UV Spectrophotometric Method for the Determination of Sirolimus in Bulk and its Dosage Form

 

S K. Mastanamma¹, S K. Reehana², Prudhvi. L³, Ravi Kiran³

¹Department of Pharmaceutical Analysis, University College of Pharmaceutical Science,

Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh (India).

²Scholar, Department of Pharmaceutical Analysis, University College of Pharmaceutical Science,

Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh (India).

³B. Pharm Student, Department of Pharmaceutical Analysis, University College of Pharmaceutical Science, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur – 522510, Andhra Pradesh (India).

 

ABSTRACT:

Objective: The main objective was to develop and validate the UV-Spectrophotometric method for the estimation of Sirolimus in bulk and pharmaceutical formulations as per ICH guidelines. Materials and methods: A simple, rapid, accurate, and economical UV-spectrophotometric method has been developed for the estimation of Sirolimus from bulk and pharmaceutical formulation. Results: The λ_max of Sirolimus in Dimethyl Sulfoxide was found to be 274 nm. The drug follows linearity in the concentration range 3–21 μg/ml with a correlation coefficient value of 0.9956. The proposed method was applied to pharmaceutical formulation and % amount of drug estimated was 101.35%. and was found to be in good agreement with the label claim. The accuracy of the method was checked by recovery experiment performed at three different levels, i.e., 75%, 100%, and 125%. The % recovery was found to be in the range of 99.30– 101.03%. The low values of % RSD are indicative of the accuracy and reproducibility of the method. The precision of the method was studied as an intraday; interday variations, and repeatability. The % RSD value < 2 indicates that the method is precise.  Conclusion: The above method was a rapid tool for routine analysis of sirolimus in the bulk and in the pharmaceutical dosage form.

 

 

 

INTRODUCTION:

Sirolimus is a macrolide compound obtained from Streptomyces hygroscopicus that acts by selectively blocking the transcriptional activation of cytokines thereby inhibiting cytokine production. It is bioactive only when bound to Immunophilins. Sirolimus is a potent immunosuppressant and possesses both antifungal and antineoplastic properties¹.

 

 

 

 

Chemically it is known as (1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28E, 30S, 32S, 35R)-1, 18-dihydroxy-12-[(2R)-1-[(1S, 3R, 4R)-4-hydroxy-3-methoxycyclohexyl]propan-2-yl]-19, 30-dimethoxy-15, 17, 21, 23, 29, 35-hexamethyl-11, 36-dioxa-4 azatricyclo [30.3.1.0^{4, 9}] hexatriaconta-16, 24, 26, 28-tetraene-2, 3, 10, 14, 20-pentone and its chemical formula is C51H79NO13². It has the chemical structure³ shown in [Fig.1]

 

Mechanism of action²: Sirolimus inhibits T lymphocyte activation and proliferation that occurs in response to antigenic and cytokine (Interleukin IL-2, IL-4, and IL-15) stimulation by a mechanism that is distinct from that of other immunosuppressants.

 

Fig.1 Chemical Structure of Sirolimus

 

Sirolimus also inhibits antibody production. In cells, sirolimus binds to the immunophilin, FK Binding Protein-12 (FKBP-12), to generate an immunosuppressive complex. The sirolimus:FKBP-12 complex has no effect on calcineurin activity. This complex binds to and inhibits the activation of the mammalian Target of Rapamycin (mTOR), a key regulatory kinase. This inhibition suppresses cytokine-driven T-cell proliferation, inhibiting the progression from the G1 to the S phase of the cell cycle.

 

Literature survey reveals that various HPLC, LC-MS^4-8 method and have been reported for the estimation of sirolimus but there was no UV method hence author made an attempt to develop a simple, accurate and precise visible UV spectrophotometric method for estimation of sirolimus by using DMSO.

 

MATERIALS AND METHODS:

Chemicals and Reagent:

Working standard of sirolimus was obtained as a gift sample from Biocon labs, Bangalore. Reagents used were Dimethyl sulfoxide (DMSO) AR Grade was purchased from merck fine chemicals (Mumbai, India).

 

Instrumentation:

Shimadzu UV -1800 double beam spectrophotometer with 1cm path length supported by Shimadzu UV-probe software, version 2.21 was used for spectral measurements with 10mm matched quartz cells. Shimadzu balance (BL-220H) was used for weighing.

 

Preparation of standard stock solution:

Accurately weighed 10mg of Sirolimus working std in 10mL volumetric flask containing 5ml of DMSO shaken for 5min then remaining volume made up with DMSO. The final concentration obtained was 1000µg/mL. It was further diluted to get concentration 100µg/ml was prepared with DMSO. From this a series of aliquots were prepared to get concentration ranging from 3-21µg/mL in 10ml vol. flask using DMSO

 

 

Construction of calibration curves for sirolimus:

For the construction of calibration curves prepare to get concentration of 3-21µg/ml of drug. ^{Construct the calibration curves by taking concentration (}µg/ml) ^{on the x-axis and average peak area on the y-axis for the drugs} [Fig.2]^{. From the calibration curves, regression equations were calculated, these regression equations were used to calculate the amount of sirolimus present in the commercial dosage form.}

 

 

 

Preparation of sample solution:

Weigh 5 tablets and crush to powdered then take powder equivalent 10 mg sirolimus into a 10 ml volumetric flask. Dissolved in sufficient quantity of DMSO and volume made up to the mark with DMSO to obtain conc. of 1000µg/ml. Filter through whatmann filter paper no 0.41. Further dilutions of the solution were made to get required concentration of 12µg/ml. This solution was estimated by above developed method. The assay procedure was repeated 6 times (n=6) the drug content was estimated using above calculated regression equation; the results of tablet dosage form shown in Table-1.

 

Table.1: Results of Marketed Formulation analysis

Drug	Labelled amount (mg/tab)	Amount found (mg/tab)	% Assay
Sirolimus	5	5.05	101.35

 

METHOD VALIDATION:

The developed method for estimation of sirolimus has been validated in accordance with the International Conference on Harmonization guidelines.

 

Table. 2: Linearity studies of Sirolimus

S. No	Concentration (μg/ml)	Absorbance
1.	3	0.186
2.	6	0.316
3.	9	0.449
4.	12	0.563
5.	15	0.692
6.	18	0.812
7.	21	0.926

 

 

Table 3: Optical characteristics of Sirolimus

Parameter	Results
λmax	274nm
Beer’s limit (µg/mL)	3-21μg/ml
Correlation coefficient (r2)	0.9956
Slope (a)	0.0431
Intercept (b)	0.0408

 

Linearity:

Several aliquots of standard stock solution of sirolimus was taken in 10 ml volumetric flask and diluted up to the mark with the mobile phase such that their final concentrations were 3-21μg/ml for sirolimus. Absorbance was plotted against the corresponding concentrations to obtain the calibration graph for compound as shown in [Table 2&3]. A good linear relationship (r=0.995). Linearity equation obtained for sirolimus was y = 0.043x + 0.0402. Figures 2, shows linearity graphs for sirolimus.

 

Accuracy:

The accuracy of the method for assay determination was achieved at three concentration levels of 75%, 100%, and 120% for Sirolimus. Known amount of standard drug concentration was added to the pre analysed sample concentration and the mean percentage recovery values are shown in [Table 4].

 

Precision:

Precision is the degree of repeatability of an analytical method under normal operating condition.

Precision is of 3types.

1    System precision.

2    Method precision.

3    Intermediate precision.

 

a. Inter- day precision:

Method precision was achieved by repeating the same procedure of preparation solution six times.

 

System precision is checked by injecting using a standard chemical substance to ensure that the analytical system is working properly. In this peak area and percentage of the drug of six determinations were measured and percentage relative standard deviation should be calculated.

 

In method precision, a homogenous sample of the single batch should be analyzed 6 times. This indicates weather a method is giving a constant result for a single batch. In this the sample is analysis six times and calculates the % RSD and the results are shown in [Table 5].

 

RESULTS AND DISCUSSION:

Linearity and range:

The linearity regression coefficient (R2) values were found to be 0.995. Regression equation obtained was y = 0.043x + 0.0402. Figures 2, shows linearity graphs for sirolimus. The method was linear over the range of 3-21 μg/ml of drug.

 

Recovery:

The percentage recovery was calculated by preparing standard drug concentrations of sirolimus with concentration levels of 75%, 100%, and 125%. A known amount of the standard drug was added to the pre analyzed concentration at each level. Good recovery of the spiked drugs was obtained at each added concentration, and the mean percentage recovery of sirolimus was achieved between 99.09–101.08 ± 0.5%. The results are given in [Table 4].

 

Precision:

Precision is expressed as the closeness of agreement between a series of measurements obtaining from multiple sampling of the same homogeneous sample. Six replicate injections of a known concentration of sirolimus 3-21 (µg/ml), have been analyzed them using a UV spectrophotometer by the different analysts and different instruments. From the results obtained, % RSD was calculated and was found to be within the limits ˂2. The results are given in [Table 5].

 

Table. 4: Recovery studies of Sirolimus

S. No	Recovery level	Preanalysed concentration (μg/ml)	Amount added (μg/ml)	Amount recovered (μg/ml)	% Recovery	Mean	SD	% RSD
1.	75%	9	6.75	15.64	99.31	100.1	0.89	0.90
2.	100%	9	9	18.19	101.08
3.	125%	9	11.25	20.23	99.093

 

Table. 5. Method Precision Studies of Sirolimus

S.No	Concentration (μg/ml)	Absorbance		Mean	SD	%RSD	Mean %RSD
		Analyst-1	Analyst-2
1.	3	0.186	0.169	0.177	0.012	6.77	2.68
2.	6	0.316	0.309	0.312	0.0049	1.58
3.	9	0.449	0.441	0.445	0.0056	1.27
4.	12	0.563	0.579	0.571	0.011	1.98
5.	15	0.692	0.705	0.698	0.0091	1.32
6.	18	0.812	0.828	0.82	0.011	1.38
7.	21	0.926	0.950	0.938	0.016	1.81

Table. 6. System Precision Studies of Sirolimus

S. No	Concentration (μg/ml)	Absorbance		Mean	SD	% RSD	Mean %RSD
		System-1	System-2
1.	3	0.18	0.169	0.174	0.007	4.46	2.24
2.	6	0.31	0.309	0.3095	0.0007	0.23
3.	9	0.470	0.441	0.455	0.020	4.5
4.	12	0.591	0.579	0.585	0.0084	1.45
5.	15	0.726	0.705	0.7155	0.014	2.08
6.	18	0.846	0.828	0.837	0.012	1.52
7.	21	0.970	0.950	0.96	0.014	1.47

 

CONCLUSION:

In this proposed UV method the selected drugs showed good linearity. Results for the recoveries of selected drugs for found within limits (98–102%). These indicate that the proposed method was accurate for the analysis. The developed UV method for the determination of Assay of selected drugs is simple, rapid, accurate, precise and economical. The mobile phase and solvents are simple and economical reliable, sensitive and less time consuming. The sample recoveries were in good agreement with their respective label claims and they suggested non interference of formulation excipients in the estimation. Since the system validation parameters of UV method used for estimation of selected drug in pure and have shown satisfactory, accurate and reproducible results (without any interference of excipients).

 

REFERENCES:

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Received on 26.11.2018           Modified on 17.01.2019

Accepted on 08.02.2019         © RJPT All right reserved