Int J App Pharm, Vol 10, Issue 1, 2018, 124-135Original Article

STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIETHYLCARBAMAZINE CITRATE, GUAIPHENESIN AND CHLORPHENIRAMINE MALEATE

PODILI BHAVANI, SEELAM MOHAN, KAMMELA PRASADA RAO*

Department of Chemistry, Bapatla Engineering College, Bapatla, Guntur (DT), AP., India
Email: prasad17467@gmail.com

Received: 22 Aug 2017, Revised and Accepted: 18 Dec 2017


ABSTRACT

Objective: The present work describes the development and subsequent validation of a simple, precise and stability–indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate in tablet dosage forms.

Methods: A simple, accurate, precise and robust RP-HPLC method was developed and validated for the estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate. The chromatographic separation of all the three active components was achieved by using luna phenyl-hexyl column (250 mmx4.6 mm, dp=5 µm) with a mobile phase consisting of isocratic method with 0.1% triethylamine as buffer along with orthophosphoric acid adjusted to PH 2.5: acetonitrile (50:50v/v) at a flow rate 1.0 ml/min and ultraviolet detection at 210 nm.

Results: The retention time of chlorpheniramine maleate, guaiphenesin and diethylcarbamazine citrate were 2.86, 4.89 and 7.76 min respectively. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines. The established method was linear in the range of 1-15, 0.6-9, 0.02-0.3 µg/ml and correlation coefficient was 0.999, 0.9991, and 0.993 for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate respectively.

Conclusion: The proposed method can be used for the quantitative analysis of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate.

Keywords: Diethylcarbamazine citrate, Guaiphenesin and Chlorpheniramine maleate


INTRODUCTION

Diethylcarbamazine citrate, chemically N, N-diethyl-4-methyl piperazine-1-carboxamide dihydrogen citrate [1] is one of the essential medicines needed in a basic health system, suggested by world health organisation (WHO) [2]. It is used in the treatment of filariasis including lymphatic filariasis, tropical pulmonary eosinophilia and loiasis.

Fig. 1: Chemical structure of diethylcarbamazine citrate

Guaiphenesin, chemically (S, S)-2-methlylamino-1-phenylpropan-1-ol hydrochloride [3, 4], mainly used as a cough remedy. It has been given to patients which have altered nasal mucociliary clearance associated with HIV. It is used to remove phlegm from the airways in acute respiratory tract infections.

Fig. 2: Chemical structure of guaiphenesin

As chlorpheniramine maleate, chemically (RS)-3-(4-chlorophenyl)-3-(pyrid-2-yl) propyl dimethylamine hydrogen maleate [5], has the relatively less sedative effect it is most commonly used as an antihistamine in small animal veterinary practices.

Fig. 3: Chemical structure of chlorpheniramine maleate

The literature survey revealed that several analytical methods have been reported for the estimation of diethylcarbamazine citrate [6], guaiphenesin [7] and chlorpheniramine maleate [8, 9] individually or in combination with other drugs by UV-visible spectro-photometry, nuclear magnetic resonance spectroscopy, high-performance liquid chromatography methods [10-13]. No method has been developed for the simultaneous determination of diethylcarbamazine citrate, guaiphenesin, chlorpheniramine maleate both in bulk and pharmaceutical dosage forms.

On the meticulous observance of the potential applications of these three active drugs, we aimed to develop and validate a new, rapid and sensitive RP-HPLC method for simultaneous estimation of diethylcarbamazine citrate, guaiphenesin, and chlorpheniramine maleate. Degradation studies (stress studies) were carried out to establish the stability characteristics of the three ingredients under heat, acid, base, peroxide, light and reductive stress conditions as recommended in the ICH guidelines Q1A (R2).

MATERIALS AND METHODS

Instrumentation

The analysis was performed on waters alliance-2695 chromato-graphic system, equipped with a quaternary pump and PDA detector-2996. Chromatographic software empower-2.0 was used for data collection and processing.

Chemicals and reagents

Acetonitrile (HPLC grade), triethylamine (HPLC grade), orthophophoric acid (HPLC grade), water (HPLC grade) were purchased from Merk (India) Ltd, Worli, Mumbai, India. All active pharmaceutical ingredients (APIs) of diethylcarbamazine citrate, guaiphenesin and chlor-pheniramine maleate as reference standards were procured from Supriya life Sciences, Goregaon (E), Mumbai, India (99.7-99.9 % purity).

Chromatographic conditions

Chromatographic analysis was done using isocratic elution and acetonitrile: 0.1% triethylamine PH adjusted to 2.5 with OPA (50:50 by volume) as a mobile phase and was filtered through 0.45µ membrane filter paper. The flow rate of the mobile phase was monitored at 1 ml/min and eluents were detected at 210 nm. Operating pressure 3000 psi was maintained at room temperature by injecting the volume 10 µl with a run time 10 min.

Selection of wavelength

By using photodiode spectrophotometer the absorption spectra of the solution of the three drugs in acetonitrile were scanned in the UV region 200-400 nm against acetonitrile as blank and spectra are shown in fig. From the fig. the spectra of the diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate shows different λmax viz. 254.5, 255.6 and 369.4 nm respectively. By considering the chromatographic parameter, sensitivity and selectivity of a method for three drugs 210 nm was selected as the detection wavelength for HPLC chromatographic method.

Fig. 4: PDA spectrum for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

Preparation of standard solution

100 mg of diethylcarbamazine citrate, 60 mg of guaiphenesin, 2 mg of Chlorpheniramine maleate (working standard) were weighed accurately and transferred into a 100 ml volumetric flask.70 ml of mobile phase was added to the above flask and then sonicated about 20 min for uniform mixing and then diluted 1 ml of the above solution to 10 ml with the mobile phase and again 1 ml of the solution was diluted to 10 ml with same mobile phase.

Preparation of sample solution

10 tablets were weighed and pulverised to powder form, from which one equivalent weight (437.5 mg) was taken into 100 ml volumetric flask.70 ml of mobile phase was added to the above flask and then sonicated about 20 min for uniform mixing. 1 ml of the above solution was diluted to 100 ml with the mobile phase and filtered through 0.45μ nylon syringe filter.

Validation

The optimized chromatographic separation was aimed to obtain a resolution above 1.5 between all components, tailing factor is less than 2.0 and plate count will be more than 2000 with respect to the stationary, mobile phase compositions, flow rate, sample volume, detection wavelength and temperature.

Validation procedure

In the present method validation was done with the aspect of system suitability, specificity, accuracy, precision, linearity, robustness, limit of detection (LOD), limit of quantitation (LOQ), forced degradation and stability according to the ICH guidelines [14-18].

Fig. 5: Typical chromatogram for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

System suitability

As per the test method the standard and check standard solutions were prepared and injected into HPLC system [10, 11], from which the evaluated system suitability parameters are found to be within the limits.

Specificity

The analyte was assessed unequivocally to know the components impurity which may be expected to be present with the help of specificity. As per test method blank was prepared and injected. No blank peak was eluted in the retention time of analyte peak. Placebo solutions were prepared in duplicate and injected as per test method. It was found that no placebo peaks were interfered at the retention time of the main peak.

Accuracy

Three different concentrations such as lower quantitation limit, medium quantitation limit, and higher quantitation limit were used to evaluate the accuracy of the RP-HPLC method. The amount of the drugs present, percentage recovery and RSD were calculated by giving a minimum of three injections from each concentration.

Precision

The precision of the test method was evaluated by considering six different concentrations. The amount of the drugs present, percentage recovery and RSD were calculated by giving a minimum of six preparations.

Linearity and range

Six series of standard solutions were selected for assessing linearity range. By using peak area versus concentration of the standard solution calibration curve was plotted and the regression equations were also calculated. The slope, intercept and the correlation coefficient was calculated by least squares method.

LOD and LOQ

By using optimized chromatographic conditions in accordance with 3.3 s/n and 10 s/n criteria, where s/n indicates signal-to-noise ratio, the LOD and LOQ were determined by injecting progressively lower concentrations of the standard solutions into the HPLC column.

Forced degradation

In chromatogram of forced degradation there should be no interference between peaks and were well separated from each other with the resolution at least 1.0 and the peak purity of the principal peaks should pass. Forced degradation studies were performed by different types of stress conditions to obtain the degradation of about 20%.

Robustness

Small changes such as±5% in the ratio of acetonitrile in the mobile phase,±0.2 ml/min in the flow rate and±5 nm in the wavelength were made to demonstrate the robustness method. The separation factor, retention time and peak asymmetry were calculated.

Stability

Standard and the sample solutions were subjected to 24 h stability studies. The stability of these solutions was studied and observed for changes in the area and retention time of the peaks which were then compared with the pattern of the chromatogram of the freshly prepared solution.

RESULTS AND DISCUSSION

Method validation

In this method system suitability, linearity, precision, accuracy, robustness, LOD (Limit detection), LOQ (Limit of quantification), forced degradation and the stability are validated for the selected diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate drugs.

System suitability

10 µl of working standard solution (10µg/ml of diethylcarbamazine citrate, 6µg/ml of guaiphenesin and 0.2µg/ml of chlorpheniramine maleate) was prepared and injected into the system. It was determined by making six replicate injections and all the parameters were found to be within the limits. The results are given table 1.

Table 1: System suitability parameters for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

System suitability parameter Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
Retention time (min) 7.850 4.715 2.900
Theoretical plate number (N) 10909 4416 5810
Tailing factor (T) 1.062 1.145 1.271
Resolution (R) 10.693 8.386 -

Linearity

The linearity of the proposed method was constructed by considering concentration on the x-axis and peak area on the y-axis. It was established by least squares linear regression analysis of the calibration curve. The calibration curve was linear in the range of 1-15, 0.6-9, 0.02-0.3 µg/ml for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate respectively. The regression equation for calibration curve was Y=561967x+13655 (r2=0.999) for diethylcarbamazine citrate, Y=474141x+21692 (r2=0.999) for guaiphenesin and Y=951864x+4648 (r2=0.999) for chlorpheniramine maleate. The results are given in table 2.

Table 2: Linearity data for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
Conc µg/ml Area counts Conc µg/ml Area counts Conc µg/ml Area counts
1.00 560581 0.60 278415 0.02 190320
2.50 1419062 1.50 687456 0.05 487956
5.00 2878887 3.00 1358742 0.10 954786
10.00 5602579 6.00 2786284 0.20 1923654
12.50 7041847 7.50 3568745 0.25 2378462
15.00 8443101 9.00 4254810 0.30 2854810
Corr Coef 0.999 Corr Coef 0.999 Corr Coef 0.999
Slope 561966.74 Slope 474141.22 Slope 9518964.73
Intercepst 13655.29 Intercept 21692.26 Intercept 4648.64

Fig. 6: Chromatogram for linearity-1

Fig. 7: Chromatogram for linearity-2

Fig. 8: Chromatogram for linearity-3

Fig. 9: Chromatogram for linearity-4

Fig. 10: Chromatogram for linearity-5

Fig. 11: Chromatogram for linearity-6

Fig. 12: Linearity plot for diethylcarbamazine citrate

Fig. 13: Linearity plot for guaiphenesin

.

Fig. 14: Linearity plot for chlorpheniramine maleate

Accuracy

In this method, accuracy was determined by recovery studies which were carried out in three different concentration levels (50%, 100% and 150%). APIs with concentration 5, 10 and 15 µg/ml of diethylcarbamazine citrate; 3, 6 and 9 µg/ml of guaiphenesin; and 0.1, 0.2 and 0.3 µg/ml of chlorpheniramine maleate were prepared. As per the test method, the test solution was injected three times for each spike level and the assay was performed. The accuracy and reliability of the developed method were established. The percentage recovery values were found to be in the range of 100.34-100.81% for diethylcarbamazine citrate and 100.51-100.18% for guaiphenesin and 100.64-100.34% for chlorpheniramine maleate. RSD values were found to be less than 2%. The results are given in table 3, 4 and 5.

Table 3: Accuracy data for diethylcarbamazine citrate

Accuracy

Amount of drug

conc µg/ml

Amount added

µg/ml

Amount obtained

µg/ml

Area counts % Recovery

Mean recovery,

±RSD

50% 10.01 5.08 5.031 1383585 100.34

100.26,

0.10

10.01 5.04 5.012 1379603 100.28
10.01 5.11 5.143 1313241 100.15
100% 10.01 10.18 10.541 2789315 100.27

100.26,

0.07

10.01 10.12 10.324 2784571 100.18
10.01 10.16 10.148 2799874 100.32
150% 10.01 15.47 15.478 4189106 100.24

100.5

0.28

10.17 15.28 15.369 4196329 100.45
10.17 15.19 15.214 4145316 100.81

Table 4: Accuracy data for guaiphenesin

Accuracy

Amount of drug

conc µg/ml

Amount added

µg/ml

Amount obtained

µg/ml

Area counts % Recovery

Mean recovery,

±RSD

50% 6.12 3.01 3.124 904105 100.51

100.37,

0.12

6.12 3.05 3.131 904979 100.35
6.12 3.14 3.214 894400 100.27
100% 6.12 6.15 6.528 1871153 100.38

100.45,

0.07

6.12 6.21 6.374 1880820 100.47
6.12 6.28 6.484 1892189 100.52
150% 6.12 9.57 9.428 2847220 100.61

100.42,

0.21

6.12 9.26 9.569 2867896 100.48
6.12 9.15 9.214 2816024 100.18

Table 5: Accuracy data for chlorpheniramine maleate

Accuracy

Amount of drug

conc µg/ml

Amount added

µg/ml

Amount obtained

µg/ml

Area counts % Recovery

Mean recovery,

±RSD

50% 0.21 0.15 0.148 254967 100.64

100.49,

0.19

0.21 0.14 0.142 257431 100.58
0.21 0.11 0.147 252908 100.27
100% 0.21 0.21 0.215 547450 100.56

100.41,

0.13

0.21 0.22 0.218 546146 100.38
0.21 0.23 0.223 541044 100.29
150% 0.21 0.30 0.347 793703 100.17

100.24,

0.08

0.21 0.31 0.311 792084 100.21
0.21 0.32 0.318 793544 100.34

Fig. 15: Chromatogram for accuracy 50%-1

Fig. 16: Chromatogram for accuracy 50%-2

Fig. 17: Chromatogram for accuracy 50%-3

Fig. 18: Chromatogram for accuracy 100%-1

Fig. 19: Chromatogram for accuracy 100%-2

Fig. 20: Chromatogram for accuracy 100%-3

Fig. 21: Chromatogram for accuracy 150%-1

Fig. 22: Chromatogram for accuracy 150%-2

Fig. 23: Chromatogram for accuracy 150%-3

Precision

Repeatability

Repeatability was calculated by injecting standard solution six times containing diethylcarbamazine citrate (10µg/ml), guaiphenesin (6µg/ml) and chlorpheniramine maleate (0.2µg/ml). Peak areas and % RSD were calculated.

Intraday precision

Six replicates of a sample solution containing diethylcarbamazine citrate (10µg/ml), guaiphenesin (6µg/ml) and chlorpheniramine maleate (0.2µg/ml) were analysed on the same day. Peak areas were calculated, which were used to calculate mean, SD and %RSD values.

Interday precision

Six replicates of a sample solution containing diethylcarbamazine citrate (10µg/ml), guaiphenesin (6µg/ml), and chlorpheniramine maleate (0.2µg/ml) were analysed on a different day. Peak areas were calculated which were used to calculate mean, SD and %RSD values. The present method was found to be precise as the RSD values were less than 2% and also the percentage assay values were close to be 100%. The results are given in table 6 and 7.

Fig. 24: Chromatogram for method precision-1

Fig. 25: Chromatogram for method precision-2

Fig. 26: Chromatogram for method precision-3

Table 6: Intraday data for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
Conc (µg/ml) Area counts % assay as is Conc (µg/ml) Area counts % assay as is Conc (µg/ml) Area counts % assay as is
10.0

2789315

100.42

6.0

1871153

100.68

0.2

547450

100.25

2788941

100.68

 

1880820

100.54

 

541665

100.37

2764650

100.54

 

1892189

100.42

 

549553

100.48

2790843

100.28

 

1892461

100.37

 

546337

100.51

2789315

100.64

 

1871153

100.26

 

547450

100.68

2789421

100.37

1892189

100.15

545149

100.75

% RSD 1.66 0.67 0.49

Table 7: Interday data for diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate

Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
Conc (µg/ml) Area counts

% assay

as is

Conc (µg/ml) Area counts

% assay

as is

Conc (µg/ml) Area counts

% assay

as is

10.0

2764851

100.56

6.0

1841523

100.43

0.2

541282

100.38

2758945

100.37

 

1847456

100.68

 

541365

100.56

2768748

100.41

 

1848752

100.36

 

541878

100.37

2775358

100.52

 

1845896

100.38

 

541758

100.58

2787486

100.78

 

1847893

100.55

 

541785

100.41

2778952

100.54

 

1847895

100.47

 

541478

100.36

% RSD 0.84 0.73 0.68

Fig. 27: Chromatogram for method precision-4

Fig. 28: Chromatogram for method precision-5

Fig. 29: Chromatogram for method precision-6

Fig. 30: Chromatogram for intermediate precision-1

Fig. 31: Chromatogram for intermediate precision-2

Fig. 32: Chromatogram for intermediate precision-3

Fig. 33: Chromatogram for intermediate precision-4

LOD and LOQ

LOD and LOQ minimum concentration level at which the analyte can be reliably detected, quantified by using the standard formulas (3.3times σ/s and 10times σ/s for LOD and LOQ respectively) were found to be 0.1 and 0.2 µg/ml for diethylcarbamazine citrate, 0.06 and 0.12 µg/ml for guaiphenesin and 0.002 and 0.004 µg/ml for chlorpheniramine maleate. The low values of LOD and LOQ indicate the high sensitivity of method. The results are given in table 8 and 9.

Table 8: Results of LOQ

Diethylcarbamazinecitrate Guaiphenesin Chlorpheniramine maleate
Conc (µg/ml) s/n Conc (µg/ml) s/n Conc (µg/ml) s/n
0.2 17 0.12 15 0.004 18

Table 9: Results of LOD

Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
Conc (µg/ml) s/n Conc (µg/ml) s/n Conc (µg/ml) s/n
0.1 5 0.06 4 0.002 6

Fig. 36: Chromatogram for LOD

Fig. 37: Chromatogram for LOQ

Forced degradation

Stress degradation conditions such as acidic, basic, oxidative, reduction, thermal, hydrolysis and photolytic stresses were attempted as per ICH guidelines Q1A (R2).

Acid degradation

Acid degradation studies were carried out by weighing 27 mg of sample and transferred to a 10 ml volumetric flask, to this add 5 ml of diluent dissolve it and add 0.1 ml of 5N HCl. The mixture was refluxed at 70 °C for 1 hour. Then the solution was neutralized with 0.1 ml of 5N NaOH and diluted with the mobile phase up to the mark and mixed well. 0.1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the HPLC system and chromatograms were recorded.

Alkali degradation

Alkali degradation studies were carried out by weighing 27 mg of sample and transferred to a 10 ml volumetric flask, to this add 5 ml of diluent dissolve it and add 0.1 ml of 5N NaOH. The mixture was refluxed at 70 °C for 1 hour. Then the solution was neutralized with 0.1 ml of 5N HCl and diluted with the mobile phase up to the mark and mixed well. 0.1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the system and chromatograms were recorded.

Peroxide degradation

Peroxide degradation studies were carried out by weighing 27 mg of sample and transferred to a 10 ml volumetric flask, to this add 5 ml of diluent dissolve it and add 0.1 ml of 15% H2O2. The mixture was refluxed at 70 °C for 30 min.1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the system and chromatograms were recorded.

Reduction degradation

Reduction degradation studies were carried out by weighing 27 mg of sample and transferred to a 10 ml volumetric flask, to this add 5 ml of diluent dissolve it and add 0.1 ml of 10% sodium bisulphate. The mixture was refluxed at 70 °C for 1 hour. 0.1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the system and chromatograms were recorded.

Hydrolysis degradation:

Hydrolysis degradation studies were carried out by weighing 27 mg of sample and transferred to a 10 ml volumetric flask, to this add 5 ml of diluent and add 0.1 ml of water and sonicated to disperse, dissolve and refluxed at 70 °C for 30 min. 0.1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the system and chromatograms were recorded.

Thermal degradation

Thermal degradation studies were carried out by weighing 27 mg of sample and exposed to a temperature of 80 °C for 72 h in hot air oven. Then the sample was transferred to a 10 ml volumetric flask, dissolves in 5 ml of diluent and diluted with mobile phase up to the mark. 1 ml of the same solution was diluted to 10 ml with the diluent. 10 µl of the above solution was injected into the system and chromatograms were recorded.

Photolytic degradation

Photolytic degradation studies were carried out by weighing 27 mg of sample and exposed to 1.2 Million lux hours of light. Then the sample was transferred to a 10 ml volumetric flask, dissolved in 5 ml of diluent and diluted with the mobile phase up to the mark. 1 ml of the same solution was diluted to 10 ml with the diluent.10 µl of the above solution was injected into the system and chromatograms were recorded.

Table 10: Results of force degradation studies of diethylcarbamazine citrate

Stress condition Time % assay % degradation Purity angle Purity threshold
Acid degradation 1h 86.8 13.2 0.12 0.25
Alkaline degradation 1h 94.8 5.2 0.14 0.28
Oxidative degradation 30 min 96.5 3.5 0.18 0.24
Reduction degradation 1h 93.6 6.4 0.16 0.30
Thermal degradation 3h 87.2 12.8 0.14 0.28
Photolytic degradation 72h 84.9 15.1 0.11 0.25
Hydrolysis degradation 30 min 91.1 8.9 0.15 0.24

Table 11: Results of force degradation studies of guaiphenesin

Stress condition Time % assay % degradation Purity angle Purity threshold
Acid degradation 1h 85.4 14.6 0.10 0.27
Alkaline degradation 1h 93.6 6.4 0.17 0.32
Oxidative degradation 30 min 95.3 4.7 0.23 0.37
Reduction degradation 1h 92.7 7.3 0.24 0.36
Thermal degradation 3h 86.5 13.5 0.25 0.45
Photolytic degradation 72h 84.2 15.8 0.16 0.28
Hydrolysis degradation 30 min 91.8 8.2 0.18 0.39

Table 12: Results of force degradation studies of chlorpheniramine maleate

Stress condition Time % assay % degradation Purity angle Purity threshold
Acid degradation 1h 85.4 13.7 0.14 0.34
Alkaline degradation 1h 93.6 5.8 0.17 0.36
Oxidative degradation 30 min 95.3 4.2 0.25 0.39
Reduction Degradation 1h 92.7 7.5 0.19 0.42
Thermal degradation 3h 86.5 12.8 0.23 0.41
Photolytic Degradation 72h 84.2 16.4 0.18 0.51
Hydrolysis Degradation 30 min 91.8 9.4 0.15 0.43

Fig. 38: Chrom for acid degradation

Fig. 39: Chrom for alkali degradation

Fig. 40: Chrom for peroxide degradation

Fig. 41: Chrom for reduction degradation

Fig. 42: Chrom for thermal degradation

Fig. 43: Chrom for photolytic degradation

Fig. 44: Chrom for hydrolysis degradation

Robustness

The proposed method was found to be Robust as the % RSD was found to be less than 2%. Slight variations were done in the optimised method parameters like flow rate (±0.2%), organic content in mobile phase (±5%), pH (±0.2) and wavelength of detection (±5%).

Flow rate variation

This study was conducted to find the effect of variation in flow rate. Standard and check standard solutions were prepared as per test method and injected into HPLC system with a flow rate of 1.0 ml/min. System suitability parameters were evaluated and found to be within the specified limits as per test method and RT of the main peak was monitored.

Organic phase variation

This study was conducted to find the effect of variation in organic phase. Standard and check standard solutions were prepared as per the test method and injected into HPLC system with mobile phases of 0.1% triethylamine as a buffer along with orthophosphoric acid adjusted to PH 2.5: acetonitrile(50:50v/v) and wavelength of 210 nm. System suitability parameters are found to be within the specified limits and RT of the main peak was monitored for 50:50 v/v (mixed 0.1% triethylamine buffer).

PH variation

This study was conducted to find the variation in PH. Standard and check standard solutions were prepared as per test method and injected into HPLC system with different buffer PH. System suitability parameters were evaluated and found to be within the specified limits as per test method and RT of the main peak was monitored.

Wavelength variation

This study was conducted to find the effect of variation in wavelength. Standard and check standard solutions were prepared as per test method and injected into HPLC system with different buffer wavelengths. System suitability parameters were evaluated and found to be within the specified limits as per test method and RT of the main peak was monitored.

Table 13: Results for robustness

Parameter Diethylcarbamazine citrate Guaiphenesin Chlorpheniramine maleate
USP plate count USP tailing USP plate count USP tailing USP plate count USP tailing
Less flow rate (0.8 ml/min) 3200 0.86 4320 0.08 5896 0.14
High flow rate (1.2 ml/min) 3450 0.78 3548 0.12 4100 0.11
Less wavelength (205 nm) 3525 0.45 3896 0.45 4752 0.86
High Wavelength (215 nm) 4272 0.52 3868 0.53 5962 0.86
Less organic phase composition (-5%) 3984 0.68 3796 0.63 4635 0.86
High organic phase composition (+5%) 3582 0.67 3863 0.45 3785 0.86
Less pH variation (-0.2) 3985 0.73 3981 0.58 3868 0.86
High pH variation (+0.2) 4584 0.59 3789 0.67 4589 0.86

Fig. 45: Chromatogram for flow plus

Fig. 46: Chromatogram for flow minus

Fig. 47: Chromatogram for org plus

Fig. 48: Chromatogram for org minus

Fig. 49: Chromatogram for wave plus

Fig. 50: Chromatogram for wave minus

Fig. 51: Chromatogram for pH plus

Fig. 52: Chromatogram for pH minus

Table 14: Results of stability studies

Stability % assay % deviation
Initial 100.2 0.00
6 h 100.8 0.06
12 h 100.4 0.02
18 h 100.3 0.01
24 h 100.5 0.03

Solution stability

Sample solutions were analysed initially to 24 h at different intervals of time at room temperature and the results were recorded. The % deviation should not be more than 5.0%. The results are given in table 14.

Fig. 53: Chromatogram for stability initial

Fig. 54: Chromatogram for stability 6 h

Fig. 55: Chromatogram for stability 12h

Fig. 56: Chromatogram for stability 18 h

Fig. 57: Chromatogram for stability 24 h

CONCLUSION

Stability indicating RP-HPLC method was developed and validated for the simultaneous estimation of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate in pharmaceutical formulations as per ICH guidelines. The developed method was found to be accurate, precise and reliable with %RSD less than 2%. Therefore, the developed method is simple, accurate, precise and robust. The present method was found to be stability indicating as the degradation of drug substance was between 5-20%. Finally, this method can be used for better analysis of pharmaceutical formulations of diethylcarbamazine citrate, guaiphenesin and chlorpheniramine maleate drug.

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. http://en.wikipedia.org/wiki/Diethylcarbamazine; 2009. [Last accessed on 20 Jul 2017].

  2. WHO. Model List of Essential Medicines. World Health Organization; 2013.

  3. Indian pharmacopoeia, Ghaziabad, Indian Pharmacopoeia; 2007. p. 558-59, 1005-6.

  4. British Pharmacopoeia. London; British Pharmacopoeia Commission; 2009. p. 2861, 5094.

  5. Indian Pharmacopoeia. The Indian pharmacopoeia commission, Ghaziabad, Govt. of India. Ministry of health and Family Welfare. Vol. II; 2007. p. 919-20, 1029-30.

  6. Michael UA, Kenneth CO, Anthony AA. Spectrophotometric and thermodynamic studies of the charge-transfer interaction between diethylcarbamazine citrate and chloranilic acid. Chem Pharm Bull 1999;47:463-6.

  7. Lee AR, Hu TM. Determination of guaiphenesin in antitussive pharmaceutical preparations containing dextromethorphan by first–and second–derivative UV spectroscopy. J Pharm Biomed Anal 1994;12:747-52.

  8. Khalode KD, Waikar SB, Pasmane SP. A validated UV spectrophotometric method for the simultaneous estimation of Dextromethorphan hydrobromide and chlorpheniramine maleate. Int J Pharm Technol 2012;4:4690-9.

  9. Joshi RS, Pawar NS, Sawant RL. Validated spectrophotometric methods for simultaneous estimation of acetaminophen, chlorpheniramine maleate and caffeine in pue and tablate dosage form. Latt Am J Pharm 2010;29:1226-30.

  10. Mahesh RJ, Jeyaprakash MR, Madhuri K, Meyyanathan SN, Elango K. A sensitive RP-HPLC method for simultaneous estimation of diethylcarbamazine and levocetirizine in tablet formulation. Indian J Pharm Sci 2011;73:320–3.

  11. Hamide C, Tuncel O. Simultaneous high-performance liquid chromatographic determination of paracetamol, phenylephrine HCl, and chlorpheniramine maleate in pharmaceutical dosage forms. J Chromatogr Sci 2002;40:97-100.

  12. Suzen S, Akay C. Cevheroglu S. Formaco 1999;54:705‐9.

  13. Kulikov AU, Aleksey GV. LC determination of lercanidipine and its impurities using DryLab software and experimental design procedures. J Chromatogr 2008;67:5-6.

  14. Anand P, Varun R, Anagha D. Simultaneous analysis of intestinal permeability markers, caffeine, paracetamol and sulfasalazine by Reverse phase liquid chromatography: a tool for standardization of rat everted gut sac model. Asian J Pharm Clin Res 2010;3:204-7.

  15. Suraj S, Prasanna KP, Sagar KM, Sabuj S. HPLC method development for simultaneous estimation of hydrochlorothiazide and perindopril in tablet dosage form. Asian J Pharm Clin Res 2012;5:136-8.

  16. Code Q2A-text on validation of analytical procedure step-3 Consensus Guideline; 1994.

  17. ICH Harmonised Tripartite Guideline; 2015.

  18. Code Q2B-Validation of analytical procedure methodology step-4 consensus guideline, ICH Harmonised Tripartite Guideline; 1994.

  19. Prakash K, Sireesha KR. Liquid chromatographic method for simultaneous determination of lomefloxacin hydrochloride and dexamethasone sodium phosphate in eye drops. Asian J Pharm Clin Res 2012;5:79-82.



About this article

Title

STABILITY INDICATING RP-HPLC METHOD FOR THE ESTIMATION OF DIETHYLCARBAMAZINE CITRATE, GUAIPHENESIN AND CHLORPHENIRAMINE MALEATE

Keywords

Diethylcarbamazine citrate, Guaiphenesin and Chlorpheniramine maleate

DOI

10.22159/ijap.2018v10i1.22180

Date

06-01-2018

Additional Links

Manuscript Submission

Journal

International Journal of Applied Pharmaceutics
Vol 10, Issue 1 (Jan-Feb), 2018 Page: 124-135

Online ISSN

0975-7058

Authors & Affiliations

Podili Bhavani
Dept. of Chemistry, Bapatla Engineering College, Bapatla, Guntur (DT), AP., India
India

Seelam Mohan
Dept. of Chemistry, Bapatla Engineering College, Bapatla, Guntur (DT), AP., India
India

Kammela Prasada Rao
Dept. of Chemistry, Bapatla Engineering College, Bapatla, Guntur (DT), AP., India
India


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