Sharma B; Satone D; Pradhan P; Jain H; Meshram D

Research Article

Austin J Anal Pharm Chem. 2017; 4(2): 1083.

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Levocloperastine Fendizoate and Chlorpheniramine Maleate in their Combined Dosage Form

Sharma B¹, Satone D², Pradhan P¹, Jain H¹ and Meshram D¹*

¹Department of Quality Assurance, Pioneer Pharmacy Degree College, India

²University Department of Pharmaceutical Sciences, RTM Nagpur University, India

*Corresponding author: Dhananjay Meshram, Department of Quality Assurance, Pioneer Pharmacy Degree College, India

Received: March 20, 2017; Accepted: April 12, 2017; Published: April 19, 2017

Abstract

A simple and rapid RP-HPLC method has been developed and validated for the simultaneous determination of Levocloperastine fendizoate and Chlorpheniramine maleate syrup formulation. Resolution of the analytes was achieved within 10min, employing a mixture of 10mM mobile phase Buffer (pH 6.5): Acetonitrile (50:50, % v/v) as isocratic mobile phase, pumped at 1.0mL/ min through a C18 column (5μm particle size). The detection wavelength for the analytes was 227nm. The system suitability parameters were found to be acceptable. The linearity of response (r2>0.999) in the appropriate ranges (from 50% up to 150% of the expected concentrations of the analytes in the formulations), method accuracy (RSD<2.0%), repeatability and intermediate precision (RSD<2.0%), were confirmed. Robustness result indicates that the methods performance can withstand small variations in method parameters. Satisfactory results obtained in terms of analyte recovery and RSD, while analyzing marketed pharmaceutical preparations. Hence the method can be useful for regular analysis of this combination in marketed syrup formulation.

Keywords: Levocloperastine fendizoate; Chlorpheniramine maleate; RPHPLC

Introduction

Chlorpheniramine maleate (CPM), 3-(p-chlorophenyl)- 3-(2-pyridyl)-N,N-dimethyl propylamine (Figure 1) [1] is a firstgeneration alkyl amine antihistamine, act by antagonizing H1- receptors. It is commonly used for symptomatic relief of the common cold and allergic rhinitis with mild sedative property [2].

Figure 1: Chemical structure of Chlorpheniramine maleate.

    
    
    Figure 1: Chemical structure of Chlorpheniramine maleate.

Levocloperastine (LCF) i.e. 1-{2-[(4-chloro-phenyl)- phenylmethoxy]- ethyl}-piperidine (Figure 2) [3], is a drug with a central antitussive effect and it is also endowed with an antihistaminic (sharing an ethylamine moiety with H1 receptor antagonists) and papaverine like activity similar to codeine, but without its narcotic effects. Pharmacological studies have revealed LCF acts on the cough center, without any depression of respiratory center in brain [4].

Figure 2: Structure of Levocloperastine fendizoate.

    
    
    Figure 2: Structure of Levocloperastine fendizoate.

Literature survey revealed few quantitative techniques like RPHPLC [5-16], UV spectroscopy [17-19], TLC [20] methods for CPM and RP-HPLC method [21], UV spectroscopy [22] for LCF are available. So far to our present knowledge, no validated analytical high performance liquid chromatography (HPLC) method was available in literature for this combination in syrup formulation. Therefore, the purpose of this study was to develop and validate a HPLC methodology for the simultaneous determination of LCF and CPM in their combined syrup formulations, which has no literature precedents.

Materials and Methods

Apparatus

The chromatographic system utilised for development of method consists of a pump (Shimadzu LC 10AT VP) with a universal loop injector (Rheodyne 7725i) with a capacity of 20μL, SPD 20A UV Detector and Hypersil BDS C18 (25cm × 4.6mm i.d. × 5μm) column. The equipment was controlled by a PC work station equipped with CLASS M 10-VP software (Shimadzu, Kyoto, Japan). A UV/ Visible double beam spectrophotometer (Shimadzu Model 1800) was employed with a spectral bandwidth of 1nm and a wavelength accuracy of 0.2nm (with automatic wavelength correction using a pair of 1cm matched quartz cells).

Chemicals and solvents

Chlorpheniramine maleate was procured from Oasis laboratory and Levocloperastine fendizoate was obtained as gift sample from LGM Pharma. HPLC grade methanol, acetonitrile and water bought from Merck India. Marketed formulation (Levotus syrup, Shasun Pharmaceuticals Pvt. Ltd, India) containing LCF 20mg and CPM 4mg, bought from local pharmacy.

Preparation of standard solutions

Standard solutions of CPM (40μg/mL), LCF (200μg/mL) were prepared in MeOH and stored in volumetric flask and utilized as stock solution. Solution was found to be stable for 15 days. Working standard solutions of CPM (4μg/mL), LCF (20μg/mL) were freshly prepared by dilution of the stock standard solutions with mobile phase. Mixture solutions containing analytes were freshly prepared, by mixing appropriate volumes of the corresponding working standard solutions in volumetric flask and made up to the mark with mobile phase.

Commercial syrups

Syrup equivalent to 4mg of CPM and 20mg of LCF was transferred to a 100ml volumetric flask and volume was made up mark with methanol. An aliquot (1ml) of the solution was transferred to 10m volumetric flask and diluted to the mark with mobile phase. The solutions were filtered through 0.45-mm Millipore filter before injection. All preparations were performed in triplicate for each brand.

Chromatographic conditions

Chromatographic separation was performed on C₁₈ (25cm × 0.46cm) Hypersil BDS column. The mobile phase comprised of buffer (pH 6.5)-acetonitrile (50:50, %v/v). The mobile phase was delivered at a flow rate of 1mlmin^-1. Analysis was performed at ambient temperature. Injection volume was 20μl and detection was carried out at 227nm.

Results and Discussion

Selection of wavelength

Figure 3, depicts the UV spectra of CPM and LCF, dissolved in the mobile phase. CPM and LCF are in the ratio found in pharmaceutical formulations; both drugs show iso-absorptive absorption at 227nm, so considered to be the most desirable detection wavelength.

Figure 3: Absorption spectra of the analytes in the 200-400nm region. CPM (4.0μg/mL), LCF (20.0μg/mL) in the optimized mobile phase.

    
    
    Figure 3: Absorption spectra of the analytes in the 200-400nm region. CPM
(4.0μg/mL), LCF (20.0μg/mL) in the optimized mobile phase.

Optimization of mobile phase

Factors such as solubility and pKa (9.47 for CPM and 8.82 for LCF) of both the drugs were utilised to develop mobile phase. Only CPM was found to be eluting in initial trials with water: methanol and water: acetonitrile. This tempted to have buffer in mobile phase. With the convention that pH of mobile phase should (ideally) be at least 2pH units below or above the sample pKa 10mM phosphate buffer of pH 6 was used [23]. With water (pH 6.0): methanol (40:60 %v/v) LCF eluted, but both peak for CPM and LCF were poorly resolved with retention time (Rt) 2.73 and 2.87min respectively. When strength of organic modifier methanol was decreased to 30% gradually, capacity factor (k) for both increases i.e. Rt increases to 3.1 and 4.5min. Selectivity (a) was increased by replacing methanol with acetonitrile, results in increased k value for LCF, Rt changes to 6.257min with tailing. On changing pH of buffer to pH 6.5 tailing decreases. Hence mobile phase Buffer (pH 6.5, 10mM): Acetonitrile (50:50, %v/v) with a flow rate of 1ml/min was finalized as it resulted in good peak shapes and a significant amount of resolution (Figure 4).

Figure 4: Chromatogram of Standard CPM and LCF in Buffer (pH 6.5): Acetonitrile (50:50, %v/v) with flow rate 1ml/min.

    
    
    Figure 4: Chromatogram of Standard CPM and LCF in Buffer (pH 6.5):
Acetonitrile (50:50, %v/v) with flow rate 1ml/min.

Method validation

The method was validated in agreement with the ICH guideline [24]. So, method linearity in the relevant working ranges, precision, accuracy, specificity, and robustness were evaluated. System suitability was also determined.

Range and linearity

Method range and linearity were evaluated with seven mixtures of standards at the concentrations: 2.0–6.0 μg/mL for CPM and 10.0- 30.0 μg/mL. Samples were injected in triplicate and calibration curves were plotted against the standard drug concentrations versus peak areas of the individual drugs. The calibration curves for both the drugs were characterized by the equations shown in Table 1; In addition, correlation coefficients > 0.999 confirms method linearity.

Table 1: Result of Linearity study.




  
    % of nominal 
    CPM 
    Mean 
    LCF 
    Mean 
  
  
    ( μg/mL) 
    AUC (n= 3) 
    ( μg/mL) 
    AUC (n= 3) 
  
  
    50 
    2 
    881.46 
    10 
    1412.37 
  
  
    75 
    3 
    1320.43 
    15 
    2116.27 
  
  
    100 
    4 
    1789.49 
    20 
    2868.67 
  
  
    125 
    5 
    2202.79 
    25 
    3531.59 
  
  
    150 
    6 
    2673.02 
    30 
    4260.36 
  
  
    Slope 
    446.55 
    142.23 
  
  
    Intercept 
    -12.75 
    -6.67 
  
  
    r² 
    0.9997 
    0.9997



Table 1:  Result of Linearity study.

Precision

Method precision was verified in its repeatability and intermediate precision. The ICH guideline Q2(R1) [24] offers two alternatives for assessing repeatability. One needs triplicate estimation with independently prepared samples at low, medium and high analyte levels or in other six replicate determinations at 100% level.

Nine independent mixed standards samples containing both analytes at 80%, 100%, and 120% of their corresponding expected concentrations in the pharmaceutical formulation were injected and the RSD (%) of their recoveries were determined. The observed RSD levels (Table 2), which were below 2%, were considered satisfactory.

For verifying intermediate precision, the samples were injected at random during three different days. In all cases results were consistent (RSD<2%), confirmed the precision of the method (Table 2).

Table 2: Result of Inter-day and Intraday precision.




  
    Drug 
    Conc. 
    Repeatability (n= 6) 
    Inter-day (n= 3) 
  
  
    (μg/mL) 
    Area 
    % R.S.D 
    Area 
    % R.S.D 
  
  
    
    Mean ± S.D. (n=6) 
    Mean ± S.D. (n=3) 
  
  
    CPM 
    2 
    
    
    888.13 ± 9.25 
    1.042 
  
  
    4 
    1777.84 ± 10.69
    0.6
    1778.12 ± 13.42 
    0.755 
  
  
    6 
    
    
    2677.29 ± 29.76 
    1.111 
  
  
    LCF 
    10 
    
    
    1425.94 ± 10.05 
    0.705 
  
  
    20 
    2843.62 ± 10.18
    0.36
    2845.13 ± 14.56 
    0.512 
  
  
    30 
    
    
    4277.60 ± 50.83 
    1.305



Table 2:  Result of Inter-day and Intraday precision.

Accuracy

Method accuracy was demonstrated by standard addition method, by evaluating declared amounts of standard drugs which was fortified to pre-assayed pharmaceutical formulation sample (2.0μg/ mL CPM and 10.0μg/mL LCF), at 80%, 100%, and 120% in triplicates. The %RSD did not exceed 2% (Table 3), meaning that essentially quantitative recoveries were achieved. This confirmed the method enables the accurate determination of the analytes.

Table 3: Result of Recovery study (n=3).




  
    Drug 
    % level 
    Spiked Conc. (�g/mL) 
    Recovered Conc.� (�g/mL) 
    Mean Recovery (%) 
    R.S.D (%) 
  
  
    CPM 
      (2.0 �g/mL) 
    80 
    1.6 
    1.61 
    100.63 
    0.63 
  
  
    100 
    2 
    1.99 
    99.33 
    1.05 
  
  
    120 
    2.4 
    2.41 
    100.28 
    1.28 
  
  
    LCF 
      (10.0 �g/mL) 
    80 
    8 
    8.04 
    100.46 
    0.58 
  
  
    100 
    10 
    9.91 
    99.1 
    0.4 
  
  
    120 
    12 
    11.97 
    99.73 
    0.93



Table 3:  Result of Recovery study (n=3).

LOD and LOQ

The ICH guidelines do not specifically need calculation of the limits of detection (LOD) and quantification (LOQ) for principal analytes in their formulations. However, to assess the confirmed concentration ranges of the analytes were above their LOQ values, the LOD and LOQ were determined employing the ICH method based on the calibration curve [24]. The LOD values were 0.108μg/ mL for CPM and 0.496μg/mL for LCF; the corresponding LOQ values, determined with the same method, were 0.327 and 1.504μg/ mL, respectively. These values fall below the lowest expected analyte concentrations in the samples (Table 4).

Table 4: Limit of Detection and Quantification.




  
     
    LOD (μg/mL) 
    LOQ (μg/mL) 
  
  
    CPM
    0.108 
    0.327
  
  
    LCF
    0.496 
    1.504



Table 4:  Limit of Detection and Quantification.

Robustness

The robustness of the method was assessed by deliberately causing small changes to the procedure. The flow rate (1mL/min) was altered by ± 2ml/min (0.8-1.2), the pH of the aqueous phase (6.5) was modified in ± 0.2 units (6.3-6.7), and the proportion of the organic mobile phase content (50) was changed in ±2 mL (48–52%). The results were repeated in triplicate and summarized in Table 5. Results indicates its reliability during normal use.

Parameter	Modification	CPM	LCF
Flow rate	0.8	3.20 ± 0.02	3.70 ±0.02
Flow rate	1.2	5.83 ± 0.06	6.27 ± 0.06
pH	6.3	1786.36 ± 9.46	1814.25 ± 7.43
pH	6.7	2870.71 ± 8.40	2909.02 ± 23.70
Organic modifier	48.0	1742.73 ± 14.28	1830.12 ± 15.39
Organic modifier	52.0	2800.49 ± 13.88	2928.82 ± 15.94

Parameter	CPM	LCF	Acceptance Limit
Parameter	Average (n=6) ± %RSD		Acceptance Limit
*Retention Time (t_r, min.)*	3.319 ± 0.14	6.118 ± 0.18	%RSD < 2 %
*Resolution (R_s)*	11.655 ± 0.76		Rs > 2
*Tailing factors (t_f)*	1.21 ± 0.05	1.32 ± 0.08	Tf = 2.0
Theoretical plates (N)	5706 ± 47.44	7282 ± 123.55	N = 2000

Analyte Dosage form	Label claim (mg)		*Assay (% of label claim)**
			Mean ± S. D.
	CPM	LCF	CPM	LCF
Levotus Syrup	4	20	99.84 ± 0.421	99.57 ± 0.416