A VALIDATED STABILITY INDICATING RP-UPLC METHOD FOR DETERMINATION OF ERYTHROMYCIN ESTOLATE IN PHARMACEUTICAL FORMULATION

A VALIDATED STABILITY INDICATING RP-UPLC METHOD FOR DETERMINATION OF ERYTHROMYCIN ESTOLATE IN PHARMACEUTICAL FORMULATION

Gonepally Surekha ^*, Narmeta Ramakrishna and Gorantla Nagamallika

Department of Pharmaceutical Chemistry, St. Mary’s College of Pharmacy, St. Francis Street, Hyderabad - 500025, Telangana, India.

ABSTRACT: A novel, sensitive and selective stability-representing RP-UPLC method was developed and validated for the quantitative determination of Erythromycin estolate in Erythromycin 250 mg capsules. The chromatographic separation was achieved on BEH C18; 50 × 2.1 mm; 1.7 µm column by using mobile phase containing a mixture of 0.002M di-potassium hydrogen phosphate and acetonitrile 53:47 v/v at a flow rate of 0.6 ml/min. The column temperature was maintained at 40 °C and detection was carried out at 210 nm. To ascertain the stability-signifying ability of the method, drug product was subjected to strain conditions of acid, base, oxidative, hydrolytic, thermal and photolytic degradation. The drug undergoes degradation at oxidative and thermal / humidity stress conditions. The resultant degrading peaks were well resolved from the drug peak. The drug was found to be stable in thermal and photolytic conditions. The proposed method was validated as per ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness and the method show excellent linearity and a correlation coefficient of more than 0.99. Therefore, the projected method can be employed for the determination of Erythromycin estolate in various pharmaceutical formulations during regular and quality-control analysis.

Erythromycin estolate, Stability studies, Capsules, RP-UPLC

INTRODUCTION: Erythromycin is a classic representative of the macrolide group of antibiotics and is produced by Streptomyces erythreus. It is extensively used in the treatment and prevention of diseases. Current indications for the drug includes respiratory infections, whooping cough, legionnaires disease and campylobacter enteritis. The side-effects are comparatively low ¹. Erythromycin is effective against penicillin - challenging Staphylococcus, Chlamydia and Mycoplasma bacteria.

Chemically Erythromycin estolate is (3R, 4S, 5S, 6R, 7R, 9R, 11R, 12R, 13S, 14R) -4-[(2, 6-Dideoxy -3- C-methyl -3 -O- methyl -α- L- ribo- hexo pyranosyl) Oxy] -14- ethyl -7, 12, 13-trihydroxy-3, 5, 7, 9, 11, 13- hexamethyl-6-[ [3, 4, 6-trideoxy- 3- (dimethylamino) -2- O- propionyl -β- D- Xylo- hexopyranosyl] Oxy] oxacyclo tetra decane-2, 10-Dione dodecyl sulfate (Erythromycin  A 2’’-propionate dodecyl sulfate).

Having a molecular formula of C₄₀H₇₁NO₁₄, C₁₂H₂₆O₄S and molecular weight is 1056 g/Mol and melting point is 135 - 140 ºC. Practically insoluble in water and dilute hydrochloric acid, freely soluble in ethanol (96 percent), soluble in acetone. The PKA value is 6.9 and stable at normal temperature and pressure ^{2, 3}. Erythromycin estolate is specified especially for those patients who are allergic or sensitive to sulfa drugs or Penicillin.

However, Erythromycin is degraded to inactive anhydrous form in acidic fluids. Several analogs of EM (Azithromycin and Clarithromycin) have been developed that have a chemical substitution at the location where internal dehydration is first initiated to prevent the degradation reaction from starting, resulting in slightly increased acidic stability and therapeutic efficacy.

Hence, ester type prodrugs are preferred such as stearate salts, which are poorly soluble in water and suitable for oral administration. Thus, the Erythromycin estolate Fig. 1 has been formulated both in liquids e.g. suspension and solid dosage forms like tablets and capsules. LC represents a tremendous growth that makes it the most popular method used in the pharmaceutical analysis. Literature review reveals numerous analytical methods for the determination of erythromycin and its analogs  either alone, or in combination with other antibacterial agent and its related substances in bulk, pharmaceutical formulations ^{4 - 16} and biological fluids ^{17 - 21}, these spectrophotometric, HPLC, LCMS / MS and FTIR techniques. The stability indicating method is a process that identifies the degradation products of analytes. A very few stability indicating LC methods were reported for ester forms of erythromycin in dosage forms and along with some alkaloids ^{22 - 27}.

FIG. 1: CHEMICAL STRUCTURE OF ERYTHROMYCINE ESTOLATE

So, the need was felt to develop an analytical technique for the estimation of Erythromycin estolate in a capsule formulation. In the present research, a very simple and rapid isocratic RP-UPLC method for the routine analysis of Erythromycin estolate in capsules was developed. The anticipated method was validated with respect to specificity, linearity, precision, accuracy, and robustness. In addition, strain testing of the drug was also conducted, as required by the International Conference on Harmonization (ICH, 2003) to support the suitability of the method ^{28, 29, 30}.

MATERIALS AND METHODS: All the reagents were of LC grade except stated or else Milli-Q-water was used throughout the research. Operational standard of Erythromycin estolate was procured from   M/S ADCOCK INGRAM, RD & I, Sabax Road, Aero ton, Johannesburg, 2013, South Africa. Dipotassium hydrogen phosphate and acetonitrile were procured from Merck, Mumbai.

Apparatus: The UPLC system consisted of high pressure pump, photodiode array detector and 10 μl capacity injector loops. The column used was BEH C18; 50 × 2.1 mm; 1.7 µm column. The output signal was monitored and processed using Empower software.

Chromatographic Conditions: BEH C18; 50 × 2.1 mm, 1.7 μm column was used for separation. Chromatographic separation was attained using a mobile phase comprising of 0.002M di-potassium hydrogen phosphate buffer and acetonitrile 53:47 v/v. The flow rate of the mobile phase was 0.6 ml/min with detection at 210 nm. The column temperature was kept at 40 ºC and the injection volume was 2 μl solution preparations.

Preparation of 0.002M of Di-Potassium Hydrogen Phosphate Buffer: Buffer solution was prepared by dissolving 0.348 gm of di-potassium hydrogen phosphate in DID water.

Preparation of Mobile Phase: 530 ml of 0.002M of di-potassium hydrogen phosphate was mixed with 470 ml of acetonitrile. The solution was degassed in an ultrasonic water bath for 5 min and filtered through 0.45 μm filter under vacuum.

Preparation of Stock Solution: Exactly weighed 400 mg of Erythromycin estolate running standard into a 50 ml volumetric flask; 35 ml of diluent was added and sonicate for 5 min to dissolve completely. Cool to room temperature; make up the volume with diluent and mix

Preparation of Standard Solution: Precisely weighed 80 mg of Erythromycin estolate operational standard into a 20 ml volumetric flask. approximately 15 ml of diluent was added and sonicate for 5 min to dissolve completely; cool to room temperature, make up to volume with diluent and mix.

Preparation of Sample Solution: Weighed and finely powdered not fewer than 20 capsules. An accurately weighed quantity of powder corresponding to about 250 mg of Erythromycin base from Erythromycin 250 mg capsule powder (about 440 mg) was transferred into 100 ml volumetric flask; approximately 75 ml of diluent was added and sonicate for 5 min to dissolve completely; cool to room temperature and makeup the volume with diluent and mix.

Diluent: 0.002M of di-potassium hydrogen phosphate buffer and acetonitrile in the ratio of 53:47 v/v was selected as diluents. Since the Erythromycin estolate is also soluble in acetonitrile.

Method Development: Any analytical method was not reported in the stability studies of Erythromycin estolate in a capsule formulation. Hence, it was noteworthy to commence the method development using Reverse Phase Liquid Chromatography as it is commonly used and C-18 columns are also available. Different columns were used with different mobile phases during the development of UPLC method suitable for the analysis of Erythromycin estolate in a capsule formulation. 0.002M of dipotassium hydrogen phosphate in water and the organic modifier acetonitrile was preferably chosen as appropriate mobile phase for ideal separation as no interference was found with the solvent. Several isocratic and gradient elution were tried to separate Erythromycin B and Erythromycin.

Finally, the mobile phase composition of 53:47% v/v of 0.002M of dipotassium hydrogen phosphate and acetonitrile was found to be most suitable for separation of Erythromycin B and Erythromycin with a resolution of greater than 2.0. The sample was injected with an injection volume of 2 µl and the injector port temperature was maintained at 40°C ± 2°C and the flow rate of 0.6 ml/min. The column BEH C18; 50 × 2.1 mm; 1.7 µm column was selected. The column was equilibrated by pumping the mobile phase through the column for at least 30 min prior to the injection of the drug solution. 2 μl of the standard, sample solutions were injected into the chromatography system and measure the area of the erythromycin estolate peak. The detection of the drug peak was monitored at 210 nm. The runtime was set at 12 min. Under these optimized chromatographic conditions, the retention time obtained from the drug was 2.69 min. A typical chromatogram showing the separation of the drug is given in Fig. 2.

Stress Degradation Study: To determine the developed analytical method was stability indicating, Erythromycin estolate standard solution was stressed under various conditions includes.

Oxidative Degradation: Erythromycin estolate solution was prepared in 3% hydrogen peroxide and kept in a mechanical shaker at at 50 °C for 1h to facilitate the oxidation of the drug.

Acid Hydrolysis: Erythromycin estolate solution was prepared in 0.01N hydrochloric acid and kept in a mechanical shaker at 50 °C for 15 min

Alkaline Hydrolysis: Erythromycin estolate solution was prepared in 0.01N sodium hydroxide and kept at room temperature for 1h.

Temperature Stress Studies: Erythromycin 250mg capsules were exposed to dry heat (105 °C) in a hot air oven for 2 h, 42 min. The drug solution was prepared and subjected to analysis

Photostability Studies: Erythromycin 250 mg capsules were exposed to light to reach greater than 1.2 million Lux hours. The drug solution was prepared and subjected to analysis

Thermal / Humidity Studies: Placebo, Erythromycin 250 mg capsules is subjected directly at 50 °C/ 75% Rh for 7 days.  The samples were analyzed.

Method Validation: The optimized method was validated as per International Conference on Harmonization (ICH) guidelines. The validated parameters were system suitability, specificity, and linearity, accuracy, precision, and robustness.

RESULTS AND DISCUSSION:

Method Optimization: A variety of chromatographic conditions were practiced to develop the stability-indicating LC method. The UPLC method was optimized through the assessment of different buffers and organic solvents. The use of  0.002M of dipotassium hydrogen phosphate and acetonitrile in a ratio of 53:47% v/v on BEH C18; 50 × 2.1 mm; 1.7 µm column was found to furnish sharp, well-defined peak with good resolution and low retention time of about 2.69 min at a flow rate 0.6 ml/min. The peak was monitored at 210 nm.

System Suitability: After equilibration of the column with mobile phase, five replicate injections of 2.0 μl of standard solution through an auto-sampler injector were injected and the chromatograms were recorded. The system suitability parameters were measured and results are revealed in Table 1. The results have been confirmed with respect to % RSD, resolution and USP tailing factor. The % RSD was less than 1. The results will make sure transferability of the method and raise the consistency of the results obtained.

TABLE 1: SYSTEM SUITABILITY RESULTS FROM STANDARD SOLUTION

FIG. 2: TYPICAL CHROMATOGRAM OF ERYTHROMYCIN ESTOLATE FROM STANDARD SOLUTION

Degradation Studies: The results of the degradation studies state the specificity of the method as there was no interference from the sample, placebo and degrading peaks and also reveal that the method was selective and stability-indicating. The % assay of the drug was calculated after exposure of Erythromycin estolate solutions to various stress conditions. The drug undergoes degradation of oxidative and thermal / humidity stress conditions. The degrading peaks were well resolved from the drug peak. The drug was stable in thermal and photolytic conditions. The results are shown in Table 2.

TABLE 2: STRESS STUDY RESULTS OF ERYTHROMYCIN ESTOLATE

Method Validation: The designed technique was validated according to ICH guidelines for specificity, linearity, accuracy, and precision, and robustness. Specificity was carried out in which no interference of the excipients was observed at retention time of the analytical peak. A calibration curve was constructed by plotting concentration vs plot area. It showed that there was a good linear relationship in the concentration range of 40% to 160% with > 0.999 as the value of correlation coefficient. The accuracy of the method was studied by analyzing the drug solutions at 80%, 100% and 120% concentration level. The mean percentage recovery was found to be 101.7% for precision the sample solution at working concentration was analyzed in replicate as per the method. The percentage relative standard deviation was found to be less than 1%. Robustness of the method shows no significant change in system suitability parameters and mean % assay for modifying chromatographic conditions from the original method.

Specificity: The Specificity of the method was established by injecting a blank, (diluent) Placebo and sample preparations into the chromatograph. No interference was observed.

This indicates that the solvent does not interfere with drug peak and shows a good resolution.

Linearity: The peak area responses of all solutions over concentration levels ranging from 40% to 160% of target concentration were measured in triplicate. A linear correlation was obtained between peak area and concentration of Erythromycin estolate for LC method. The linearity was validated by the value of correlation coefficients of the regression (r = 0.9999). The curves are shown in Fig. 3.

FIG. 3: LINEARITY GRAPH FOR ERYTHROMYCIN ESTOLATE FROM LINEAR CURVE STANDARD SOLUTIONS

Accuracy: The accuracy of the method was determined by recovery experiments and was performed in triplicate by standard addition method at 80%, 100% and 120% of test concentration and analysis precision was expressed as % RSD. A known amount of measuring analyte was added to placebo preparations and was   subjected   to   the proposed UPLC method. Results of recovery studies are shown in Table 3. The mean percentage recovery was 101.7% for Erythromycin estolate and % RSD was found to be less than 1.0%. The slope of Erythromycin estolate was 1.02 from 10% to 150% of accuracy levels and the confidence level 95% of accuracy of the method was 0.32. The results are shown in Table 3.

TABLE 3: ACCURACY RESULTS OF ERYTHROMYCIN ESTOLATE

Precision: Method repeatability (intra - day precision) was evaluated by assaying six injections of sample preparation of same the batch. The mean % assay was 104.78% and was within the acceptance criteria. The % RSD was found to be 0.41%. The results are shown in Table 4. The difference in the assay of Erythromycin estolate in Erythromycin 250 mg capsules between the preparations are less than 2.0% of % RSD. The results are given away in Table 3.

TABLE 4: PRECISION RESULTS OF ERYTHROMYCIN ESTOLATE

Robustness: Robustness of the method was established by determining the assay of  a sample under small but deliberately modified chromatographic conditions specified under the method like flow rate, column temperature, pH of buffer or buffer strength in % v/v, mobile phase composition and wavelength on lower and higher side of the actual values.

TABLE 5: ROBUSTNESS STUDY RESULTS FOR SYSTEM SUITABILITY STUDIES

TABLE 6: ROBUSTNESS STUDY RESULTS FOR % ASSAY

The drug concentration was analyzed under these changed experimental conditions. There was no significant change in the retention time and assay of the drug when the flow rate and composition of the mobile phase were changed. The results are illustrated in Table 5.

CONCLUSION: In view of the fact that the Erythromycin peak was not found in the existing HPLC method, will pose less of a problem using the proposed UPLC method because it is extracted in the solvent. The proposed UPLC method for the assay of Erythromycin estolate in Erythromycin 250 mg capsules was accurate, precise, robust, specific, and stability-indicating thus demonstrating a quality by design approach to method development.

Due to its shorter run time, use of an economical and readily available mobile phase, optimized UV detection the method has been proven to be a significant importance compared with the reported methods and is compliant with current regulatory requirements. Owing to shorter run time this method enable rapid quantification of many samples in routine and quality-control analysis of various formulations containing Erythromycin estolate.

ACKNOWLEDGEMENT: This work was supported by St. Mary`s College of Pharmacy and St. Paul`s College of Pharmacy. The authors would like to thank for providing research facilities.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Carter BL, Woodhead JC, Cole JK and Mlavetz G: Gastrointestinal side effects with Erythromycin preparations. Drug Intelligence and Clinical Pharmacy 1987; 21: 734-736.
2. Erythromycin estolate, Ph. Eur. monograph 0674.
3. Erythromycin estolate, USP32-NF27, 2959.
4. Rattanapoltaveechai R, Vongkom W and Worapot: Simple and rapid spectrophotometric method for the analysis of Erythromycin in pharmaceutical dosage forms. Journal of Food and Drug Analysis 2007; 15(1): 10-14.
5. Finete VLM, Arissawa M and AucélioI RQ: Fluorimetric method for the determination of Erythromycin using a photochemical derivatization approach. J Braz Chem Soc 2008; 19(7): 1418-1422.
6. Tsuji K, Kane MP: Improved High-Pressure Liquid Chromatographic method for the analysis of Erythromycin in solid dosage forms. Journal of Pharmaceutical Sciences 1982; 71(10): 1160-1164.
7. Mostafavi A, Abedi G and Jamshidi: Quantitative analysis of Erythromycin by Reversed-Phase Liquid Chromato-graphy using column-switching. Journal of Pharma-ceutical and Biomedical Analysis 1991; 9(7): 547-55.
8. Hwang YH, Lim JH, Park BK and Yun HI: Simultaneous Determination of various Macrolides by Liquid Chromato-graphy / Mass Spectrometry. Journal of Veterinary Science 2002; 3(2): 103-108.
9. Lahane SB, Deokate UA and Ahire SK: Available analytical method for Macrolide Antibiotic. Int J Pharm Sci Rev Res 2014; 26(2), 44: 256-261.
10. Ali M, Sherazi STH and Mahesar SA: Quantification of Erythromycin in pharmaceutical formulation by transmission Fourier Transforms Infrared Spectroscopy. Arabian Journal of Chemistry. 2014; 7(6): 1104-1109.
11. Sharmin N, Shanta NS and Bachar SC: Spectro-photometric analysis of Azithromycin and its pharma-ceutical dosage forms: Comparison between spectro-photometry and HPLC. J Pharm Sci 2013; 12(2): 171-179.
12. Gharia T, Kobarfard F and Mortazavia SA: 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. Iranian Journal of Pharmaceutical Research 2013; 12 (S): 57-63.
13. Tzouganaki Z and Koupparis M: Development and validation of an HPLC method for the determination of the Macrolide antibiotic Clarithromycin using Evaporative Light Scattering Detector in raw materials and pharmaceutical formulations mediterranean. Journal of Chemistry 2017; 6(4): 133-141.
14. Sonia L, Hassib T, Farag AE and Elkad EF: Liquid chromatography and spectrophotometric methods for the determination of Erythromycin stearate and Trimethoprim in tablets. Bulletin of Faculty of Pharmacy, Cairo University 2011; 49(2): 81-89.
15. Murali D and Rambabu C: Stability-indicating HPLC method for the simultaneous estimation of Erythromycin and Sulfafurazole in bulk and oral suspension. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2016; (2): 1788-1798.
16. Wardrop J, Ficker D, Franklin S and Gorski RJ: Determination of Erythromycin and related substances in enteric-coated tablet formulations by Reversed-Phase Liquid Chromatography. J Pharm Sci 2000; 89(9): 1097-1105.
17. Stubbs C, Haigh JM and Kanfer: Determination of Erythromycin in serum and urine by High-Performance Liquid Chromatography with Ultraviolet detection. J Ph Sciences 1985; 74(10): 1126-1128.
18. Stubbs C and Kanfer I: High-Performance Liquid Chromatography of Erythromycin propionyl ester and Erythromycin base in biological fluids. J Chromatography 1988; 427(1): 93-101.
19. Jangid AG, Tale RH and Vaidya VV and Robu A: Simple and rapid validated method for estimation of Erythromycin ethylsuccinate in human plasma by Liquid Chromato-graphy - tandem Mass Spectrometry and its Application to Clinical Study IJPFA 2011; 2(1): 1-7.
20. Ahmed MU, Islam MS, Sultana TA, Mostofa AGM, Sayeed MSB, Nahar Z and Hasnat A: Quantitative determination of Azithromycin in human plasma by Liquid Chromatography-Mass Spectrometry and its Application in Pharmackokinetic Study. J Pharm Sci 2012; 11(1): 55-63.
21. Alvi SN, Dgither SA and Hammami MM: Rapid determination of Clarithromycin in human plasma by LCMS/MS Assay. Pharm Anal Chem 2016; 2(1): 1-5.
22. Stubbs C and Kanfer I: A stability-indicating High-Performance Liquid Chromatographic assay of Erythromycin estolate in pharmaceutical dosage forms. International Jour of Pharmaceutics 1990; 63 (2): 113-119.
23. Kamareiab F, Movagharia F, Ghaffariac A, Bozchalooid IS, Zamania A and Jabbaria A: Development of a stability-indicating High Performance Liquid Chromatography method for assay of Erythromycin ethyl succinate in powder for oral suspension dosage form. Arabian Journal of Chemistry 2014; 7(6): 1079-1085.
24. Wahba MEK: Liquid Chromatographic determination of Roxithromycin: Application to stability studies. Journal of Chromatographic Science 2013; 51(1): 1:44-52.
25. Krishna VR, Krishna KBR and Babu BH: Development and validation of stability-indicating Liquid Chromato-graphic method for the simultaneous estimation of Azithromycin, Fluconazole and Ornidazole in the combined dosage form. Indian Journal of Pharmaceutical Science and Research 2014; 4(3): 176-186.
26. Okaru AO, Abuga KO, Kamau FN, Ndwigah SN and Lachenmeier DW: A robust Liquid Chromatographic method for confirmation of drug stability of Azithromycin in bulk samples. Tablets and Suspensions Pharmaceutics 2017; 9(11): 1-11.
27. Janakiraman K: Compatibility and stability studies of Erythromycin estolate and piperine mixture. Journal of Pharmacy Research 2011; 4(10): 3405-3408.
28. RD-QC-046 – Validation of Analytical Methods; RD-QC-050 – Development of Analytical Methods
29. USP < 1225 > Validation of Compendia Procedures.
30. ICH guidelines Q2 (R1) – Validation of Analytical Procedures: Text and Methodology.
    

    ---

    How to cite this article:

    Surekha G, Ramakrishna N and Nagamallika G: A validated stability indicating RP-UPLC method for determination of Erythromycin estolate in pharmaceutical formulation. Int J Pharm Sci & Res 2018; 9(12): 5287-93. doi: 10.13040/IJPSR.0975-8232.9(12).5287-93.

    ---

    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.

    ---