Development and Validation of UV Spectrophotometric Method for Determination of Tacrolimus in Bulk and Capsule Dosage Form

Introduction

Ultraviolet and visible spectroscopy deals with the interpretation of absorption of radiations within the ultraviolet, visible regions of electromagnetic spectrum extending from UV region 10 to 400 nm. The visible region extends from 400 to 800 nm.¹ Validation of any analytical procedure can be defined as the process by which it is accepted that the performance characteristic of an analytical procedure meets the requirements for intended analytical applications in laboratory studies. The main objectives of the analytical measurement are to get reliable, consistent and accurate data. The results from the method validation can also be used to inspect the consistency, quality and reliability of analytical results, which is the integral part of a good analytical practice.²

Tacrolimus is a macrolide lactone with alpha, beta-diacetamide hemiacetal incorporated within the 23-membered ring structure. It is extracted from Streptomyces tsukubaensis, has potent immunosuppressive activity, and is employed clinically for the prevention of organ transplant rejection, such as liver, kidney, heart, pancreas and bone marrow. The drug is additionally used for the therapy of atopic dermatitis, eczema, psoriasis and vitiligo. Tacrolimus is highly soluble in lipids and few organic solvents, slightly soluble in saturated hydrocarbons and insoluble in water. Pharmaceutical dosage forms like capsule, injection and ointment are available for clinical use. Tacrolimus has the below mentioned structure (Figure 1).

According to International Conference on Harmonization (ICH) guidelines, “the objective of validation of any analytical procedure is to signify that the method is appropriate for its intended purpose.” It is mandatory within the process of drug development to provide the validation data to responsible authorities. Guidelines for analysis of method validation includes ICH and USP guidelines.^3,4

Method validation is the process of establishing documented evidence which gives high degree of assurance that the product or equipment would meet the requirements for the intended applications.⁵

The existing method for the determination of Tacrolimus is high-performance liquid chromatography (HPLC); however, it requires longer time and is not economical. Hence an attempt was made to develop UV method using aqueous medium i.e. Phosphate buffer (pH 6.8) as diluent. UV method does not require elaborate procedures that are usually related to chromatographic method.

Materials and Methods

Instrumentation: UV-1601 UV-VISIBLE spectrophotometer (Make: Shimadzu).

Chemicals and Reagents

Tacrolimus was supplied by Dr. Reddy’s Laboratories, Acetonitrile was purchased from SDFCL. All other chemicals used were of analytical grade. 

Determination of λ_max

For the optimization of optimum λ_max, 2 µg/mL of Tacrolimus solution was scanned in UV wavelength range of about 200-400 nm using Phosphate buffer (pH 6.8) solution as blank. The drug showed maximum absorbance at 215 nm, which was considered as λ_max for further method development. The spectrum of the drug solution was recorded and is shown in figure 2.

Preparation of primary standard stock solution

Accurately weighed, 20 mg of Tacrolimus was dissolved in 10 mL of acetonitrile resulting in a concentration of 2 mg/mL.

Preparation of secondary standard stock solution

0.5 mL of primary standard stock solution was mixed with 0.5 mL of Sulphuric acid in a 10 mL volumetric flask and was then diluted with Phosphate buffer (pH 6.8) up to the mark. The concentration of the obtained solution was 100 µg/mL.

Method Validation

The developed method was validated for the parameters given below:

a) Linearity: Analytical procedure performed should be linear so that the acquired test results should be directly proportional to concentration of the analyte in the sample.3 Aliquots of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mL were withdrawn from the secondary stock solution and diluted to 10 mL with Phosphate buffer (pH 6.8) solution to obtain 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 µg/mL respectively. Absorbance of each aliquot was measured by UV spectrophotometer at 295 nm using Phosphate buffer pH 6.8 solution as a blank. The regression correlation coefficient [R² ] was found to be 0.999, with a y-intercept 0.004 and the slope of the linearity curve was 0.055 (Figure 3) and the linearity data is shown in Table 1.  

b) Precision: Precision of the method was determined by intraday and interday variation studies.3 Nine different solutions of the same concentration were measured for absorbance on the same day (intraday) and on the next day (interday) and % RSD was calculated as shown in Table 2.

c) Accuracy: Accuracy of the method was assessed by determination of the recovery of method at three different concentrations (80%, 100% and 120% concentrations) by addition of known amount of standard to the placebo. For each concentration, three sets were prepared.6 The results are mentioned in Table 3.

d) Robustness: Robustness of the UV method development was determined by analyzing appropriate concentrations (6 µg/mL) at different wavelengths (295±2 nm) and values of % RSD were calculated using regression coefficient and the calculations are represented in Table 4.

e) Ruggedness: Ruggedness is the measure of reproducibility of any test results under normal and expected operational conditions such as from analyst to analyst and instrument to instrument. Appropriate concentrations of Tacrolimus were analyzed using different UV spectrophotometry equipment by a different analyst to record absorbance values. % RSD was calculated using regression coefficients obtained and the results are shown in Table 5. 

Assay of Marketed Formulation

Ten marketed formulations i.e., capsules of Tacrolimus containing 2 mg drug per each capsule were taken, emptied into a mortar and pestle. Weighed amount of the capsule powder, which was equivalent to 20 mg of Tacrolimus was transferred into a 10 mL volumetric flask, to which 1 mL acetonitrile was added and the solution was sonicated for 3 minutes on bath sonicator. It was extracted with acetonitrile and filtered. The prepared solution was found to be clear and with the strength 2000 μg/mL. From this solution, 0.5 mL solution was transferred to 100 mL volumetric flask and was diluted with pH 6.8 Phosphate buffer to obtain 10 μg/mL solution. From this solution, aliquots were prepared and were analyzed at 295 nm⁷ and the results are summarized in Table 6.

Discussion

The developed method was validated as per the ICH guidelines. The method validation for each parameter was carried out. The linearity studies showed a good linear relationship over the concentration range of 1-10 µg/mL.

The drug solutions were analyzed for accuracy by the proposed method and the % recovery was found to be 135.27, 118.14, 108.56% with the % RSD of <2 which indicated no interference of the excipients used in the formulation. This indicates the accuracy of the developed method which can be used for the determination of Tacrolimus both in bulk and in formulation compared to the more tedious and time consuming HPLC method.

Robustness was analysed by making small changes in analytical wavelength by ± 2 nm i.e., 213 nm and 217 nm. The % RSD was found to be 0.330 and 0.419 respectively signifying the robustness of the developed method.

The ruggedness was obtained by different analysts i.e, analyst 1 and analyst 2. The % RSD was found to be 0.53, 0.80 respectively which is not more than 2, indicating that the method is rugged enough to variation in the analysts.

Marketed formulations were analysed by developed method at 295 nm. The drug concentration was calculated from the calibration curve of the drug and the % amount was found to be 99.1%.

Conclusion

The developed analytical method was validated as per ICH Q2 (R1) guidelines and it met the acceptance criteria for each parameter. The developed analytical method showed linearity in the concentration range of 1 to 10 µg/mL. The λmax was found to be at 215 nm with percentage relative standard deviation of 1.731 and 1.243% respectively. It can be concluded that the developed method is specific, linear, precise, accurate, robust and sensitive in analyzing Tacrolimus in bulk and in commercial dosage form. The main advantage of the developed UV method over HPLC method is that it is less time consuming and also economical. Thus, the developed method can be used for routine analysis of Tacrolimus in pure form as well as in the pharmaceuticals.

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication.