INTRODUCTION

Analytical chemistry is the branch of chemistry involved in separating, identifying and determining the relative amounts of the components making up a sample of matter. It is mainly involved in the qualitative identification or detection of compounds and the quantitative measurement of the substances present in bulk and pharmaceutical preparation.⁽¹⁾It is the subject of science, which deals with interaction of radiation and matter. All atoms and molecules are capable of absorbing energy in accordance with certain restrictions, these limitations depending upon the structure of the substance. Spectroscopic analytical methods are based on measuring the amount of radiation produced and absorbed by molecular or atomic species. The kind and amount of radiation absorbed depends upon the number of molecules interacting with the radiation. The study of these dependencies is called absorption spectroscopy. Absorption spectroscopy is one of the most valuable analytical techniques; its advantages include simplicity, speed, specificity and sensitivity⁽²⁾. The parts of the molecule, that is, the atoms or groups of atoms, may move with respect to each other called as vibration and energy called as vibrational energy. The molecule may rotate about an axis; such rotation is characterized by the rotational energy. This mode of movement molecules possesses an electronic energy.⁽³⁾

E= E trans+ Evib+ Erot+ Eelect

Fingolimod is available as capsule at the dose of 0.5 mg in the market under the brand name of Gilenya. Fingolimod is a sphingosine 1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from contributing to an autoimmune reaction.⁽⁴⁾ Chemically it is 2-amino-2-[2-(4-octylphenyl) ethyl] propane-1,3-diol with molecular formula C₁₉H₃₃NO₂ and is presented in Figure No: 1.

Figure No: 1. Shows structure of Fingolimod⁽⁵⁾

Literature review ^(6-9) for Fingolimod analysis revealed there are very few methods based on different techniques. However there is no method reported for the detection of Fingolimod in bulk and pharmaceutical formulation by UV- Vis spectrophotometry. The aim of present work is to develop simple, sensitive, specific, spectrophotometric method and validation is to be carried out as per ICH guidelines.

MATERIALS AND METHODS:

Fingolimod working standard was supplied by Reddy Labs Ltd, Hyderabad. All other chemicals used in the analysis were AR grade. A double – beam spectrophotometer Labindia UV 3200 was used for the detection of absorbance, Afcoseter-200A (weighing balance) and Analytical Technologies Limited- Ultrasonic cleaner were used for experimental purpose.

Method:

Preparation of standard:

Accurately weigh and transfer 10 mg of Fingolimod standard into a 100 ml volumetric flask, add about 70 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.8ml of the Fingolimod stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. Water was used as diluent.

Preparation of sample:

Accurately weigh and transfer 10 mg of Fingolimod sample into a 100 ml volumetric flask, add about 70 ml of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent (Stock solution). Further pipette 0.8ml of the Fingolimod stock solution into a 10ml volumetric flask and dilute up to the mark with diluent. Water was used as diluent.

Figure No: 2. Shows spectrum for standard of Fingolimod

Method validation:

Method validation for Fingolimod in bulk drug was carried out as per ICH guideline.

Linearity:

The method was validated according to ICH Q2B guidelines for validation of analytical procedures in order to determine the linearity, precision and accuracy of the analyte. For Fingolimod, five point calibration curves were generated with the appropriate volumes of the working standard solutions for UV methods.

Precision and accuracy:

Precision is the degree of repeatability of an analytical method under normal operational conditions. The precision and accuracy were determined with standard quality control samples (in addition to calibration standards) prepared in triplicate at different concentration levels covering the entire linearity range. The precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) and reported as RSD % for a statistically significant number of replicate measurements. The intermediate precision was studied by comparing the assays on three different days and the results are documented as the standard deviation and RSD %. Accuracy is the percent of analyte recovered by assay from a known added amount.

Limit of detection:

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from back-ground levels. In this study, LOD is calculated from calibration curve.

Recovery study:

Recovery of the analyte of interest from a given matrix can be used as a measure of the accuracy or the bias of the method. The same range of concentrations, as employed in the linearity studies, was used.

RESULTS AND DISCUSSION:

The development of a simple, rapid, sensitive and accurate analytical method for the routine quantitative determination of samples will reduce unnecessary tedious sample preparations and the cost of materials and labour. Fingolimod is a UV-absorbing molecule with specific chromophores in the structure that absorb at a particular wavelength and this fact was successfully employed for their quantitative determinations using the UV spectrophotometric method. The λ Max of the drug for analysis was determined by taking scans of the drug sample solutions in the entire UV region. The λ Max of the drug was found to be 220nm.

Calibration curve data were constructed in the range of the expected concentrations of 4µg/ mL to 12µg/ mL. Beer’s law was obeyed over this concentration range. The correlation coefficient (r) of the standard curve was found to be 0.999. The stock solutions and working standards were prepared in water. Calibration curve and Linearity table is presented in Figure No: 3 and Table No: 1 respectively.

Table No: 1 Linearity study of Fingolimod

Linearity Level	Concentration	Absorbance
I	4µg/ml	0.1578
II	6µg/ml	0.2333
III	8µg/ml	0.3167
IV	10µg/ml	0.3932
V	12µg/ml	0.4726
Correlation Coefficient		0.999

Table No: 2 Data for Precision

Reading	Absorbance
Reading-1	0.3156
Reading-2	0.3152
Reading-3	0.3154
Reading-4	0.3156
Reading-5	0.3154
Reading-6	0.3149
Average	0.31535
Standard deviation	0.000266
%RSD	0.08

Table No: 4 Summary for assay of Fingolimod UV spectroscopy

S. NO	Validation parameters	Acceptance criteria	Observation
1.	Linearity range (µg/ml)	-	4-12
2.	Correlation coefficient	NLT 0.999	0.999
4.	Method precision	% RSD (NMT 2%)	0.12
5.	System precision	% RSD (NMT 2%)	0.08
	Intermediate precision	% RSD (NMT 2%)	0.13
7.	% Recovery	98% to 102%	98.9
8	Limit of Detection (µg)	NMT 3	0.16µg/ml
9.	Limit of Quantification (µg)	NMT 10	0.49µg/ml

Figure No: 3 Shows calibration curve for linearity of Fingolimod

Performing replicate analyses of the standard solutions was used to assess the accuracy, precision and reproducibility of the proposed methods. The selected concentration within the calibration range was prepared in water and analyzed with the relevant calibration curves to determine the intra- and inter day variability.

The accuracy of the method was shown by analyzing the model mixtures contained 50, 100 and 150% of Fingolimod within the linearity ranges were taken. After the injection of the standard solution, Accuracy -50%, Accuracy -100% and Accuracy -150% solutions, the Amount found, Amount added for Fingolimod, individual recovery and mean recovery values were calculated. The mean percentage recoveries were found to be 98.5%, 99% and 99.3% for 50%, 100% and 150% respectively. Accuracy data were present in Table No: 3.

The intra- and inter day precision were determined as the RSD %. Intraday precision (repeatability) study was done by measuring absorbance of 6 samples of same concentration, % RSD value was 0.08 which is less than 2% and well accepted according to guideline data is given in Table No: 2. Other parameter of assay of Fingolimod by UV-Vis Spectroscopy is given in Table No: 4.

The proposed methods can be successfully applied for Fingolimod assay in bulk drug form without any interference. The obtained results demonstrate the validity and accuracy of the proposed method for the determination of Fingolimod was within limit. These results reveal that the developed method have an adequate precision and accuracy and consequently, can be applied to the determination of Fingolimod.

CONCLUSION:

The developed Spectrophotometric method was simple, sensitive, and specific, for the detection of Fingolimod in bulk drug form. It could be precisely quantify and LOD was found to be 0.16µg/ml . All the calibration curves show a linear relationship between the absorbance and concentration and coefficient correlation was found to be 0.999. % RSD in Precision of the method was found to be 0.08. The percentage recovery was found to be 98.5-99.3%. The proposed method will be suitable for the analysis of Fingolimod in bulk drug form.

Bibliography:

1. Becket and Stenlake. Practical pharmaceutical chemistry. CBS publications and distributors, New Delhi 2005; 24^th ed: pp 157-168.

2. Sethi PD. HPLC quantitative analysis of pharmaceutical formulations. CBS publications and distributors, New Delhi, 2001; 1^st ed: pp 69-70.

3. Willard H. Instrumental method of analysis. CBS Publishers and Distributors, New Delhi,, 1986; 7^th ed: pp 28-34.

4. Available at: http://www.drugbank.ca/search?utf8=%E2%9C%93andquery=fingolimodandcommit=Search, retrieved on 4^th February 2013.

5. Available at: http://www.chemspider.com/Chemical-Structure.97087. html, retreived on 4^th February 2013

6. C. Emotte, F. Deglave, O. Heudi, F. Picard, O. Kret, Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with liquid chromatography–tandem mass spectrometry, Journal of Pharmaceutical Biomedical Analysis, 2012, 102-105.

7. K. Kathiresan, M. Bhagath Kumar Reddy, C.Moorthi, N. Ahamed Dawood Sha, Kiran Krishnan, R. Manavalan,Formulation and evaluation of Fingolimod capsules, International Journal of Pharmacy and Pharmaceutical sciences, 2011, 4(1), 282-292.

8. SN. Razzaq, IU. Khan, I. Mariam and SS. Razzaq, Stability indicating HPLC method for the simultaneous determination of Prednisolone and moxifloxacin in Pharmaceutical formulation, Chemistry central Journal, 2011, 1-10.

9. H. O. Kaila, M. A. Ambasana, R. S. Thakkar, H. T. Saravaia and A. K. Shah, A New Improved RP-HPLC Method for Assay of Rosuvastatin Calcium in Tablets, Indian Journal of Pharmaceutical Sciences, 2010, 72(5), 592-598.

Received on 05.11.2013 Modified on 07.12.2013

Asian J. Research Chem 7(1): January 2014; Page 55-57

%Concentration (at specification Level)	Absorbance	Amount Added (µg/ml)	Amount Found (µg/ml)	% Recovery	Mean Recovery
50%	0.1552	4	3.93	98.5	98.9%
100%	0.3120	8	7.92	99
150%	0.4696	12	11.91	99.3