DEVELOPMENT AND VALIDATION OF A NEW ISOCRATIC RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF DEXTROMETHORPHAN HYDROBROMIDE, PHENYLEPHRINE HYDROCHLORIDE AND TRIPROLIDINE HYDROCHLORIDE IN THEIR COMBINED LIQUID DOSAGE FORM

DEVELOPMENT AND VALIDATION OF A NEW ISOCRATIC RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF DEXTROMETHORPHAN HYDROBROMIDE, PHENYLEPHRINE HYDROCHLORIDE AND TRIPROLIDINE HYDROCHLORIDE IN THEIR COMBINED LIQUID DOSAGE FORM

Vivek Kumar Pandey, Gulzar Alam * and Jai Narayan Mishra

Kailash Institute of Pharmacy & Management, GIDA, Gorakhpur, Uttar Pradesh, India.

ABSTRACT: A new isocratic HPLC method based on reverse phase separation has been developed for simultaneous estimation of Dextromethorphan hydrobromide (DMP), Triprolidine hydrochloride (TPN), and Phenylephrine hydrochloride (PHE) in their mixed liquid dosage form. The chromatographic separation was accomplished on Agilent TC-C18 (250 x 4.6 mm, 5× m) column with a flow rate of 1 ml/min, UV detection (271 nm). Mobile phase consisting of 5 g DOSS, methanol, tetrahydrofuran, and water in the ratio of (70:10:20) pH 4.0 adjusted by acetic acid. The method was found to be selective for PHE, TPN, and DMP with retention time 4.7 min, 5.9 min, and 6.9 min, respectively. The linearity was established over the concentration range of 160-240 mcg/ml, 40-60 mcg/ml and 320-480 mcg/ml for PHE, TPN and DMP, respectively. Limit of detection and Limit of quantification for PHE was found to be 37.87 μg/ml, and 114.77 μg/ml, respectively, for TPN was found to be 10.19 μg/ml and 30.89 μg/ml, respectively and for DMP was found to be 57.50μg/ml and 174.26 μg/ml, respectively. The developed method was further validated in compliance with ICH guidelines. The statistical result showed that the method was precise, accurate, reproducible, and specific for the analysis of PHE, TPN and DMP.

Keywords: Phenylephrine hydrochloride, Triprolidine hydrochloride, Dextromethorphan hydrobromide, ICH guidelines, RP-HPLC, Mehtod validation

INTRODUCTION: Dextromethorphan Hydro-bromide (DMP), Triprolidine (TPN) and Phenylephrine Hydrochloride (PHE) in combined liquid dosage form, has gained increasing acceptance in temporarily reliving symptoms due to common cold, hay fever (allergic rhinitis) and other respiratory allergies. Dextromethorphan chemically (+)-3-methoxy-17-methyl-(9α,13α,14α)-morphinan is an NMDA (N-methyl-D-aspartic acid) anta-gonist, non-competitive channel blocker, which is one of the most widely used antitussive¹.

Triprolidine hydrochloride, chemically [(E)-2-[3-(l-pyrrolidinyl)-l-p-tolylpropenyl] pyridine mono-hydrochloride] is a histamine H1-receptor anta-gonist ^2-3. Phenylephrine hydrochloride, chemi-cally 3 - (1 – hydroxyl - 2- methyl amino-ethyl) phenol is indicated for general cold-related distress or short-term stuffy nose caused by allergic rhinitis.

FIG. 1: CHEMICAL STRUCTURE OF DEXTRO-METHORPHAN, TRIPROLIDINE AND PHENYL-EPHRINE

As a vasoconstrictor, Phenylephrine has both indirect and direct sympathomimetic activity Fig. 1 ^4-5. A detailed investigation revealed that analytical methods exist for estimating specified drugs with other combinatorial by RP-HPLC ^6-16.

There were few RP-HPLC methods for the estimation of DMP, PHE, and TPN simultaneously, for specified combination in solid dosage form 17 and for combined liquid dosage form ¹⁸.

The objective of the present approach was to develop and validate a reversed-phase high-performance chromatographic technique for simultaneous estimation of PHE, TPN and DMP in their combined liquid dosage form. The developed method was later validated in terms of accuracy, inter-day and intra-day precision, specificity, linearity, LOD and LOQ values.

MATERIALS AND METHODS:

Chemicals and Reagents: Pharmaceutical grade Dextromethorphan Hydrobromide, Triprolidine Hydrochloride, and Phenylephrine Hydrochloride were provided as gift samples by Apple International Pharmaceuticals Pvt. Ltd. Nepal. HPLC grade water (Thermo Fischer Scientific), Tetrahydrofuran (Thermo Fischer Scientific), HPLC Methanol (Thermo Fischer Scientific).

Instrument: Chromatography was performed on Shimadzu HPLC L201055, equipped with a UV detector. For Intermediate Precision Shimadzu HPLC L204350 equipped with UV detector, by using Agilent TC-C18 (250 x 4.6 mm, 5mm) column.

Chromatographic Condition: For method development, a reverse phase C18 column (250 x 4.6 mm, 5 mm), a mobile phase consisting of 5 g DOSS, Methanol, Tetrahydrofuran and Water in the ratio of (70:10:20 % v/v/v) pH 4.0 adjusted by Acetic acid, the flow rate of 1.0 ml/min at a wavelength of 271 nm (UV detection).

Preparation of Standard Solution:

Dextromethorphan HBr Stock Solution: Fifty milligrams of Dextromethorphan HBr raw material was weighed and transferred to a 25 ml volumetric flask and then dissolved with solvent mixture sonicating for 10 min in sonicator. The volume was then made up to 25 ml with the same solvent.

Phenylephrine HCl Stock Solution: Twenty-five milligram of Phenylephrine HCL raw material was weighed and transferred to a 25 ml volumetric flask and then dissolved with solvent mixture sonicating for 10 min in a sonicator. The volume was then made up to 25 ml with the same solvent.

Triprolidine HCl Stock Solution: Twenty-five milligram of Triprolidine raw material was weighed and transferred to a 100 ml volumetric flask and then dissolved with solvent mixture sonicating for 10 min in a sonicator. The volume was then made up to 100 ml with the same solvent

Mixed Standard Solution: Five milliliters of Dextromethorphan stock solution, five milliliters of Phenylephrine stock solution, and five milliliters of Triprolidine stock solution was pipetted in 25 ml volumetric flask and volume made up to 25 ml with the solvent mixture to obtain PHE (200 μg/ml), TPN (50 μg/ml) and DMP (400 μg/ml).

Sample Solution: 11.972 grams of syrup was weighed in 50 ml volumetric flask and then dissolved with solvent mixture sonicating for 10 minutes in a sonicator. The volume was then made up to 50 ml with the same solvent.

Method Validation: The developed method was subjected to validation under various criteria such as linearity and range, precision, accuracy and robustness in accordance with the international conference of harmonization Guidelines ¹⁹.

Linearity: Suitable aliquots of PHE, DMP, and TPN working standard solutions were taken in different 10 ml volumetric flask and diluted up to the mark with mobile phase to obtain a final concentration of 160, 180, 200, 220, and 240 mg/ml of PHE, 40, 45, 50, 55 and 60 mg/ml of TPN and 320, 360, 400, 440 and 480 mg/ml of DMP respectively. The calibration curve was constructed by drawing the mean peak area versus concentrations, and regression equations for all three drugs were calculated.

Precision: The repeatability (Intraday) study was performed by estimating the response of PHE, TPN, and DMP at three different concentrations (160, 200 and 240 mg/ml), (40, 50 and 60 mg/ml) and (320, 400 and 480 mg/ml), three replicates each respectively. Intermediate precision, i.e., inter-day and equipment change studies for PHE, TPN and DMP, were calculated by assaying the sample solution on different days and on different equipment, and the result were reported in terms of relative standard deviation.

Accuracy: The accuracy of the developed method was determined by calculating recoveries of PHE, TPN, and DMP by standard additions method. Known amounts of PHE (60, 100 and 140 mg/ml), TPN (15, 25 and 35 mg/ml) and DMP (120, 200, and 280 mg/ml) were added to a pre-selected sample solution and by measuring the peak areas amounts of PHE, TPN and DMP were estimated. Recovery (%) and RSD (%) were calculated for each contraction.

Limit of Detection (LOD) and Limit of Quantitation (LOQ): The LOD and LOQ of PHE, TPN, and DMP were calculated by using the following equation as per ICH guidelines.

LOD = 3.3× σ / S

LOQ=10×σ/S

Where s is the standard deviation of y-intercepts of regression line and S is the slope of calibration curve.

Robustness: The robustness of this method was investigated by purposefully changing experimental conditions such as flow rate and pH. The study was carried out by changing 0.2 units in pH and 0.2 ml/min of flow rate.

System Suitability: During method development and validation, system suitability is essential-function.5 repeated injections were studied for the analysis of PHE, TPN and DMP in reference to their retention time, tailing factor, theoretical plates and asymmetry factor.

RESULTS AND DISCUSSION:

Selection of Mobile Phase: Numerous Mobile Phase compositions were attempted to figure out the peak of PHE, TPN, and DMP. The ultimate mobile phase containing Doss (5.5 g); Methanol, THF and water (70: 20: 10) adjusted pH 4.0 by using Acetic acid, since it was able to resolve peaks of PHE (4.66 ± 0.03), TPN (5.82 ± 0.04) and DMP (6.80 ± 0.05) with resolution factor of 5.33 and 4.17. For quantification λ was set to 271 nm.

Specificity and Selectivity: By using the mobile phase as a solvent, a mixture of PHE, TPN, and DMP (200, 50, and 400 μg/ml) was prepared and introduced in the column and retention time was inspected. While comparing the results with the blank, it was found that there is no interference at the retention time. The method was found specific and precise. An illustrative chromatogram of PHE, TPN and DMP standard and sample is demo-nstrated in Fig. 2 and 3.

FIG. 2: CHROMATOGRAM OF STANDARD PHE, TPN AND DMP                  

FIG. 3: CHROMATOGRAM OF TEST PHE, TPN AND DMP

Linearity: With a focus on linearity range of developed HPLC method peak areas versus concentrations were plotted. An excellent linear relationship (r² = 0.998), (r² = 0.997) and (r² = 0.998) were witnessed for PHE, TPN and DMP respectively. The harmonious relation eq. was y = 10594x + 17103 for PHE, y = 30642x – 32669 for TPN and y = 4386.7x + 66901 for DMP. The data are expressed in the Table.1 calibration curves are drawn in Fig. 4, 5 and 6.

TABLE 1: REGRESSION DATA OF CALIBRATION CURVE OF PHE, TPN AND DMP

FIG. 4: CALIBRATION CURVE OF PHE

FIG. 5: CALIBRATION CURVE OF TPN

FIG. 6: CALIBRATION CURVE OF DMP

Accuracy: For the proposed method, accuracy was evaluated by calculating % recoveries of PHE, TPN and DMP by pattern of standard additions (in terms of target assay concentration).A sound recoveries were achieved between the necessary criteria (98.0-102.0 %) as illustrated in Table. 2, 3, and 4.

TABLE 2: ACCURACY DATA OF PHE

TABLE 3: ACCURACY DATA OF TPN

TABLE 4: ACCURACY DATA OF DMP

Precision: Precision was determined in terms of repeatability (intra-day precision) and intermediate precision (inter-day precision). For repeatability, an assay of three concentrations (80%, 100%, and 120%), three replicates each were performed in a single day. Intermediate precision (different day & equipment change) was performed at 100% concentration by evaluating assay of the freshly prepared test solution and the same solution after 24 h and also in different equipment. The % RSD of PHE, TPN, and DMP was found to be less than 2.0 in all the determinations Table 5 and 6.

TABLE 5: INTRA DAY PRECISION (REPEATABILITY) OF THE PROPOSED METHOD

TABLE 6: INTER DAY PRECISION OF THE PROPOSED METHOD

System Suitability: In order to verify that the system is functioning properly during a test, system suitability parameters need to be checked. Five replicates of the optimized standard were injected and method performance data comprising column efficiency (N), resolutions separating nearest peaks (Rs) and asymmetry factor (As) were put on the list in Table 7. All the parameters were in accordance with the fundamentally prescribed ones.

TABLE 7: RESULT OF SYSTEM SUITABILITY TEST

Limits of Detection and quantification (LOD and LOQ): LODs and LOQs were identified using the ICH method based on the calibration curve to assess that the validated concentration ranges of the analytical sample were above their LOQ values 19. The LOD and LOQ values for PHE (37.87 μg/ml and 114.77μg/ml), TPN (10.19μg/ml and 30.89μg/ml) and DMP (57.50μg/ml and 174.26μg/ml)

Robustness: The optimized HPLC factors were revised slightly to investigate the robustness of the method. Robustness was studied using Plackett-Burmann design, keeping pH and flow rate as two variables (pH 3.8, flow rate 0.8), (pH 3.8, flow rate 1.2), (pH 4.2, flow rate 0.8) and (pH 4.2, flow rate 1.2). At higher flow rates, analytes do not have enough time to interact with the stationary phase as compared to lower flow rates. Therefore, analytes elute faster, and retention times are shorter. The theoretical plate number is directly proportional to the retention time. Therefore, as the flow rate increases, the number of theoretical plates decreases.

The method was observed to be robust to peak parameters, as the number of Theoretical Plates exceeded 2000, the Tailing Factor was less than two and the resolution was greater than 2 in all conditions.

TABLE 8: RESULT OF ROBUSTNESS

^aRetention time; btailing factor; ^ctheoretical plate

CONCLUSION: A credible and instantaneous liquid chromatography method for simultaneous estimation of PHE, TPN, and DMP in liquid dosage form has been developed and validated. The chromatographic run time of 15 minutes enables analysis of stacks of samples in a short time. The experimental design was applied for rational robustness study and presentation of its suitability for the desired objective. The results suggest that the method is linear, sensitive, precise, accurate, and robust in response to the mixture under examination. The optimized and validated HPLC procedure was found to be simple, responsive, precise, and accurate. Subsequently, it can be used for routine analysis of PHE, TPN and DMP in the liquid dosage form.

ACKNOWLEDGEMENT: The author would like to thank the Director (Dr. J. N. Mishra) and Dr. Gulzar Alam, Kailash Institute of Pharmacy & Management, Gorakhpur, (UP), India, and Apple International Pharmaceuticals Pvt. Ltd, Nepal, for providing gift sample Dextromethorphan, Phenylephrine, and Triprolidine.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Pandey VK, Alam G and Mishra JN: Development and validation of a new isocratic RP-HPLC method for simultaneous estimation of dextromethorphan hydrobromide, phenylephrine hydrochloride and triprolidine hydrochloride in their combined liquid dosage form. Int J Pharm Sci & Res 2021; 12(9): 4920-26. doi: 10.13040/IJPSR.0975-8232.12(9).4920-26.

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