Development and validation of UV Spectrophotometric method for quick estimation of milnacipran HCl in dissolution media.

 

Nayan C. Ratnakar¹*, K. N. Patel², D. B. Doshi³

¹Research Scholar, Singhania University, Jhunjhunu, Rajasthan.

²SAL Institute of Pharmacy, Ahmedabad.Gujarat.

³L. M. College of Pharmacy, Ahmedabad, Gujarat.

*Corresponding Author E-mail: nayanratnakar@gmail.com

 

 

 

INTRODUCTION:

Milnacipran HCl (racemic mixture with the chemical name: (±)-[1R ( S ) , 2 S ( R ) ] - 2 - (a m i n o m e t h y l ) - N , N -diethyl-1-phenylcyclopropanecarboxamide hydrochloride)  is a selective serotonin and norepinephrine reuptake inhibitor (SNRI) indicated for the management of fibromyalgia. It inhibits norepinephrine uptake with greater potency than serotonin. ¹ Fibromyalgia (FM) is a multisymptom condition characterized by chronic widespread pain, functional disability, physical deconditioning, and a variety of other symptoms.²

 

Analysis is an important component in the formulation development of any drug molecule. A suitable and validated method has to be available for the analysis of drug(s) in the bulk, in drug delivery systems, from release dissolution studies and in biological samples.  If a suitable method, for specific need, is not available then it becomes essential to develop a simple, sensitive, accurate, precise, rapid and reproducible method for the estimation of drug samples.³

 

Milnacipran is not official in any pharmacopoeia. Literature review reveals, there is no report of UV-Visible Spectrophotometric method for its estimation. Only few analytical techniques including high performance liquid chromatography (HPLC) with fluorescence detection in human plasma and few chiral HPLC methods are available.^4,5 So, an attempt was made to develop a simple, accurate, precise and rapid Spectrophotometric method for the estimation of the milnacipran HCl in bulk and pharmaceutical tablet dosage form in phosphate buffer pH 6.8. In this study, a simple UV Spectrophotometric method was developed and validated as per International Conference on Harmonization (ICH) guidelines

 

INSTRUMENT AND MATERIALS:

Materials

Milnacipran HCl was gifted from Torrent Research Centre (Gandhinagar, Gujarat, India). All chemical and reagents used were of analytical grade. Two marketed tablets of Milnacipran HCl were procured from local market.

 

Instrument

A double-beam Shimadzu UV- Visible spectrophotometer, 1700 with spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm and a pair of 1-cm matched quartz cells was used to measure absorbance of the resulting solution.

 

Method

Preparation of phosphate buffer pH 6.8 Solution

Solution of phosphate buffer pH 6.8 was prepared according to Indian Pharmacopoeia 2007.⁶

 

Preparation of milnacipran HCl standard solutions

Accurately weigh 10 mg milnacipran HCl was transferred in 100 mL volumetric flask. It was dissolved and diluted up to mark with phosphate buffer pH 6.8 to obtain stock solution (100μg/mL). The standard solutions in concentration range of 2-40 μg/mL were prepared by dilutions of the standard stock solution with phosphate buffer pH 6.8. The determination was conducted five times at room temperature.

 

Preparation of Sample Solution

Twenty tablets were weighed to obtain the average tablet weight, which were then powdered Powder equivalent to 10 mg of milnacipran HCl was weighed and transferred to 100 ml  volumetric flask containing 70 ml phosphate buffer pH 6.8. This mixture was sonicated for 20 minute to ensure complete solubility of the drug and filtered through Whatman filter paper No. 41. The volume was made up to mark with phosphate buffer pH 6.8 to get solution having milnacipran HCl 100μg/mL.

 

Scanning for l_max

The standard solution of milnacipran HCl was scanned in the wavelength range of 400 nm to 200 nm using UV Spectrophotometer.

 

Preparation of Calibration curve:

A calibration curve was constructed over a concentration range 2-40 μg/mL. Absorbance of each solution was measured at the wavelength of 223 nm. Calibration curve was constructed for milnacipran HCl by plotting concentration (μg/mL) vs. absorbance at 223 nm. The determination was conducted five times.

 

Validation of Proposed UV Spectrophotometric method

The developed UV Spectrophotometric method was validated for linearity range, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) parameter as per ICH guideline.⁷

Linearity range

The linear response of the drug was verified at 2-40 μg/mL concentrations with five separate series of solutions of milnacipran HCl. The calibration graphs were obtained by plotting concentration (μg/mL) vs. absorbance data and were treated by linear regression analysis.

 

Accuracy

Accuracy of the proposed method was studied by recovery experiments. The recovery experiments were performed by adding known amounts to tablet. The recovery was performed at three levels of drug concentrations lower concentration (80%), intermediate concentration (100%) and higher concentration (120%) of milnacipran HCl standard concentration. The recovery samples were prepared in afore mentioned procedure. Three samples were prepared for each recovery level. The solutions were then analyzed by the proposed UV Spectrophotometric method and the percentage recoveries were calculated from the linearity curve.

 

Precision

The reproducibility of the proposed method was determined by performing assay at different time intervals (morning, afternoon and evening) on same day (Intra-day assay precision) and on three different days (Inter-day precision). Result of intra-day and inter-day precision is expressed in % RSD.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of milnacipran HCl were determined by using standard deviation of y-intercept and slope approach as defined in International Conference on Harmonization (ICH) guideline.

 

RESULT AND DISCUSSION:

The λ_max of drug in phosphate buffer pH 6.8 was determined using UV‐Spectrophotometer. The λ_max was determined by scanning standard solution of drug in the test medium in the range of 400‐200 nm. The λ_max was found to be 223 nm.

 

Figure 1 UV spectrum of milnacipran HCl in phosphate buffer pH 6.8

 

Figure 2 Linearity curve of milnacipran HCl in phosphate buffer pH 6.8

 

The content of drug was calculated from the equation y = 0.026x + 0.085. These solutions obeyed Beer-Lambert’s law in concentration range of 2-40 μg /mL with regression coefficient (r2) values 0.997 which indicates excellent linearity of the proposed UV Spectrophotometric method.

 

Accuracy can also be associated with the term bias. A biased estimate is systematically either higher or lower than the true value. Thus, for accuracy, recovery studies were carried out and the percentage recovery was found to be in the range of 99.82-101.23 %.Result of recovery study close to 100 % indicates good accuracy of the proposed UV Spectrophotometric method.

 

 

The precision of an analytical method or a test procedure is referred to as the degree of closeness of the result obtained by the analytical method. Repeatability was found to be 0.41%. The intraday and inter day precision were found to be 0.64-0.78% and 0.73-1.30% respectively. The values obtained were below 2% indicating the proposed UV Spectrophotometric method was sufficiently precise.

 

Limit of detection (LOD) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under a stated experimental condition and the limit quantification. (LOQ) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. These two parameters are required for assay validation as per ICH Q2A guidelines. LOD & LOQ values were found to be 0.27 μg /mL and 0.80 μg /mL respectively. This data shows that this method is sensitive for the determination of milnacipran HCl.

 

Validation Parameter

Table No 2: Validation Parameters

Sr. No	Parameter	Result
1	Wavelength Maxima(nm)	223
2	Linearity range(μg /mL)	2-40
3	Standard Regression Equation	y = 0.026x + 0.085.
4	Regression  coefficient (r2)	0.997
5	LOD	0.27 μg /mL
6	LOQ	0.80 μg /mL
7	Intra day precision(%RSD)	0.64-0.78%
8	Inter day precision(%RSD)	0.73-1.30%
9	Repeatability(%RSD)	0.41

 

CONCLUSION:

The developed method was found to be simple, sensitive, accurate, precise, reproducible and economic and can be used for routine quality control analysis of milnacipran HCl in bulk and pharmaceutical formulation

 

REFERENCES:

1.       Forest Pharmaceuticals, Highlights of Prescribing Information about SAVELLA® Tablets, Available from: URL: http://www.frx.com/products/.

2.       Kathryn L, Fibromyalgia and Savella (milnacipran HCl): An Overview for the Pharmacist. Pharmacytimes, 2010:202-210

3.       Shelke Santosh et al, Development and Validation of UV Spectrophotometric Method of  Cefuroxime Axetil in Bulk and Pharmaceutical Formulation., Asian Journal of  Research in Chemistry. 2(2): April.-June, 2009

4.       Angela P et al, Chiral HPLC analysis of milnacipran and its FMOC-derivative on cellulose-based stationary phases, Chirality 20:2007: 63 – 68.

5.       Marie Lecoeur L, Raphael D, Jean Paul R and Philippe M, Chiral analysis of milnacipran by a nonchiral HPLC - circular dichroism: Improvement of the linearity of dichroic response by temperature control. J Separ Sci 31:2007: 3009 – 3014.

6.       Indian pharmacopoeia 2007,the controller of India, New Delhi, India.

7.       International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, ICH harmonised tripartite guideline. Validation of analytical procedures: methodology (cited 1996) Available     from: http://www.ich.org

 

 

 

 

Received on 11.02.2012         Modified on 05.03.2012

Accepted on 18.03.2012         © AJRC All right reserved