Introduction
Erythromycin, a macrolide antibiotic produced by Streptomyces erythraea, functions as an antibacterial agent effective against various bacterial infections. Chemically, Erythromycin is characterized as 6-{[(2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy}-14-ethyl-7,12,13-trihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dione (C37H67NO13, molecular weight 733.92 g/mol). Its mechanism of action involves inhibition of bacterial protein synthesis by binding to the 50S ribosomal subunit, thereby inhibiting peptidyl transferase activity and interfering with amino acid translocation during protein translation and assembly. Isotretinoin, chemically designated as 3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoic acid (C20H28O2, molecular weight 300.43 g/mol), exerts its therapeutic effects through modulation of the cell cycle, cell division, proliferation, and apoptosis. These actions collectively reduce sebum production, prevent pore blockage, and inhibit the proliferation of acne-causing microorganisms. Unlike other retinoids, Isotretinoin exhibits minimal affinity for retinol-binding proteins (RBPs) and retinoic acid nuclear receptors (RARs). It promotes apoptosis in sebocytes, leading to decreased sebum production, and reduces comedone formation by decreasing hyperkeratinization through an unknown mechanism. Although not directly bactericidal, Isotretinoin creates a less favorable microenvironment for acne-causing bacteria by reducing the size of sebum ducts.
Literature Review and Research Gap
A comprehensive literature survey revealed that no previously reported HPLC method exists for the simultaneous estimation of Isotretinoin and Erythromycin in pharmaceutical formulations. Previous studies have focused on individual analysis or alternative techniques for these compounds. For instance, Rathore et al. (2010) developed an HPTLC method for simultaneous estimation, while Roy and Chakrabarty (2013) investigated RP-HPLC methods for tretinoin degradation products. This research gap necessitates the development of a novel, rapid, accurate, and precise RP-HPLC method for simultaneous quantification of these analytes in tablet dosage forms.
Materials and Methods
Instrumentation
The chromatographic analysis was performed using a Waters 2695 HPLC system equipped with an auto sampler and UV-Vis detector. An Inertsil ODS C18 column (4.6 × 250 mm, 5 μm particle size) was employed for separation. A 20 μL Rheodyne injector port was used for sample injection, and data analysis was conducted using Empower 2 software. pH measurements were performed using a Thermo Scientific pH meter.
Chemicals and Reagents
Working standards of Isotretinoin and Erythromycin were provided as gift samples by Dr. Reddy’s Laboratories (Hyderabad, India). Marketed tablet formulations containing Isotretinoin (10 mg) and Erythromycin (20 mg) were procured from local pharmacies. HPLC-grade water and acetonitrile were purchased from E. Merck (India) Ltd. (Mumbai, India), while methanol and dipotassium hydrogen phosphate of analytical reagent grade were obtained from Rankem Chemicals Ltd. (Mumbai, India).
Chromatographic Conditions
The optimized mobile phase consisted of phosphate buffer (0.05 M, pH 4.6) and acetonitrile in a 30:70 (v/v) ratio. The pH of the buffer was carefully controlled between 2-8, as values below 2 could lead to siloxane linkage dissociation, while values above 8 might cause silica dissolution. The mobile phase was filtered through a 0.45 μm membrane filter and sonicated prior to use. The flow rate was maintained at 1.0 mL/min, with an injection volume of 10 μL. The column temperature was set at 30°C, and the detection wavelength was 260 nm. The run time was programmed for 7 minutes, with the column equilibrated by pumping the mobile phase for at least 30 minutes before sample injection.
Method Validation
The developed method was comprehensively validated according to ICH guidelines, evaluating parameters including linearity, accuracy, precision, specificity, limit of detection (LOD), limit of quantification (LOQ), and solution stability.
Results and Discussion
Optimization of Chromatographic Conditions
The detection wavelength was optimized by scanning individual and mixed standard solutions (10 μg/mL) in the UV range of 200-400 nm. The overlay spectrum revealed maximum absorbance at 260 nm for both compounds, which was selected as the detection wavelength. The optimized chromatogram demonstrated well-resolved peaks for Erythromycin (retention time: 2.399 min) and Isotretinoin (retention time: 3.907 min), with theoretical plate counts of 5105 and 3788, and tailing factors of 1.3 and 1.4, respectively (Table 1).
Table 1: Optimized Chromatogram Parameters
| PEAK NAME | RETENTION TIME (MIN) | AREA | HEIGHT | USP PLATE COUNT | TAILING FACTOR | RESOLUTION |
|---|---|---|---|---|---|---|
| Erythromycin | 2.399 | 946124 | 155429 | 5105 | 1.3 | 8.1 |
| Isotretinoin | 3.907 | 111541 | 13239 | 3788 | 1.4 | 1.46 |
Linearity
The linearity of the method was evaluated by analyzing five concentration levels for each analyte. For Erythromycin, concentrations ranged from 10-50 μg/mL, while Isotretinoin concentrations ranged from 10-50 μg/mL. Excellent correlation coefficients (r² = 0.999) were obtained for both analytes, indicating strong linear relationships between concentration and peak area (Tables 2 and 3).
Table 2: Linearity Results for Isotretinoin
| S.NO | CONCENTRATION (ΜG/ML) | RETENTION TIME (MIN) | AREA | HEIGHT |
|---|---|---|---|---|
| 1 | 10 | 4.304 | 1164173 | 74586 |
| 2 | 20 | 4.323 | 1342555 | 87689 |
| 3 | 33 | 4.214 | 1556824 | 101999 |
| 4 | 40 | 4.524 | 1774565 | 117084 |
| 5 | 50 | 4.218 | 1956421 | 129409 |
Table 3: Linearity Results for Erythromycin
| S.NO | CONCENTRATION (ΜG/ML) | RETENTION TIME (MIN) | AREA | HEIGHT |
|---|---|---|---|---|
| 1 | 10 | 2.309 | 1810101 | 145867 |
| 2 | 20 | 2.322 | 2044873 | 176895 |
| 3 | 30 | 2.324 | 2367122 | 206674 |
| 4 | 40 | 2.336 | 2602248 | 228475 |
| 5 | 50 | 2.340 | 2869772 | 259345 |
Precision
Precision was evaluated through repeatability and intermediate precision studies. Repeatability was assessed by analyzing six replicate injections of standard solutions, yielding %RSD values of 0.31% for Erythromycin and 0.38% for Isotretinoin (Tables 4 and 5). Intermediate precision was determined at three different concentration levels (50%, 100%, and 150%), with mean recoveries of 100.1% for Erythromycin and 100.4% for Isotretinoin, and %RSD values of 0.12% and 0.15%, respectively (Tables 6 and 7).
Table 4: Repeatability Results for Erythromycin
| S.NO | RETENTION TIME (MIN) | AREA | HEIGHT |
|---|---|---|---|
| 1 | 2.320 | 2265419 | 196958 |
| 2 | 2.341 | 2204588 | 197584 |
| 3 | 2.356 | 2247569 | 195874 |
| 4 | 2.344 | 2258741 | 194583 |
| 5 | 2.325 | 2258967 | 194587 |
| Mean | 2.337 | 2255501 | 195917 |
| Standard Deviation | 0.014 | 6545.5 | 1176.6 |
| %RSD | 0.61 | 0.31 | 0.60 |
Table 5: Repeatability Results for Isotretinoin
| S.NO | RETENTION TIME (MIN) | AREA | HEIGHT |
|---|---|---|---|
| 1 | 4.302 | 1401475 | 100274 |
| 2 | 4.305 | 1401345 | 100078 |
| 3 | 4.325 | 1402415 | 98425 |
| 4 | 4.315 | 1404775 | 98165 |
| 5 | 4.312 | 1408614 | 98154 |
| Mean | 4.312 | 1403725 | 99019 |
| Standard Deviation | 0.009 | 5882.5 | 935.4 |
| %RSD | 0.21 | 0.38 | 0.94 |
Table 6: Precision Results for Isotretinoin
| % CONCENTRATION | AREA | AMOUNT ADDED (MG) | AMOUNT FOUND (MG) | % RECOVERY |
|---|---|---|---|---|
| 50% | 2332744 | 5 | 5.10 | 101.8 |
| 100% | 3132697 | 10 | 9.99 | 99.9 |
| 150% | 3918997 | 15 | 14.9 | 99.1 |
| Mean Recovery | 100.4% |
Table 7: Precision Results for Erythromycin
| % CONCENTRATION | AREA | AMOUNT ADDED (MG) | AMOUNT FOUND (MG) | % RECOVERY |
|---|---|---|---|---|
| 50% | 3538675 | 5 | 5.0 | 101.3 |
| 100% | 4735088 | 10 | 9.94 | 99.4 |
| 150% | 5911798 | 15 | 14.8 | 99.2 |
| Mean Recovery | 100.1% |
Accuracy
The accuracy of the method was determined by recovery studies at three concentration levels (50%, 100%, and 150%). The mean recoveries were 100.1% for Erythromycin and 100.4% for Isotretinoin, with %RSD values below 2%, confirming the accuracy of the method.Specificity
System suitability parameters were evaluated by varying the flow rate (0.8, 1.0, and 1.2 mL/min). The method demonstrated robustness, with theoretical plate counts ranging from 883.3 to 1234.1 for Erythromycin and 1748.4 to 1948.1 for Isotretinoin, and tailing factors between 1.2 and 1.6 (Tables 8 and 9).
Table 8: System Suitability for Erythromycin
| FLOW RATE (ML/MIN) | USP PLATE COUNT | TAILING FACTOR |
|---|---|---|
| 0.8 | 883.3 | 1.55 |
| 1.0 | 1234.1 | 1.2 |
| 1.2 | 969.1 | 1.6 |
Table 9: System Suitability for Isotretinoin
| FLOW RATE (ML/MIN) | USP PLATE COUNT | TAILING FACTOR |
|---|---|---|
| 0.8 | 1748.4 | 1.23 |
| 1.0 | 1548.3 | 1.3 |
| 1.2 | 1948.1 | 1.2 |
Limit of Detection (LOD) and Limit of Quantification (LOQ)
LOD and LOQ values were calculated based on signal-to-noise ratios of 3:1 and 10:1, respectively. For Erythromycin, LOD was 2.94 μg/mL and LOQ was 9.87 μg/mL. For Isotretinoin, LOD was 3.03 μg/mL and LOQ was 10.1 μg/mL Assay of Marketed Formulation
The assay of a commercial tablet formulation containing Erythromycin (46 mg) and Isotretinoin (66 mg) equivalent to 100 mg was performed. Six replicate injections of sample and standard solutions were analyzed, yielding assay values of 100.6% for Erythromycin and 101.3% for Isotretinoin (Figure 7).
Conclusion
This research successfully developed and validated a novel, rapid, specific, accurate, and precise RP-HPLC method for the simultaneous estimation of Erythromycin and Isotretinoin in tablet dosage forms. The method utilized an Inertsil ODS C18 column with a mobile phase of phosphate buffer (pH 4.6) and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL/min. Detection was performed at 260 nm with retention times of 2.395 min and 3.906 min for Erythromycin and Isotretinoin, respectively. The method demonstrated excellent linearity (r² = 0.999), accuracy (mean recovery 100.1-100.4%), precision (%RSD ≤ 0.38%), and sensitivity (LOD 2.94-3.03 μg/mL, LOQ 9.87-10.1 μg/mL). The robustness of the method was confirmed through system suitability studies. Given its compliance with ICH validation guidelines, this method is suitable for routine quality control analysis of pharmaceutical formulations containing these active pharmaceutical ingredients.
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