Stress degradation studies and development of stability-indicating TLC-densitometry method for determination of prednisolone acetate and chloramphenicol in their individual and combined pharmaceutical formulations
© Musharraf et al; 2011
Received: 5 November 2011
Accepted: 22 January 2012
Published: 22 January 2012
A rapid and reproducible stability indicating TLC method was developed for the determination of prednisolone acetate and chloramphenicol in presence of their degraded products. Uniform degradation conditions were maintained by refluxing sixteen reaction mixtures for two hours at 80°C using parallel synthesizer including acidic, alkaline and neutral hydrolysis, oxidation and wet heating degradation. Oxidation at room temperature, photochemical and dry heating degradation studies were also carried out. Separation was done on TLC glass plates, pre-coated with silica gel 60F-254 using chloroform: methanol (14:1 v/v). Spots at Rf 0.21 ± 0.02 and Rf 0.41 ± 0.03 were recognized as chloramphenicol and prednisolone acetate, respectively. Quantitative analysis was done through densitometric measurements at multiwavelength (243 nm, λmax of prednisolone acetate and 278 nm, λmax of chloramphenicol), simultaneously. The developed method was optimized and validated as per ICH guidelines. Method was found linear over the concentration range of 200-6000 ng/spot with the correlation coefficient (r2 ± S.D.) of 0.9976 ± 3.5 and 0.9920 ± 2.5 for prednisolone acetate and chloramphenicol, respectively. The developed TLC method can be applied for routine analysis of prednisolone acetate and chloramphenicol in presence of their degraded products in their individual and combined pharmaceutical formulations.
Prednisolone acetate (1), is a corticosteroid, used in polychemotherapy of cancer and also as an immunosuppressive to treat allergic disorders and hypersensitivity reactions [1, 2]. Chloramphenicol (2), an antibiotic, possesses broad spectrum antibacterial activity and is used for the treatment of rickettsial and chlamydial diseases, gram + ve and gram -ve bacterial infections and topically for superficial conjunctivial infections . A number of corticosteroid and antibiotic combinations are frequently used as antibacterial agents to cure infections particularly associated with the eye. These combinations are available in different formulations including eye ointment, eye drops and ophthalmic suspensions. Ophthalmic preparations of prednisolone acetate along with chloramphenicol are widely practiced for the treatment of superficial eye infections. However, this combination is not official with British Pharmacopoeia or US Pharmacopoeia. Many analytical methods have been reported for the determination of prednisolone acetate in cosmetics, Chinese medicinal preparations, human serum, urine and pharmaceutical preparations [4–6]. HPLC and UPLC methods are also reported for the estimation of chloramphenicol in aquatic products, pharmaceutical preparations, cosmetics comedo cream, blood, gastric contents, urine, tissues and cerebrospinal fluid [7–11].
Simultaneous determination of prednisolone, chloramphenicol and a degraded product, 2- amino-1-(4-nitrophenyl)propane-1,3-diol in ophthalmic solutions through HPLC is also reported . Few HPTLC methods have also been reported for the determination of chloramphenicol in combination with benzocaine and 2-amino-1-(4-nitrophenyl) propane-1,3-diol (ANPD), and for different corticosteroids including hydrocortisone, hydrocortisone acetate, prednisolone, betamethasone-17-valerate, prednisolone sodium phosphate, dexamethasone sodium phosphate and betamethasone sodium phosphate . No report for the simultaneous determination of prednisolone acetate and chloramphenicol in the presence of their degraded products through TLC-densitometry has been found so far.
Intensive consideration and attention is being paid to the development of TLC-densitometry stability-indicating assay as it is fast, reliable and accurate and involves simultaneous analysis of many samples by using small quantity of mobile phase, thus minimizing analysis time and cost per analysis. Stress testing provides evidence that how the quality of a drug substance varies with time under the influence of various environmental factors (temperature, light, humidity, etc.) and helps to establish shelf life and recommended storage conditions for the drug .
Taking ICH guidelines Q1A in consideration, present study describes a simple and a validated TLC-densitometry method  for the simultaneous determination of prednisolone acetate and chloramphenicol in presence of their degraded products formed under the applied stress conditions. As all the pharmaceutical products are supposed to be assayed for potency, a validated TLC-densitometry method, demonstrating no interferences of degraded products with the drug active components can be useful in measuring these components in routine analysis.
Instrumentation and chromatographic system
Stress degradation studies were performed using parallel synthesizer (Smart Start Synthesizer, Chem Speed Ltd., Switzerland) with sixteen reaction vessels. Planar chromatography was performed by spotting the sample on TLC glass plate, pre-coated with silica gel 60F-254 (20 × 10 cm) with the aid of CAMAG microliter sample syringe using CAMAG automatic TLC Linomat V applicator (Muntenz, Switzerland). A constant sample application rate of 0.1 μL/s was adopted and the distance between the two bands was 6 mm. 15 mL of mobile phase (chloroform: methanol, 14:1 v/v) was used for linear ascending development and chromatogram was allowed to move to a distance of 8 cm, in twin trough glass chamber (CAMAG). The chamber saturation time for mobile phase was 8 minutes at 25 ± 2°C with relative humidity 42 ± 5%. The developed TLC plate was dried with the help of air dryer for 4 min. Densitometric scanning was performed on CAMAG Reprostar scanner III in the reflectance absorbance mode at multiwavelength (λmax, 243 nm for prednisolone acetate and λmax, 278 nm for chloramphenicol) by utilizing deuterium lamp as the source of radiation. Quantitative evaluation was performed via peak areas by WinCats software (version 1.2.3). Densitometric scanning parameters were as follows: bandwidth: 10 mm, slit width: 0.45 mm, slit length: 5 mm, scanning speed: 10 mm/s.
Preparation of standard solutions and pharmaceutical samples
Three stock solutions were prepared by dissolving 100 mg of each prednisolone acetate and chloramphenicol in 100 mL methanol, individually and in combination. Working standard solutions were prepared by dilution of stock solution with methanol to give solutions in concentration range of 30-1000 μg/mL for calibration curve. Six-point calibration curve was formed by spotting 6 μL of each standard solution in concentration range of 200-6000 ng/spot containing both components, each concentration was spotted thrice on six replicate plates. For sample preparation, 1 mL ophthalmic samples (P1, P2, P3 and P4) containing 10 mg of prednisolone acetate were diluted in 20 mL volumetric flasks with methanol, separately. Similarly, 1 mL of spersinicol and chloroptic containing 0.5% chloramphenicol was diluted in 10 mL volumetric flasks with methanol. Chloramphenicol capsules, equivalent to 2500 mg (chlormycetin) were placed in 500 mL of volumetric flask and after addition of 100 mL water, heated on steam bath till the capsules weredisintegrated. After further addition of 300 mL water, it was again heated on steam bath with mixing. After cooling to room temperature, it was diluted to volume with water. 5 mL of the resulting solution was transferred to 100 mL volumetric flask and diluted with methanol [16, 17]. 1 mL ophthalmic samples (prednisynth and prednicol) containing 5 mg of prednisolone acetate and 2 mg chloramphenicol were diluted in 10 mL volumetric flasks with methanol [4, 12]. In similar manner, solutions for expired spersinicol and prednisynth were also prepared (1 mL in 10 mL volumetric flask). 6 μL of each sample was applied on TLC plate for chromatographic analysis.
The developed method was validated as per the requirements of the ICH guidelines. Linearity was evaluated by determining six standard working solutions at a concentration 200-6000 ng/spot. Peak area and concentration was subjected to the least square linear regression equation to calculate the regression data and correlation coefficients. In order to calculate S/N ratio for LOD and LOQ, the formulae used were 3.3 δ/S and 10 δ/S, respectively where δ is the residual error and S stands for slope of calibration curve. In order to check the robustness, following parameters were deliberately changed within the range of ± 5% at three different concentration levels (200, 400 and 800 ng); amount of mobile phase, mobile phase composition, time from spotting to chromatography, time from chromatography to scanning and chamber saturation time. Intra-day and inter-day precisions were determined with the standards and degraded reaction mixtures. For method repeatability, assay at three different concentration levels (200, 400 and 800 ng) was repeatedly performed six times on the same day (intra-day). For reproducibility, same samples at three concentration levels (200, 400 and 800 ng) were analyzed in different days (inter-day) and results were statistically evaluated in terms of % R.S.D. For recovery studies, pre analyzed pharmaceutical drugs containing prednisolone acetate (P1), chloramphenicol (C1) and both in combination (PC1) were spiked with extra 25, 50 and 75% of prednisolone acetate and chloramphenicol. The specificity of the proposed method was analyzed by overlapping the densitogram of the standard and samples and comparing it at peak start, peak apex and peak end positions.
Preparation of forced degradation products
Methanolic stock solutions (1 mg/mL) of both prednisolone acetate (set 1) and chloramphenicol (set 2) were prepared, separately and in combination (set 3) to perform forced degradation studies in parallel synthesizer by refluxing the reaction mixtures for two hours at 80°C. After the reactions were completed, all the solutions were preserved at -80°C till analysis. Average peak areas of active components were analyzed after triplicate analysis.
For acidic hydrolysis, 1N and 5N HCl were used, for alkaline hydrolysis, 0.1N, 1N and 5N NaOH were used while for neutral hydrolysis Milli Q water was used. 3 mL of each concentration of acidic and alkaline solutions and Milli Q water were added into 3 mL (1 mg/mL) stock solutions of all three sets. To study wet heating degradation, 3 mL (1 mg/mL) of stock solution of each set 1, set 2 and set 3 was subjected to degradation. Oxidation was carried out by adding 1 mL of H2O2 (35% v/v) in 3 mL stock solution of each set. All the resultant solutions were refluxed for two hours at 80°C in parallel synthesizer. 1 μL (500 ng/spot) of 1N HCl, 0.5 μL (250 ng/spot) of 5N HCl, 4 μL (2000 ng/spot) of 0.1N, 1N and 5N NaOH treated solutions, 4 μL (2000 ng/spot) of neutral hydrolysis, 6 μL (6000 ng/spot) from wet heat degradation mixture and 4 μL (3000 ng/spot) from oxidation mixture were applied on TLC plate in triplicate for chromatographic analysis.
Dry heat degradation was conducted by taking standard prednisolone acetate and chloramphenicol and heated in oven at 90°C for 4 hrs. 1 mg of each treated standard was dissolved in 1 mL of methanol and 4 μL (4000 ng/spot) of resultant solution of each, prednisolone acetate, chloramphenicol and both in combination was applied on TLC plate in triplicate for chromatographic analysis. For oxidation reaction at room temperature, 3 mL stock solution of each set was added with 1 mL of H2O2 (35% v/v) and the resultant solutions were kept for 24 hours at room temperature. 4 μL (3000 ng/spot) of each treated solution was applied on TLC plate in triplicate for chromatographic analysis. In order to evaluate photochemical degradation of prednisolone acetate, chloramphenicol and both in combination, stock solution of each set was directly exposed to the sunlight for three days from 8 to 18 hrs at 30 ± 2°C. 6 μL (6000 ng/spot) of each treated solution was applied on TLC plate in triplicate for chromatographic analysis.
Results and discussion
Optimization of TLC system and method validation
Linear regression data and validation parameters of prednisolone acetate (1) and chloramphenicol (2).
(at λmax 243 nm)
(at λmax 278 nm)
Correlation coefficient, r2 ± SD
0.9966 ± 3.5
0.9920 ± 2.5
Slope ± SD
2.42 ± 0.5
2.74 ± 0.34
Intercept ± SD
1106 ± 0.48
4183 ± 2.15
Y = mx + c
2.42x + 1106
2.738x + 4183
Intra-day (n = 3), % RSD
Inter-day (n = 3), % RSD
Limit of detection
Limit of quantification
Stability indicating property in prednisolone acetate
76.2% of prednisolone acetate was decomposed in 1N HCl and 100% in 5N HCl. Three additional peaks at Rf 0.01, 0.03, and 0.29 were commonly generated in both acidic reaction mixtures. In addition to those peaks, three additional peaks at Rf 0.17, 0.19 and 0.73 were only generated in 1N HCl reaction mixture while four additional peaks at Rf 0.02, 0.58, 0.78 and 0.80 were only obtained under strong acidic condition (5N HCl). Moreover, 100% degradation was observed under alkaline conditions. Degraded peaks were observed at Rf 0.01, 0.02 and 0.50. In addition to common peaks, some additional peaks were also observed. Three peaks were at Rf 0.17, 0.36 and 0.78 in 0.1N NaOH, and peaks at Rf 0.03 and 0.29 in 1N and 5N NaOH treated solutions, respectively. Prednisolone acetate showed 77.3% degradation by neutral hydrolysis with four additional peaks at Rf 0.01, 0.02, 0.17 and 0.29. Prednisolone acetate degradations under wet and dry heating were 95.8 and 18.5%, respectively. Both conditions showed common degraded peaks at Rf 0.01, 0.17, 0.26 and 0.47, while additional peaks were observed at Rf 0.29, 0.46 and 0.80 for wet heating and Rf 0.78 for dry heating conditions. Both oxidation mixtures, refluxed for 2 hours at 80°C and 24 hours at room temperature, showed 21.05% and 5.47% degradation, respectively with three common peaks at Rf 0.05, 0.14, and 0.29. Under photochemical conditions, 100% degradation was observed with the degraded peaks at Rf 0.01, 0.29, 0.33, 0.47, 0.58, and 0.73.
Summary of stress degradation studies of prednisolone acetate (1).
Rf of degraded products
0.01, 0.03,0.17, 0.19, 0.29, 0.73
0.01, 0.02, 0.03, 0.29, 0.58,0.78,0.80
0.01, 0.02, 0.17, 0.36, 0.50,0.78
0.01, 0.02, 0.03, 0.29, 0.50
0.01, 0.02, 0.29, 0.50
0.01, 0.02, 0.17, 0.29
0.01, 0.17, 0.26, 0.29, 0.46, 0.47, 0.80
0.01, 0.17, 0.26, 0.47, 0.78
0.01, 0.29, 0.33, 0.47, 0.58, 0.73
35%v/v H2O2 a
0.05, 0.14, 0.29
Oxidation at room temp
0.05, 0.14, 0.29
Stability indicating property in chloramphenicol
Chloramphenicol was 23.7% degraded in 1N HCl and 69.3% in 5N HCl. Common degraded peaks were observed at Rf 0.01 and 0.04, while in strong acidic medium an additional peak generated at Rf 0.81. Under alkaline conditions, chloramphenicol was 100% degraded and a common degraded peak at Rf 0.01 was also generated. Under all alkaline conditions, additional degraded products were observed at Rf 0.44 and 0.79, while peaks at Rf 0.81 were found in 1N NaOH treated solution. Three additional peaks at Rf 0.02, 0.04 and 0.48 were found in 5N NaOH treated solution. A single degraded product was found under neutral hydrolysis at Rf 0.01. In wet and dry heat degradation conditions, two common additional peaks were found at Rf 0.01 and 0.79. Under oxidation conditions, three additional peaks at Rf 0.01, 0.12 and 0.31 were generated, while seven additional peaks at Rf 0.01, 0.09, 0.15, 0.27, 0.44, 0.81 and 0.85 were found by photochemical degradation.
Summary of stress degradation studies of chloramphenicol (2).
Rf of degraded products
0.01, 0.04, 0.81
0.01, 0.44, 0.79
0.01, 0.04, 0.81
0.01, 0.02, 0.04, 0.48
35%v/v H2O2 a
0.01, 0.12, 0.31
Oxidation at room temp
0.01, 0.12, 0.31
Stability indicating property of prednisolone acetate and chloramphenicol in combination
In comparison with the individual standard solutions, a combined standards solution showed two additional peaks under acidic (1N HCl) condition at Rf 0.78 and 0.81 while three degraded products of prednisolone acetate at Rf 0.03, 0.19 and 0.73 were missing. 5N HCl stress condition showed two additional peaks at Rf 0.17 and 0.38. Peak at Rf 0.81 corresponding to the degraded product of chloramphenicol while degraded products of prednisolone acetate with Rf 0.03 and 0.80 were missing. Moreover, under both acidic conditions, prednisolone acetate was 100% degraded.
Under alkaline conditions, chloramphenicol and prednisolone acetate were completely degraded. Two common additional peaks were generated under all alkaline conditions at Rf 0.01 and 0.02. Under 0.1N NaOH stress condition, two additional peaks were found at Rf 0.29 and 0.85 while chloramphenicol degraded peak at Rf 0.44 and prednisolone acetate degraded peaks at Rf 0.36 and 0.78 were missing. Moreover, under 1N NaOH stress condition, an additional peak was observed at Rf 0.44 while chloramphenicol degraded products at Rf 0.04 and 0.81 and prednisolone acetate degraded product at Rf 0.50 were missing. In 5N NaOH treated solution, comparison showed an additional peak at Rf 0.31 while chloramphenicol degraded products at Rf 0.04 and 0.48 and prednisolone acetate degraded products at Rf 0.29 and 0.50 were missing. In neutral hydrolysis reaction mixture, an additional peak was generated at Rf 0.79, while peak for prednisolone acetate at Rf 0.02 was missing.
Similarly an additional degraded product at 0.50 was observed in combined sample under wet heat degradation while degraded products of prednisolone acetate at Rf 0.47 and 0.80 were missing. Under dry heat degradation condition, comparison showed an additional peak at Rf 0.73 while prednisolone acetate degraded peaks at Rf 0.17 and 0.78 were missing. Under wet heat degradation condition, prednisolone acetate was 17% degraded while chloramphenicol was degraded up to 36.9%.
For oxidation reaction, two conditions were applied, under oxidation condition 1 (refluxed for two hours at 80°C), additional peak at Rf 0.55 while two additional peaks at Rf 0.23 and 0.55 were observed under oxidation condition 2 (reaction mixture kept for 24 hours at room temperature). Prednisolone acetate degraded peaks at Rf 0.05, 0.14 and 0.29 were missing.
Summary of stress degradation studies of prednisolone acetate and chloramphenicol in combination.
% Degradation of 1
% Degradation of 2
Rf of degraded products
0.01, 0.04, 0.17, 0.29,0.78,
0.01, 0.02, 0.04, 0.17, 0.29,
0.1 N NaOH
0.01, 0.02, 0.17,0.29, 0.50, 0.79,0.85
0.01, 0.02, 0.03, 0.29,0.44
0.01, 0.02, 0.31
0.01, 0.17, 0.29, 0.79
0.01, 0.17, 0.29, 0.50,0.79
0.01, 0.26, 0.47,0.73,0.79
0.01, 0.47, 0.49,0.50,0.58, 0.69,0.73
35%v/v H2O2 a
0.01, 0.12, 0.31, 0.55
At room temp
0.01, 0.12, 0.23,0.31, 0.55
Analysis of marketed samples
Analysis of pharmaceutical products.
Conc. (in ng)
Conc. (in ng)
A validated TLC method for routine analysis to determine the stability of prednisolone acetate and chloramphenicol in pharmaceutical dosage forms has been developed. Current study demonstrates the degradation susceptibility of drugs to different stress conditions and thus helps in determining the changes in chemical, physical and microbiological properties of the drug samples with time. It also helps in understanding the mechanism and pathway of degraded products formation and in developing a profile reflecting the changes in identity, purity and potency of the product. The comparison of stress degradation in individual and combined formulations reveals the effect on % degradation and the number of degraded products. The developed stability indicating TLC method is simple, reproducible and can be used for the simultaneous analysis of two active components (chloramphenicol and prednisolone acetate). It appears suitable for routine analysis of these components selectively in presence of their degraded products in individual and in combined pharmaceutical formulations.
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