Modified Simultaneous Spectrophotometric Method Development for the Estimation of Duloxetine and Methylcobalamin in Bulk, Tablet and Capsule Dosage Forms

 

Sengamalam Radhakrishnan^1, Dr. S. Meena², Ravindran Muthukumarasamy³*

¹E.G.S. Pillay College of Pharmacy, Nagapattinam, Tamilnadu, India.

²College of Pharmacy, Shaqra University, Dawadmi, Kingdom of Saudi Arabia.

³Universiti Kuala Lumpur, Royal College of Medicine Perak, Faculty of Pharmacy and Health Sciences, Ipoh, Perak, Malaysia.

 

ABSTRACT:

Duloxetine is chemically N – methyl-3-(1-naphthyloxy)-3-(thiophen-2-yl)-propan-1-amine, is a balanced and potent reuptake inhibitor of serotonin and norepinephrine. Methylcobalamin is chemically Methyl-5, 6-dimethyl benzimidazolylcobalamin, is one of the two coenzyme forms of vitamin B₁₂ in the enzyme methionine synthase which functions to transfer methyl groups for the regeneration of methionine from homocysteine. The combination of these two drugs is used in relieving the complications of diabetic peripheral neuropathy. The aim of the current study is to develop a modified simultaneous spectrophotometric determination (Q-Absorbance ratio method) of duloxetine and methylcobalaminin bulk and pharmaceutical dosage form (tablet and capsule). Duloxetine has exhibited maximum absorbance at 288 nm and it transparent at 326 nm. The isobestic point was selected at 254 nm as both the drugs recorded maximum absorbance, also it was found wavelength difference is higher when compared to 278 nm. In replicate analysis (n=5) of the two drugs by the proposed UV method, the % label claim and % RSD of duloxetine and methylcobalamin were appreciably low for tablet and capsule formulation. The correlation between absorbance and concentration in terms of correlation coefficient (r) was found to be 0.9956 and 0.9979. The validation parameters observed proved that this method simple, sensitive, accurate and precise. Hence, the developed new method can be employed for the routine analysis of these two drugs in combined dosage forms.

 

 

 

INTRODUCTION:

Establishment of suitable assay procedure for the quantification of each ingredient of complex dosage formulation containing several therapeutically and chemically compatible drugs with identical chemical nature is a critical process. The presence of excipients, additives and decomposition products further complicates the analysis. Methods are developed for new products when no official methods are available and for already existing products to reduce the cost and time for better precision and ruggedness.¹

 

 

The Q analysis method is a modification of the simultaneous equation method. The ratio of absorbance of two different solution at two wavelength is constant. This constant is termed as ‘Q’ value. It depends on the property that, for a substance which obeys Beer’s Law at all wavelengths, the ratio of absorbances at any two wavelengths is a constant value which is independent of concentration or pathlength and therefore can be applied to assess the purity of compounds. The quantitative assay of two components in an admixture by the absorbance ratio method, the absorbances are measured at two wavelengths one being the l_max of one of the components (l₂) and the other being a wavelength of equal absorptivity of the two components (l₁) ie. iso-absorptivity point.^2-7

 

Duloxetine (Fig. 1), chemically N – methyl-3-(1-naphthyloxy)-3-(thiophen-2-yl)-propan-1-amine,⁸ is a balanced and potent reuptake inhibitor of serotonin (5- hydroxy tryptamine, 5-HT) and norepinephrine, (SSNRI-selective serotonin norepinephrine reuptake inhibitor). Duloxetine a novel compound binds selectively with high affinity to both norepinephrine and serotonin transporters and lacks affinity for monoamine receptor within the central nervous system. This helps to prolong the “mood lightening” effect of any released serotonin and norepinephrine. DULO (Duloxetine) also used to treat nerve pain in the feet, legs or hands due to nerve damage caused by poorly controlled diabetes. Therefore, it enhances the nerve signals within the central nervous system which naturally inhibit pain. However, it does not treat the underlying nerve damage but can help to reduce the pain.

 

Methylcobalamin (Fig. 2), chemically Methyl-5, 6-dimethylbenzimidazolylcobalamin,⁹ is one of the two coenzyme forms of vitamin B₁₂ (the other being adenosylcobalamin). It is a cofactor in the enzyme methionine synthase which functions to transfer methyl groups for the regeneration of methionine from homocysteine.¹⁰

 

 

Fig. 1: Chemical structure of Duloxetine

 

Fig. 2: Chemical structure of Methylcobalamin

Oral administration of CH₃B₁₂(Methylcobalamin) resulted in subjective improvement in burning sensations, numbness, loss of sensation and muscle cramps. An improvement in reflexes, vibration sense, lower motor neuron weakness and sensitivity to pain was also observed.

 

There is not much information on this combination however, there were studies published as individual determination. In a study using UV spectrophotometric method was developed for the assay of duloxetine hydrochloride in raw material and capsule was found to be simple, sensitive and accurate. Validation of the method yielded good results concerning range, linearity, precision and accuracy. The absorbance was measured at 290 nm for duloxetine solution. The linearity range was found to be 5-50 mg/mL for the drug. ¹¹Furthermore, the information was more on individual drug estimation using UV spectroscopy and High Performance Liquid Chromatography. Thus, the current study was designed inorder to fill the gap for future researchers by studying the modified simultaneous spectrophotometric method for the estimation of duloxetine and methylcobalaminin tablet and capsule dosage forms.

 

METHODOLOGY:

Preparation of standard stock solution:

Standard stock solution (1000 mg/mL) of duloxetine and methylcobalamin were prepared with 100 mg of accurately weighed drug substance, transferred to two separate 100 mL volumetric flask. Both drugs were dissolved completely with 25 mL of water and was diluted upto the mark with more water. 10 mL of the stock solution from each drug sample was further diluted to 100 mL using water to obtain working stock solution (100 mg/mL).¹²

 

Determination of isoabsorptive point and wavelength of maximum absorbance:

Working stock solution containing 10 mg/mL of duloxetine and 40 mg/mL of methylcobalamin were prepared separately and was scanned between 200nm to 400 nm against water as blank. The overlying spectrum was also obtained to determine the isoabsorptivepoint.¹³

 

Analysis of mixed standards using bulk drug:

Two individual mixed standards were prepared in the ratio of 40:1 containing 20, 40 mg/mL duloxetine and 0. 5:1 mg/mL methylcobalamin from standard stock solutions. The absorbances of mixed standard solutions were measured at 288 nm and 254 nm. The concentrations of duloxetine and methylcobalamin present in mixed standard were calculated by using Q analysis method to counter check whether proportionate results are suitable to carry out in formulation.

 

Analysis of tablet formulations:

Twenty tablets of duloxetine and methylcobalmin in combination were weighed, average weight of tablet was determined and powdered. The tablet powder equivalent to 20 mg of duloxetine and 0. 5 mg of methylcobalamin was weighed accurately and transferred to 100 mL volumetric flask, 60mL of water was added, vortexed for 5 minutes. Final volume was adjusted with more water. The solution was suitably diluted to get the concentration of 20 mg/mL and 0. 5 mg/mL duloxetine and methylcobalamin respectively.

 

Analysis of capsule formulations:

20 capsules of duloxetine and methylcobalamin in combination were weighed. Average content in a capsule was determined as capsule powder equivalent to 30 mg of duloxetine and 1. 5 mg of methylcobalamin was weighed, transferred to 100 mL volumetric flask and dissolved in 50ml of water. The contents were shaken in vortex mixer for 5 min finally volume was made up to the mark with water. The solution was filtered through Whatmann filter paper No. 1.

 

Recovery Studies for Tablet and CapsuleFormulations¹⁴:

The recovery studies of tablet and capsule formulations were performed individually using standard addition method. A known amount of standard duloxetine and methylcobalamin corresponding to 80%, 100% and 120% of label claim was added to the powder sample of tablet dosage from separately, absorbance was measured at 288 nm and 350 nm respectively.

 

RESULTS AND DISCUSSION:

The solutions of 10 mg/mL of duloxetine and 40 mg/mL of methylcobalamin were analyzed and two analytical wavelengths were selected, one being the isoabsorptive point (254 nm) and other being the wavelength of maximum absorption of one of the two components (288 nm ie., l_max of Duloxetine)The results were shown in Table no. 1. Duloxetine has exhibited maximum absorbance at 288 nm and it transparent at 326 nm. The isobestic point at 254 nm was selected because both of them have maximum absorbance and also wavelength difference is higher when compared to 278 nm.

 

The results on various optical characteristics determination of duloxetine and methylcobalamin by UV spectrophotometric method were shown in Table no. 2 which show the sensitivity, validity and reproducibility of the method. Table no. 3 shows the report of data of analysis for mixed standards.

 

Table no: 1 Selection of isobestic point and wavelength of maximum absorbance.

Duloxetine	288 nm
Isobestic point of Duloxetine and Methylcobalamin	254 nm, 326 nm, 278 nm

 

Table. no:2. Data for Optical Characteristics Of duloxetine and methylcobalamin by UV-Spectrophotometric Method.

S. No.	Parameters	Duloxetine	Methylcobalamin
1.	l max (nm)	288 nm	270 nm, 350 nm, 520 nm
2.	Molar absorptivity	182	142, 182, 59. 46
3.	Sandel’s Sensitivity	0. 0000182	0. 0000182
4.	Molar extinction Coefficient (l/mol. cm)	5412. 97	24467. 716
5.	Slope	0. 018358	0. 220276
6.	Intercept	0. 0110593	0. 41809
7.	Linearity (µg/mL)	10-80	10-80
8.	LOD	1. 4267	0. 5346
9.	LOQ	4. 323	1. 6202
10.	Regression Coefficient	0. 9956	0. 9979

 

In replicate analysis (n=5) of the two drugs by the proposed UV method, the % label claim and % RSD of duloxetine and methylcobalamin were appreciably low for tablet and capsule formulation. The lower values of RSD indicate that the method is precise and accurate. The % mean and % RSD recovery studies of duloxetine and methylcobalamin are appreciably low for tablet and capsule. The average percentage recoveries obtained by the proposed method ranged from 97. 77 to 99. 95 % which indicates adequate accuracy and precision, also free from any positive and negative interference of the excipients. The low value of standard deviation obtained confirms the precision of the method. The assay results obtained by the proposed method are in good agreement with the labelled amount. The results were shown in Table no. 4.

 

The regression equation was calculated by the method of least square, each of which was the average of five independent determinations. The correlation between absorbance and concentration in terms of correlation coefficient (r) was 0. 9956 and 0. 9979. Various statistical validations of duloxetine and methylcobalamin was shown in Table no 5 and Table 6.

 

 

Table: 3 - Data for analysis of mixed standards.

DULO: Duloxetine; CH₃B₁₂ – Methylcobalamin.

Note: The values shown were on the mean of 5 determinations.

Label claim (Tablet) – 20 mg (DULO) + 5 mg CH₃B₁₂.

Label Claim (Capsule) - 30 mg (DULO) + 1. 5 mg CH₃B₁₂.

Table no. 4: Recovery studies for Tablet and Capsule formulation. Qm = 1. 5443

* mean of 5 determinations.

DULO: Duloxetine; CH₃B₁₂ – Methylcobalamin.

 

Table no. 5: Data for Statistical Validation of Duloxetine.

 

Table no. 6: Data for Statistical Validation of Methylcobalamin.

 

By observing the validation parameters, the method was found to be simple, sensitive, accurate and precise. Hence the method reported can be employed for the routine analysis of these two drugs in combined dosage forms.

 

CONCLUSION:

The reported UV spectrophotometric method is simple, rapid, economical, reproducible and accurate for modified simultaneous spectrophotometric method estimation of duloxetine and methylcobalamin in bulk and combined oral dosage formulations using Q absorbance ratio method. It does not suffer from any interference due to common excipients present in pharmaceutical preparations and it can be conveniently adopted for quality control analysis.

 

REFERENCES:

1.       Snyder LR, Glajch JL and Kirkland JJ, Practical HPLC Method Development. John Wiley2^nd. 1988.

2.       Beckett AH and J. B. Stenlake JB. Practical Pharmaceutical Chemistr. CBS Publishers and Distributers. 2005.

3.       Balyejjusa, SA Dome RO and Musoke D. Spectrophotometric determination of Sulphamethoxazole and Trimethoprim (Co-trimoxazole) in binary mixtures and in tablets. African Health Sciences. 2002; 2(2):56–62.

4.       Ahmed MMJ and Uddin MN. A simple spectrophotometric method for the determination of cobalt in industrial, environmental, biological and soil samples using bis(salicylaldehyde)orthophenylenediamine. Chemosphere, 2007; 67: 2020-2027.

5.       Erk N. Three new spectrophotometric methods applied to the simultaneous determination of hydrochlorothiazide and irbesartan. Pharmazie. 2003; 58, 543-548.

6.       Erk N. Simultaneous determination of fosinopril andhydrochlorothiazide in pharmaceutical formulations by spectrophotometric methods. J. Pharm. Biomed. Anal. 2002; 27: 901-912.

7.       J. B. Cashtledine, A. F. Fell, R. Modin and B. Sellberg., J. Pharm. Biomed. Anal, 1991, 9, 619-624.

8.       Duloxetine Hydrochloride” http://wiki. medpedia. com/Clinical: Cymbalta_(Duloxetine_hydrochloride), January 2013.

9.       Drug profile of Methylcobalamin through www. Wikipedia. com

10.     Snyder LR, Kirkland JJ and Glaichy J. Practical HPLC Method Development. John Wiley. 1997.

11.     Kamila MM, Mondal N and Ghosh LK. A validated UV spectrophotometric method for determination of duloxetine hydrochloride. Pharmazie. 2007; 62, 414-415.

12.     Sengamalam R, Ravindran M, Manish Gunjan, Meena S. Analytical method development and dissolution profile of Duloxetine and Methylcobalamin by Vierodt’s Method. Journal of Pharmacy Research. 2011; 4(2): 449-451.

13.     Beckett AH and Stenlake JB. Practical Pharmaceutical Chemistry. CBS Publishers and Distributers. 2005.

14.     Ahuja S and Scypinski S. Handbook of Modern Pharmaceutical Analysis. Academic Press. 2001.

 

 

 

Received on 19.12.2018          Modified on 16.01.2019

Accepted on 09.02.2019        © RJPT All right reserved