APPLICATION OF DOE AND STATISTICAL ANALYSIS FOR DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR CHLROHEXIDINE GLUCONATE AND CETRIMIDE IN ITS BULK AND PHARMACEUTICAL DOSAGE FORMS
HTML Full TextAPPLICATION OF DOE AND STATISTICAL ANALYSIS FOR DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR CHLROHEXIDINE GLUCONATE AND CETRIMIDE IN ITS BULK AND PHARMACEUTICAL DOSAGE FORMS
S. J. Rajput^{*} and M. A. Sathe
Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara - 390020, Gujarat, India.
ABSTRACT: The present work includes development of analytical method for simultaneous estimation of Chlorhexidine gluconate and Cetrimide by RP-HPLC method. The chromatographic conditions were successfully optimised for the separation of Chlorhexidine gluconate and Cetrimide by using Hypersil BDS C18 column (4.6 × 150 mm) 5µ, flow rate of 0.8 ml/min, mobile phase ratio of (30:55:15 v/v/v) ACN: methanol: phosphate buffer (KH_{2}PO_{4} and K_{2}HPO_{4}) phosphate pH 3 (pH was adjusted with orthophosphoric acid) and detection wavelength used was 210 nm. The retention times were found to be 3.10 min and 3.9 min for Cetrimide and Chlorhexidine gluconate respectively. The % purity of Chlorhexidine gluconate and Cetrimide was found to be 99.92% and 100.45% respectively. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1). The linearity study of Chlorhexidine gluconate and Cetrimide was found in concentration range of 3 µg - 18 µg and 30 µg - 180 µg. DOE was applied in validation for checking robustness of the developed method using Box-Behnken design and also normal distribution of data was verified by Anderson-Darling normality test. The method developed was found to be specific, selective, and robust and can be applied for routine analysis of marketed formulation in laboratory premises.
Keywords: |
Chlorhexidine gluconate, Cetrimide, RP-HPLC method, DOE, Anderson - Darling normality test
INTRODUCTION: Chlorhexidine gluconate is an antiseptic and antibacterial drug with molecular weight of 897.75716 g/mol and pKa value of 10.3.^{1 }It was approved by USFDA on 19 December 2003. A RP - HPLC analytical method along with its impurity Para chloroaniline is available in literature for its estimation ^{2}. Cetrimide (Cetrimonium bromide) is a local infective agent with molecular weight 364.44 g/mol ^{3}. It was approved by USFDA on 30 June 2006. Due to absence of any significant chromophoric group in its structure till date no analytical method is available for its estimation.
Also no analytical method is available for estimation of Chlorhexidine gluconate and Cetrimide in combination which is found in various marketed formulations like Savlon antiseptic solution ^{4} and Aceptic Lotion.
FIG. 1: STRUCTURE OF CHLORHEXIDINE GLUCONATE ^{1}
FIG. 2: STRUCTURE OF CETRIMIDE ^{3}
The main aim of the study was to develop a RP- HPLC method for the determination of Chlorhexidine gluconate and Cetrimide bulk and its formulation as no such method for is available in literature till date. The robustness of the developed method is then validated using DOE approach using Box-Behnken design of Response surface methodology and also normal distribution of the data is checked by application of Anderson-Darling normality test for statistical analysis.
MATERIALS AND METHODS:
Apparatus and Software: The liquid chromato-graphic system was of Waters, Ahmedabad and consisting of following components a gradient pump, PDA detector, a manual injection facility with 20 μl fixed loop. The chromatographic analysis was performed using Empower 3 software on a Hypersil BDS C18 column (250 × 4.6 mm, 5 µm particle size).
Materials: Chlorhexidine gluconate and Cetrimide was kindly supplied as a gift sample by Mil Laboratories Pvt. Ltd., Vadodara.
Reagents and Chemicals: Methanol and Acetonitrile used were of HPLC grade and were purchased from Fisher Scientific Pvt. Ltd., Double distilled water was prepared at the laboratory premises. All other reagents and chemicals used were of analytical grade.
Preparation of Mobile Phase Buffer: 20 mM phosphate buffer was prepared by dissolving 0.272 g of potassium dihydrogen orthophosphate in sufficient water to produce 100 ml. The pH was adjusted to 3 using orthophosphoric acid. The buffer was filtered through 0.22 μ membrane filter, stored at ambient temperature.
Preparation of Mobile Phase: (Phosphate Buffer (pH 3): ACN: Methanol (15:30:55) v/v/v): The appropriate volumes of phosphate buffer, acetonitrile and methanol were transferred into a reagent bottle, mixed thoroughly, sonicated for 10 min and filtered through 0.22 µm membrane filter and used as mobile phase.
Preparation of Stock Solutions (1000 ppm): 10 mg each of CH and CET were weighed accurately and transferred into a 10 ml volumetric flask containing 1 ml Acetonitrile. DDW was added up to the mark to produce a stock solution containing 1000 μl/ml of CH and CET respectively.
Preparation of Working Standards and Calibration Curve Solutions: For preparation of working standard solution, 2.5 ml each of CH and CET transferred into a 25 ml volumetric flask containing 2.5 ml Acetonitrile. DDW was added up to the mark to produce a stock solution containing 100 μl/ml of CH and CET respectively. Considering the ratio of CH and CET in commercial formulation to be 1:10 appropriate aliquots of CH and CET working standard solutions were taken in different 6 ml volumetric flasks each and diluted up to the mark with mobile phase to obtain final concentrations of 3 - 18 μl/ml and 30 - 180 μl/ml respectively.
Method Development: For development of liquid chromatographic method, various parameters were considered like 1) Selection of appropriate λ_{max} for detection. For this parameter, whole UV range was scanned by PDA detector ^{2}) Mobile phase selection was based on one factor at a time method (on trial and error basis). The pH of buffer was selected based on pKa values of drugs ^{3}) For selection of diluents of final samples, various solvents were tried ^{4}) The flow rate was also selected based on one factor at a time method (on trial and error basis).
Applicability of the Method: The developed HPLC method was applied for analysis of its formulation available in market. “Savlon antiseptic solution” manufactured by ITC was procured from local pharmacy. 0.5 ml of the sample formulation was withdrawn in a 50 ml volumetric flask and diluted up to the mark using Acetonitrile and DDW to produce a clear solution. The resulting solution was again diluted by withdrawing 1 ml and making up to 10 ml to give the final solution for analysis. The final solution was analyzed and chromatogram was recorded. Concentrations of both analytes were then calculated from the calibration graph. Six replicate samples were used for analysis.
Method Validation: ^{5 }
Linearity and Range: The proposed RP-HPLC method showed good linearity in the concentration range of 3 - 18 µg/ml for CH and 30 - 180 µg/ml for CET.
Precision: Inter-day and intra-day precision for the method were measured in terms of % RSD. The experiment was repeated 3 times in a day (Intraday precision) and the average % RSD values of the results were calculated. Similarly the experiment was repeated on 3 different days (Inter day precision) and the average % RSD value for absorbance of CH and CET were calculated. The low value of SD obtained confirms the precision of the method.
LOD and LOQ: Calibration curve was repeated for 9 times and the standard deviation (SD) of the intercepts was calculated. Then LOD and LOQ were measured as follows. LOD = 3.3 * SD/slope of calibration curve, LOQ = 10 * SD/slope of calibration curve where SD = Standard deviation of intercepts
Accuracy: Accuracy of the method was confirmed by recovery study from marketed formulation at 3 level of standard addition (80%, 100%, and 120%) of label claim. Recovery greater than 98% with low SD justified the accuracy of the method
Robustness: The robustness of the method was determined by using DOE approach. In it Box-Behnken design of response surface methodology was utilized. The factors considered for development of design were flow rate, λ_{max} and mobile phase ratio whereas the responses considered for the study were retention time, theoretical plates and resolution between the peaks of CH and CET. The plots and the statistical analysis implicate the robustness of the method.
Statistical Analysis: ^{6, 11 }In order to verify whether a statistical procedure follows a normal distribution or not, 3 common types of Normality tests are performed namely 1) Anderson-Darling Test 2) Ryan-Joiner Test 3) Kolmogorov-Smirnov Test. As Anderson-Darling Test is especially effective in detecting departure from normality in low and high values of distribution it is used in our study to verify the normality of data distribution.
RESULTS AND DISCUSSION: The present investigation is aimed to develop a new method for the simultaneous estimation of Chlorhexidine gluconate and Cetrimide by RP-HPLC method, its validation by application of DOE approach and verifying the normality of data by statistical Anderson Darling normality test. Literature reveals that there is no analytical method reported for the simultaneous estimation Chlorhexidine gluconate and Cetrimide by RP-HPLC method. Hence, it was felt that, there is a need of new analytical method development for the simultaneous estimation of Chlorhexidine gluconate and Cetrimide in pharmaceutical dosage form.
FIG. 3: SELECTION OF WAVELENGTH MAXIMA FOR COMBINATION DOSAGE FORM = 210 nm
FIG. 4: OPTIMIZED PEAK AND CALIBRATION CURVE FOR CETRIMIDE AND CHLORHEXIDINE GLUCONATE
For optimization of chromatographic conditions for combined dosage form, various combinations of solvents, dilutions solvents, pH, l_{max} were tried and finally chromatographic conditions selected for analysis of sample included detection wavelength of 210 nm, mobile phase ratio of (30:55:15 v/v/v) ACN: methanol: phosphate buffer (KH_{2}PO_{4} and K_{2}HPO_{4}) phosphate pH 3 (pH was adjusted with ortho-phosphoric acid) using Hypersil BDS C18 column (4.6 × 150 mm) 5µ, flow rate of 0.8 ml/min and diluents for sample preparation was selected to be mobile phase itself.
FIG. 5: CALIBRATION CURVE OF CHLORHEXIDINE GLUCONATE
FIG. 6: CALIBRATION CURVE OF CETRIMIDE
TABLE 1: SUMMARY OF VALIDATION PARAMETERS OF RP-HPLC METHOD
Parameter | Chlorhexidine gluconate | Cetrimide |
Analytical wavelength (nm) | 210nm | 210nm |
Retention time (min) | 3.8 | 3.2 |
Linearity range (µg/ml) | 3-18 | 30-180 |
Regression equation | Y = 75,152.4000x – 35884.5333 | Y= 9132.4200x+452776.0667 |
Correlation coefficient | 0.9991 | 0.9993 |
Intraday precision (%RSD) | 0.046% | 0.893% |
Inter day precision (%RSD) | 1.06% | 0.568% |
LOD (µg/ml) | 0.256 | 1.475 |
LOQ (µg/ml) | 0.768 | 4.435 |
Accuracy (% Mean Recovery) | 98-102% | 98- 102 % |
TABLE 2: SYSTEM SUITABILITY TEST (SST) PARAMETERS
Parameter | Data obtained for Cetrimide | Parameter | Data obtained for Chlorhexidine gluconate |
Retention time (min) ± SD | 3.2 ± 0.0784 | Retention time (min) ± SD T | 3.8 ± 0.1893 |
Theoretical plate ± SD | 8100 ± 228.492 | Theoretical plate ± SD | 2103 ± 103.380 |
Tailing factor ± SD | 1.23 ± 0.29 | Tailing factor ± SD | 1.19 ± 0.11 |
Resolution | 3.2 ± 0.264 |
(*Data obtained from 6 replicate Injections)
TABLE 3: APPLICABILITY OF METHOD: FORMULATION ANALYSIS (SAVLON SOLUTION: 3% w/v OF CH AND 30% w/v IN 100 ml SOLUTION, MFG. BY: ITC)
Actual conc. (mg in 10 ml) | Amount of CH found (mg in 10 ml) | %
Label claim |
SD | %
RSD |
Actual conc. (mg in 10 ml) | Amount of CET found (mg in 10 ml) | %
Label claim |
SD | %
RSD |
3 | 3.018 | 100.600% | 0.6751 | 0.6744% | 30 | 30.002 | 100.006% | 0.8318 | 0.8288% |
3 | 3.029 | 100.966% | 30 | 29.929 | 99.763% | ||||
3 | 3.012 | 100.400% | 30 | 30.129 | 100.430% | ||||
3 | 2.978 | 99.266% | 30 | 29.982 | 99.940% | ||||
3 | 2.982 | 99.400% | 30 | 30.600 | 102.000% | ||||
3 | 3.001 | 100.033% | 30 | 30.010 | 100.033% |
TABLE 4: APPLICATION OF DOE FOR ROBUSTNESS OF METHOD: BBD OF RSM ^{7, 9, 10}
Factors | Responses |
Flow rate | RT of CET |
Wavelength | RT of CH |
pH | TP of CET |
TP of CH | |
Resolution |
TABLE 5: EXPERIMENTAL RUNS
Std. | Run | Block | Factor 1 A: Flow rate ml/min | Factor 2B: Wavelength nm | Factor 3
C: pH unit |
Response 1 Rt of CET min | Response 2 Rt of CH min | Response 3 TP of CET | Response 4 TP of CH | Resolution |
7 | 1 | Block 1 | 0.7 | 210 | 3.2 | 3.21 | 3.9 | 2104 | 8102 | 3.25 |
9 | 2 | Block 1 | 0.8 | 207 | 208 | 3.2 | 3.85 | 2202 | 8001 | 3.14 |
11 | 3 | Block 1 | 0.8 | 207 | 3.2 | 3.2 | 3.8 | 2196 | 8110 | 3.09 |
6 | 4 | Block 1 | 0.9 | 210 | 2.8 | 3.19 | 3.81 | 2099 | 8053 | 3.34 |
3 | 5 | Block 1 | 0.7 | 213 | 3 | 3.2 | 3.86 | 2058 | 8201 | 3.01 |
8 | 6 | Block 1 | 0.9 | 210 | 3.2 | 3.3 | 3.79 | 2150 | 8103 | 3.16 |
4 | 7 | Block 1 | 0.9 | 213 | 3 | 3.1 | 3.8 | 2100 | 8194 | 3.12 |
5 | 8 | Block 1 | 0.7 | 210 | 2.8 | 3.18 | 3.8 | 2083 | 8120 | 3.11 |
12 | 9 | Block 1 | 0.8 | 213 | 3.2 | 3.2 | 3.82 | 2209 | 8102 | 3.06 |
10 | 10 | Block 1 | 0.8 | 213 | 2.8 | 3.19 | 3.81 | 2100 | 8013 | 3.30 |
2 | 11 | Block 1 | 0.9 | 207 | 3 | 3.21 | 3.82 | 2085 | 8038 | 3.28 |
1 | 12 | Block 1 | 0.7 | 207 | 3 | 3.2 | 3.8 | 2108 | 8024 | 3.24 |
DOE PLOTS:
FIG. 7: 3D CONTOUR PLOT
FIG. 8: NORMAL PLOT OF RESIDUAL
FIG. 9: 3D CONTOUR PLOT
FIG. 10: NORMAL PLOT OF RESIDUAL
FIG. 11: 3D CONTOUR PLOT
FIG. 12: NORMAL PLOT OF RESIDUAL
FIG. 13: 3D CONTOUR PLOT
FIG. 14: NORMAL PLOT OF RESIDUAL
FIG. 15: 3D CONTOUR PLOT
FIG. 16: NORMAL PLOT OF RESIDUA
TABLE 6: MODEL SUMMARY STATISTICS
Statistical parameters | R1 | R2 | R3 | R4 | R5 |
SD | 0.034 | 0.071 | 50.86 | 65.47 | 0.12 |
Mean | 3.2 | 3.83 | 2131.92 | 8088.42 | 1.18 |
CV% | 1.06 | 1.86 | 2.39 | 0.81 | 9.79 |
R-Squared | 0.0000 | 0.0000 | 0.0000 | 0.000 | 0.0000 |
Adjusted R^{2} | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 |
Predicted R^{2} | -0.1901 | -0.1901 | -0.1901 | -0.1901 | -0.1901 |
F value | 1.15 | 5.06 | 2586.99 | 4285.72 | 0.013 |
p-value | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
PRESS | 0.015 | 0.066 | 33866 | 5603.97 | 0.18 |
TABLE 7: ANDERSON DARLING NORMALITY TEST FOR CHECKING NORMAL DISTRIBUTION OF DATA FOR CHLORHEXIDINE GLUCONATE
Data | Sorted | Count | F1i | 1-F1i | F2i | Si | N Plt Line | N Plt Line |
363678 | 363678 | 1 | 0.09323358 | 0.90676642 | 0.0922127 | -4.7563052 | 377662.5 | 0.1 |
545997 | 545997 | 2 | 0.2102753 | 0.7897247 | 0.202243 | -9.4728691 | 603319.8 | 0.26 |
728291 | 728291 | 3 | 0.38594605 | 0.61405395 | 0.399137 | -9.3525411 | 759408.7 | 0.42 |
921163 | 921163 | 4 | 0.60086299 | 0.39913701 | 0.614054 | -6.9794258 | 902180 | 0.58 |
1125552 | 1125552 | 5 | 0.79775701 | 0.20224299 | 0.7897247 | -4.158199 | 1058269 | 0.74 |
1300085 | 1300085 | 6 | 0.90778734 | 0.09221266 | 0.9067664 | -2.1407708 | 1283926 | 0.9 |
TABLE 8: TEST STATISTICS
0.1434 | AD test statistic |
0.17023 | AD* test statistic |
0.932945 | P-value |
Inference: p- value greater that alpha value of 0.05 signifies normal distribution of data
TABLE 9: ANDERSON DARLING NORMALITY TEST FOR CHECKING NORMAL DISTRIBUTION OF DATA FOR CETRIMIDE
Data | Sorted | Count | F1i | 1-F1i | F2i | Si | N Plt Line | N Plt Line |
730687 | 730687 | 1 | 0.09206744 | 0.90793256 | 0.0860661 | -4.8378735 | 754570.3 | 0.1 |
1011844 | 1011844 | 2 | 0.21775591 | 0.78224409 | 0.2121202 | -9.2249475 | 1081808 | 0.26 |
1269729 | 1269729 | 3 | 0.39094947 | 0.60905053 | 0.4128188 | -9.1196179 | 1308160 | 0.42 |
1524638 | 1524638 | 4 | 0.58718123 | 0.41281877 | 0.6090505 | -7.1979306 | 1515200 | 0.58 |
1821408 | 1821408 | 5 | 0.78787976 | 0.21212024 | 0.7822441 | -4.3559842 | 1741553 | 0.74 |
2111775 | 2111775 | 6 | 0.91393389 | 0.08606611 | 0.9079326 | -2.0524044 | 2068790 | 0.9 |
FIG. 17: ANDERSON DARLING NORMALITY PLOT
FIG. 18: ANDERSON DARLING NORMALITY PLOT
TABLE 10: TEST STATISTICS
0.1315 | AD test statistic |
0.156108 | AD* test statistic |
0.954926 | P-value |
Inference: p- value greater that alpha value of 0.05 signifies normal distribution of data
CONCLUSION: A simple RP-HPLC method was developed for the combined dosage form of Chlorhexidine gluconate and Cetrimide. The retention times were found to be 3.10 min and 3.9 min for Cetrimide and Chlorhexidine gluconate respectively. The % purity of Chlorhexidine gluconate and Cetrimide was found to be 99.92% and 100.45%. It was validated as per ICH guidelines and robustness study was done using DOE approach.
The only significant factor affecting the robustness of method was pH of mobile phase having impact on response of Retention time of Chlorhexidine gluconate. All other parameters were robust. Normal distribution of data was inferred by application of Anderson darling normality test.
ACKNOWLEDGEMENT: The authors are thankful to MIL Laboratories Pvt. Ltd., Vadodara, for kindly providing the gift samples of API Chlorhexidine gluconate and Cetrimide.
COMPETING INTERESTS: We hereby declare that the work submitted in this manuscript has not been published or under consideration in any other journal.
CONFLICT OF INTEREST: Nil
REFERENCES:
- https://pubchem.ncbi.nlm.nih.gov/compound/29089/Chlorhexidinegluconate.
- Havlikova L, Matysova L, Novakova R and Hajkova P: HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment. Journal of Pharmaceutical and Biomedical analysis 2007; 3(43): 1169-1173.
- https://pubchem.ncbi.nlm.nih.gov/compound/Cetrimonium(15.06.2016).
- https://www.drugs.com/uk/savlon-antiseptic-liquid-spc-8506.html(30/06/2016).
- ICH Validation of Analytical procedures: Text and Methodology Q2 (R1) International conference on Harmonization 2005.
- KeyaRani D and Rahmatullah I:A Brief Review of Tests for Normality. American Journal of Theoretical and Applied Statistics 2016; 5(1): 5-12.
- Gyorgy S, Bruno H, Marco G and Carlos A: Experimental design for the optimization and robustness testing of a liquid chromatography tandem mass spectrometry method for the trace analysis of the potentially genotoxic 1, 3-diisopropylurea. Drug Test Anal 2014; 6(9):898-908.
- Asghar G and Saleh Z: Tests for Statistical Analysis: A Guide for Non-Statisticians. Int J Endocrinol Metab 2012; 10(2): 486-489.
- Danaher M, O'Keeffe M and Glennon JD: Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up. Analyst 2000; 125(10): 1741-1744.
- Isabela D, Costa C and Gerson AP: Robustness evaluation of the chromatographic method for the quantitation of lumefantrine using Youden’s test. Brazilian Journal of Pharmaceutical Sciences 2009; 45(2).
- Scholz F and Stephens W: K-sample Anderson–Darling Tests.Journal of the American Statistical Association 1987; 82(399): 918-924.
- Flavia A, Masquio F, Marcos AC, Herida R and Nunes S: Analytical Methods for the Determination of Chlorhexidine: A Review. Critical Reviews in Analytical Chemistry 2010; 40(2): 89-101.
- Liljana B, Sehmedin S, Mirjana P, Aneta D, Liljana U and Rumenka P: Development and validation of RP-HPLC assay of chlorhexidine in gingival crevicular fluid. Arh. farm 2014; 64: 69-82.
- Manranjan VC, Yadav DS, Jogia HA and Chauhan PL: Design of Experiment (DOE) Utilization to Develop a Simple and Robust Reversed-Phase HPLC Technique for Related Substances’ Estimation of Omeprazole Formulations. Sci Pharm 2013; 81(4): 1043-1056.
- Christian JR, Patel K and Gandhi TR: Validation and experimental design assisted robustness testing of RPLC method for the simultaneous analysis of Brinzolamide and Brimonidine tartrate in an ophthalmic dosage form. Indian J Pharm Sci 2016; 78(5): 631-640.
- Gyorgy S, Bruno H, Marco G and Carlos A: Experimental design for the optimization and robustness testing of a liquid chromatography tandem mass spectrometry method for the trace analysis of the potentially genotoxic 1, 3-diisopropylurea. Drug Testing and Analysis 2014; 6(9): 898-908.
How to cite this article:
Rajput SJ and Sathe MA: Application of doe and statistical analysis for development and validation of analytical method for chlrohexidine gluconate and cetrimide in its bulk and pharmaceutical dosage forms. Int J Pharm Sci Res 2018; 9(7): 2800-06. doi: 10.13040/IJPSR.0975-8232.9(7).2800-06.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
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2800-2806
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English
IJPSR
S. J. Rajput * and M. A. Sathe
Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India.
sjrajput@rediffmail.com
28 October, 2017
19 December, 2017
25 December, 2017
10.13040/IJPSR.0975-8232.9(7).2800-06
01 July, 2018