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
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2018v10i1.22180
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
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