1. INTRODUCTION:

Famotidine is an antiulcer drug and is chemically N-(aminosulfonyl)-3-[[[2-[(diaminomethylene) amino]-4-thiazolyl] methyl] thio] propanimidamide.¹ Famotidine is potent histamine (H₂)-receptor antagonist that binds to the H₂ receptor in a competitive reversible. The H2RAs are reversible, competitive antagonists at histamine type-2 receptors, primarily on the parietal cells within the gastrointestinal mucosa².

It is official in and European Pharmacopoeia.³A literature survey revealed there is UV spectrophotometry, HPLC, HPTLC^4-9 methods for estimation of Famotidine in Pharmaceutical formulation. Dicyclomine hydrochloride is an anticholinergic drug and is chemically [Bicyclohexyl]-1-carboxylic acid, 2-(diethyl amino) ethyl ester hydrochloride which shows beneficial effects on anticholinergic effect (antimuscarinic) at the acetylcholine-receptor sites and second is a direct effect upon smooth muscle (musculotropic).¹⁰ A literature survey revealed that RP-HPLC^11-14method for the estimation of Dicyclomine Hydrochloride in Pharmaceutical formulation. The chemical structures of both drugs are shown in given figures.1

Fig.: 1(A) Chemical structure of Famotidine

Fig.: 1(B) Chemical structure of Dicyclomine HCl

The review of literature stated that various analytical methods involving spectrophotometry, HPLC, and HPTLC have been reported for famotidine in single form and in combination with other drugs. Several analytical methods have been reported for dicyclomine hydrochloride in single form and in combination with other drugs including HPLC, HPTLC methods. This paper is in continuation with our work ^17-21 where we studied spectrophotometric method for single or multicomponent drugs. However no references have been found for simultaneous equation method for estimation of famotidine and dicyclomine hydrochloride in combined tablet dosage form. The developed method was validated as per ICH guidelines and successfully applied for the assay of famotidine and dicyclomine hydrochloride in their combined tablet dosage form.

2. MATERIALS AND METHODS

2.1Chemicals and Reagents:

Famotidine was kindly gifted by Nakoda Chemical Ltd., Hyderabad while dicyclomine HCl was kindly gifted by Palam Pharma Pvt. Ltd., Ahmadabad. Tablet of famotidine (20mg) and dicyclomine hydrochloride (10mg) in combined dosage form (Emetec-Spas) was kindly gifted by Oyster Lab. Ltd., Haryana.

2.2. Instruments:

Double beam UV- visible spectrophotometer (Shimadzu, Model UV-1800) having two matched quartz cells with 1cm light path and loaded with UV probe software. Electronic Analytical balance (Anamed). Ultrasonicator (HMG India).

3. METHOD DEVELOPMENT:

3.1. Famotidine standard Stock Solution:

Accurately weighed reference standard of FAM (10 mg) was transferred to 100 ml volumetric flask and dissolved in 100 ml methanol as solvent, to obtain standard stock solution (100µg/ml) of drug. For the preparation of working standard, suitable aliquots of stock solution were pipette out and volumes were made up to the mark with solvent methanol to get required concentrations. The spectra of FAM is Shown in figure 2.

Fig. 2 spectrum of FAM

3.2. Dicyclomine Hydrochloride standard stock solution:

Accurately weighed reference standard of DIL (10 mg) was transferred to 100 ml volumetric flask and dissolved in100 ml methanol and sonicated for 10 min to obtain standard stock solution (100µg/ml) of drug. For the preparation of working standard, suitable aliquots of stock solution were pipette out and volumes were made up to the mark with methanol to get required concentrations. The spectra of DIL is shown in figure 3.

Fig.3 spectrum of DIL

3.3. Determination of λmax:

An absorbance maximum is determined of both API stock solutions separately to get concentration of 10μg/ml of FAM and DIL respectively with methanol and run the spectra in the range of 200-400 nm separately. The overlain spectrum is shown in figure 4.

Fig. 4 Overlay Spectrum of Famotidine and Dicyclomine HCl

3.4. Preparation of calibration curve

Stock solutions of 1mg/ml of FAM and 1mg/ml of DIL respectively were prepared in methanol. Appropriate aliquots of FAM and DIL from stock solution further diluted with methanol to obtain 20-120 µg/ml concentrations of FAM and 50-100 µg/ml of DIL. The calibration curve of FAM and DIL are shown in figure 5and 6 respectively.

Fig. 6: Calibration curve of DIL

Fig. 5: Calibration curve of FAM

3.5. Development of simultaneous equation

Absorbances of both the drugs were taken on selected 292 nm and 218 nm wavelength respectively. The absorptivity values were determined of both the drugs on both respective wavelengths. The concentration of drugs in sample solution is determined by following formula.

At 292 nm A₁= ax₁ C_F+ ay₁ C_D……………………. (1)

At 218 nm A₂= ax₂C_F+ ay₂ C_D……………………… (2)

Where, C_F, C_D, is the concentration of FAM and DIL respectively. A₁ and A₂ are absorbance’s of sample solutions on 292 and 218 nm respectively, ax₁ and ax₂ are absorptivity values of FAM at 292 and 218 nm accordingly, ay₁ and ay₂ are absorptivity values of DIL at 292 and 218 nm.

3.6. Determination of absorptivity coefficients at analytical wavelengths

The absorptivity coefficients for the two drugs were determined at both the selected wavelength. The value obtained as a mean of six independent determinations were used for forming the simultaneous equations. The simultaneous equation formed were

A₁=5.854×C₁+0.072×C₂------at 292nm (For FAM)

A₂=4.384×C₁+6.936×C₂------at 218nm (For DIL)

Where A₁ and A₂ are the absorbance of sample solution at 292 nm and 218 nm respectively and C₁ and C₂ are the concentration of FAM and DIL respectively (gm/lit) in the sample solution. By solving the two simultaneous equation, the concentration of FAM (C₁) and DIL (C₂) in a sample solutions can be obtained.

3.7. Analysis of marketed formulations

Accurately weighed twenty tablets were accurately weighed and average weight was calculated, triturated in mortar and pestle to form fine powder. The amount equivalent to 20 mg of FAM and 10 mg of DIL were weighed and dissolved in 100ml of methanol. Filter through Whatmann filter paper No. 41 and suitably diluted to get concentration of 20μg/ml and 10 μg/ml of FAM and DIL with methanol respectively. Its drug content was estimated from simultaneous equation method at 292 nm and 218 nm respectively.

4. METHOD VALIDATION:

The proposed method has been extensively validated according to ICH guidelines.

4.1Linearity

Solutions of FAM ranging from 20-120µg/ml and solutions of DIL ranging from 50-100µg/ml. The absorption spectra of above solutions were recorded in the range of 200 to 400 nm using methanol as blank. Linearity observed of FAM at 292nm and DIL at 218nm. The results are shown in table 1and 2 respectively.

Table 1: Linearity study data of FAM

Sr. No.	Conc. ( µg/ml)	Absorbance at 292nm
1	20	0.423
2	40	0.5828
3	60	0.8034
4	80	0.9726
5	100	1.188
6	120	1.381

Table 2: Linearity study data of DIL

Sr. No.	Conc. ( µg/ml)	Absorbance at 218nm
1	50	1.3785
2	60	1.5227
3	70	1.6831
4	80	1.8258
5	90	1.9886
6	100	2.1593

4.2. LOD and LOQ

LOD and LOQ were determined as per ICH guidelines, LOD was found to be 0.6944 and 0.4446μg/ml for FAM and DIL respectively and LOQ was found to be 2.1044 and 1.3474μg/ml for FAM and DIL accordingly. The results are shown in table 3.

Table 3: LOD and LOQ

Name of the drug	LOD (µg/ml)	LOQ (µg/ml)
FAM	0.6944	2.1044
DIL	0.4446	1.3474

4.3. Repeatability

It was done on concentrations of 20µg/ml and 10µg/ml for FAM and DIL respectively by suitably diluting the stock solutions with methanol. The % RSD was found to be 0.140 and 0.180 for FAM and DIL respectively. The % RSD is less than 2 indicate the given method can be repeatable.

4.4. Intermediate precision (Reproducibility)

Precision studies were carried out for intraday and inter-day variations of the responses. Study was carried out at concentrations of 20µg/ml and 10µg/ml for FAM and DIL respectively by suitably diluting the stock solutions with methanol. The inter-day and intraday precision was carried out and the results were found to be within limits. The results are shown in table 4 and 5.

Table 4: Statistical validation of intra-day precision data

Name of the drug	Mean*	SD*	%RSD*
FAM	100.0133	0.13051	0.130
DIL	99.986	0.032146	0.032

* Indicates average of six determinations

Table 5: Statistical validation of inter-day precision data:

Name of the drug	Mean*	SD*	%RSD*
FAM	100.003	0.1674	0.167
DIL	99.9966	0.047258	0.047

* Indicates average of six determinations

4.5. Recovery (Accuracy) Studies

To ascertain the accuracy of the proposed methods, recovery studies were carried at three different levels (80%, 100% and 120%) as per ICH guidelines. The percent recovery for FAM and DIC was found to be in range. The results are shown in table 6.

Table 6: Accuracy data of FAM and DIL

Level of Recovery	Amount present (mg)		Added concentration(mg)		Amount recovered (mg)		% Recovery
	FAM	DIL	FAM	DIL	FAM	DIL	FAM	DIL
	20	10	16	8	35.1	17.97	98	99.22
80%	20	10	16	8	35.7	18.11	99.44	100.62
	20	10	16	8	36.1	18.05	100.31	100.28
	20	10	20	10	39.6	20.02	99.14	100.14
100%	20	10	20	10	39.32	20	98.06	100
	20	10	20	10	39.15	20.01	99.72	100.09
	20	10	44	12	42.93	24.05	98	100.2
120%	20	10	44	12	43.2	23.77	98.18	99.04
	20	10	44	12	43.06	23.97	98	99.89

5. Forced Degradation Study

The stability indicating assay method is a method that is employed for the analysis of stability samples in pharmaceutical industry¹⁵. Stress testing of a drug substance can help in turns to establish the degradation pathways and the intrinsic stability of the molecule.¹⁶The quality of the finished product is very important from the point of view of its safety, acceptability and efficacy. Thus stability is considered as one of the most important criteria in pharmaceutical quality control as stable preparations would promise delivery of the drug to the patient. Force degradation studies carried out as per ICH guidelines to check the stability property of method. Various stress condition is applied on FAM and DIL were shown in table 7.

5.1. Acid Hydrolysis

Accurately weighed 20mg of FAM and 10mg of DIL, transferred to two separate 100mL volumetric flasks, added 50mL methanol and 10ml 0.1N HCl. These flasks were heated on water bath at room temp for 6hr. Solutions were cooled and neutralized with 0.1N NaOH. Finally these solutions were diluted with methanol to get 20µg/ml of FAM and 10µg/ml of DIL and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively.

5.2. Alkali Hydrolysis

Accurately weighed 20mg of FAM and 10mg of DIL, transferred to two separate 100mL volumetric flasks, added 50ml methanol and 10ml 0.1N NaOH. These flasks were heated on water bath at room temp for 6hr. Solutions were cooled and neutralized with 0.1N Hcl. Finally these solutions were diluted methanol to get 20µg/ml of FAM and 10µg/ml of DIL and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

5.3. Neutral hydrolysis

Accurately weighed 20mg of FAM and 10mg of DIL, transferred to two separate 100mL volumetric flasks, added 50ml methanol and 10ml Water. These flasks were heated on water bath at room temp for 6hr. Then these solutions were diluted with methanol to get 20µg/ml of FAM and 10µg/ml of DIL and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

5.4. Oxidative degradation

Accurately weighed 20mg of FAM and 10mg of DIL, transferred to two separate 100mL volumetric flasks, added 50ml methanol and 10ml H₂O₂ Solution (3%). These flasks were heated on water bath at room temp for 6hr. Then these solutions were diluted with water to get 20µg/ml of FAM and 10µg/ml of DIL and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

5.5. Photostability

Pure drugs were exposed to UV radiations for 6hrs. The samples after exposure to light were diluted with methanol to get FAM (20mg/ml) and DIL (10mg/ml) and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

5.6. Thermal degradation

Thermal degradation was carried out by exposing pure drugs to dry heat at 60^oC for 6hrs. The samples after exposure to heat were diluted with methanol to get FAM (20mg/ml) and DIL (10mg/ml) and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

5.7. Sunlight degradation

Sunlight degradation was carried out by exposing pure drugs in sunlight for 4hrs. The samples after exposure to sunlight were diluted with methanol to get FAM (20mg/ml) and DIL (10mg/ml) and absorbance was measured at 292 and 218 nm for FAM and DIL, respectively. Finally absorbance of sample was compared with standard absorbance and percent degradation was calculated.

Table 7: Force degradation study data

Sr. No.	Condition	% Degradation		% Assay
Sr. No.	Condition	FAM	DIL	FAM	DIL
1.	Acid hydrolysis (0.1N HCl, room temp,6hrs)	31.49%	17.4%	68.51%	84.25%
2.	Base hydrolysis (0.1N NaOH, room temp,6hrs)	13.61%	9.76%	86.38%	90.23%
3.	Neutral hydrolysis (H₂O,room temp 6hrs )	50.51%	8.3%	49.48%	91.69%
4.	Oxidative degradation (3% H₂O_2,room temp, 4hr)	31.06%	10.52%	68.93%	89.44%
5.	Photolytic degradation (UV-radiation, room temp,6 hrs)	42.36%	16.79%	57.63%	83.20%
6.	Thermal degradation (60ºC,room temp, 6hrs)	49.72%	10.09%	50.27%	89.91%
7.	Sunlight degradation ( keep under sunlight,4hr )	32.94%	13.04%	67.05%	86.95%

6. RESULT AND DISCUSSION:

As both the drugs are soluble in methanol, therefore estimation was carried out in methanol as solvent. The λ_max for detection of FAM and DIL were selected as 292nm and 218nm respectively. Absorbances of both the drugs were found to be 292 and 218 wavelengths. FAM and DIL solutions individually follow Beer-Lambert’s law over concentration range 20-120μg/ml and 50-100μg/ml respectively at selected wavelengths, with r²= 0.998 for FAM and r²=0.999 for DIL. The % assay of FAM was found to be in the range of 98.54 –99.2% with % S.D. 0.260 and % assay of DIL was found in the range of 101.7- 102% with% S.D. 0.1389. The % assay of both drugs in tablet formulation was found within acceptable limit (i.e. 98-102% as per ICH guidelines). Recovery studies were performed by adding a known amount of standard drug to pre-analysed samples and contents were pre-analysed by proposed method. The selected drugs FAM and DIL were estimated by using simultaneous estimation method as per ICH guidelines. The method was validated as per ICH guidelines. Limit of detection was found to be 0.6944µg/ml for FAM and 0.4446µg/ml for DIL. Limit of Quantification was found to be 2.1044µg/ml for FAM and 1.3474µg/ml DIL. The % RSD for intraday and inter-day precision was <2%. The method has been validated as per ICH guideline. The accuracy of the method was validated by recovery studies and was found to be significant and it is within specification limits, with % recovery 98-99.25. % for FAM and 99.71-100.04% for DIL (i.e. within acceptable range of 98-102%).

7. CONCLUSION:

The new UV spectrophotometric method for simultaneous estimation of Famotidine and Dicyclomine HCl was developed and it was found to be simple, sensitive, accurate, precise, and economic which can be used for routine analysis of Famotidine and Dicyclomine HCl in combine solid tablet dosage form. The developed method was successfully validated as per ICH guidelines. Hence it can be conveniently used as tool for quality control analysis of Famotidine and Dicyclomine HCl in combine pharmaceutical solid tablet formulations.

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Received on 14.10.2016 Modified on 06.11.2016

Research J. Pharm. and Tech. 2017; 10(2): 408-413.

DOI: 10.5958/0974-360X.2017.00082.8