Degradation Kinetic Study of Melatonin in Alkaline and Acidic Medium by Validated Stability Indicating HPTLC Method

 

Shubhangi V. Sutar*¹, Veerendra C. Yeligar²

Ashokrao Mane College of Pharmacy, Peth-Vadgaon

 

ABSTRACT:

 

 

1. INTRODUCTION:

 

 

Melatonin, N-acetyl -5-methoxy tryptamine, MT, is a neuroharmone produced mainly at night by the pineal gland, subsequently decreasing to minimum during the day.

 

It helps to treat sleep disorders with diminishing latency of sleep inception, effective as free radicals remover and seeing that endogenous antioxidant. The MT has been use with magnificent therapeutic results in Alzheimer treatment, intended for the neurotoxicity induced by glutamate and throughout jet lag treatment. MT is found available as tablets and capsules for human consumption and is sold without medical prescription in many countries, including Canada and United States of America and off the shelves even in nutrition supplement stores.

 

However, there are no reported methods intended for determination of degradation kinetics of Melatonin in Alkaline and Acidic medium. Now days there are various methods for determining Melatonin, its pharmaceutical dosage form, such like HPLC method, spectrophotometric method and thin layer chromatography scanning method and so on^1-11.

 

The main intention of the work was to establish a rapid and stability indicating method intended for analysis of MT in the presence of its degradation products for estimation of the purity of the bulk drug furthermore the stability of its dosage forms. The method was validated in accordance with ICH guidelines. The method was used to explore the kinetics of base and Acid induced degradation of the drug^12-15.

 

2. MATERIAL AND METHODS:

2.1 The HPTLC system:

Camag Linomat IV was used as the application device. A horizontal development chamber and TLC scanner III was also provided by Camag. Silica gel 60 F254 Plates were used for the study. Scanner was combined with WinCAT software version 1.4.3.6336.

 

2.2 Procurement of standard drug as gift sample:

In HPTLC Method, Toluene, Methanol, Formic Acid, NaOH, HCl, H₂O₂ etc. solvents of AR grade was used for analysis.

 

2.3 Marketed Formulation used for Method Development and Validation:

The investigated product (Tablet) was over the counter product (dosage:3 mg,Meloset)

 

2.4 Preparation of Standard Stock Solution:

Accurately weighed 3mg of Melatonin transferred in to volumetric flask and dissolved in AR grade methanol by ultrasonicating for 10 min and volume level was made up to the mark to give stock solution of concentration 300 µg/ml.

 

2.5 Selection of Mobile Phase:

Appropriate dilutions of stock solutions were prepared and applied on TLC plates in the form of band (band size: 6mm) and the plates were run in different solvent system. Different mobile phase system consist of toluene: methanol, Toluene: methanol: chloroform, Toluene: methanol: GAA, Methanol: 1%ammonium acetate solution, Chloroform: dimethyl amine, Toluene: methanol: GAA: TEA was tried in different ratios in order to determine best condition for the effective separation. Among the different mobile phase combinations tested, Toluene: Methanol: Formic acid (7:3:0.1) was selected as it gives good resolution and peak symmetry for Melatonin. The Rf value for MT was found to be 0.67.

 

2.6 Selection of Analytical Wavelength for Densitometric Evaluations:

Standard stock solution was applied on TLC plate with the help of CAMAG LINOMA-V automatic sample applicator, in order to determine the absorbance maxima. The plate was developed in twin-through glass chamber saturated with mobile phase for 10 minutes. Subsequent to chromatographic development, the plate was removed and dried out. The bands observed on the TLC plate were scanned over the wavelength range of 200-700 nm. Melatonin showed absorbance and good resolution at 290 nm which is selected as analytical wavelength.

 

2.7 Optimized Chromatographic conditions:

The following chromatographic conditions were optimized by trial and error for Densitometric analysis of Melatonin.

 

Stationary Phase: Aluminium plates precoated with silica gel 60

 

Mobile Phase: Toluene: Methanol: Formic acid (7:3:.0.1)

 

Plate size: 10cm×10cm

 

Mode of application: Band

 

Band Size:6mm

 

Sample applicator volume: 0.5µl

 

Development Chamber: Twin–through glass chamber, 10cm×10cm with stainless steel lid

 

Saturation Time: 10 minutes

 

Scanning Mode: Absorbance /Reflection

 

Slit Dimensions: 5×0.45

 

Scanning Wavelength: 290mm

 

2.8 Degradation Kinetics:

Degradation Kinetics of Melatonin was studied in Acidic, Alkaline and oxidative by the validated stability indicating method.

 

2.8.1 Alkaline and Acidic Degradation Kinetic Study:

Preparation of standard stock Solution:

Accurately weighed 3mg of Melatonin was transferred to 10ml of volumetric flask and dissolved in AR grade methanol by ultrasonicating for 10 minutes and volume prepared up to the mark using the methanol to give stock solution of concentration of 300µg/ml.

 

 

 

 

 

Preparation of Sample for Alkaline and Degradation Study:

0.1N, 0.5N, 1N NaOH and 0.1N, 0.5N, 1N HCl was prepared and accurately weighed 3mg of Melatonin transferred   in 10ml volumetric flask.3ml of various strengths HCL and NaOH were added to volumetric flasks containing weighed quantity of Melatonin. These Mixtures were heated under reflux at 60⁰C for 2hrs. From each degradation solutions, sample was collected initially and after1hr time interval up to 2hrs was diluted with methanol up to the mark. The resulting solutions were applied over TLC plate and analyzed using optimized chromatographic conditions. Graph was plot of percent log remaining against time. The First order plots for alkaline degradation of melatonin at temp 60⁰C at varying time interval are elucidated in Fig no.3. The Alkaline degradation kinetics and its observed rate constant (k_obs), half lives are given in the table no.5. The First order plots for Acidic degradation of melatonin at temp 60⁰C at varying time interval are elucidated in Fig no.4. The Acidic degradation kinetics and its observed rate constant (k_obs), half lives are given in the table no.6.

 

Calculate half life and rate constant for the drug using following equation

 

Slope= -K/2.303

 

T_1/2 =    0.693/ K

 

Energy of Activation (Ea):

A plot of In k as a function of 1/T referred to as the Arrhenius plot is linear if Ea is independent of temperature. The slope of line obtained from the plot of In k versus 1/T is equal to –Ea/R    (–Ea/R=Slope)

 

2.8.2 Oxidative Degradation Kinetic Study:

3% H₂O₂ was prepared and accurately weighed 3mg of Melatonin transferred in 10ml volumetric flask. 3ml of 3% H₂0₂ was added to volumetric flask containing weighed quantity of Melatonin. The above reaction mixture was heated in precession water bath at 60⁰C. The reaction mixture was well kept in dark to avoid photo- oxidation effect.

 

From these solutions, sample was collected initially and after 1hr.time interval up to 2hrs. The volume was made up to the mark by means of methanol. The slope of the individual straight lines in the plot is the negative of the rate constant (k) of the reaction of interest. The oxidative degradation kinetics and its observed rate constant (k_obs),half lives are given in the table no.7.

 

3. RESULT AND DISCUSSION:

The TLC plates were prewashed through methanol and were dried in an oven to eradicate any adsorbed or else volatile impurities. From various proportions tried, the mixture of Toluene: Methanol: Formic acid (7:3:.0.1) yielded good resolution and compact spots with Rf value 0.63 for Melatonin. It was observed with the purpose of drying the TLC plate under IR lamp after spotting also presaturation of TLC chamber with mobile phase for 10 minutes ensured good reproducibility of value. Wavelength maximum of MT (290nm) was selected for detection of drug as well as degradation products at the same wavelength.

 

3.1 Method Validation:

The proposed method was validated by studying parameters such as accuracy, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and robustness.

 

3.1.1 Preparation of Calibration Curve:

Standard stock solution of Melatonin prepared by dissolving 3mg of Melatonin in 10ml volumetric flask, volume was made up to the mark with methanol to obtain the final concentration 300µg/ml. Above solution of Melatonin was applied on the TLC plates in the range of 0.3to 0.8 µl with the help of Hamilton syringe by using LINOMAT-V automatic sample applicator. The Plate was afterwards developed in optimised mobile phase. Densitometric evaluations of the drug were performed at 290 nm. Peak area was recorded. The peak areas were plotted against the corresponding concentration to obtain the calibration graph. The standard calibration curve for Melatonin is shown in Fig. No.01 and data shown in Table No.01

 

Fig.No.1. 3D Graph of Melatonin Linearity at 290nm

 

Table No. 1 Linearity study of Melatonin in physical mixture

Sr. No.	Concentration (ng)	Peak Area*
1	30	956.65
2	60	1317.44
3	90	1675.08
4	120	2054.71
5	150	2353.7
6	180	2740

*Average of Three determinations

 

Fig.No.2. Calibration Curve for Melatonin (y=609.080+11.81×) (r²=0.999)

 

3.1.2 Accuracy:

To ascertain the accuracy of the proposed method, recovery studies were carried out by standard addition method at 80, 100 and 120% of the test concentration as per ICH guidelines. All the solutions were sonicated for 20 minutes and volume was made up to the mark with methanol. The solution was individually mixed and filtered through the whatmann paper no. 42. On the TLC plate, band of standard solution and sample solution, 0.5µl each, were applied and the plate was developed and scanned under the optimum chromatographic condition. After scanning, the peak obtained for standard and sample bands were integrated. Total amount of drug estimated in sample was obtained by comparing the peak area of sample bands with that of the standard bands.

 

3.1.3 Precision:

Repeatability: To check the degree of repeatability of the method, suitable statistical evaluation was carried out. Six sample of the marketed tablets preparation were analyzed as per the procedure given under the analysis of the same .The standard deviation (S.D.), % Relative standard deviation (%RSD) was calculated and was given in the table.3

 

Intermediate precision (intra-day and Inter day precision):

 

Results of intraday and interday precision were shown in Table no.3

 

 

Table. No.2 Results of recovery studies

Sr. No.	Level of Recovery	Actual Concentration level	Amount of Drug present ng/band	Amount of Drug Recovered (ng/band)	% Recovery	% Mean Recovery	S.D.	% RSD
1	80	2.4mg	120	120.78	100.65	100.10	0.748	0.748
			120	120.5	100.41
			120	119.01	99.25
2	100	3mg	150	150.45	100.3	100.0	0.651	0.651
			150	150.69	100.46
			150	148.90	99.26
3	120	3.6mg	180	180.20	100.11	99.87	0.830	0.831
			180	178.20	98.95
			180	181.01	100.56

 

Table No.3 Results of Intraday and Interday precision of HPTLC Method

Precision parameters	% Label claim*	S.D.	% RSD
Repeatability	99.67	0.1	0.167
Intraday precision	98.48	0.083	0.085
Interday precision	99.40	0.734	0.739

 

3.1.4 Limit of detection and limit of Quantitation:

The LOD and LOQ were separately determined which is based on the standard deviation of response of the calibration curve. The standard deviation of y-intercept and slope of the calibration curves were used to calculate the LOD and LOQ. Results of LOD and LOQ are 2.83 ng/band and 9.83 ng/band respectively.

 

3.1.5 Robustness:

To evaluate the robustness of the proposed method, small but deliberate variations in the optimized method parameters such as composition of the mobile phase, chamber saturation time, time from spotting to the development, development to scanning, volume of the mobile phase and stability of solution were done.

 

 

Table.No.4 Results of Robustness Study

Factor	Level	Peak Area	Rf	Factor	Level	Peak Area	Rf
Mobile Phase Composition (±0.1ml )				Spotting to Development(±5min)
6:4:0.1	-1	2552.1	0.66	5 min	-	2553.2	0.65
7:3:0.1	0	2457.71	0.67	10 min	-	2457.7	0.68
8:2:0.1	+1	2343.3	0.68	15 min	-	2343.4	0.67
	RSD	0.581	1.51		RSD	0.5631	1.52
Duration For Chamber Saturation(±2min)				Volume of Mobile Phase(±1ml)
8min	-2	2553.3	0.66	9.0	-1	2552.2	0.68
10 min	0	2457.71	0.68	10.0	0	2469.61	0.68
12min	+2	2387.52	0.68	11	+1	2487.74	0.67
	RSD	0.565	1.51		RSD	0.5721	1.53

3.2 Results of Alkaline Kinetic Degradation Study:

Table No. 5 Alkaline Degradation Kinetic Study

Strength of NaOH	Time	%Drug Estimation	Log of % Remaining	% Degradation	Degradation Rate Constant K (per Hrs.)	Half Life (hr.)	Order of Reaction
0.1 N NaOH	Initial	99.01 %	1.99	1.00%	0.080	8.66	First
	1Hour	92.00 %	1.97	8.00%
	2Hour	84.00 %	1.92	16.01%
0.5 N NaOH	Initial	95.00 %	1.97	5.00%	0.1842	3.67
	1Hour	78.00 %	1.89	22.2%
	2Hour	65.06 %	1.81	34.94%
1 N NaOH	Initial	89.4 %	1.95	10.6%	0.5527	1.25
	1Hour	49.4 %	1.69	50.6%
	2Hour	29.6 %	1.47	70.7%

 

Fig. No.3 Comparison of Degradation of Melatonin in 0.1, 0.5 and 1.0N NaOH

3.2.1 Energy of Activation (Ea):

A plot of In k as a function of 1/T referred to as the Arrhenius plot is linear if Ea is independent of temperature. The slope of line obtained from the plot of In k versus 1/T is equal to –Ea/R (Ea/R=Slope}. Energy of activation found in Kcal/m is 0.15,0.366 and 1.09in 0.1,0.5,1.0 N NaOH respectively

 

 

3.3 Results of Acidic Kinetic Degradation Study:

Table No.6 Acidic Degradation Kinetic Study

Strength of HCl	Time	% Drug Estimation	Log of % Remaining	% Degradation	Degradation Rate Constant K (per hr)	Half Life (hr)	Order of Reaction
0.1 N HCl	Initial	99.1 %	1.99	1.00%	0.0460	15.06	First
	1Hour	94.8 %	1.97	5.2%
	2Hour	90.2 %	1.95	9.8%
0.5 N HCl	Initial	96.6 %	1.98	3.4%	0.1266	5.470
	1Hour	81.7%	1.91	18.3%
	2Hour	74.9 %	1.87	25.1%
1 N HCl	Initial	91.9 %	1.96	8.1.%	0.3569	1.941
	1Hour	63.2 %	1.8	36.8%
	2Hour	44.8 %	1.65	55.2%

 

Fig.No.4 Comparison of Degradation of Melatonin in 0.1N, 0.5 N and 1.0 N HCl

 

3.4 Results of Oxidative Degradation Kinetic Study:

Table No.7 Oxidative Degradation Kinetic Study

Strength of H2O2	Time	%  Drug Estimation	Log of % Remaining	% Degradation
3% H2O2	Initial	99.2 %	1.99	0.8%
	1Hour	95.8 %	1.98	4.2%
	2Hour	92.8%	1.96	7.2%

 

Fig. No.5 Comparative 3D Spectra of Melatonin in 3% H₂O₂, O.1N HCl, 0.1NNaOH

 

4. DISCUSSION:

Due to the varying lability and subsequent degradation behaviour of Melatonin, it was held essential to confirm the rate of degradation. It was also known that kinetic degradation act as additional validation step in stability testing.

It was observed that Melatonin degraded in acidic, alkaline and oxidative stress conditions. However, it showed higher lability in alkaline degradation. Acid, alkaline and oxidative degradation reactions studied to determine the rates of the reaction and susceptibility of Melatonin.

 

The degradation kinetics of Melatonin for acid, alkaline and oxidative degradation was investigated in 0.1N NaOH, 0.5N NaOH, 1NNaOH, 0.1NHCl, 0.5NHCl, 1NHCl and 3%H₂O₂, since the decomposition of the drug at lower strengths of the acid, alkali and peroxide was too slow to obtain the reliable kinetic data.

 

Solutions of Melatonin (300µg/ml) were degraded in HCl, NaOH and H₂O₂ at 60⁰C at definite interval of time. The chromatograms obtained revealed that peak areas of the drug decreased with increasing time.

 

The logarithmic plot of concentration of melatonin against time indicated apparent first order degradation behaviour. The observed first order degradation rate constant, k_obs were calculated from the slop of the logarithmic plots of the drug fraction remaining versus time.

 

The values clearly indicated that the rate of acid degradation and oxidation degradation was comparatively slower than the rate of alkaline degradation.

 

5. CONCLUSION:

The introduction of HPTLC into pharmaceutical analysis was major step in quality assurance. The HPTLC technique established in this work is precise, specific, accurate and stability indicating statistical analysis proves the method is suitable for analysis of Melatonin. Melatonin degraded in acidic, alkaline, oxidative stress conditions. Alkaline and acidic degradation of Melatonin followed first order kinetics and higher degradation was found in 1N NaOH and 1N HCl. Acid, alkaline and oxidative degradation reactions studied to determine the rates of the reaction and susceptibility of Melatonin.

 

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Accepted on 28.08.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):523-528.