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

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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)

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

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)

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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