INTRODUCTION:

Lamotrigine is an epileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters^[^1]. IUPAC name of Lamotrigine is 3, 5-diamino-6-2, 3(dichlorophenyl)-1, 2, 4 triazine^[^2].Clozapine is benzodiazepine derivative and use in treatment of schizophrenia^[3]. The IUPAC name of Clozapine is 8-Chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo [b, e] [1, 4] diazepine^[4]. It acts by inhibiting presyneptic voltage sensitive sodium channels and excitatory neurotransmitter release. Dopamine is one of the important neurotransmitter and plays a significant role in the functioning of central nervous system ^[5]. The inhibition of dopamine transmission through blockade of dopamine D2 receptors is considered to be essential for antipsychotic efficacy, but it is postulated that modulation of glutamate transmission may be equally important. In support of this, symptoms similar to schizophrenia can be induced in healthy volunteers using N-methyl-D-aspartate (NMDA) antagonist drugs that are also known to enhance glutamate transmission^.[^6]

Furthermore, Lamotrigine, which can modulate glutamate release, may add to or synergise with atypical antipsychotic drugs, some of which may themselves modulate glutamate transmission^[7].So, Lamotrigine use with Clozapine in treatment of schizophrenia.

Chemical structure of Lamotrigine and Clozapine

Fig-1 Lamotrigine^[^8]

Fig-2 Clozapine^[^9]

MATERIAL AND METHODS:

Apparatus and instrument:

A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 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 (Borosilicate)

All instruments and glass wares were calibrated.

Reagents and material:

Lamotrigine raw material was received as gift sample from Praveen Laboratories.

Clozapine raw material was received as gift sample from ZCl Pharmaceuticals Pvt. Ltd.

Methanol AR Grade (Rankem), Distilled Water, 0.1 N HCl, 0.1 N NaOH were used for development purpose.

For simultaneous equation method preparation of standard solution:

Standard solution of Lamotrigine (LAMO):

Preparation of stock solution of LAMO:

Accurately weighed quantity of LAMO 10 mg was transferred to 100ml volumetric flask, dissolved, and diluted up to mark with methanol to give a stock solution having strength 100µg/ml.

Standard solution of Clozapine (CLO):

Preparation of stock solution of CLO:

Accurately weighed quantity of CLO 10 mg was transferred into 100 ml volumetric flask, dissolved and diluted up to mark with methanol to give a stock solution having strength 100µg/ml.

Preparation of Standard Mixture Solution (LAMO + CLO):

1ml of standard stock solution of LAMO (100μg/ml) and 6 ml of standard stock solution of CLO (100μg/ml) was pipette out into two 10ml volumetric flasks and volume was adjusted to the mark with methanol to get 10μg/ml of LAMO and 60μg/ml of CLO.

Preparation of test solution:

The preparation of synthetic mixture was as per patent:

· Lamotrigine: 10mg

· Clozapine: 60mg

· Sodium Starch Glycolate: 10 mg

· Starch: 10 mg

· Magnesium stearate: 10 mg

All the excipients were mixed in 100ml volumetric flask then make up the volume with methanol and sonicated for 15min.after sonicate make up the volume up to 100 ml with methanol. The solution was filtered through Whatman filter paper No. 42. Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

Validation of proposed method:

Parameters to be considered for the validation of methods are:

1) Linearity and Range:

Procedure:

● Calibration curves for Lamotrigine:

The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of 1-5μg/ml OF LAMO .This series consisted of five concentrations of standard LAMO solution ranging from 1-5 μg /ml. The solutions were prepared by pipetting out Standard LAMO stock solution (0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order spectrum, measured the absorbance at 308 nm against a reagent blank solution (Methanol).

● Calibration curve for Clozapine:

The linearity response was determined by analyzing 6 independent levels of calibration curve in the range of 6-30 μg /ml CLOZ. This series consisted of five concentrations of standard CLOZ solution ranging from 6-30 μg/ml. The solutions were prepared by pipetting out Standard CLOZ stock solution (0.6ml, 1.2ml, 1.8ml, 2.4ml, and 3.0ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order spectrum measured the absorbance at 259.50 nm against a reagent blank solution (Methanol).

2) Precision:

I. Intraday precision:

Procedure:

The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1,3,5μg/ml for LAMO and 6,18,30 μg/ml for CLO and three replicate (n=3)each on same day.

For zero order spectra was measured at 308 nm for LAMO and 259.50 nm for CLO.

The % RSD value of the results corresponding to the absorbance was expressed for intra-day precision.

II. Interday Precision:

Procedure:

The precision of the developed method was assessed by analyzing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1,3,5 μg/ml for LAMO and 6,18,30μg/ml for CLO and three replicate (n=3)each on different day.

For zero order spectra was measured at 308 nm for LAMO and 259.50 nm for CLO.

The % RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

3) Accuracy:

It was determined by calculating the recovery of LAMO and CLO by standard addition method.

Accuracy was done by adding both API standard solution and test solution. Total concentration was as per table.1

Table 1: Solution for accuracy study

Concentration of Formulation (µg/ml)		Concentration of API in spiking solution (µg/ml)		Total concentration of (μg/ml)
LAMO	CLO	LAMO	CLO	LAMO	CLO
2	12	1.8	10	3.8	22
2	12	2	12	4	24
2	12	2.2	14	4.2	26

Procedure:

Each solution was taken and diluted with methanol up to 10ml volumetric flask and scanned between 200nm to 400nm against methanol as a blank. The amount of LAMO and CLO was calculated at each level and % recoveries were computed.

4) Limit of detection and quantitation:

The Limit of detection and quantitation of the developed method was assessed by analyzing ten replicates of standard solutions containing concentrations 1 μg/ml for LAMO and 6 μg/ml of CLO.

The LOD and LOQ were calculated as LOD = 3.3σ/S, and LOQ = 10σ/S, where σ is the standard deviation of the lowest standard concentration and S is the slope of the standard curve.% RSD was calculated.

5) Robustness and ruggedness:

Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the:

Change in Stock Solution Preparation,

Stock-1(10mg LAMO in 100ml methanol-100μg/ml and10mg CLO in 100ml methanol –100 μg/ml)

Stock-2(10mg LAMO in 50ml methanol-200μg/ml and 10mg CLO in 50ml methanol - 200 μg/ml)

Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450),

Three replicates were made for the concentration (1, 3, 5 μg/ml of LAMO and 6, 18, 30 μg/ml of CLO) with different stock solution preparation.

5) Analysis of EDA and ARG in synthetic mixture (assay):

Composition of synthetic mixture:

The preparation of synthetic mixture was as per patent:

Lamotrigine : 10mg

Clozapine :60mg

Sodium Starch Glycolate : 10 mg

Starch : 10 mg

Magnesium stearate : 10 mg

RESULT AND DISCUSSION:

Selection of wavelength and method development for determination of lamotrigine and clozapine

Fig -3 Overlain zero order spectra of Lamotrigine and Clozapine

The Simultaneous Equation Method Can be done as per Zero Order Spectra was overlay.

Spectra were taken in solvent Methanol in the range of 1-5 µg/ml for LAMO and 6-30 µg/ml for CLOZ.

Validation parameters:

1. Linearity and Range:

The Zero order spectra (fig.7.1) showed linear absorbance at 308 nm for LAMO (1-5 µg/ml) and 259.50 nm for CLO (6-30 µg/ml) with correlation coefficient (r²) of 0.998 and 0.998 for LAMO and CLO, respectively.

This method obeyed beer’s law in the concentration range 1-5 µg/ml and 6-30µg/ml for LAMO and CLO, respectively. (Table 7.2)

Correlation coefficient (r²) form calibration curve of LAMO and CLO was found to be 0.998and 0.998, respectively (figure 7.2)

The regression line equation for LAMO and CLO are as following,

y = 0.0362X+0.0044 for LAMO_____________ (1)

y = 0.0641 + 0.0294 for CLO _______________ (2)

Table 2:Calibration data for LAMO and CLO at 308 nm and 259.50 nm, respectively. *(n=6)

Lamotrigine Conc.	Absorbance ± SD*	Clozapine Conc.	Absorbance ± SD*
1	0.042 ± 0.0012	6	0.436 ± 0.0019
2	0.076 ± 0.0012	12	0.796 ±0.0011
3	0.113 ± 0.0006	18	1.155 ±0.0016
4	0.146 ± 0.0016	24	1.548 ± 0.0008
5	0.188 ±0.0021	30	1.984 ±0.0010

Fig-4 Calibration data for LAMO and CLO at 307 nm and 360 nm, respectively.*(n=6)

Table -3: Regression analysis data and validation parameter

Sr. No.	Statistical Parameter	Lamotrigine	Clozapine
1.	Concentration (µg/ml)	1-5 µg/ml	6-30 µg/ml
2.	Straight line equation	Y=0.0362x+0.0044	Y=0.0641x+0.0294
	Slope	0.0362	0.0641
	Intercept	0.0044	0.0294
3.	Correlation coefficient (r²)	0.998	0.998

Mixture linearity [for simultaneous equation]:

Fig-5 Mixture Linearity in range of 1-5 µg/ml of LAMO and 6-30 µg/ml CLO (1:6 Ratio)

Table-4

Sr.no	Concentration in mixture (LAMO:CLO) (1:6)µg/ml	Absorbance ( 308 nm)	Absorbance (259.50 nm)
1	1 : 6	0.231	0.521
2	2 : 12	0.432	0.916
3	3 : 18	0.642	1.446
4	4 : 24	0.844	1.801
5	5 : 30	1.027	2.243

_{Table-5: Intraday precision data for estimation of LAMO
and
CLO}_*(n=3)

Precision	Conc.	LAMO (308 nm)	Conc.	CLO (259.50nm)
Intraday (n=3)
Abs. ±% RSD	1µg/ml	0.232± 0.53	6 µg/ml	0.522 ± 0.23
	3 µg/ml	0.642 ± 0.26	18 µg/ml	1.445 ± 0.19
	5 µg/ml	1.025 ± 0.25	30 µg/ml	2.244 ± 0.11

_{Table-6: Interday precision data for estimation of LAMO
and
CLO}_*(n=3)

Precision	Conc.	LAMO(308 nm)	Conc.	CLO(259.50nm)
Interday (n=3)
Abs. ± % RSD	1 µg/ml	0.234 ± 0.60	6 µg/ml	0.535 ± 0.84
	3 µg/ml	0.644 ± 0.31	18 µg/ml	1.456 ± 0.28
	5 µg/ml	1.026 ± 0.24	30 µg/ml	2.244 ± 0.12

_{Table 7:
Recovery data of LAMO *
and CLO* (n=3)}

Initial conc. ( µg/ml)		Level of recovery	Quantity of Std. Added (µg/ml)		Total Amount (µg/ml)		Result of recovery study
Initial conc. ( µg/ml)		Level of recovery	Quantity of Std. Added (µg/ml)		Total Amount (µg/ml)		Total Quantity Found* (µg/ml)± %RSD		% recovery ± %RSD
LAM	CLO		LAM	CLO	LAM	CLO	LAM	CLO	LAM	CLO
2	12	0 %	-	-	2	12	2.03±0.81	12.10±0.38	101.97±0.18	100.85±0.30
2	12	80 %	1.8	10	3.8	22	3.82±0.12	22.21±0.03	100.84±0.23	100.91±0.38
2	12	100 %	2	12	4	24	4.05±0.20	24.44±0.14	101.11±0.44	101.73±0.12
2	12	120 %	2.2	14	4.2	26	4.23±0.44	26.26±0.09	100.54±0.28	100.98±0.20
Mean of 3 Determination									100.74%	101.91%

Table-10: %Assay of synthetic mixture analysis (n=3)

Drugs	Result of Synthetic Mixture Analysis (n=3)
Drugs	Amount of Drug	%Assay± S.D.	%R.S.D.
LAMO	2 µg/ml	101.97±0.034	0.036
CLO	12 µg/ml	100.85±0.037	0.039

Application of the proposed method for analysis of EDA and ARG in synthetic mixture (ASSAY)

In that synthetic mixture (100 mg) the excipient were like Sodium Starch Glycolate, Starch, Magnesium Stearate were taken as per the required weight. With the Lamotrigine and Clozapine with the ratio (1:6) dissolved in Methanol with small volume of Solvent, Sonicate for 15 min, then make up to 100ml with methanol and filter it.

Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

Table 11: Summary of validation parameter

Sr.No	PARAMETER	Lamotrigine	Clozapine
1	Wave length Max.	308 nm	259.50 nm
2	Linearity (µg/ml) (n=6)	1-5 µg/ml	6-30 µg/ml
3	Regression equation	Y=0.0362x + 0.0044	0.0641x + 0.0294
4	Correlation coefficient (r²)	0.998	0.998
5	Accuracy(%Recovery) (n=3)	100.74 %	101.91%
6	Precision Intra-day (%RSD)(n=3) Inter-day (%RSD)(n=3)	0.25-0.53 0.24-0.60	0.16-0.70 0.11-0.32
7	LOD (µg/ml) (n=10)	0.289	0.108
8	LOQ (µg/ml) (n=10)	0.877	0.329
9	Robustness and Ruggedness (%RSD)	0.12-0.53	0.08-0.23
10	Assay	101.97 %	100.85%

CONCLUSION:

All the parameters are validated as per ICH guidelines for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are in fair agreement.

ACKNOWLEDGEMENT:

We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat, for providing us Infrastructure facilities and moral support to carry out this research work. We are also thankful to SDPARC for giving us their special time and guidance for this research work. We also thank our colleagues for their helping hand.

REFERENCES:

1. Goa KL, Ross SR and Chrisp P. Lamotrigine. A review of its pharmacological properties and clinical efficacy in epilepsy. DRUGS, 1993 Jul; 46(1):152-76.

2. Cohen AF, Land GS, Breimer DD, Yuen BC, Winton PC and Peak AW. Lamotrigine, a new anticonvulsant: Pharmacokinetics in normal humans. Clinical Pharmacological Therapeutics, 1987 June; 42:535-541.

3. Manoj G Tyagi. Estimation of Clozapine in Human Plasma by High – Performance Liquid Chromatography and detection by UV-VIS detector. Asian Journal of Biochemical and Pharmaceutical Research, 2012 Apr; 2(2):49-53.

4. IUPAC Name available from :Clozapine _ C₁₈H₁₉ClN₄ _ ChemSpider.htm

5. Manjunatha JG, Kumara Swami BE, Mamatha GP, Umesh C, Niranjana E and Sherigara BS. Cyclic Voltammetric Studies of Dopamine at Lamotrigine andTX-100 Modified Carbon Paste Electrode. International Journal of Electrochemical Science, 2009 Feb; 4:187-196.

6. James WJ. Lamotrigine in Psychiatry Pharmacology and Therapeutics.CNS Spectrum, 2005; 10(3):224-232.

7. Large CH, Webster EL and Goff DC. The potential role of Lamotrigine in schizophrenia. Psychopharmacology, 2005 Sep; 181(3):415-436.

8. Maurice D, Chao C. Lamotrigine: Chemistry, Bioinformation and Pharmacokinetics. Antiepileptic Drugs, 2002; 5:370-385.

9. Somayeh J, Francesca F, Xu-Feng H. Structural Contribution of antipsychotic drugs to their therapeutic profiles and metabolic side effect. Journal of Neurochemistry, 2012; 120:371-384.

10. International Conference on Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and Methodology, ICH Q2 (R1), 2005.

Received on 04.03.2015 Accepted on 01.04.2015

Asian J. Pharm. Ana. 5(2): April-June 2015; Page 79-85

DOI: 10.5958/2231-5675.2015.00013.7

Conc. (μg/ml)	Lamotrigine (Mean* ±% RSD) (n=3)
	Instrument		Stock
	UV-2450	UV-1800	Stock – 1	Stock – 2
1	0.232± 0.53	0.235± 0.52	0.233± 0.53	0.237± 0.42
3	0.642 ± 0.19	0.645± 0.12	0.643± 0.25	0.648± 0.12
5	1.012 ± 0.12	1.014± 0.16	1.019± 0.21	1.024± 0.12
Clozapine (Mean* ±% RSD) (n=3)
6	0.524 ± 0.15	0.520± 0.20	0.524± 0.23	0.528± 0.17
18	1.444 ± 0.08	1.446± 0.11	1.446± 0.22	1.447± 0.14
30	2.214 ± 0.11	2.216± 0.13	2.211± 0.11	2.218± 0.14

Parameter	LAMO*	CLO*
LOD (µg/ml)	0.289	0.108
LOQ (µg/ml)	0.877	0.329