INTRODUCTION:

Alosetron hydrochloride (AST) chemically is 2, 3, 4, 5-tetrahydro-5-methyl-2-[(5-methyl-1H-imidazol-4-yl) methyl]-1H-pyrido [4,3-b]indol-1-one,Monohydrochloride. Alosetron is achiral and has the empirical formula of C₁₇H₁₈N₄O.HCl, representing a molecular weight of 330.8gms/mol. It is a potent and selective antagonist of the serotonin 5-HT₃ receptor type. Activation of these receptors affects the regulation of visceral pain, colonic transit, and GI secretions. By blocking the action of serotonin on the intestinal system, the receptors are able to effectively control Irritable Bowel Syndrome (IBS). This reduces the cramping, stomach pain, stomach discomfort, urgency, and diarrhea caused by IBS. 5-HT₃ receptors are nonselective cation channels that are extensively distributed on enteric neurons in the human gastrointestinal tract, as well as other peripheral and central locations.

AST inhibit activation of non-selective cation channels, results in the modulation of the enteric nervous system in neuronal depolarization affect^1-6.

The review of literaturerevealed that no analytical methods have been reported on Alosetron in Pharmaceutical dosage forms so far. Hence author has attempted to develop a novel, simple, precise and rapid UV Spectrophotometric method for the estimation of Alosetron hydrochloride(AST) in bulk and pharmaceutical dosage forms and the developed method was validated in accordance with ICH guidelines ⁷and which can successfully used for the assay of AST in the Pharmaceutical dosage form.

Figure: 1 Structure of Alosetron hydrochloride (AST)

MATERIALS AND METHODS:

Equipments:

ELICO SL 210 UV Double beam Spectrophotometer having Spectra Treats (3.11.1) Software with a pair of 1cm matched quartz cells was employed to measure absorbance of all solutions. Digital balance (Essae) of 1 mg sensitivity was used for weighing.

Chemicals and Reagents:

Alosetron hydrochloride was received as a gift sample from MSN Laboratories Ltd., Hyderabad. The marketed tablet dosage form of Lotronex 0.5mg and 1mg tablets manufactured by Prometheus Laboratories Inc. was procured from foreign pharmacy. Double distilled water (in house laboratory) was used in the following experimental work.

ANALYTICAL METHOD DEVELOPMENT:

Preparation of Standard Solution of Alosetron hydrochloride (AST):

Accurately weighed 10 mg of reference standard of AST was transferred into a 10 ml volumetric flask and dissolved in double distilled water. Then volume was adjusted up to the mark with double distilled water to obtain stock standard concentration of 1mg/ml. Further 1ml of the above stock solution was diluted to 10 ml with double distilled water to obtain 100 μg/ml concentration of working standard solution. Then it was further diluted into 10 ml volumetric flasks to obtain linearity solutions of concentration ranging from 2-10 µg/ml.

Preparation of Sample Solution from Tablet Dosage Form:

20 tablets were weighed and crushed to a fine powder. A quantity of the tablet powder equivalent to 10 mg of AST was accurately weighed and transferred to 10 ml volumetric flask and dissolved in double distilled water. The resulting solution was filtered through Whatmann filter paper no. 41. Further dilutions were carried out with double distilled water to obtain a solution of 10µg/ml concentration respectively.

Figure: 2 Absorption spectrum of AST (10µg/ml in Distilled water)

Estimation of absorption maxima of AST:

The absorption maxima of Alosetron hydrochloride (AST) was measured by scanning standard solution of concentration 10µg/ml in the wave length range from 200-400nm. The λ_maxof AST was found to be 218 nm as shown in figure 2. The absorbance of linearity solutions were recorded at the selected wavelength (218 nm) to obtain overlay spectra as shown in figure 3.

Figure: 3 Overlay spectrum of AST (2-10 μg/ml at 218 nm)

RESULTS:

METHOD VALIDATION:

The developed method has been validated as per ICH Q2 (R1) guidelines by means of the following parameters:

Linearity:

The linearity was evaluated by analyzing different concentrations of AST solutions. Appropriate aliquots (0.1-1.0 ml) of AST working standard solution (100µg/ml) were transferred into the series of 10ml volumetric flasks and the volume was made up to the mark with distilled water. The absorbance of the above linearity solutions were measured at 218 nm against distilled water as blank solvent. A calibration curve was constructed by plotting absorbance versus concentration. The graph was found to be linear as shown in Figure 4 and regression equation was calculated. The results were shown in Table 1.

Figure: 4 Calibration curve of AST at 218 nm

Table 1: Linearity Studies of AST

S.No	Parameters	AST at 218 nm
1	Beer’s law limit (µg/ml)	2-10µg/ml
2	Regression equation	Y= 0.128x+0.0134
3	Correlation coefficient (r)	0.9994
	Intercept (a)	0.0134
	Slope (b)	0.128

Precision

The repeatability of the proposed method was ascertained by performing six independent assays of test sample. The intra-day study was carried out on the same day at an interval of two hours whereas inter-day study was carried out on three different days. The results of statistical analysis were given in Table 2.

Table 2: Precision Studies of AST

Drug

Repeatability

(n=5) %

RSD

Inter-day precision

(n=5)

% RSD

Intra-day

Precision

(n=5)% RSD

Alosetron hydrochloride

0.6582

1.22

0.62

Accuracy:

The accuracy of the proposed method was determined by recovery studies using standard addition method. The % recovery studies of AST were carried out in three different concentration levels (50, 100 and 150%) by spiking previously analyzed samples of the tablet with standard drug solutions. The results of accuracy studies were shown in Table 3 and assay in Table 4.

Table 3: Accuracy Studies of PVT

Drug	% Spike Level	% Recovery ± SD^*	% RSD*
AST	50	100.2.±0.002	0.365
	100	100.7±0.0076	0.985
	150	99.95±0.0073	0.731

*Mean of three determinations.

Table 4: Assay of Alosetron hydrochloride (AST) in Formulation

Drug	Labeled Amount (mg)	Amount found (mg)	% Assay
AST	0.5mg	0.502	100.4

The optical characteristics such as Beer’s law limit, molar absorptivity, Sandell’s sensitivity, Correlation coefficient, slope and intercept, % Relative Standard Deviation (Precision) , % Range of error (at 0.05 and 0.01 confidence limits) were calculated and are summarized in Table 5.

DISCUSSION:

In this proposed method the absorbance of all solutions were measured at 218 nm against distilled water as blank. AST obeyed Beer-Lambert’s law in the concentration range of 2-10 µg/ml with correlation coefficient (r) of 0.9994. %RSD for inter-day and intra-day precision were found to be 1.22 and 0.62, respectively. %RSD for repeatability was found to be 0.6582 for AST. As the %RSD values were <1% the method was found to be highly precise. %Recovery values of AST were found to be 99.95-100.7 % respectively which indicates that the proposed method is accurate. Tablet dosage form was analyzed and the result of assay of the drug was in good agreement with the label claim of formulation as indicated by % assay with 100.4% for AST. All the results were found to be within the limits and therefore the proposed method was found to be free from interferences from excipients from the tablet dosage form.

Table 5. Optical characteristics and Precision of proposed UV Spectrophotometric method for Alosetron hydrochloride (AST)

Parameter	Results
λ max	218 nm
Beer’s law limit (mg/mL)	2-10
Apparent Molar absorptivity (L mole^-1 cm^-)⁾¹) (L mole^-1 cm^-1)	0.41x10⁵
Correlation coefficient (r)	0.9994
Slope (b)	0.128
Intercept (a)	0.0134
Regression equation (Y = a + bC)	0.128x+0.0134
System precision (%RSD)* Relative standard deviation deviation (%)*	0.6582
Inter-day precision (%RSD)*	1.22
Intra-day precision (%RSD)*	0.62
Sandell’s sensitivity (mg cm^-2 / 0.001 Abs unit)	0.00776
Limit of Detection (LOD)	0.1015 µg/ml
Limit of Quantitation (LOQ)	0.3078 µg/ml

*Mean of six determinations

CONCLUSION:

A simple, sensitive, rapid and economic UV spectrophotometric method has been developed and validated for the assay of Alosetron hydrochloride (AST) in tablet formulation. This method yielded high recoveries with good linearity and precision. It can be concluded that the proposed method is a good approach for obtaining reliable results and found to be suitable for the routine analysis of Alosetron hydrochloride (AST) in tablet formulation.

ACKNOWLEDGEMENT:

The author is thankful to Bapatla College of Pharmacy for providing facilities required for the research. The author is also thankful to Aurobindo Pharma Ltd., Hyderabad for providing the gift sample of Alosetron hydrochloride (AST).

REFERENCE:

1 Camilleri M, Northcutt AR, Kong S, Dukes GE, McSorley D, Mangel AW. Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised, placebo-controlled trial. Lancet .355, 2000: 1035-1040.

2 Corrigan B, Manzo J, James C, et al. The effect of repeat dosing on the pharmacokinetics of alosetron 1mg BID. Pharm.Sci. 1, 1998: 149.

3 Houghton LA, Foster JM, Whorwell PJ. Alosetron, A 5-HT3 receptor antagonist, delays colonic transit in both patients with irritable bowel syndrome and healthy volunteers. Aliment Pharmacol Ther. 14, 2000: 775-782.

4 Markowitz MA, Bagby BA, Gordon SH, et al. Satisfactory control of bowel urgency and global improvement in IBS with Lotronex (alosetron HCl) therapy. Am J Gastroenterol. 95, 2000: 2543.

5 Mangel AW, Northcutt AR. The safety and efficacy of alosetron, A 5-HT3 receptor antagonist, in female irritable bowel syndrome patients. Aliment Pharmacol Ther. 13 (Suppl.2), 1999: 77–82.

6 Camilleri M, Mayer EA, Drossman DA ,et al. Improvement in pain and bowel function in female irritable bowel patients with alosetron, a 5-HT3 receptor antagonist. Aliment Pharmacol Ther. 13(9), 1999: 1149–59.

7 ICH, Q2 (R1) Validation of Analytical Procedures: text and methodology; 2005.

Received on 16.04.2014 Modified on 30.04.2014

Asian J. Research Chem. 7(8): August 2014; Page 748-750