1. INTRODUCTION:

Mesalazine and Rifaximin use for Inflammatory Bowel Disease. Mesalazine is used as in anti-inflammatory agent, Steroidal. Rifaximin is used in Gastrointestinal Agents, Anti-infective agent. The use of Rifaximin in combination with Mesalazine has been proved to provide beneficial effect in inflammatory bowel disease. The mechanism of Mesalazine and Rifaximin is quite different. Mesalamine and Rifaximin are two different types of drugs offering some symptomatic relief to the IBD patients. Mesalamine treats inflammation, whereas, Rifaximin reduces bio burden. Mesalazine and Rifaximin novel combination used in treatment of Inflammatory Bowel Disease Patented by Lupin Ltd, WIPO WO2009047801 A₁.^[1]

Mesalazine

Mesalamine is a salicylate, its therapeutic effect does not appear to be related to cyclooxygenase inhibition; indeed, traditional non-steroidal anti-inflammatory drugs actually may exacerbate inflammatory bowel disease. Although the mechanism of action of mesalazine is not fully understood, it appears to be topical rather than systemic. Mucosal production of Arachidonic acid metabolites, both through the cyclooxygenase pathways, i.e., prostanoids, and through the lipoxygenase pathways, i.e., leukotrienes and hydroxyeicosatetraenoic acids, is increased in patients with chronic inflammatory bowel disease, and it is possible that mesalazine diminishes inflammation by blocking cyclooxygenase and inhibiting prostaglandin production in the colon. Mesalamine appears to diminish inflammation by inhibiting cyclooxygenase and lipoxygenase, thereby decreasing the production of prostaglandins, and leukotrienes and hydroxyeicosatetraenoic acids (HETs) respectively. It is also believed acts as a scavenger of oxygen-derived free radicals, which are produced in greater in patients with IBD.^[2]

Mesalazine is chemically 5-Amino-2-Hydroxybenzoic acid. It is a appears as off white to gray, having molecular weight 153.14 g/mol.^(3-5)

FIG. 1 Chemical Structure of Mesalazine

Rifaximin

Rifaximin is a semisynthetic, rifamycin-based non-systemic antibiotic, meaning that the drug will not pass the gastrointestinal wall into the circulation as is common for other types of orally administered antibiotics. It is used to treat diarrhea caused by E. coli. Rifaximin acts by inhibiting RNA synthesis in susceptible bacteria by binding to the beta-subunit of bacterial deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase enzyme. This results in the blockage of the translocation step that normally follows the formation of the first phosphodiester bond, which occurs in the transcription process.⁽⁶⁾

Rifaximin is chemically (7S,9E,11S,12R,13S,14R,15R, 16R,17S,18S,19E,21Z)-2,15,17,36-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22,30-octamethyl-6,23-dioxo-8,37-dioxa-24,27,33-triazahexacyclo[23.10.1.1⁴,⁷.0⁵,³⁵.0²⁶,³⁴.0²⁷,³²]heptatriaconta1,3,5(35),9, 19,21,25(36),26(34),28,30,32-undecaen-13-yl acetate^.It is a Red Orange Crystalline Powder having molecular weight 785.87 g/mol.^(7-8)

FIG. 2 Chemical Structure of Rifaximin

The review of literature regarding quantitative analysis of Mesalazine and Rifaximin revealed that no Simultaneous Equation method attempt was made to develop analytical methods for Mesalazine and Rifaximin. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual and combination of drugs. The focus of the present study was to develop and validate a rapid, stable, specific, and economic Spectroscopic method for the estimation of Mesalazine and Rifaximin in Synthetic Mixture.

Theory^[9]

We can find out concentration of both the drug from combination mixture using the simultaneous equation method. In this method using the absorbance of both the drug and mixture at their wavelength and put this value in following equation and we can find out the concentration of drugs present in combination.

(A₂ × Ay₁) – (A₁ × Ay₂)

Cx = --------------------------------- ------------------------ (1)

(Ay₁ × Ax₂) – (Ay₂ × Ax₁)

(A₁ × Ax₂) – (A₂ × Ax₁)

Cy = --------------------------------- --------------------- (2)

(Ax₂× Ay₁) – (Ax₁ × Ay₂)

Where,

Cx = Concentration of drug A

Cy = Concentration of drug B

A1 = Absorbance of mixture at wavelength 1

A2 = Absorbance of mixture at wavelength 2

Ax1 = Absorptivity of drug A at wavelength 1

Ax2 = Absorptivity of drug A at wavelength 2

Ay1 = Absorptivity of drug B at wavelength 1

Ay2 =Absorptivity of drug B at wavelength 2

2. MATERIALS AND METHODOLOGY

2.1. Apparatus

· A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2nm, 1 cm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software.

· An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples.

· Sonicator (D120/2H, TRANS-O-SONIC)

· Class ‘B’ volumetric glassware were used (Borosillicte)

· All instruments and glasswares were calibrated.

2.2. Reference samples

MESA and RIFA reference standard are kindly supply by CTX Life Sci. Sachin, Surat and Lupin Ltd, Ankleshwar as a gift sample respectively.

2.3.Materials and reagents

Methanol AR Grade (Finar), Distilled Water, NaOH AR Grade (Ranchem), Distilled water, HCl (Astron) was used for development purpose.

2.4.Preparation of Standard Solution and Synthetic Mixture

2.4.1 Preparation of stock solution of Pravastatin:

An accurately weighed quantity equivalent to 10mg of Mesalazine was transferred to 100 ml volumetric and dissolved and diluted to the mark with the 0.01 N NaOH to obtain standard solution having concentration of MESA (100 μg/ml).

2.4.2 Preparation of standard stock solution of Valsartan:

An accurately weighed quantity equivalent to 10 mg of Rifaximin was transferred to 100 ml volumetric flask and dissolved and diluted to the mark with 0.01 N NaOH to obtain standard solution having concentration of RIFA (100μg/ml).

2.4.3.Preparation of Standard Mixture Solution (MESA+ RIFA):

4.0 ml of working standard stock solution of MESA (100 μg/ml) and 1.0 ml of standard Stock solution of RIFA (100μg/ml) were pipetted out into 10ml volumetric flask and volume was adjusted to the mark with 0.01 N NaOH to get 40μg/ml of MESA and 10μg/ml of RIFA.

2.4.4 Preparation of Test Solution

The preparation of synthetic mixture was as per patent:

Ingredients	Quantity
Mesalazine	800mg
Rifaximin	200mg
Na.CMC	544mg
MCC	416mg
Mg. Stearate	q. s.
	Total=2000mg

From that powder equivalent to 100 mg of synthetic mixture in 100ml vol. flask dissolve in 25 ml 0.01 N NaOH. Sonicate for 15min diluent up to the 100ml with 0.01 N NaOH and Shake Vigoursly filter the solution. and Filter the solution and further dilute. Withdraw 0.1 ml and make up to 10ml that give 40ppm and 10ppm of MESA and RIFA respectively.

3. RESULT AND DISCUSSION

3.1 Selection of Wavelength for Estimation of Mesalazine and Rifaximin

The Standard Stock Solutions of Mesalazine and Rifaximin were scanned in the range of 200 to 400nm against 0.01 N NaOH as a blank. Maximum absorbance was obtained at 328nm and 292nm for Mesalazine and Rifaximin, respectively. But Spectra were no overlay.

FIG.3 Overlain Zero order spectra of MESA and RIFA in4:1 ratios, respectively with the combination solution (4:1)

FIG.4 Overlain Zero Order Spectra of MESA and RIFA (1:1) Ratio, Respectively

4. VALIDATION PARAMETERS ^[10]

4.1 Linearity

Five point calibration curves were obtained in the concentration range of 10-50μg/ml for Mesalazine and 10-50μg/ml for Rifaximin. The response of drug was found to be linear in investigation range and the regression equations was found to be y = 0.0199x-0.0008 for MESA (n=5) and y = 0.0178x+0.0367 for RIFA (n=5), with the correlation coefficient 0.9992 and 0.9991 (n=5) respectively, is listed in Table 1.

Table.1 Calibration Data for MESA and RIFA at 328nm and 292nm, Respectively *(n=6)

Conc. (µg/ml)	MESA at 328nm(n=6)		RIFA at 292nm(n=6)
Conc. (µg/ml)	Abs ±SD	%RSD	Abs ±SD	%RSD
10	0.189± 0.0018	0.992	0.142± 0.0013	0.964
20	0.399 ±0.0032	0.820	0.316± 0.0025	0.816
30	0.608 ±0.0046	0.769	0.509± 0.0024	0.477
40	0.795 ±0.0021	0.269	0.665± 0.0051	0.769
50	0.982 ±0.0090	0.921	0.859± 0.0058	0.678

Calibration curves for MESA:

This series consisted of five concentrations of standard MESA solution ranging from 10 to 50μg/ml. The solutions were prepared by pipetting out Standard MESA stock solution (100μg/ml). Then pipetting out (1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with 0.01 N NaOH. A zero order spectrum of the resulting solution was recorded, measured the absorbance at 328 nm against a reagent blank solution (0.01 N NaOH). Calibration curve was prepared by plotting absorbance versus respective concentration of MESA.

Calibration curve for RIFA:

This series consisted of five concentrations of standard RIFA solution ranging from 10 to 50μg/ml. The solutions were prepared by pipetting out Standard RIFA stock solution (1.0 ml, 2.0 ml, 3.0 ml, 4.0ml, and 5.0 ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with 0.01 N NaOH. A zero order spectrum of the resulting solution was recorded, measured the absorbance at 292 nm against a reagent blank solution (0.01 N NaOH). Calibration curve was prepared by plotting absorbance versus respective concentration of RIFA.

FIG. 5 Calibration curve for MESA at 328nm

FIG. 6 Calibration curve for RIFA at 292nm

4.2 Precision

The precision of the method was evaluated in terms of inter-day and intra-day by carrying out independent assays of three concentrations chosen from range of the standard curves (10, 30, and 50 μg/ml of MESA and 10, 30, 50μg/ml of RIFA) and the %RSD of assay (inter-day and intra-day) was calculated. The results of study are shown in Table 2 and 3.

Table 2.Intraday Precision data for estimation of MESA and RIFA *(n=3)

Conc. (μg/ml)	At 328nm		At 292nm
MESA + RIFA	Mean	%RSD	Mean	%RSD
10:10	0.190	0.802	0.143	0.694
30:30	0.613	0.163	0.515	0.194
50:50	0.994	0.153	0.865	0.115

Table3.Interday Precision data for estimation of MESA and RIFA *(n=3)

Conc. (μg/ml)	At 328nm		At 292nm
MESA + RIFA	Mean	%RSD	Mean	%RSD
10:10	0.191	0.523	0.144	0.694
30:30	0.615	0.248	0.514	0.298
50:50	0.995	0.100	0.864	0.115

4.3 Accuracy

Composition of synthetic mixture

The preparation of synthetic mixture was as per patent:

Ingredients	Quantity
Mesalazine	800mg
Rifaximin	200mg
Na.CMC	544mg
MCC	416mg
Mg. Stearate	q. s.
	Total=2000mg

From the Synthesis Mixture weigh accurately equivalent about 100mg of RIFA. Take Four 100ml Volumetric Flask and in each flask add synthetic mixture equivalent to 10mg of RIFA. Flask 1 form as a Placebo and remaining flask 2, 3,4 spike with 80, 100,120% of solid API. Repeat the same procedure for MESA as per Table. Take content in 100 ml volumetric flask dissolved in 25 ml 0.01 N NaOH and Sonicate for 15min. make up the volume with 0.01 N NaOH up to 100 ml. The solution was filtered through Whatman filter paper No. 42.With the Mesalazine and Rifaximin with the ratio 4:1dissolved in 0.01 N NaOH with small volume of Solvent, Sonicate for 15 mins, then make up to 100ml with 0.01 N NaOH with all excipients.Now, this solution was used for further dilution.

Amt. of API in synthetic mixture (mg)		Amt. of API Spiking (mg)		Total Amt. (mg)
MESA	RIFA	MESA	RIFA	MESA	RIFA
40	10	-	-
40	10	32	8	72	18
40	10	40	10	80	20
40	10	48	12	88	22

Take total amt. of formulation in 100ml of volumetric flask and add 25 ml 0.01 N NaOH, Sonicate, filter it; make up to mark with 0.01 N NaOH. From it 0.5 ml Dilute to 10 ml with 0.01 N NaOH of 0%, 80%, 100% and 120% solution this Accuracy method done by spiking method. Data from nine determinations over three concentration levels covering the specified range was determined and % recovery was calculated. The% recovery values are tabulated in Table 4.The value of %RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

4.3Limit of Detection and Limit of Quantification

The limit of detection (LOD) and limit of quantitation (LOQ) of the method were evaluated by standard deviation of response and slope method. LOQ and LOD were calculated by the equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where “N” is standard deviation of the absorbance, and “B” is the slope of the corresponding calibration curve. The limit of detection (LOD) were found to be 0.215 μg/ml for MESA and 0.214μg/ml for RIFA and respectively and limit of quantitation (LOQ) were found to be 0.652μg/ml for MESA and 0.648μg/ml for RIFA presented in Table 5.

4.5 Robustness and Ruggedness

Robustness was done by different instrument and difference in preparation of stock solution. The result was decided by %RSD which is in the limit which is mentioned in table no 6.

_{Table 4 Recovery data
of MESA and RIFA*(n=3)}

Level of recovery	Total Conc. (µg/ml)		Result of recovery study
	Total Conc. (µg/ml)		Total Quantity Found (µg/ml)		% Recovery ± %RSD
	MESA	RIFA	MESA	RIFA	MESA		RIFA
0%	20	5	20.09	5.02	100.4	0.174	100.5	0.304
80 %	36	9	36.08	9.00	100.5	0.164	100.2	0.381
100 %	40	10	39.98	10.01	99.92	0.202	100.3	0.502
120 %	44	11	43.97	11.01	99.90	0.061	100.1	0.255
Mean of 3 Determination					100.2	0.314	100.3	0.170

_{Table 5LOD
and LOQ data of}MESA and RIFA_*(n=10)

Parameters		At 328nm (MESA+RIFA)		At 292nm (MESA+RIFA)
		Abs	%RSD	Abs	%RSD
ROBUSTNESS
Different instrument	Inst.1	0.404	0.377	0.319	0.475
Different instrument	Inst.2	0.405	0.376	0.321	0.623
Different Analyst	Analyst 1	0.407	0.375	0.320	0.476
Different Analyst	Analyst 2	0.406	0.490	0.322	0.473
RUGGEDNESS
Change wavelength	326and290nm	0.391	0.511	0.306	0.498
Change wavelength	330and294nm	0.414	0.368	0.318	0.314
Change Ratio	1:1	0.404	0.377	0.319	0.478
	4:1	0.796	0.227	0.144	0.694
	1:4	0.191	1.087	0.672	0.227
Solvent change	0.012N NaOH	0.475	0.295	0.353	0.458
Solvent change	0.009N NaOH	0.312	0.309	0.295	0.242

Table 6 Robustness and Ruggedness data of MESA and RIFA *(n=3)

Conc. (μg/ml)		*Avg. abs ± SD (328.00nm) MESA**	% RSDDD	*Avg. abs±SD (292.00nm) RIFA RFC**	% RSD
MESA	RIFA
20	20	0.403 ± 0.001	0.313	0.318 ± 0.001	0.363
LOD (μg/ml)		0.215		0.214
LOQ (μg/ml)		0.652		0.648

Table 7 Analysis data of Synthetic Mixture*(n=3)

Sr. No.	Formulation (synthetic mixture)		Absorbance* (328.00nm) MESA	%Assay MESA ±SD	Absorbance* (250.00nm) RIFA	%Assay RIFA ±SD
	MESA	RIFA
1	40	10	0.909	100.93 ± 0.05	0.426	101.70 ± 0.20
2			0.911		0.425
3			0.908		0.427

Summary Table

Table. 8 Summary of Validation Parameters

SR. NO.	Parameters	Mesalazine	Rifaximin
1	Wave length Max.	328.00nm	292.00nm
2	Linearity (µg/ml) (n=6)	10-50 µg/ml	10-50 µg/ml
3	Regression equation	y = 0.0199x -0.0008	y = 0.0178x +0.0367
4	Correlation coefficient (r²)	0.9992	0.9991
5	Accuracy(%Recovery) (n=3)	100.2%	100.3%
6	Precision Intra-day (%RSD)(n=3) Inter-day (%RSD)(n=3)	0.153-0.802 0.100-0.523	0.115-0.699 0.115-0.694
7	LOD (µg/ml) (n=10)	0.215	0.214
8	LOQ (µg/ml) (n=10)	0.652	0.648
9	Robustness (%RSD)(n=3) Change in instrument Change in Analyst	0.376-0.377 0.375-0.490	0.475-0.623 0.473-0.476
10	Ruggedness (%RSD)(n=3) Change Solvent Change in Wavelength	0.295-0.309 0.368-0.511	0.242-0.458 0.314-0.498
11	Assay	100.93	101.70