DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR SIMULTANEOUS ESTIMATION OF LAMOTRIGINE AND CLOZAPINE IN SYNTHETIC MIXTURE BY ABSORPTION CORRECTION METHOD

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ABOUT AUTHORS:
Priyanka P. Atodariya*, Hasumati A. Raj, Vineet C. Jain
*Shree Dhanvantary Pharmacy Collage, Kim,
Surat, Gujarat, India
*atodariya.priyanka@yahoo.com

ABSTRACT:
The simple spectroscopic method has been developed for simultaneous estimation of Lamotrigine and Clozapine in synthetic mixture. Absorbance Correction Method involves the measurement of absorption at two wavelengths 307 nm (l_maxfor Lamotrigine) and 360 nm (l_max for Clozapine). The method was found linear between the range of 1-5 µg/ml for Lamotrigine and 6-30 µg/ml for Clozapine for method. The accuracy and precision was determined and validated statistically. Both the method showed good reproducibility and recovery with %RSD less than 1. The method was found to be rapid, specific, precise and accurate and can be successfully applied for the routine analysis for Lamotrigine and Clozapine in bulk and combined dosage form.

REFERENCE ID: PHARMATUTOR-ART-2346

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

MATERIAL AND METHODS
APPARATUS ANDINSTRUMENT
- 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 ABSORBANCE CORRECTION 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 What man filter paper No. 42. Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

VALIDATION OF PROPOSED METHOD^[10]
Parameters to be considered for the validation of methods are:

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 307 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 360 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 307 nm for LAMO and 360 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 307 nm for LAMO and 360 nm for CLO.
- The % RSD value of the results corresponding to the absorbance was expressed for inter-day precision.

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

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.

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.

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.

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

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 what man filter paper No. 42. Finally the solution had concentration 100μg/ml for LAMO and 600μg/ml for CLO.

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RESULTAND DISCUSSION

SELECTION OF WAVELENGTH AND METHOD DEVELOPMENT FOR DETERMINATION OF LAMOTRIGINE AND CLOZAPINE

Overlain zero order spectra of Lamotrigine and Clozapine

- From spectra at 360 nm (λmax of Clozapine) Lamotrigine has zero absorbance so Clozapine is directly estimate at 360 nm.
- At 307 nm (λmax of Lamotrigine) both drugs have some absorbance so Lamotrigine is estimate at 307 nm using absorbance correction method.

VALIDATION PARAMETERS
Linearity and Range
- The Zero order spectra (fig.7) showed linear absorbance at 307 nm for LAMO (1-5 µg/ml) and 360 nm for CLO (6-30 µg/ml) with correlation coefficient (r²) of 0.997 and 0.997 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.
- Correlation coefficient(r²) form calibration curve of LAMO and CLO was found to be 0.998and 0.998, respectively.

The regression lineequation for LAMO and CLO areas following
Y=0.0367X+0.0035 for LAMO---------- (1)
Y=0.0067+0.0034 for CLO------------- (2)

Table 2: Calibration data for LAMO and CLO at 307 nm and 360nm, respectively. *(n=6)

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

* MIXTURE LINEARITY

[FOR ABSORPTION CORRECTION METHOD]

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

Table 3: Mixture Linearity in range of 1-5 µg/ml of LAMO and 6-30 µg/ml CLO (1:6 Ratios)

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

Precision

I. Intraday precision
The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations 1, 3, 5 μg/ml for LAMO and  6, 18, 30 μg/ml for CLO. Three replicate (n=3) each on same day for intraday

Table-4: Intraday precision data for estimation of LAMO and CLO*(n=3)

II. Interday Precision
The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations  1, 3, 5 μg/ml  for LAMO and  6, 18, 30 μg/ml  for CLO. Three replicate (n=3) each on different day for interday precision.

Table-5: Interday precision data for estimation of LAMO and CLO*(n=3)

ACCURACY
• Accuracy of the method was determined by recovery study from synthetic mixture at three levels (80%, 100%, and 120%) of standard addition.
• Percentage recovery for LAMO and CLO  by this method was found in the range of 99.96 to 101.65%and 99.77-101.90%, respectively,
• 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.

Table 6: Recovery data of LAMO* and CLO* (n=3)

LIMIT OF DETECTION AND QUANTITATION

Table 7: LOD andLOQdataofLAMO andCLO*(n=10)

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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 Wavelength
LAMO at 306.80nm and 307.20 and CLO at 359.80nm and 360.20nm
• 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.

Table 8: Robustness and Ruggedness data of  LAMO and CLO*(n=3)