INTRODUCTION:

Psoriasis is a chronic inflammatory and disabling auto-immune disease that affects millions of individual world-wide. In general, psoriasis may occur at any age but it is most common in the age group of 50 to 69 years¹. The basic parts of the body involved in psoriasis are skin and nails along with a number of comorbidities. It is clinically characterized by erythematous, sharply demarcated papules and rounded plaques covered by silvery micaceous scale. Epidermal hyper proliferation and immune-mediated dermal inflammation in psoriasis lead to profound adverse effects on patient’s physical, social and mental well-being².

Drugs such as silymarin, calcipotriol, betamethasone dipropionate, sirolimus, cyclosporine, dithranol, acitretin, capsaicin, mycophenolate mofeteil etc are used orally and topically to treat the disease. Mycophenolate mofetil (MMF) is very effective among them. It is 2-(morpholin-4- yl) ethyl (4E)-6-(4hydroxy- 6-methoxy-7-methyl-3-oxo l, 3 dihydroisobenzofuran- 5-yl)-4- methylhex-4-enoate (Fig. 1). It belongs to BCS Class II of drugs characterized by a strongly pH-dependent solubility profile³. It is an immune-suppressant and prodrug of mycophenolic acid (MPA), extensively used to prevent rejection in organ transplantation⁴. MPA, a product of a Penicillium fungus, was originally isolated in 1896. MPA has anti-neoplastic, anti-viral, antifungal and immunosuppressive activity⁵. MMF has recently been added to therapeutic regimens for psoriatic skin disorder⁶. It is commercially available as capsules (250 mg), immediate-release tablets (500mg), delayed-release tablets (180 and 360mg) and oral suspension (200mg/ml). Its oral use is associated with side effects such as nausea, leucopenia, sepsis and diarrhoea⁷. That’s why chronic use of the drug for psoriasis is not advisable for the patients.

Fig. 1: Structure of Mycophenolate Mofetil

Topical treatment of psoriasis is more effective than oral treatment as it reduces undesirable side effects associated with the drugs⁸. A lot of novel topical formulations such as liposome, noisome, microemulsion based gel etc are being researched for effective treatment of psoriasis. Baboota et al.⁹ and Ali et al.¹⁰ reported microemulsion based formulations for treatment of psoriasis.

Development of any microemulsion at preformulation stage requires solubility study of drug in various oils, surfactants, co-surfactants or solubilizers. Additionally, characterization of formulations at development stage need estimation of drug in formulations. Use of HPLC and other sophisticated instruments for the same is quite expensive and time consuming considering the analysis of huge numbers of samples as well as chemicals and solvents requirements. That’s why, development and validation of a simple and accurate UV-spectrometric method is quite essential. There are few published methods for estimation of MMF by UV-spectrophotometric method. Reddy et al. reported one such method using first and second derivatives UV which is complicated and analysis is not straight forward and conventional (zero order) UV method¹¹. Other UV methods were used for estimation of MMF in tablet formulations^12,13. However, commonly used excipients for tablet formulation are different from the excipients used for microemulsion formulation. It is important to ensure there is no interference of excipients with active drug for its estimation by UV method during development of microemulsion formulation. No UV method was reported for microemulsion formulation of MMF.

So, the main objective of this work was to develop and validate a simple and sensitive UV spectrophotometric method in order to estimate MMF useful for development of its microemulsion formulation. Various analytical parameters such as linearity, precision, accuracy, robustness, limit of detection (LOD) and limit of quantification (LOQ) were evaluated as per ICH Q2 (R1) guidelines¹⁴.

METHODOLOGY:

Materials:

SHIMADZU UV-1800 double beam UV-Vis spectrophotometer equipped with 1cm matched pair of rectangular quartz cells was used for the present study. Mycophenolate mofetil pure drug was obtained as a gift sample from Conchord Biotech, Ahmedabad, India. Labrasol, transcutol P, lauroglycol 90 and capryol 90 were obtained as gift samples from Gattefosse India Private Limited, Mumbai. Super refined oleic Acid, medilan super-SO, super refined NOVOL and arlasolve were obtained as gift samples from Croda, USA. Various oils such as black seed oil, fish liver oil, linseed oil, shisham oil, almond oil, olive oil and lavender oil were purchased from local market, Bathinda, Punjab, India. Tween 80 was obtained from Loba Chemie Pvt. Ltd., India. Ethanol and methanol were obtained from Merck, Germany. All the chemicals used were of analytical grade.

Preparation of Calibration samples

Standard stock solution of MMF was prepared by dissolving 50mg of MMF in 50ml of methanol which gave a concentration of 1000µg/ml. The secondary stock solution of 100µg/ml was prepared by taking 10ml from standard stock solution and diluted upto 100ml with methanol. Volumes of 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5 ml, 3.0ml and 3.5ml were pipetted out into separate 10 ml volumetric flasks and final volume was made upto 10ml with methanol to produce concentrations of 5µg/ml, 10µg/ml, 15µg/ml, 20µg/ml, 25µg/ml, 30µg/ml and 35µg/ml, respectively.

Linearity:

The linearity of an analytical procedure is its ability to acquire test results which are directly proportional to the concentration of analyte in samples within a given range within the purview of Beer-Lambert law. The various aliquots ranging from 5-35µg/ml were prepared from secondary stock solution (100µg/ml). Concentration of 10µg/ml was scanned in UV-Vis Spectrophotometer against methanol as blank to find out λmax. All the samples were prepared in triplicate and the absorbances of respective concentrations were measured at 250 nm (λmax).

Calibration curve was constructed with absorbance (y-axix) against concentration (x-axix) followed by estimation of coefficient of correlation using Microsoft excel. The overlay spectrum containing all the calibration samples was also obtained.

LOD and LOQ:

Limit of detection (LOD) is the lowest amount of analyte in the sample that can be detected, but not necessarily quantitated as an exact value, under the stated conditions of the test. Limit of quantification (LOQ) is the lowest amount of analyte in the sample that can be quantitatively determined with an acceptable precision and accuracy under the stated conditions of the test. The LOD and LOQ of the proposed method were determined from calibration standards by using following equations:

Equation 1

Equation 2

Where,

σ is standard deviation of intercepts of calibration curve equations.

S is the mean of slopes of the related calibration curve equations.

Accuracy and Precision:

Accuracy and precision were determined at three different levels of target concentration: 16µg/ml (80%), 20µg/ml (100%) and 24µg/ml (120%). A total of 3 replicates of each of those samples were analysed. Precision is expressed as % coefficient of variation (%CV) while accuracy is measured as % nominal as per the following equations.

Equation 3

Equation 4

Where,

S.D. is Standard Deviation.

Robustness:

Robustness of the proposed method was determined by carrying out analysis of 20µg/ml with different wavelengths (245nm, 250nm, 255nm) while other working parameters of the UV-Spectrophotometer remains the same.

Stability Study:

These include testing of samples that may result in some changes during storage and are likely to influence quality, safety and efficacy of the desired product. Stability of the proposed method was checked on 20µg/ml by storing it for 24 hours followed by measuring its absorbance. The study was repeated in triplicate (n=3).

Solubility studies:

The solubility studies of MMF in various oils (oleic acid, blackseed oil, fish liver oil, linseed oil, shisham oil, almond oil, olive oil and lavender oil), surfactants (tween 80, labrasol and acconon MC8-2) and cosurfactants/ solubilizers (capryol-90, lauroglycol, arlasolve, transcutol P and ethanol) were carried out. In this, the excess amount of MMF was added in 1ml of each selected oils, surfactants and cosurfactants in a centrifuged tube and mixed by vortex shaker for 5-10 minutes. Then the tubes were placed in a mechanical shaker and allowed to mix them for 72 hours. Then, the samples were centrifuged at 4000 rpm for 10 minutes to remove excess amount of undissolved drug. The supernatant was filtered through filter paper. From each filtered samples, 0.1ml was withdrawn and diluted upto 10ml with methanol. Further, 0.1ml was taken from above sample solution and diluted upto 10ml with methanol. The samples were prepared in triplicate and the absorbance of each of selected oils, surfactants and cosurfactants/ solubilizers were determined by UV Spectrophotometer at 250nm.

RESULTS:

Analytical procedure explains the method by which analysis can be performed and describes the steps necessary for analytical test. Validation of an analytical method establishes documented evidence and provides a high degree of assurance that the method will consistently produce a desired result meeting its predetermined specifications and quality characteristics. The representative sample (10µg/ml) of MMF showed three major peaks at 215nm, 250 nm, and 304 nm. In spite of maximum absorbance as compared to other two peaks, 215 nm was not selected for our study assuming it might have interaction with microemulsion compositions. Between 250 nm and 304 nm, 250 nm was chosen as λ_max due to its higher absorbance value. The overlay graph of all the calibration samples is shown in Fig. 2.

Fig. 2. Overlay spectra of Mycophenolate mofetil using UV Spectrophotometer

Linearity, LOD and LOQ:

The calibration curve was prepared within a range of 5µg/ml to 35µg/ml by plotting concentration in x-axis and absorbance in y-axis. The obtained absorption values of individual calibration standards for three replicates are presented in Table 1. The constructed calibration curve (Fig.3) was found to be linear within the investigated concentration range with equation y = 0.0219x - 0.006 and coefficient of correlation value 0.9989. The LOD and LOQ for the developed UV method were found to be 0.796µg/ml and 2.412µg/ml, respectively.

Table 1: Linearity of Mycophenolate mofetil by UV Spectroscopy

Concentration (µg/ml)	Absorbance 1	Absorbance 2	Absorbance 3	Mean
5	0.091	0.103	0.099	0.097
10	0.227	0.225	0.218	0.223
15	0.317	0.323	0.328	0.322
20	0.433	0.412	0.421	0.422
25	0.523	0.548	0.555	0.542
30	0.643	0.658	0.68	0.660
35	0.754	0.750	0.761	0.755

Accuracy and Precision:

Accuracy & precision were determined by 3 samples at 80%, 100% and 120% levels. Nominal % results represent accuracy data while % CV results are used for precision. Accuracy of the developed UV method ranged from 100.95% to 102.54%, whereas precision results were within a range of 1.02 to 1.14%. Both the accuracy and precision results were within the limits of ICH guidelines. The results of accuracy and precision for the developed UV method are given in Table 2.

Fig. 3: Calibration curve of Mycophenolate Mofetil

Table 2: Accuracy and precision data by UV method

Actual conc. (µg/ml)	Observed conc. (µg/ml)			Mean conc. (µg/ml)	S.D.	Nominal %(accuracy)	CV% (precision)
16 (80%)	16.174	16.435	16.609	16.406	0.219	102.54	1.33
20 (100%)	19.957	20.261	20.348	20.189	0.205	100.95	1.02
24 (120%)	24.348	24.435	24.870	24.551	0.280	102.30	1.14

Table 3: Robustness data at different wavelengths in UV Spectroscopy

Wavelength (nm)	Actual Conc. (µg/ml)	Observed Conc. (µg/ml)			Mean conc. (µg/ml)	% deviation
245	20	18.652	19.218	18.913	18.928	7.03
250	20	19.957	20.261	20.348	20.189	Considered 100%
255	20	17.435	17.609	17.304	17.449	13.57

CONCLUSION:

The developed UV method was validated and all the validation parameters were within the acceptable limits of ICH Q2 (R1) guidelines. The method can be applied successfully for estimation of mycophenolate mofetil in bulk and solubility samples required at preformulation stage for microemulsion formulation of the drug. The method is easy and economical compared to expensive and time consuming HPLC method.

ACKNOWLEDGEMENTS:

Authors acknowledge contributions of gift samples from Conchord Biotech, Ahmedabad, India; Gattefosse India Private Limited, Mumbai, India and Croda Inc, USA.

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest regarding publication of this article.

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Received on 29.05.2019 Modified on 19.06.2019

Research J. Pharm. and Tech. 2019; 12(10):4777-4781.

DOI: 10.5958/0974-360X.2019.00824.2