ABSTRACT:
A simple, reversed phase high performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of Azithromycin and Dexamethasone in their combination formulation. The method was developed by using GRACE ODS C18, 250 x 4.6 mm, 5 µm , L1 packing, maintained at an ambient temperature at 230 nm wavelength maxima. The mobile phase used in this method was a mixture of Methanol and 0.0335M Phosphate Buffer (pH 7.5) in the ratio of (80:20 v/v), flow rate of mobile phase was set 1.2 ml/min. Azithromycin and Dexamethasone peaks were eluted at 3.832 min and 4.798 min respectively with good resolution. The above method was validated in terms of System suitability, linearity, accuracy, precision, Limit of Detection (LOD), Limit of Quantification (LOQ) in accordance with ICH guidelines. The results which are obtained from the method validation were within the acceptable limit. In conclusion a method has been developed for the simultaneous estimation of Azithromycin and Dexamethasone in combined dosage form. The developed method was simple, rapid, precise, economical and suitable for routine quality control analysis and quantification of these drugs in combined dosage form
KEYWORDS: Azithromycin (Azasite) and Dexamethasone sodium phosphate (Decadron), Analytical method development, validation, Ayzeecon-D, RP-HPLC.
INTRODUCTION
Azithromycin is a bacteriostatic drug acts by inhibiting protein synthesis. It binds to 50S ribosomal subunits of the 70S bacterial ribosomes, and therefore inhibits RNA-dependent protein synthesis in bacterial cells. Azithromycin interferes with transpeptidation and translocation thus there is inhibition of protein synthesis and hence inhibition of cell growth. It is used to treat certain eye infections.It is a macrolide antibiotic that works by stopping the growth of bacteria. Its effects may be bacteriostatic or bactericidal depending of the organism and the drug concentration. Its long half life, which enables once daily dosing and shorter administration durations, is a property distinct from other macrolides. A semisynthetic antibiotic belonging to the macrolide subgroup of azalides, is used to treat STDs due to chlamydia and gonorrhea, community-acquired pneumonia, pelvic inflammatory disease, pediatric otitis media and pharyngitis, and Mycobacterium avium complex (MAC) in patients with advanced HIV disease. Similar in structure to erythromycin. azithromycin reaches higher intracellular concentrations than erythromycin, increasing its efficacy and duration of action[1, 4].
Fig.1.Structure of AzithromycinDexamethasone is a steroid that prevents the release of substances in the body that causes inflammation. It is a glucocorticoid agonist. Unbound dexamethasone crosses cell membranes and binds with high affinity to specific cytoplasmic glucocorticoid receptors. This complex binds to DNA elements (glucocorticoid response elements) which results in a modification of transcription and hence protein synthesis in order to achieve inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, suppression of humoral immune responses, and reduction in edema or scar tissue. The antiinflammatory actions of dexamethasone are thought to involve phospholipase A2 inhibitory proteins, lipocortins, which control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes. Dexamethasone eye drops are used to treat short term inflammatory eye conditions .They contain a corticosteriod which helps to relieve inflammation, redness and irritation[2, 4].
Fig.2. Structure of DexamethasoneBoth the drugs are marketed as combined dose Eye drops formulation in the ratio of 10:1 mg Ayzeecon-D. Literature survey revealed that there are some methods reported for the simultaneous estimation of these drugs, some methods for estimation of individual drugs[5, 6], UV-spectrophotometry[7], and RP-HPLC[8-11] . HPLC method is more sensitive compared to UV. Therefore, there is a challenge to develop RP-HPLC method for the simultaneous estimation of Azithromycin and Dexamethasone Well and up dated developed and validated analytical methods are quite essential for quality control of the drugs available in the market. So the proposed method provides fast separation with effective resolution, good peak shape, use of lesser sample volumes and buffer volumes, providing cost effective. The proposed established method was validated with respect to specificity, linearity, precision, accuracy, Limit of detection and Limit of quantitation subject to ICH Q2B guidelines 1996 and Q2 (R1) guidelines 2005[12-13].
MATERIALS AND METHODS
Methanol (HPLC grade) was obtained from Merck specialties private limited, Mumbai, India. Acetonitrile (HPLC grade) was obtained from Merck specialties private limited, Mumbai, India and Potassium di hydrogen Phosphate. Pure drugs of Azithromycin and Dexamethasone were obtained as gift samples. Ayzeecon-D Eye drops manufactured by Indiana ophthalmics purchased from pharmacy is used for the analysis. The label claim states that this formulation contains 10mg of Azithromycin and 1mg of Dexamethasone of each eye drops.
Instrumentation and chromatographic condition
HPLC method development and validation were done on SHIMADZU (Japan) liquid chromatograph equipped with LC-10AD pump, SPD-10A UV/Vis detector and Rheodyne 7725i injection with a 20 µL loop. For instrument control, data acquisition and processing, the chromatographic system was interfaced to empower N-2000 solutions software. Other instruments included are Contech Electronic balance CAS-44, Citizen-Digital ultrasonic cleaner.
The column used for chromatographic separations was reverse phase GRACE ODS C18, 250 x 4.6 mm, 5 µm column The analytical wave length was set as 214 nm and samples of 20 µL were injected.
METHOD DEVELOPMENT
Preparation of mobile phase
The chromatographic separations were accomplished using mobile phase consisting of Methanol:Phosphate buffer (80:20 % v/v), filtered through 0.45 µm filter using vacuum pump and deaerated in fast clean ultrasonic cleaner. Mobile phase was pumped in isocratic mode at a flow rate of 1 ml/min at room temperature.
Preparation of standard solutions
Stock solution was prepared by dissolving 10mg of Azithromycin and 10mg of Dexamethasone in 10 ml of methanol. This was 1000μg/ml solution.
Fig.3. Chromatogram of standard solution (10µg/ml Azithromycin and 10µg/ml Dexamethasone )
Preparation of working standard solutions
From above solution 0.1 ml was taken into 10ml of volumetric flask. The volume was made upto the mark with methanol. The concentration of each drug in solution was 10μg/ml..Preparation of sample solutions for assay Ayzeecon-D each containing 10 mg of azithromycin and 1 mg of dexamethasone solution taken and extracted in to methanol, and filtered. 1 ml from filtrate was taken in to 10ml volumetric flask , volume was made up to mark with diluents,. The 0.1 ml from above filtrate pipetted in 10 ml of volumetric flask, volume was made up to mark with mobile phase. It was sonicated for 10minutes and injected.
METHOD VALIDATION
The developed method was validated for linearity, Range, Precision, Accuracy, LOD and LOQ as per the ICH guidelines.
RESULTS AND DISCUSSION
Method Development
For the RP-HPLC, chromatographic conditions were optimized to get best resolution and peak shape. The selection of mobile phase was based on peak parameters like symmetry, theoretical plates and capacity factor. Symmetrical peaks with good separation (retention time for Azithromycin and Dexamethasone were found to be 3.832 min and 4.798 min ) were obtained with reverse phase GRACE ODS C18, 250 x 4.6 mm, 5 µm column. The mobile phase containing Methanol:Phosphate buffer (75:25 % v/v) was used at a flow rate of 1.0 ml/min. The optimum wavelength for detection and quantification was at 214 nm, at which good detector response was obtained for both. The results are given in Table 1.
Method Validation:
The proposed method was validated as per International Conference on Harmonization (ICH) guidelines.
Linearity and Range
Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 0.1-12μg/ml for Azithromycin, 0.1-8μg/ml for Dexamethasone. Peak areas were plotted versus respective concentrations and linear regression analysis was performed on the resultant curves. Correlation coefficients were found to be 0.9931 and 0.9949 for Azithromycin, and Dexamethasone respectively. Calibration graph of Azithromycin, and Dexamethasone by RP-HPLC was shown in Fig.4 and Fig.5.The results are given in Table 1.
Linearity of Azithromycin S.NO Concentration (ppm) Peak area
1 0.1 2545
2 0.2 6542
3 0.4 12568
4 0.8 30254
5 1 48569
6 2 88456
7 4 171759
8 8 336584
9 10 365894
10 12 401524
Fig. 4. Calibration graph of Azithromycin by RP-HPLC
Linearity of DexamethasoneS.NO Concentration (ppm) Peak area
1 0.1 13563
2 0.2 13584
3 0.4 13695
4 0.8 13999
5 1 14562
6 2 16587
7 4 19546
8 8 27550
Fig. 5. Calibration graph of Dexamethasone by RP-HPLC
Precision
The precision of the analytical method was studied by multiple sampling of the homogenous sample. The precision was done at two levels (intraday and inter day). Intraday precision was done by analyzing the intermediate concentration of each drugs (4 µg/ml Azithromycin and 4 µg/ml Dexamethasone ) for six times. Interday precision was measured over three consecutive days for the same drug concentrations for six times. The %RSD values were found to calculated for each of them and the low RSD values indicate that the method is precise. The results were found within in the acceptable criteria of %RSD NMT 2.The results are given in Table 1.
S.NO AzithromycinDexamethasone1 16587 171759
2 16496 170679
3 16507 170896
4 16579 16978
5 16580 170796
6 16591 171268
Average :
SDV:
% RSD: 16556.67
43.10298
0.260 170846
694..0844
0.404
Accuracy
Recovery studies were carried out by applying the method to drug sample to which known amount of standard Azithromycin and Dexamethasone corresponding to 80, 100 and 120 % of label claim had been added. At each level of the amount three determinations were performed. The % recovery found within the acceptable criteria (98-102%). The results are given in Table 1.
ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
AZITHROMYCIN
50% 16587
16496
16507 14562
14463
14369 31118
30988
31897 102 %
AVERAGE :
SDV:
%RSD: 16530
49.669
0.300 14464.67
96.51079
0.666 31334.56
491.4525
1.568 ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
AZITHROMYCIN
100% 18987
18890
18798 14562
14463
14369 33356
33298
33348 99.8%
AVERAGE :
SDV:
%RSD: 18891.67
94.511
0.500 14464.67
96.51079
0.666 33334
31.432
0.094 ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
AZITHROMYCIN
150% 20989
20908
20896 14562
14463
14369 35395
35401
34989
99.3%
AVERAGE :
SDV:
%RSD: 20931
50.589
0.241 14464.67
96.51079
0.666 35261
236.155
0.669 ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
DEXAMETHASONE
50% 171759
170679
170896 365894
360986
359989 533400
532906
531962 99.6%
AVERAGE :
SDV:
%RSD: 171111.3
571.2963
0.338 362289.7
3160.998
0.872 532756
730.6408
0.137 ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
DEXAMETHASONE
100% 201897
201818
202212 365894
360986
359989 564199
564098
565175 100.3%
AVERAGE :
SDV:
%RSD: 201909
97.555
0.048 362289.7
3160.998
0.872 564490.7
594.797
0.105 ACCURACY LEVEL STANDARD TEST SPICKED % RECOVERY
DEXAMETHASONE
150% 269866
265468
268658 365894
360986
359989 630287
631254
630989 100.2 %
AVERAGE :
SDV:
%RSD: 267997
2272.215
0.847 362289.7
3160.998
0.872 630843
499.686
0.079 Sensitivity
LOD and LOQ decide about the sensitivity of the method. LOD is the lowest detectable concentration of the analyte by the method while LOQ is the minimum quantifiable concentration. LOD and LOQ were calculated by standard calibration curves. The results are given in Table 1.
System suitability studies
System suitability parameters like number of theoretical plates (N) (NLT 2000), peak asymmetry factor (As), tailing factor (NMT 2), resolution (NLT 2) were studied. The results are given in the table..
S. no Theoretical plates Tailing factor Resolution Asymmetry Retention time
Azithromycin 2658 1.4 2.6 1.2 3.83
Dexamethasone 2965 0.8 2.9 1.5 4.79
Table 1: Summary of validation parameters
Parameters AzithromycinDexamethasoneLinearity range (μg/ml)
Correlation coefficient
Slope
Intercept
LOD (μg/ml)
LOQ (μg/ml)
Recovery %
50%
Recovery % ± SD
100%
Recovery % ± SD
120%
Recovery % ± SD
Precision
Intraday (n=6) (%RSD)
SD
Inter day (n=6) (%RSD)
SD
Theoretical plates
Tailing factor
Resolution 0.1-12
0.9931
36088
7610
1.60
4.84
102% 99.6%
99.8% 100.3%
99.3% 100.2%
0.653
100.3% ±0.08
0.293
108.26
0.272
100.58
2488.7
0.876
5.502 0.1-8
0.9949
1786
12951
0.88
2.67
0.388
562.10
0.375
543.26
3027.1
1.778
* RSD and SD denote relative standard deviation and standard deviation
Analysis of marketed formulation
The proposed procedures were successfully applied for the simultaneous estimation of Azithromycin and Dexamethasone in the formulation and the drug contents in each sample were calculated by comparision with the appropriate standard solution of the drug. The results obtained were in agreement with label claim. The results of analysis are given in (Table 2) (Fig. 6).
Table. 2. Analysis of marketed formulation
Drug Amount labelled(mg) Amount % Assay %RSD
found (mg)
Azithromycin
Dexamethasone10
0.1
2.498 102 0.66
25.01 99.6 0.87
*Mean of three observations
Fig.6. Chromatogram of formulation (2.5 µg/ml Azithromycin and 25 µg/ml Dexamethasone )
CONCLUSION
RP-HPLC methods enable the quantitation of Azithromycin and Dexamethasone in eye drop with good accuracy and precision, either in laboratory prepared samples or in pharmaceutical dosage forms. The good recoveries were obtained in all cases as well as the reliable agreement with the reported procedure proved that the proposed methods could be applied efficiently for determination of Azithromycin and Dexamethasone in in eye drop with satisfactory precision. This method is considered simple, reliable, economical providing satisfactory accuracy, precision with lower limits of detection and quantification more sensitive. More over the shorter duration of analysis for Azithromycin and Dexamethasone makes the reported method suitable for routine analysis in mixed dosage form.
Hence we believe that the future analytical methods will use this method for the estimation of Ayzeecon-D in combined dosage form with no modification.
RECOMMENDED FUTURE RESEARCH:
Future research is recommended in any other pharmaceutical dosage form as this method is optimized to elute the analytes.
REFERENCES
http://www.drugbank.ca/drugs/DB00207
http://www.drugbank.ca/drugs/ DBSALT000843
Tripathi KD. Essential of Medical Pharmacology, 5th Ed., Jaypee Brothers Medical Publishers(P) Ltd., 2004
H.P. Rang and Dale et al., Pharmacology , Edition 2006,
S.N.Waghule et al, Method development and validation of HPLC method for determination of azithromycin.. Der Pharma Chemica 5(4):166-172 · January 2013
A. A. Heda, et al, Development and Validation of RP-HPLC Method for Simultaneous Determination of Granisetron and Dexamethasone 2011 Nov-Dec; 73(6): 696–699.
Tayebeh Ghari et al, Development of a Simple RP-HPLC-UV Method for Determination of Azithromycin in Bulk and Pharmaceutical Dosage forms as an Alternative to the USP Method 2013.
Ranjith singh, HPLC Method Development and Validation- an Overview, J Pharm. Educ. Res.4 (2013) 26-33.
Ankita J. Patel et al, First Order Derivative Method Development And Validation For Simultaneous Estimation Of Azithromycin And Dexamethasone From Eye Drops. ejpmr, 2017,4(06), 449-454
Ravindra N Kamble et al RP-HPLC Analytical Method Development and Validation for Azithromycin and Levofloxacin in Tablet Dosage Form. International Journal of Pharmaceutical Sciences Review and Research 31(2 27):162-165 · March 2015
Irin Dewan et al, Development And Validation Of A New Hplc Method For The Estimation Of Azithromycin In Bulk And Tablet Dosage Form International Journal Of Pharmaceutical Sciences And Research
International Conference on Harmonization Q2B, Validation of Analytical procedure: Text and Methodology, 1996.
International Conference on Harmonization, Q2 (R1) Validation of Analytical procedure: Text and Methodology, 2005.