated that the intermediate precision of this method was acceptable for three components.

Robustness:

some chromatographic conditions have been changed to evaluate the ruggedness of this method such as pH, temperature, wavelength and flow rate. Each condition has been studied alone and the tables (6-7-8-9) below show that changes happened to the analytical procedure and the acceptance criteria, which is RSD % is not more that 5 % to the evaluating response, has been met at the modification of temperature and PH and it hasn't been met at the modification of flow rate and wavelength.

 

Table 6: HPLC method robustness: Variation of flow rate.

 

Table 7: HPLC method robustness: Variation of detection wavelength.

Table 8: HPLC method robustness: Variation of column temperature.

 

Table 9: HPLC method robustness: Variation of pH mobile phase.

 

The evaluating response by changing the flow rate and temperature is the retention time (see fig. 7), and the peak area for changing wavelength and pH (see fig. 8). So the method is robust at pH and temperature modification with a well resolution. The standard concentration was used in all injections.

 

System Suitability:

According to the robustness test results, the wavelength and the flow rate can't be changed and it's important to be controlled and calibrated to before applying this method as a validated one.

 

Specificity:

The analyzed components were separated very well with a good resolution and without interferences between the peaks, that means this method is specific. and after applying it on a commercial syrup, there weren't any interfaces between the analyzed components and the excipients used in this syrup.

 

 

 

 

LOD and LOQ:

Although, the percent method has been analyzed quantitatively three active components. Enhancement calculated LOD (limit of detection) and LOQ (limit of quantitation) for the three components are shown in Table 10.

 

Table 10: LOD and LOQ values of three analysts.

Analyst	LOD	LOQ
Pseudoephedrine hydrochloride	3 µg/ml	7.5 µg/ml
Chlorpheniramine maleate	1 µg/ml	2.5 µg/ml
Dextromethorphan hydrobromide	1.5 µg/ml	3.75 µg/ml

 

Assay of a commercial syrup:

Shifa Pharmaceutical Industries produces a "Tusil" syrup which contains Pseudoephedrine hydrochloride 15 mg/5ml, Chlorpheniramine maleate 1mg/5ml and Dextromethorphan hydrobromide 7.5mg/ml. this syrup is used to evaluate the effective of this validated method by assaying its three components and table 11 shows the assay results. This analysis is not only specific and accurate, it also takes only 7 minutes which could be used in pharmaceutical industries.

 

Table 11: Results of pharmaceutical formulation (Tusil) obtained of recent method.

Active ingredient	Pseudoephedrine hydrochloride	Chlorpheniramine maleate	Dextromethorphan hydrobromide
Labled amount	15 mg/5ml	1 mg/5ml	7.5 mg/5ml
Amount found	15.2	0.99	7.55
Amount found % ± (SD)	101.33 (± 0.23)	99 (± 0.85)	100.66 (± 0.44)

 

 

 

CONCLUSION:

A reversed phase HPLC method without using ion-pair was developed and validated for simultaneous assay of Pseudoephedrine hydrochloride, Chlorpheniramine maleate and Dextromethorphan hydrobromide in 7 minutes with robust, accurate and precise estimation which could be used in a routine assay of these three drugs in both cases bulk and finish product in pharmaceutical control.

 

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Accepted on 21.09.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):831-839.