INTRODUCTION:

Prednisolone is a synthetic glucocorticoid (corticosteroid). It is a steroid medication which possess anti-inflammatory and immunosuppressive effect^1,2. It is used in the treatment of allergies, inflammatory conditions, auto-immune disorders, acute and chronic obstructive pulmonary disease, rheumatoid arthritis, systemic lupus erythematosus, dermatitis, eye inflammation, asthma and multiple sclerosis^3,4,5.Few types of Rheumatoid arthritis are Seropostitive, seronegative and juvenile arthritis⁶.Rheumatoid arthritis is a disorder in which body’s own immune system attacks the body tissues,especially at joints and cartilages, this condition is characterized by swelling at the joints, sleep disturbances, morning stiffness, grinding at joints etc^7,8.Arthritis is more prevalent in females due to deficient of estrogen hormone levels⁹. Systemic lupus erythematosus is a multisystem autoimmune inflammatory disorder characterized by tissue injury due to accumulation of immune response factors¹⁰.

Prednisolone is white or almost white, crystalline, hygroscopic powder. It is very slightly soluble in water, soluble in ethanol and methanol, sparingly soluble in acetone, slightly soluble in methylene chloride. It shows polymorphism. Specific optical rotation is in dioxan solvent is +96 to +102 on dried basis¹¹. Its molecular formula is C₂₁H₂₈O₅(anhydrous), molecular weight is 360.45 and chemically it is described as Pregna-1,4-diene-3,20-dione, 11,17,21-trihydroxy-, (11 β) or 11β,17,21-Trihydroxypregna-1, 4-diene-3, 20-dione (anhydrous)¹². log P value is Prednisolone is 1.66, hence it is polar(hydrophilic) in nature¹³.

Prednisolone is available in various dosage forms such as tablets, parenteral and cream. It is an intermediate acting drug¹⁴.Prednisolone exhibits its mechanism of action by binding to glucocorticoid receptor. It crosses the cell membrane and binds to glucocorticoid receptor to form complex, complex formed translocates to the nucleus and undergoes gene transcription by interacting with DNA. This causes expression of inflammatory genes, it undergoes transcription and inhibits prostaglandin synthesis. Prostaglandin synthesis is inhibited through arachidonic acid pathway. Prednisolone firstly inhibits transcription factors (AP- and NF-κB) involved in regulation pro-inflammatory proteins and secondly it increases the synthesis of anti-inflammatory protein annexin-I, which inturn inhibits phospholipase A2 enzyme activity, which produces arachidonic acid. It undergoes urinary excretion. It reduces airway inflammation⁵.Possible side effects of Prednisolone are Osteoporosis, weight gain, nausea, hyperglycemia, indigestion, heartburn⁴. Prednisolone improves short term survival of patient diagnosed with alcoholic hepatitis¹⁵.

Spectrophotometry works on the principle of Beer Lambert’s Law. It states that when a beam of light passes through a transparent cell containing a solution of an absorbing species, intensity of light decreases with increase in concentration of absorbing species or thicness¹⁶. As per literature survey, analytical methods such as UV, HPLC and LC-MS were employed. Mr. Singh and Mr. Rohan performed determination of Prednisolone in formulation and compared Spectrophotometric method with reverse phase HPLC¹⁷. Few papers performed method development and validation of Prednisolone acetate in combination with Ofloxacin in eye drop by Spectrophotometric technique^18,19. Mr.Yilmaz et al., performed quantitative determination of Prednisolone in human serum and tablets by electrochemical oxidation using glassy carbon electrode²⁰.Mr. Raval Kashyap et.al., reported estimation of Prednisolone in bulk and dosage form by UV spectroscopy using acetonitrile and methanol as solvent²¹.Mr. Somsubhra Ghosh et.al., developed method for Prednisolone and validated it by Reverse Phase HPLC technique using methanol and water mobile phase, against an internal standard²².Mr.Rajendra and Ms. Sunita carried out estimation of Prednisolone in human body fluid using Voltammetric method²³.Parag et.al, and Sohan et.al. Performed spectrophotometric estimation of Prednisolone in combination with Salbutamol sulphate^24,25. Farheen et.al formulated and evaluated colon targeted Prednisolone Osmotic tablets²⁶.

Figure 1 Chemical structure of Prednisolone¹²

MATERIAL AND METHODS:

Chemicals and reagents:

Analytical Grade Ethanol was used of Merck Grade. Prednisolone standard, active pharmaceutical ingredient was a gifted sample. Prednisolone dispersible commercial tablets (WYSOLONE tab.5 mg) manufactured by Pfizer Limited, India containing 5 mg of Prednisolone. Whatman filter paper made up of cellulose grade 1 manufactured of Merck Grade.

Instrumentation:

Instruments used for this method are Double beam Shimadzu UV-Visible Spectrophotometer of UV 1800 model, Cuvettes made up of glass quartz of 1 cm. Weighing was done using Analytical Calibrated Electronic Microbalance (Mettler Toledo). Calibrated glass wares such as Volumetric flask, measuring cylinder, pipettes, test tubes and beakers were used made up of borosilicate glass of Class-A grade. Ultrasonic Bath XUBA series sonicator was used for sonication. Fisher Scientific Mortar and Pestle made up of Porcelain was used for powdering tablets. BSCO Hot air oven was used for drying of glass wares.

Solubility and Physical Apperance Determination:

Small quantity of Prednisolone standard was taken in a dried test tube and to this small volume of water was added and its solubility was checked. It was found to be insoluble in water and solution was not clear.

Then a very small quantity of Prednisolone standard was taken in a dried test tube and to this small volume of ethanol was added and its solubility was checked. It was found to be soluble in ethanol and solution was clear.

After 24 hrs its appearance was checked again visually, the solution was found to be clear.

Preparation of Standard Stock Solutions of Prednisolone for Linearity:

Prednisolone Stock Solution (about 500 μg/mL):

Accurately weighed and transferred about 5 mg of Prednisolone in a 10 mL of dried volumetric flask, Small volume of ethanol was added and sonicated for about 2min and then the volume was made up to 10 mL with ethanol.

Prednisolone Standard Solution-I (about 2.5 μg/mL):

0.05 mL was pipette out from Prednisolone stock solution and carefully transferred to a 10 mL volumetric flask. Volume was made up to 10 mL with ethanol and then Prednisolone solution was shaken well after placing the lid.

Prednisolone Standard Solution-II (about 5 μg/mL):

0.1 mL was pipette out from Prednisolone stock solution and carefully transferred to a 10 mL volumetric flask. Volume was made up to 10 mL with ethanol and Prednisolone solution was then shaken well after placing the lid.

Prednisolone Standard Solution-III (about 10 μg/mL):

0.2 mL was pipette out from Prednisolone stock solution and carefully transferred to a 10 mL volumetric flask. Volume was made up to 10 mL with ethanol and Prednisolone solution was then shaken well after placing the lid.

Prednisolone Standard Solution-IV (about 15 μg/mL):

0.3 mL was pipette out from Prednisolone stock solution and carefully transferred to a 10 mL volumetric flask. Volume was made up to 10 mL with ethanol and Prednisolone solution was then shaken well after placing the lid.

Prednisolone Standard Solution-V (about 20 μg/mL):

0.4 mL was pipette out from Prednisolone stock solution and carefully transferred to a 10 mL volumetric flask. Volume was made up to 10 mL with ethanol and Prednisolone solution was then shaken well after placing the lid.

Determination of λ Max-Result:

Prednisolone working standard solution-III was scanned from 400 to 200 nm. The spectrum is represented in Figure 2. Lambda max was found to be 243.1 nm.

Figure 2: Spectrum of Prednisolone in ethanol

Linearity Result:

For a concentration range of about 2.5 µg/mL to 20 µg/mL, the developed method was found to be linear as correlation coefficient was found to be 1.00, as depicted in Figure 3.

Figure 3: Calibration Curve of Prednisolone at 243 nm.

Procedure for Assay of Prednisolone Tablets:

Weighed and transferred twenty tablets of Prednisolone (WYSOLE tablets) into a dried mortar. Tablets were crushed to a fine powder with pestle. Weighed accurately powder containing an amount equivalent to 5 mg of Prednisolone and transferred to a 10 mL volumetric flask. About 5 mL of ethanol was and the flask was subjected for sonication for about 30 min and then volume was made up to 10 mL using ethanol. The contents of Prednisolone tablet solution were mixed well and the solution was filtered through a Whatman filter paper. From the filtrate 0.2 mL was pipette out and transferred to a 10 mL volumetric flask, volume was made up to 10mL with ethanol. The sample absorbance was measured at wavelength of 243 nm against a blank(ethanol).Concentration of drug was determined using Beer-Lambert’s law by applying specific absorbance as 415.Then the amount of drug and % Label claim was calculated.% Label claim was reported and checked with that of the limits.

Limits:

Prednisolone tablets contain not less than 90 % and not more than 110 % of the stated amount of Prednisolone²⁷.

Specific Absorbance:

Specific absorbance of Prednisolone in ethanol as solvent, A^1%_1cm is 415 (in 1 gm/100 mL)²⁷.

Calculation of Concentration of drug by using Beer-Lambert’s Law Equation, and subsequent calculation of Amount of the drug and % Label Claim:

The greatest advantage of this method of calculation is that concentration of the drug can be calculated even without any standard graph or standard solutions preparation, by directly applying the measured absorbance value in equation by using specific absorbance as 415.Calculation is depicted in Figure 4.

Beer-lamberts Law Equation:

Where A = Absorbance

= Specific absorbance (=415>1gm/100 mL for Prednisolone in ethanol at 243 nm)

b = Path length of radiation through sample (1 cm)

c = Concentration of solute in solution

Assay Result:

Percent label claim of Prednisolone was found to be within limits. And % RSD of percent label claim was found to be not more than 2% for five replicates of samples. Hence the developed method was found to be accurate. As per this Precision- Repeatability was found to be passed as per ICH guidelines²⁸.

Figure 4: Calculation of Concentration of drug based on Beer-Lambert’s Law Equation.

Validation of Proposed Method and It’s Results:

The developed method was validated as per ICH guidelines.

Accuracy:

Accuracy or recovery was performed at three different levels by spiking appropriate known amount of standard solution to tablet mixture. Recovery was performed at three different levels with that of the target concentration (10 µg/mL). It was done by selecting 10 % of target concentration, 100 % of target concentration and 120 % of target concentration. Recovery was performed at three different levels with three replicates of each level. Amount added and percent recovery was calculated. Average % recovery of three samples at each level was calculated and tabulated in table 2.

Table 1: Results of Assay/Determination of Prednisolone (Precision):

Sample	Concentration of drug (μg/mL)	Amount of the drug (Prednisolone) found (mg)	% Label Claim	% RSD of % Label claim of five samples
Sample-1	9.93	4.97	99.3 %	1.9 %
Sample-2	9.61	4.81	96.1 %
Sample-3	10.1	5.05	101 %
Sample-4	9.76	4.88	97.6 %
Sample-5	9.82	4.91	98.2 %

Table 2: Results of Accuracy:

S.No.	Recovery Level	Sample	Amount added,µg/mL	Amount Found,µg/mL	% Recovery	Average % Recovery
1	50 %	Sample-1	4.9	4.7	95.9 %	99.3 %
		Sample-2	4.9	4.9	100.0 %
		Sample-3	4.9	5.0	102.0 %
2	100 %	Sample-1	10.4	10.4	100.0 %	101.0 %
		Sample-2	10.4	10.5	101.0 %
		Sample-3	10.4	10.6	101.9 %
3	120 %	Sample-1	12.6	12.9	102.4 %	101.9 %
		Sample-2	12.6	12.7	100.8 %
		Sample-3	12.6	12.9	102.4 %

Based on above results, accuracy at three different levels was found to be passed, as it is within limits as per ICH guidelines for Assay of tablets.

Table 3: Results of Intraday Precision:

Time	% Label Claim
Time	Sample-1	Sample-2	Sample-3	Average	SD	% RSD
Morning	98.20	100.20	102.00	100.13	1.90	1.9
Afternoon	99.60	98.20	101.00	99.60	1.40	1.4
Average % RSD						1.65

Based on above results Intraday Precision was found to be passed as per ICH limits for Assay of tablets.

Table 4: Results of Ruggedness-Different Analyst:

Analyst	% Label Claim
Analyst	Sample-1	Sample-2	Sample-3	Average	SD	% RSD
Analyst-1	99.60	99.90	100.20	99.90	0.30	0.30
Analyst-2	109.0	108.5	106.0	107.83	1.61	1.49
Analyst-3	100.00	100.10	99.90	100.00	0.10	0.10
Average % RSD						0.63

Based on above results ruggedness on different analyst was found to be passed as per ICH limits for Assay of tablets.

Table 5: Results of Ruggedness-Different Instrument:

Instrument	% Label Claim
Instrument	Sample-1	Sample-2	Sample-3	Average	SD	% RSD
Equipment-1	100.0	101.00	100.90	100.63	0.55	0.55
Equipment-2	98.60	99.00	100.00	99.20	0.72	0.73
Average % RSD						0.64

Based on above results ruggedness on different instrument was found to be passed per ICH limits for Assay of tablets.

Precision-Intraday Precision:

Intraday precision was performed by analyzing samples in morning and afternoon, in a replicate of three (n=3), by following the same procedure as developed for assay of tablets mentioned above. Amount of drug and percent label claims were calculated. Based on this % RSD were calculated for samples (n=3). Results are tabulated in table 3.

Ruggedness-Different Analyst

Ruggedness was performed by three different analysts in a replicate of three samples (n=3), by following the same procedure as developed for assay of tablets mentioned above. Amount of drug and percent label claims were calculated. Based on this % RSD were calculated for samples (n=3) by one analysts and even for three different analysts. Results are tabulated in table 4.

Ruggedness-Different Equipment:

Ruggedness was performed by two different equipment of same model, in a replicate of three samples (n=3),by following the same procedure as developed for assay of tablets mentioned above. Amount of drug and percent label claims were calculated. Based on this % RSD were calculated for samples (n=3).Results are tabulated in table 5.

RESULTS:

Parameters	Observations
Linearity Range	2.5 – 20 µg/mL
Correlation Coefficient (r)	1.00
Slope	0.041
Intercept	0.001
Regression equation	Y = 0.041x – 0.001
Precision of Assay Samples	% RSD was found to be within limits
% Recovery	% Recovery was found to be within limits

CONCLUSION:

Prednisolone is widely used steroid drugs which possess anti-inflammatory and immunosuppressive actions. Assay, Dissolution and Related substances are few quality control tests performed repeatedly for new processed formulation and stability batches. Regular performance of this test acquires various chemicals, solvents and analytical instruments. Usually when a method is developed easy and economical procedure is opted.

Prednisolone is a potent drug and usually available in very low doses such as 5mg, 10mg and 15mg.As it is a potent drug, working with this drug in analytical department is quite difficult, as we are supposed to work on lower concentrations. Hence the developed method detects and quantitates lower concentration of drug by Spectrophotometric method. The developed method for Assay of Prednisolone tablets is a simple, eco-friendly, undemanding, uncomplicated and economical. Primarily in this method only one solvent i.e, Ethanol is used, which is a eco-friendly green solvent. Performance of this method causes less harm to environment and analysts. The analytical technique used is very simple i.e, UV spectrophotometer and doesn’t require costly analytical instruments. The calculation method for estimation of percent label claim involved is very simple. Using Beer-Lambert’s law equation concentration of drug in sample can be analysed without use of any standard solution or standard graph and by just applying specific absorbance value as explained above in calculation part. Percent label claim for Prednisolone tablets were found to be within limits as per mentioned in Pharmacopoeia. Linearity using standard solution was performed, so that we can assure the developed method is linear and passes acceptance criteria. Other Validation parameters were performed and based on results we conclude that the developed method is simple, accurate, linear, precise and rugged. Finally we conclude, that the developed method is a simple, eco-friendly and economical.

ACKNOWLEDGEMENT:

We are thankful for the management of JNTU University for providing us instruments and laboratory to perform our study.

CONFLICT OF INTEREST:

We declare no conflicts of interest.

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Received on 18.09.2020 Modified on 21.10.2020

Asian Journal of Research in Chemistry. 2021; 14(3):155-160.

DOI: 10.52711/0974-4150.2021.00029