DEVELOPMENT AND VALIDATION OF UV-VISIBLE SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF TACROLIMUS IN BULK AND PHARMACEUTICAL NANOPARTICLES

An accurate, simple, reproducible and cost-effective UV visible spectrophotometric method was developed and validated for the determination of Tacrolimus in nanoparticles. The optimum conditions for the analysis of the drug were established. The maximum wavelength (λmax) was found to be 291 nm. The response was linear in the range of 0.2-1.8 mg/ml (r2 = 0.9989). The relative standard deviations for intraand inter-day precision studies were found to be less than 2% which indicates method is precise.


INTRODUCTION
Tacrolimus (TAC), a macrolide agent, derived from Streptomyces Tsukubaensis, inhibits Tlymphocyte activation through a process that is thought to involve it binding to an intracellular protein, FKBP-12. [1] Tacrolimus is primarily used in post organ transplant patients to prevent organ rejection. [2] It is also used in a topical preparation in the treatment of severe atopic dermatitis, severe refractory uveitis after bone marrow transplants, and the skin condition vitiligo. [3] It is insoluble in water, slightly soluble in saturated hydrocarbons, and highly soluble in lipids and other organic solvents. [4] Pharmaceutical dosage forms such as capsules, injection and ointment are available for clinical use. In this study, we developed and validated a new UV visible spectrophotometric method for quantitation of tacrolimus in nanoparticles. The method was validated by following the analytical performance parameters suggested by the ICH guidelines. The aim of our investigation was to develop and validate an UV visible spectrophotometric method for the determination of tacrolimus in pharmaceutical dosage forms.

CHEMICALS AND REAGENTS
Tacrolimus was procured from Centurion laboratories private limited (Vadodara, India). Methanol was purchased from the Merck (India).

DETERMINATION OF λmax
First step in development of UV spectroscopic method is screening of each formulation component expected to be present in sample prepared for estimation of drug content in formulation and diffusion media over the entire UV range. Excipients should not interfere with drug peak at absorption maxima (λmax) of drug and if any excipients interfere with drug peak, then method should be modified accordingly.
The λmax (maximum absorbance) of Tacrolimus was determined by screening of 1.8 mg/ml drug solution respectively in methanol over entire UV range 200 -400 nm.

CALIBRATION CURVE OF TACROLIMUS DRUG IN METHANOL
Calibration curve was plotted for Tacrolimus drug in methanol (as a solvent) using UV-Visible spectrophotometer at 291 nm (λmax). It shows the linearity in the range 0.2 mg/ml -1.8 mg/ml with regression coefficient of 0.9989. Various values are shown in Table 1 and represented graphically in

METHOD VALIDATION
The method was validated in terms of parameters like specificity, precision, accuracy, linearity and range, LOD and LOQ. For all the parameters percentage relative standard deviation values were calculated. The proposed UV visible spectrophotometric method was validated as per ICH guideline.

ACCURACY
Accuracy was determined by adding the three different quantities [5,6]

PRECISION
The precision of an analytical procedure defines the degree of closeness of agreement between a series of measurements obtained from multiple samplings of homogenous sample under prescribed conditions. Precision of the method was reported as RSD% at different levels-repeatability, Intraday precision and Inter-day precision. [5,6] The results are shown in table 2.

REPEATABILITY
Repeatability was determined on 6 replicates of each concentration of the standard solution. [5] The results are shown in table 2.

LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION
The limit of Detection (LOD) and limit of Quantification (LOQ) of the developed method were determined by low concentrations of the standard solutions using the developed UV visible spectrophotometric method. [5] The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD for tacrolimus found to be 0.09 mg/ml.
The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ was 0.2 mg /ml for tacrolimus.

RESULT AND DISCUSSION
The UV visible spectrophotometric method described in this research article was developed to provide a rapid quality control determination of tacrolimus in any pharmaceutical nanoparticles.

CONCLUSION
A convenient and rapid UV visible spectrophotometric method has been developed for analysis of tacrolimus in nanoparticles. The assay provides a linear response across a wide range of concentrations. Low intra-day and inter-day %RSD coupled with excellent recoveries. The proposed method is simple, economic fast, accurate and precise for the quantification of tacrolimus in dosage form as well as bulk drugs for quality control purpose.