SIMULTANEOUS QUANTITATION AND VALIDATION OF PHENYLEPHRINE HYDRO-CHLORIDE, AMBROXOL HYDROCHLORIDE AND LEVOCITRIZINE HYDROCHLORIDE IN SYRUP FORMULATION BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

SIMULTANEOUS QUANTITATION AND VALIDATION OF PHENYLEPHRINE HYDRO-CHLORIDE, AMBROXOL HYDROCHLORIDE AND LEVOCITRIZINE HYDROCHLORIDE IN SYRUP FORMULATION BY REVERSE PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Padmakana Malakar*¹, Arup Ratan Deb ² and S. K. Veliyath ²

Azad College of Pharmacy ¹, Moinabad, R.R. District, Hyderabad, Andhra Pradesh, India

Moonray Institute of Pharmaceutical Sciences ², Shadnagar, MBNR District, Andhra Pradesh, India

ABSTRACT:A simple, precise and accurate RP-HPLC method was developed and validated for the estimation of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride in syrup formulation. This method is based on HPLC separation of the three drugs on Octadecyl Silane C18 column (250 mm × 4.6 mm, 5.0μ), with isocratic conditions and mobile phase containing 0.01M Sodium dihydrogen phosphate monohydrate buffer [pH 3.0, adjusted with Ortho Phosphoric Acid and 1.1 gm of Octane sulfonic acid sodium salt]: Acetonitrile: Methanol (60:30:10) at a flow rate of 1 ml/min, using UV detection at 230 nm. The retention time of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride in syrup formulation were found to be around 3.47 min, 11.8 min and 32.6 min respectively. The method was validated for linearity, accuracy, precision, ruggedness, robustness and solution stability as per ICH guidelines. The linearity of Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride solutions ranged from 0.104 mg/ml to 0.303 mg/ml; 0.6 mg/ml to 1.804 mg/ml and 0.051 mg/ml to 0.150 mg/ml, respectively, equivalent to 50% to 150% of the working concentration. Statistical analysis showed that the proposed method is repeatable and selective for the estimation of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocitrizine hydrochloride in syrup formulation.

Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride, RP- HPLC, Simultaneous quantification, Validation

INTRODUCTION:The chemical name of Phenylephrine hydrochloride is (R)-(α)-m-Hydroxy-α-[(methyl amino) methyl] benzyl alcohol hydrochloride. The structure of Phenylephrine hydrochloride is given in the Fig.1. Phenylephrine acts predominantly by direct stimulation o alpha adrenergic receptors, which constrict resistance and capacitance of blood vessels, resulting in increased total peripheral resistance: increased systolic and diastolic blood pressure.

Alpha-adrenergic effects include action on the dilator muscle of the pupil and local decongestant action in the arterioles.

The decongestant works by constricting blood vessels and reducing swelling in the nasal passages.Phenylephrine does not stimulate beta receptors except in large doses.

FIG.1:    STRUCTURE OF PHENYLEPHRINE HYDROCHLORIDE

Chemically Ambroxol hydrochloride (Fig. 2) is Trans-4-(2-Amino-3, 5-dibrombenzylamino)-cyclo hexanolHydrochloride. Ambroxol is an active N-desmethyl metabolite of the mucolytic bromhexine. Although its mechanism of action has not been fully defined, it may increase the quantity and decrease the viscosity of tracheobronchial secretions. It may also act as an expectorant, increasing mucociliary transport via stimulation of cilliary motility.

Ambroxol may stimulate the synthesis and secretion of pulmonary surfactant; the drug has been referred to as a “surfactant activator”. It was postulated that ambroxol decreased airway hyper-reactivity by either increasing lysophosphatidyl-choline turnover and/or modifying epithelial secretions ^{1, 2, 3}.

FIG. 2: STRUCTURE OF AMBROXOL

Levocetirizine, the (R) enantiomer of cetirizine (Fig. 3), is a potent and selective antagonist of peripheral H1-receptors. Binding studies revealed that levocetirizine has high affinity for human H1-receptors (Ki = 3.2 nmol/l). Levocetirizine has an affinity 2-fold higher than that of cetirizine (Ki = 6.3 nmol/l). Levocetirizine dissociates from H1-receptors with a half-life of 115 ± 38 min. After single administration, levocetirizine shows receptor occupancy of 90% at 4 hours and 57% at 24 hours. The onset of action of levocetirizine 5 mg in controlling pollen-induced symptoms has been observed at 1 hour post drug intake in placebo controlled trials in the model of the allergen challenge chamber ^{3, 4, 5}.

FIG. 3: STRUCTURE OF LEVOCETIRIZINE HYDROCHLORIDE

Literature review reveals that methods have been reported for analysis of phenylephrine hydrochloride, ambroxol hydrochloride and levocitrizine hydrochloride either alone or in combination with other drugs ^6-18.

To date, there have been no published reports about the simultaneous quantitation of phenylephrine hydrochloride, ambroxol hydrochloride and levocitrizine hydrochloride by chromatographic method in syrup formulation.

This present study reports for the first time simultaneous quantitation of the same drugs by RPHPLC in syrup formulation dosage form. The proposed method is validated as per ICH guidelines ¹⁹.

MATERIALS AND METHODS: Working standards of pharmaceutical grade Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocitrizine hydrochloride were obtained from Malladi Drugs, Alchymar ICM Sm Pvt. Ltd. and Auctus Pharma respectively. They were used without further purification and certified to contain 98.5-100.5%, 99.0-101.0% and 98.0-102.0% on dry weight basis for Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocitrizine hydrochloride, respectively. All the chemicals were of HPLC grade. Water used was double distilled and filtered through 0.45μm filter.

Instrumentation: The HPLC system consisted of Intelligent HPLC pump model (Shimadzu Prominence) with sampler programmed at 10μl capacity per injection. The detector consisted of a UV/ VIS (SPD-20A/20AV Series). Data was integrated using LC Solution system. The column used was, Octadecyl Silane C18 column (250 mm × 4.6 mm, 5.0μ), with isocratic conditions.

Mobile phase containing 0.01M Sodium dihydrogen phosphate monohydrate buffer [pH 3.0, adjusted with Ortho Phosphoric Acid and 1 gm of Octane sulfonic acid sodium salt]: Acetonitrile: Methanol (60:30:10) at a flow rate of 1 ml/min, using UV detection at 230 nm. The mobile phase was filtered through a 0.45 micron membrane filter and degassed. The injection volume was 10μl and analysis was performed at ambient temperature.

Preparation of Standard Solution: About 20 mg of Phenylephrine hydrochloride, 120 mg of Ambroxol hydrochloride and 10 mg of Levocitrizine hydrochloride working standards were dissolved in 10ml of diluents in three separate 100ml volumetric flask and sonicated, then volume was made up to the mark with diluents.

Preparation of Mixed Standard Solution: About 20 mg of Phenylephrine hydrochloride, 120 mg of Ambroxol hydrochloride and 10 mg of Levocitrizine hydrochloride working standards were dissolved in 10ml of diluents in a 100ml volumetric flask and sonicated, then volume was made up to the mark with diluents.

Preparation of Sample Solution:  About 5 g of syrup was dissolved in 10ml of diluents in a 25ml volumetric flask and sonicated,  then volume was made up to the mark with diluents.

TABLE 1: SUMMARY OF CHROMATOGRAPHIC CONDITION

Optimization of HPLC Method:All drugs were subjected to chromatographic analysis using mobile phases of differing pH at mentioned chromatographic conditions (Table 1). The changes in the retention time of all drugs were noted as a function of changing mobile phase, pH, strength and selectivity.After all the trials it was found that Sodium dihydrogen phosphate monohydrate buffer (pH 3 adjusted with orthophosphoric acid and 1.1g of octane sulfonic acid sodium salt) : Acetonitrile : Methanol in the ratio of (60:30:10) at flow rate of 10 µl/min gave acceptable retention time of 3.366, 11.605 and 32.159 with number of plates (N) 3058, 8502 and 9721 and good resolution (22.06 between Phenylephrine Hydrochloride and Ambroxol Hydrochloride, 22.74 between Ambroxol Hydro-chloride and Levocetirizine Hydrochloride) for Phenylephrine hydrochloride, Ambroxol hydro-chloride and Levocitrizine hydrochloride, respectively (Fig. 4).

 FIG. 4: CHROMATOGRAM OF PHENYLEPHRINE HCL (RT =3.366), AMBROXOL HCL (RT =11.605) AND LEVOCETIRIZINE HCL (RT =32.159)

Validation of the method: Validation of the optimized HPLC method was carried out with respect to the following parameters.

1. Specificity: Specificity was analyzed with the placebo (200mg/ml) and the drug substance [Phenylephrine hydrochloride (0.40mg/ml), Ambroxol hydrochloride (1.20mg/ml), Levocetirizine hydrochloride (0.40mg/ml)] separately at different concentrations.
2. System Suitability: A standard solution was prepared containing 0.2 mg/ml Phenylephrine hydrochloride, 1.2 mg/ml Ambroxol hydrochloride, 0.1 mg/ml Levocetirizine hydrochloride. Percentage relative standard deviation, tailing factor and theoretical plates of the Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride peak in standard solution was calculated.
3. Linearity and Range: Linearity was performed by injecting the standard solution in duplicate ranging from 50% to 150% of working concentration of Phenylephrine hydrochloride, Ambroxol hydrochloride, and Levocetirizine hydrochloride by covering at least six points. The regression analysis was performed and the linearity range for determining assay was reported.
4. Accuracy: The accuracy of the method was determined by analyzing the solutions containing Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride at approximately 50%, 100% and 150% of the working strengths spiked with placebo. Each level was analyzed in triplicate.
5. System Precision: System precision was performed by injecting the standard solution (0.2 mg/ml Phenylephrine hydrochloride, 1.2 mg/ml Ambroxol hydrochloride and 0.1 mg/ml Levocetirizine hydrochloride in diluents) in six times and the percentage relative standard deviation of six replicate injections of standards were determined.
6. Method Precision: The method precision was performed by analyzing a sample solution of Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride at working concentration six times (six replicate sample preparation).
7. Ruggedness (Intermediate Precision): The ruggedness of the method was determined by analyzing a standard solution five times followed by a sample in duplicate with varying following parameters (Table 2):

TABLE 2: PARAMETERS FOR OPTIMIZING INTERMEDIATE PRECISION

8. Robustness: The robustness of the method was determined by analyzing a standard solution five times followed by a sample in duplicate with varying HPLC conditions as described below (Table 3):

TABLE 3: HPLC CONDITIONS FOR DETERMINING THE ROBUSTNESS OF THE METHOD

Solution Stability: The stability of Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride sample was measured at 100% of the target concentration by keeping the solution up to forty-eight hours at room temperature. The sample was analyzed at different intervals (12hrs, 24hrs, 48hrs), using freshly standards every time.

RESULTS AND DISCUSSION: The results of validation studies on simultaneous estimation method developed for Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocitrizine hydrochloride in the current study involving mobile phase Sodium dihydrogen phosphate monohydrate buffer (pH 3 adjusted with OPA) : Acetonitrile : Methanol in the ratio of (60:30:10) at flow rate of 10 µl/min are given below;

1. Specificity:Placebo solution was prepared separately at a concentration of 200mg/ml. A solution of placebo was spiked with Phenylephrine hydrochloride, Ambroxol hydrochloride, and Levocetirizine hydrochloride at working concentration. A mixed standard solution was prepared and these solutions were analyzed as per the HPLC method described in the protocol. The study shows that the placebo and API were accurately resolved (Fig. 5).

FIG. 5: CHROMATOGRAM OF PLACEBO (200mg/ml) WITH PHENYLEPHRINE HCl (0.40mg/ml), AMBROXOL HCl (1.20mg/ml) AND LEVO-CETIRIZINE HCl (0.40mg/ml) 

2. System Suitability: Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride at working concentration was prepared and analyzed as per the HPLC method described in the protocol. Table 4 shows the %RSD,tailing factor and theoretical plate of the peaks.

TABLE 4: SYSTEM SUITABILITY STUDIES

 Linearity and Range: The linearity of Phenylephrine hydrochloride, Ambroxol hydrochloride, Levocetirizine hydrochloride solutions were ranging from 0.104 mg/ml to 0.303 mg/ml; 0.6 mg/ml to 1.804 mg/ml and 0.051 mg/ml to 0.150 mg/ml, respectively which is equivalent to 50% to 150% of the working concentration. Five standard solutions at concentration within the mentioned range were prepared and analyzed as per method.The linearity results obtained are shown in the Table 5 and Figure 6-8, the line of best fit for peak area versus weight of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride respectively. The HPLC method was shown to be linear of 50% to 150% of the working standards concentration with a regression coefficient 0.999. The range of the HPLC method for determining the assay is 50% to 150% of the working strength.

TABLE 5: LINEARITY STUDIES

FIG. 6: LINEARITY GRAPH OF PHENYLEPHRINE

FIG. 7: LINEARITY GRAPH OF AMBROXOL

FIG. 8: LINEARITY GRAPH OF LEVOCETIRIZINE

4. Accuracy: The percentage recovery values were in the range of 98.0% to 102.0% which is within the acceptance criteria. The percentage recovery results obtained were listed in Table 6.
5. Method Precision: The method precision was performed by analyzing a sample solution at working concentration for six times. Results of percentage relative standard deviation of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydro-chloride are summarized in Table 7.

TABLE 6: PERCENTAGE RECOVERY STUDIES

TABLE 7: PERCENTAGE RELATIVE STANDARD DEVIATION OF THREE DRUGS

6. 
   System Precision: The %RSD for peak area variation of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride was 1.89, 1.91 and 1.73 respectively at the working concentration (Figure 9).

FIG. 9: CHROMATOGRAM FOR SYSTEM PRECISION

7. Method Precision: The method precision was performed by analyzing a sample solution at working concentration for six times. Results of percentage relative standard deviation of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride are summarized in Table 8. The percentage relative standard deviation for assay value of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydro-chloride was 0.58, 0.67 and 0.57 respectively.

  TABLE 8: PERCENTAGE RELATIVE STANDARD DEVIATION OF THREE DRUGS

8. 
   Ruggedness: The ruggedness is assessed by evaluating the variability of the results obtained by the analysis of sample syrup formulation by different analyst on different days with different instruments, different reagents and different columns. The results are given in the Table 9.

TABLE 9: RUGGEDNESS STUDIES

 Robustness: Robustness of the method was tested by slightly changing the parameters like pH, flow rate and mobile phase ratio. The following tables 10 (A), (B) and (C) show the results for the variation of parameters that were altered to test the robustness of the method. System suitability was performed along with the sample analysis to assess if changes had a significant effect on the chromatography and assay values.

TABLE 10 (A): ROBUSTNESS TESTING

TABLE 10 (B): ROBUSTNESS TESTING

TABLE 10 (C): ROBUSTNESS TESTING

FIG. 10: CHROMATOGRAMS FOR DRUGS AT VARIOUS PARAMETERS (A) FOR PH 2.90, (B) FOR PH 3.10, (C) FOR FLOW RATE 0.9ML/MIN, (D) FOR FLOW RATE 1.1ML/MIN, (E) FOR MP 58:42 AND (F) FOR MP 62:38

The tailing factor was observed to be not more than 2.0 for Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride and theoretical plates were within the acceptance criteria.

The retention time of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride were affected by the pH change and flow rate change and mixing of mobile phase composition (Figure 10).

Hence, the system suitability criteria for the method were fulfilled and the assay values obtained were comparable.

1. Solution Stability: A solution of sample formulation at 100% of working concentration was kept at room temperature. The solutions were analyzed at different intervals (i.e. 12 hrs, 24 hrs and 48 hrs).The assay values obtained for sample solutions (Figure 11) were within the label claim and % RSD is within acceptance criteria i.e. not more than 2.0%. Therefore, sample solutions were stable up to 48 hours at room temperature.

FIG. 11: CHROMATOGRAMS OF SAMPLE SOLUTION AT DIFFERENT TIME INTERVAL (A) AT 12 HRS, (B) AT 24 HRS AND (C) AT 48 HRS INTERVAL

CONCLUSIONS: The combination syrup of Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride was subjected to simultaneous determination by reverse phase HPLC method. Chromatographic figures, calibration data and recovery of the drug from the spiked concentrations were determined to assess the validity of the method. The results obtained by repeatability test, confirms the precision of the method. The proposed RP HPLC method gives good resolution between Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride using a time 45 min and can be used for routine quality control analysis.

ACKNOWLEDGEMENT: The authors would like to thank Fourrts (India) Laboratories Pvt. Ltd. (Kelambakkam, Chennai, India) for providing gift samples of standard Phenylephrine hydrochloride, Ambroxol hydrochloride and Levocetirizine hydrochloride. The authors would like to thank, Mr. M. Ramalingam, Sr. General Manager (R&D), Fourrts (India) Laboratories Pvt. Ltd., Chennai, India for providing necessary facilities to carry out the work.

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    How to cite this article:

    Malakar P, Deb AR and Veliyath SK: Simultaneous Quantitation and Validation of Phenylephrine hydro-chloride, Ambroxol hydrochloride and Levocetrizine hydrochloride in Syrup formulation by Reverse Phase High Performance Liquid Chromatography. Int J Pharm Sci Res 2013: 4(8); 3028-3038. doi: 10.13040/IJPSR. 0975-8232.4(8).3028-38

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