Gradient RP-HPLC Method development for simultaneous estimation of Dextromethorphan hydrobromide, Phenylephrine hydrochloride, and Triprolidine hydrochloride in Liquid Dosage Form

 

Uttam Singh Baghel¹*, Himani Sharma², Anamika Chouhan³, Abhay Sharma³,

Mohammad Mukim³, Deeksha Singh^4*

¹Department of Pharmacy, University of Kota, MBS Marg, Swami Vivekanand Nagar, Kota 324005, Rajasthan, India.

²Department of Pharmaceutical Analysis, ASBASJSM College of Pharmacy, BELA (Ropar)

Punjab-140111, India.

³Kota College of Pharmacy, SP-1, Industrial Area, Jhalawar Road, Ranpur, Kota, 325003, Rajasthan, India.

⁴ESI Hospital, Jhalawar Road, Vigyan Nagar, Kota, Rajasthan, India.

 

ABSTRACT:

A novel, sensitive, and specific gradient RP-HPLC method with UV detection is proposed for the simultaneous quantification of multicomponent liquid dosage form. The separation of the three drugs was achieved using a C₁₈ column (250 mm×4.0 mm, 5 µ) as a stationary phase. No preliminary extraction procedure is required for liquid formulations and a very simple extraction procedure is required for tablets and creams. A mixture of two mobile phases was used with gradient time programing at a flow rate of 1 ml/min. Detection was performed by using PDA detector at 270 nm. Phenylephrine HCl (RT=7.705 min), triprolidine HCl (RT=26.510 min) and dextromethorphan HBr (RT=29.166 min) were separated with good resolution in a single chromatographic run of 50 min. Each drug obeys Beer-Lambert’s law in the linearity range of 0.080-0.12 mg/ml for dextromethorphan HBr with a correlation coefficient of 0.998, 0.040–0.060 mg/ml for phenylephrine HCl with a correlation coefficient of 0.996, and 0.010–0.015 mg/ml for triprolidine HCl with a correlation coefficient of 0.998. The recoveries for dextromethorphan HBr and phenylephrine HCl were found to be 99.35% and 99.53%, respectively and for triprolidine HCl was 98.88%. Validation results show satisfactory linearity, precision, accuracy and specificity.

 

 

1. INTRODUCTION:

Cough is a protective reflex, its purpose being expulsion of respiratory secretions or foreign particles from air passages. It occurs due to stimulation of mechano- or chemoreceptors in throat and respiratory passages or stress receptors in the lungs.^[1]

 

Currently, the most commonly prescribed medications for cough is a combination of dextromethorphan hydrobromide (DXM), phenylephrine hydrochloride (PHN) and triprolidine hydrochloride (TRP) as cough–cold syrup. DXM is an anti-tussive drug^[2] which acts as a cough suppressant and chemically it is ent-3-methoxy-9ᾳ-methyl morphinanhydrobromide with molecular formula C₁₈H₂₅NO.HBr.H₂O (figure1(a)).^[3,4] PHN is a nasal decongestant and an expectorant. Chemically, it is (R)-1-(3-hydroxyphenyl)-2-methylamino hydrochloride with a chemical formula C₉H₁₃NO₂.HCl (figure1(b)).^[3] TRP is a first generation histamine H₁ antagonist used in allergic rhinitis, asthma, and urticaria. It is a component of cough and cold medicines. ^[5] It is chemically, (E)-2-(3-pyrrolidine-1-yl-1(4-tolyl)prop-1-enyl)pyridine hydrochloride with a molecular formula  C₁₉H₂₂N₂.HCl (figure1(c)).^[3] In this respect, a method for the analysis of this combination is needed.

 

 (a)

 (b)

 

 (c)

Figure 1 Chemical structures of (a) Dextromethorphan HBr (DXM), (b) Phenylephrine HCl (PHN) and (c) Triprolidine HCl (TRP).

 

Several UV ^[6-8], electrophoresis ^[9], LC-MS^[10], derivative method^[11], HPLC^[12-18] and GC/IT-MS^[19,20] has been reported for single drug as well as for the combination with any other drugs. Most of them are tedious and time consuming involving complex sample preparation such as equilibrium dialysis, ultra filtration, solid-phase extraction and liquid-liquid extraction. All the methods which are reported previously either have costly instruments or have inferior sensitivity for detection, so that the cost of analysis will increase or compromise with the quality. While the proposed study have both quality as well as sensitivity with comparatively reduced analysis price. Validation of the current method will be performed according to the requirements of USP^[21] & ICH ^[22] guidelines.

 

2. EXPERIMENTAL:

2.1. Reagents and chemicals:

Pure drug sample of dextromethorphan HBr, phenylephrine HCl and triprolidine HCl were received as a gift sample from Glenmark pharmaceuticals, Nalagarh. All the chemicals and reagents were of analytical grade procured from Merck chemicals, Mumbai, India. Water was distilled and deionised by passing through water purification system and milli-Q filter. Further water was filtered through 0.45 µ Millipore nylon filter. HPLC grade methanol was purchased from Merck Chemicals, Mumbai, India. The marketed formulation (Ascoril D) used for analysis was purchased from the local pharmacy.

 

2.2. Mobile phase preparation:

2.2.1. Preparation of buffer for mobile phase A:

2.0 g ammonium acetate and 2.5 g of pentane sulphonic acid sodium salt were dissolved in 1000 ml of water and the pH was adjusted to 4.0 (±0.1) with orthophosphoric acid. It was then passed through nylon filter paper of 0.45 µ.

 

2.2.2. Preparation of buffer for mobile phase B:

1.0 g of pentane sulphonic acid sodium salt was dissolved in 1000 ml methanol with the aid of sonication. It was also passed through nylon filter paper of 0.45 µ.

 

2.2.3. Diluent preparation:

Diluent was prepared by mixing water and the methanol in the proportion of 80:20% v/v.

 

2.3. Chromatographic conditions:

Analysis was performed on a Shimadzu HPLC (LC-2010) equipped with Prominence LC-Gradient quaternary pump (LC-20AD), auto sampler, sampler cooler and a SPD-20A prominence PDA  detector which was set at 270 nm of detection wavelength. The analytical column used was inertsil ODS (250 mm×4.6 mm i.d, 5 µ). A gradient flow with time programming (Table I) was consisting mobile phase A and mobile phase B in gradient elution mode at a flow rate of 1 ml/min. Operation data acquisition and analysis were performed by using LC solution software. Mobile phase was filtered through 0.45 µ Millipore nylon filter under vacuum and degassed by ultrasonication.

 

Table I Time programming for gradient elution by HPLC

Time (min)	Flow	Mobile Phase A (%)	Mobile Phase B (%)
0.01	1.00	75.0	25.0
6.0	1.00	75.0	25.0
16.0	1.00	45.0	55.0
40.0	1.00	45.0	55.0
42.0	1.00	75.0	25.0
50.0	1.00	75.0	25.0

 

 

2.4.    Standard and sample preparation:

Standard solution of the three active ingredients of the drug was prepared in the following manner:

2.4.1. Standard (A):

50 mg TRP working standard was weighed and transferred into volumetric flask. 30 ml of diluent was added and sonicated for 5 min and the volume was made up with diluent.

 

2.4.2. Mixed standard:

40 mg of DXM, 20 mg of PHN working standard were accurately weighed and transferred into 100 ml volumetric flask. 70 ml of diluent was added and sonicated to dissolve the content. 5 ml of standard (A) was transferred and the volume was made up to the mark with diluent.

 

2.4.3. Sample preparation:

10 ml of sample was pipetted out in a volumetric flask, containing 30 ml of diluent. Pipette was rinsed twice with the diluent and the rinsate was added to the flask.  Pipette was rinsed once again with the fresh diluent .The sample was sonicated for 10 min, and then the volume was made up with diluent. The above solution was filtered through 0.45 µ nylon filter paper discarding first few millilitres.

 

3. RESULTS AND DISCUSSION:

3.1. Method optimisation:

Reversed-phase LC-method was employed in the current work for the separation of these three analytes. To this end, reversed phase C₁₈ column using a mixture of organic solvents (methanol and water) and aqueous buffer as a mobile phase were tested. Firstly the column used was inertsil ODS 3V (C₁₈), 150 x 4.6 mm, 5 µ and the flow rate was maintained at 0.8 ml/min which does not show enough resolution between analytes. Then the length of the column was increased to inertsil ODS 3V (C₁₈), 250 x 4.6 mm, 5 µ and the flow rate was increased to 1.0 ml/min which showed adequate resolution between DXM, TRP and PHN. In order to optimise the chromatographic parameters, the effect of changing the composition of mobile phase with gradient time programming system was studied on the capacity factor (k’), peak asymmetry, theoretical plates, retention time and resolution. Therefore, the selection of the concentration of ammonium acetate in buffer (pH 4.0) and the composition of mobile phase was based on providing good baseline, adequate separation, and sharp peaks in a minimum run time. Detection wavelength used at 270 nm in PDA detector. The injection volume was 20 µl as well as column temperature was kept ambient with the run time of 50 min.

 

3.2. System suitability tests

The results of the system suitability tests, recorded in Table II, assure the feasibility and adequacy of the proposed method for simultaneous estimation of the three drugs in routine pharmaceutical application.

 

The system suitability tests performed verified the peak area, peak asymmetry, capacity factor, theoretical Plates, resolution, retention time and repeatability of the chromatographic system and ensured that the equipment, electronics, and analytical operations for the samples analysed could be constituted as an integral system that can be evaluated as a whole. The RSD of peak areas of five consecutive injections was found to be less than 2%, thus showing good injection repeatability, and excellent chromatographic and environmental conditions.

 

Table II System suitability tests

^a Standard deviation; ^bRelative Standard deviation; ^cStandard error of mean; ^dPercentage range of error

 

Figure 2 Typical chromatogramof dextromethorphan HBr, phenylephrine HCl and triprolidineHCl.

 

Table III Linearity studyⁿ

^aStandard deviation; ^bCoefficient of variance; ^c Standard error of mean; ^dCorrelation Coefficient; ⁿFive times

 

The resolution (Rs) between the peaks was found to be greater than 2, indicating good separation of the drug from each other as shown in figure 2. The values for theoretical plate number (N) and capacity factor (k´) demonstrated good column efficiency.

 

3.3. Validation study:

3.3.1. Linearity and range:

The linearity of an analytical procedure is its ability (within a range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.  A series of combination dilutions and standard curves were prepared over a concentration range of all the three drugs. The data of peak area versus drug concentration was treated by linear least square regression analysis, whereby the slope, intercept, and the correlation coefficient were determined.

 

Standard stock of DXM with a concentration of 0.4 mg/ml was prepared by dissolving 40 mg of DXM in 100 ml of diluent. Five different concentrations of DXM were prepared in the following manner: 2, 2.25, 2.5, 2.75, 3 ml of the standard stock solution was diluted to 10 ml with diluent. The same procedure was repeated for TRP and PHN. The results obtained (Table III) show that the linearity range is 0.080-0.12 mg/ml for DXM with a correlation coefficient of 0.997, 0.040-0.060 mg/ml for PHN with a correlation coefficient of 0.996, and 0.010-0.015 mg/ml for TRP with a correlation coefficient of 0.996.

 

3.3.2. Accuracy:

The accuracy of the analytical procedure expresses the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. It is sometimes termed as trueness.

 

For recovery study, 100 ml of simulated syrup was prepared by dissolving 40 mg DXM, 5 ml of TRP, and 20 mg of PHN in the required excipients of the drug formulation. A dilution was performed, and 20 µl were injected into the column. Percent recovery of 80%, 100%, and 120% was determined with the concentration of 2, 2.5 and 3 ml respectively, as shown in Table IV. The peak areas resulting were compared with that of the standard.

 

Table IV  Accuracy (Recovery study)

^aStandard deviation;  ^bstandard error of mean;  ^crelative standard deviation

 

 

Table V Precision (Intra-day and inter-day study)

^arelative standard deviation; ^bstandard deviation; ^cstandard error of mean

 

Table VI Robustness study

 

3.3.3. Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision may be expressed as inter-day and intra-day.

 

Inter-day and intra-day study was assayed by performing the determination of six replicates of working standard solutions in two successive days. Coefficient of variance (RSD) of six determinations is less than 1% which shows that the developed method is précised and ready for the quality control of DXM, PHN and TRP. The results are shown in Table V.

 

3.3.4. Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. In case of liquid chromatography, typical variations are influence by variations in flow rate, temperature, pH, mobile phase composition, buffer concentration and by using different columns.

 

The robustness of method was established by making deliberate minor variation in the flow rate and wavelength. Method was performed twice first by same method as described in proposed assay method and second time by changing the flow rate from 1.0 ml/min to 0.9 and 1.1 ml/min, and the wavelength was changed from 270 to 268 nm and 272 nm. Then the % deviation was calculated as shown in Table VI.

 

 

3.3.5. Specificity:

In case of the assay, demonstration of specificity requires that it can be shown by the presence of impurities or excipients. Placebo (sample without analyte) was prepared in the same way as the sample under the conditions prescribed in the assay method and duplicate injection was taken. The excipients mixture of the syrup shows no specific peak at the retention time of the analyte peak. This shows that the excipients do not interfere with the analyte peak. This demonstrates that the assay is specific for DXM, PHN and TRP.

 

4. CONCLUSION:

The proposed developed method was found to be sensitive, specific and accurate for routine simultaneous analysis of the formulations without prior separation. The precision of the proposed method was found to be satisfactory which is evidenced by low values of standard deviation, percent relative standard deviation and standard error of mean. The percent recovery and specificity obtained indicates non-interference from the excipients used in the formulation. Thus the method developed in the present investigation found to be sensitive, specific, accurate and precise and can be successfully applied for the simultaneous estimation of DXM, PHN, TRP in syrup. It can, therefore, be concluded that the reported method could find practical application as an economical and rapid quality-control tool with good separation for simultaneous analysis of the three drugs from their combined dosage forms in both research and industrial quality-control laboratories.

 

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Accepted on 31.07.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):583-588.