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

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)

 Lamotrigine Conc. Absorbance ± SD* Clozapine Conc. Absorbance ± SD* 1 0.042 ± 0.0010 6 0.046 ± 0.0011 2 0.075 ± 0.0012 12 0.084 ± 0.0009 3 0.115 ± 0.0012 18 0.120 ± 0.0015 4 0.146 ± 0.0012 24 0.162 ± 0.0006 5 0.190 ± 0.0009 30 0.208 ± 0.0006

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)

 SR.NO CONCENTRATION IN MIXTURE (LAMO:CLO) (1:6)µg/ml ABSORBANCE ( 307 nm) ABSORBANCE  (360 nm) 1 1 : 6 0.214 0.052 2 2 : 12 0.456 0.104 3 3: 18 0.680 0.146 4 4 :24 0.890 0.194 5 5 : 30 1.106 0.224

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

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)

 Precision Conc. LAMO(307 nm) Conc. CLO(360 nm) INTRADAY (n=3) Abs. ±% RSD 1µg/ml 0.215±0.57 6 µg/ml 0.052±0.90 3 µg/ml 0.681±0.24 18 µg/ml 0.142±0.33 5 µg/ml 1.104±0.19 30 µg/ml 0.227±0.71

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)

 Precision Conc. LAMO(307 nm) Conc. CLO(360 nm) INTERDAY (n=3) Abs.±% RSD 1 µg/ml 0.216±0.75 6 µg/ml 0.051±0.91 3 µg/ml 0.682±0.31 18 µg/ml 0.148±0.84 5 µg/ml 1.106±0.15 30 µg/ml 0.228±0.35

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)

 Initial conc. (µg/ml) Level of recovery Quantity of Std. Added (µg/ml) Total Amount (µg/ml) Result of recovery study Total Quantity Found* (µg/ml)±%RSD % recovery ± %RSD LAM CLO LAM CLO LAM CLO LAM CLO LAM CLO 2 12 0 % - - 2 12 1.98 ±0.81 12.03 ±0.38 99.03 ±0.18 100.26 ±0.30 2 12 80 % 1.8 10 3.8 22 3.7 ±0.19 22.23 ±0.09 99.03 ±0.20 101.08 ±0.38 2 12 100 % 2 12 4 24 4.00 ±0.28 24.09 ±0.14 100.24 ±0.39 100.37 ±0.19 2 12 120 % 2.2 14 4.2 26 4.21 ±0.46 25.75 ±0.19 100.23 ±0.29 99.06 ±0.55 Mean of 3 Determination 100.72% 100.37%

LIMIT OF DETECTION AND QUANTITATION

 Parameter LAMO* CLO* LOD (µg/ml) 0.259 0.205 LOQ (µg/ml) 0.786 0.679

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

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