1. INTRODUCTION:

Mesalamine (MSA) 5-amino-2-hydroxybenzoic acid (Figure1) is lightly pink powder, slightly soluble in water, insoluble in alcohol and soluble in acetone. It acts as an antiulcer drug. It is official in Indian Pharmacopoeia^[1]. Literature survey reveals that many analytical methods such as HPLC^[2-6], Spectrophotometric^[7-11], UPLC^[12,13] have been reported in biological fluid and pharmaceutical formulation. HPTLC method^[14] for mesalmine and for its impurities has been reported.

Practically MSA not soluble in alcohol^[15]. Sample preparation in volatile solvent is primary requirement for HPTLC method development^[16].The proper sample preparation method has given in Indian Pharmacopeia^[1]. None of the reported methods have the ability to determine MSA by HPTLC using appropriate solvent for solubility of MSA.

Fig .1: Chemical structure of Mesalamine

The objective of the analysis is to develop a simple, precise and accurate stability indicating TLC/ Densitometric method for the quantification of MSA in bulk and pharmaceutical formulation. The developed method has the advantage over the reported HPTLC methods that it provides proper solvent for the sample preparation of MSA as the MSA is not directly soluble in alcohol and shows more sensitivity.

The method can successfully determine the drugs in presence of their degradation products. The high percentage recoveries with low % RSD for the recovery study add to the advantage of the proposed method.

2. EXPERIMENTAL:

2.1. Materials and reagent:

MSA was obtained from Sun Pharma Ltd, Mumbai, India. HPLC grade solvent has been used. Toulene and methanol were obtained from Merck India Ltd, Mumbai, India and triethylamine was purchased from Rankem RFCL Ltd, New Delhi, India.

2.2. Instrument and chromatographic condition:

A Camag TLC system (Muttens, Switzerland) attached with Linomat 5 automatic sample applicator, 100 µl Hamilton syringe, Camag TLC scanner 3, with winCATS software (1.3.0), twin trough Camag chamber (20 x 10 cm) and ultra sonicator was used during the study. TLC plates used were pre-coated silica gel aluminium plate 60F₂₅₄ [(20 × 10 cm) having 250 µm thickness, E. Merck, Darmstadt, Germany, provided by Anchrom Technologists, Mumbai, India]. The pre-washing of plate was done by methanol and prior to the chromatography activation of plates which was carried out at 110 ⁰C for 5 min. A fixed application rate of 150 nL s^-1 was used and distance between two bands was kept 15.4 mm. The dimension for slit kept at 6 mm × 0.45 mm. The mobile phase composed of Toulene: Methanol: Triethylamine: (05:05:0.1 v/v/). Development of plate in linear ascending manner was carried out in 20 cm× 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) the chamber was saturated with mobile phase for 20 min at temperature (25⁰C) and relative humidity (60 % ± 5%). The plates were developed for 8cm length and were air dried up. Densitometric scanning was carried out at 325 nm. Deuterium lamp was used as a source of radiation. Estimation was performed by means of peak area with linear regression.

2.3. Preparation of stock standard solution of MSA for study of linearity curves:

Stock standard solution was prepared by dissolving 10 mg of MSA in 50 mL equal mixture of acetic acid and water in 100 mL flask and final volume make up by methanol that gives concentration of 0.1 mg/mL.

2.4. Validation of method^[17]:

Developed HPTLC method was validated as per International Conference on Harmonization (ICH) guidelines Q2 (R1).

2.4.1.Precision:

Precision studies for the MSA were done by using drug standard solution containing concentrations in the calibration range. The precision of the method by means of intra-day variation (% RSD) was carried out by analyzing standard drug solutions in calibration range, three times on same day. Inter-day precision (%RSD) was calculated by analyzing the drug standard solutions in calibration range three times on three different days in one week period.

2.4.2. Robustness and ruggedness:

The robust nature of analytical methods is to find out its potential to stay unchanged by small, but intentional changing in the method parameters and gives its consistency in normal routine usage.

The robustness of MSA was established by studying different parameters like change in volume of mobile phase, composition of mobile phase, relative humidity, distance for development, saturation period, change in time from spotting to chromatography and from chromatography to scanning were considered and its effects on the results were studied.

The ruggedness of the developed method was studied by two different analysts using same environmental and experimental conditions.The methods robustness was carried at 1200 ng/spot and ruggedness study was carried out at concentration 800 ng/spot.

2.4.3. Limit of detection (LOD) and limit of quantification (LOQ):

In order to find detection and quantification limit, concentration in the lower part of the linear range of the calibration curve used. The amount of MSA by spot versus peak area was graphed and the equation for this was obtained, the average of standard deviation calculated. Detection limit was determined by (3.3 x A.S.D)/b and Quantification limit was determined by (10 x A.S.D)/b where b correspond to slope obtained in graph.

Fig.4: Overlain spectra of MSA sample [a] and MSA Standard[b] scanned at the peak start, peak apex, and peak end position of the band

2.4.4. Specificity:

The specificity of the developed method was confirmed by analyzing the sample solutions containing standards and marketed tablets for MSA in relation to interferences from formulation ingredients. The spot of MSA in the sample was confirmed by comparing Rf value of the spot with that of the spot of the standard(Figure2).

2.4.5. Recovery:

Recovery study was carried out at levels of 80 %, 100 % and 120 %.By over spotting 80 %, 100 % and 120% of the standard drug solution of MSA and the mixtures were re-analyzed by the projected method. Experiment was carried out in triplicate. This study was finished to make sure the recovery of the drug at different levels in the tablet formulations.

2.5. Analysis of MSA in marketed tablet formulation:

To estimate the content of MSA in tablet formulation (label claimed 400 mg); twenty tablets were accurately weighed and the average weigh was calculated. The tablets were powdered and a quantity of powder equivalent to 10 mg of MSA was transferred in to 50 mL volumetric flask containing 20 mL equal mixture of glacial acetic acid and water, shaken manually for 30 min and diluted to mark with methanol. The resulting solution was filtered using 0.45 µm filter (Millifilter, Milford, MA, USA) and 4 µL equivalent to 800 ng MSA was applied to a plate, developed and scanned as described in section 2.2. The same analytical procedure was repeated for six times. MSA produced distinct peaks at R_f0.63 ± 0.02, when scanning was done at 325 nm. The results were shown in Table1 specify that there was no interference from the excipients usually present in the tablet formulation.

Table 1: Assay of tablet formulation^a

Component	Label claim (mg/Tablet)	% Amount found	% RSD
MSA	400	100.24	1.54

^a Average of six estimations

3. FORCED DEGRADATION STUDY^[18,19]

Force Forced degradation studies was conducted according to ICH guidelines.

Acid and base-induced degradation was done by separately adding 10 mg of MSA in 10 mL each of 0.1 N HCl and 0.5 N NaOH solutions. These solutions were kept for 3 h at room temperature in the dark. The solutions (1 mL) were neutralized and diluted to 10 mL with methanol. A constant volume 12 µL of the resulting solutions (1200 ng/band) were applied to a TLC plate and developed. For oxidative degradation, 10 mg of MSA added in 10 mL of 3% (v/v) hydrogen peroxide solution. These mixtures were kept for 6 h. The solution (1 mL) were diluted to 10 ml with methanol and treated as described for acid and base-induced degradation. Photochemical degradation was studied by taking 10 mg of MSA in 10 mL equal mixture of acetone,water and methanol in10 mL volumetric flask and by exposing to sun light for 8 h. The resulting 1.2 µL solution was applied on plate (1200 ng/band) and chromatogram was developed in said mobile phase. For dry heat study MSA 10 mg was stored at 55ºC for 2 h in oven separately, and then transferred to 10 ml volumetric flask containing mixture of acetone, water and methanol. 1.2 µl (1200 ng/band) was applied on TLC plate in triplicate and run as described in section 2.2.

4. RESULTS AND DISCUSSION:

4.1. Optimization of TLC/densitometric method:

The selection of the mobile phase to develop the TLC/Densitometry method for the estimation of MSA was carried out on the basis of polarity. Initially, combination of various proportions of solvents Ethylacetate-methanol, n-hexane- methanol and Toulene-methanol were tried as mobile phase but tailing was observed in Toulene: methanol. To get the dense and compact spot without tailing, 0.1 ml of triethylamine was added as modifier. Finally, mobile phase composed of Toulene :Methanol: Triethylamine: (05: 05:0.1 v/v/v) was established as observed to well resolved and spot with R_f value 0.63± 0.03 when plate was developed and scanned at 325 nm (Figure3). The chamber was saturated with the mobile phase for 20 min at room temperature and plates were activated at 110 ºC for 5 min to obtain well defined spots.

Fig. 3: Densitogram of Mesalamine Rf (0.63) at 325 nm

4.2. Calibration

The linear regression data for the calibration plots (n = 6) were significance of a good linear relationship between peak area and amount in range 400-1600 ng per spot of MSA. The correlation coefficient was found to be 0.9993.

4.3. Method validation:

4.3.1. Precision:

Precision of the developed method was calculated by intra-day and inter-day variation (% RSD). For Intra-day precision (% RSD) analysis of standard drug in calibration range was carried out on same day at three times. % RSD was observed 0.68 – 1.43. For Inter-day precision (% RSD) analysis of drug solution in calibration range was carried out three times on three different days in period of one week. The % RSD of MSA was found 0.19 -1.67. These values established the sufficient preciseness for the developed HPTLC method.

4.3.2. Robustness and Ruggedness:

As shown in Table2, RSD [%] of peak areas was calculated at every change in condition and RSD ˂ 2% indicated the method to be robust. The % RSD of results of analysis by two analysts were calculated and were found to be 1.09 and 1.24. There was no significant difference statistically observed between each analyst results shows ruggedness of the developed method for MSA.

Table 2: Results from assessments of the robustness of the method

Condition	RSD [%]
Mobile phase composition (± 0.3 mL)	1.10
Mobile phase volume (± 2 mL)	0.38
Development distance (± 0.5 cm)	1.21
Plate saturation time (± 5 min)	0.98
Relative humidity (± 5%)	1.34
Activation of TLC plates previously developed with methanol and dried at 60º C (±2 min)	0.49
Time from application to chromatography (±10 min)	1.14
Time from chromatography to scanning (±10 min)	0.83

^a Results are averages from six determinations

4.3.3. Limit of Detection (LOD) and limit of Quantification (LOQ):

The calculation of LOD and LOQ was done as described in Section 2.4.3. The limit of detection and quantification limit were found to be be 37.66 ng and 114.14 ng. This showed the enough sensitivity for the developed method.

4.3.4. Recovery:

The results concluded from estimation of recovery were listed in Table3; the low values of % RSD suggested the accuracy of the developed method.

Table 3: Results of recovery studies ^a

Component	Labe]l claim(mg/Tablet)	% amount of standard drug added	%drug recovered	% RSD
MSA	400	80	99.76	1.75
		100	99.20	0.86
		120	101.63	0.72

^aMean of three estimations at each level

4.4. Force degradation study:

MSA was exposed to different stress degradation conditions. Peaks obtained from the samples degraded by acid, alkali, hydrogen peroxide and dry heat treatment showed well separated spots of the pure drugs and few degradation products spots at various R_fvalues. MSA showed degradation products peak under acid, alkali and oxidation conditions but no degradation was observed in dry heat sunlight. R_fvalues of the degradation peaks with percentage recovery of the drugs were listed in Table 4.The degradation peaks developed under various stress condition for MSA were well separated from the peaks of the intact drugs. The peaks of MSA were not remarkably shifted in the presence of the degradation peaks, which indicates the stability-indicating nature of the developed method.

Table 4: Force degradation study

Sample exposure condition	No. of degradation products (Rf value)	% of drug remaining after degradation
0.1 N HCl	2(0.12, 033)	81.25
0.5 N NaOH	2(0.11,0.27, 0.67)	85.39
H₂O₂ 3%	2(0.11,0.15, 0.19)	83.12
Photo 8 hr	No Degradation	99.25
Heat 2hr 55^oC	No degradation	98.91

5. CONCLUSION:

A new TLC/Densitometric Stability-indicating has been developed and validated for the identification and quantification of MSA in bulk and in tablet formulation. Use of proper solvents for solubility of MSA for the development of TLC method is uniqueness of this method. The validation of the method is done as per ICH guideline. All the validated data achieved are in good agreement with the ICH guidelines Q2 (R1) (ICH, 2005)^[17]. Once developed method compared with the reported method^[15]exhibit more sensitivity. The LOD and LOQ were 37.66 and 114.14 ng respectively. Good recovery observed in the range of 99.63 to 101.63 (Table 3) in marketed sample proves the accuracy of the method. Good enough precision, low cost, and faster analysis are the important features of this method. Reproducibility and specificity of method are proved by statistical analysis. The developed method thus can be recommended for the quality control purpose in pharmaceutical laboratories.

6. CONFLICT OF INTEREST:

All Authors have none to declare

7. ACKNOWLEDGEMENT:

The authors are thankful to Dr. S. J. Surana, Principal of R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur (M.S.), India for providing the necessary facilities to carry out this research work

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Received on 06.07.2018 Modified on 12.08.2018

Asian J. Research Chem. 2018; 11(6):871-875.

DOI: 10.5958/0974-4150.2018.00152.9