STABILITY – INDICATING SPECTROPHOTOMETRIC METHODS FOR DETERMINATION OF LAMOTRIGINE IN PURE FORM AND PHARMACEUTICAL PREPARATIONS

Three simple, sensitive, accurate, and precise methods were developed for determination of lamotrigine (LTG) in bulk powder and pharmaceutical preparations. The first method depends on charge transfer complexing of LTG with chloranilic acid (CA) as will as with 2,3-Dichloro-5,6-Dicyano-1,4-benzoquinone (DDQ) to produce purple color measured at 520 and 588 nm for CA and DDQ respectively. Beer’s low was obeyed in the range of 25 – 200 and 642 μg ml -1 with LOD of 3.06 and 0.498 μg ml -1 and LOQ of 10.19 and 1.66 μg ml -1 respectively. The second method depends on ion pairing of LTG with bromothymol blue (BTB), bromophenol blue (BPB) and bromocresol green (BCG) to produce red colored complexes measured at 419, 417 and 417 nm for the three reagents respectively. Beer’s low was obeyed in the range of 2 – 12 μg ml -1 for all reagents. LOD was found to be 0.56, 0.18 and 0.13 μg ml -1 while LOQ was 1.66, 1.86 and 0.53 respectively. The third method is the adopting of the first derivative spectrophotometry at 291 nm for direct determination of lamotrigine in presence of its degradation product. A linear relationship between peak height and drug concentration was obtained in the range of 10 – 70 μg ml -1 . LOD and LOQ were found to be 0.68 and 2.26 μg ml -1 respectively. The percent recoveries ± RSD% of these methods were 99.52-100.36 ± 0.78-1.34, 99.65-100.48 + 0.85-1.38, and 99.62100.26  1.17-1.22 % respectively. The obtained results were compared with those of the reported method and no significant difference was observed regarding accuracy and precision.

Lamotrigine C 9 H 7 Cl 2 N 5 M.wt.256.1 Lamotrigine, a phenyltriazine compound with anticonvulsant activity is used as monotherapy and as an adjunct to treatment with other anti-epileptics for partial seizures and primary and secondarily generalized tonic-clonic seizures.It is well absorbed from the gastrointestinal tract and peak plasma concentrations occur approximately 2.5 hours after oral doses.It is widely distributed in the body and is reported to be about 55% bound to plasma proteins.It is extensively metabolized in the liver and excreted almost entirely in urine, principally as a glucuronide conjugate (2) .Several methods have been reported for the determination of lamotrigine in pure sample, pharmaceutical preparations and biological fluids including spectrophotometric methods in ultraviolet region in pure and dosage forms at 305 nm (3) .Also, difference spectroscopy in bulk drug and in it's tablet was described by Rajput and Patel (4) .Capillary zone electrophoresis was utilized for determination of lamotrigine in human serum and plasma (5,6) .Several HPLC procedures have been reported for the analysis of lamotrigine in pure form, in its pharmaceutical formulations as will as in biological fluids (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) .

Pharmaceutical Preparations:
Lamotrine® tablets: The product of Apexpharma Company, Egypt.It is labeled to contain 100 mg of lamotrigine per tablet (Batch no .480206).Lamictal® tablets: The product of Glaxosmithkline, England.It is labeled to contain 200 mg per tablet.(Batch no.0301048)

Degraded Sample:
100 mg of pure lamotrigine powder were refluxed with 50 ml 1N HCl for 30 hours.After cooling the solution was neutralized by 3M NaOH, evaporated to dryness under vacuum, the residue was extracted three times with 25 ml methanol, filtered into 100 ml volumetric flask then the volume was adjusted by the same solvent.The obtained solution was labeled to contain (1 mg ml-1).

Standard Solutions: For method A (Charge Transfer Complex Technique):
Solution of drug (1 mg ml -1 ) was prepared by dissolving 100 mg of lamotrigine in 100 ml acetonitrile.Appropriate concentrations were obtained by diluting using the same solvent.

For method B (Ion-Pair Technique):
Stock solution (0.2 mg ml -1 ) of lamotrigine was prepared by dissolving 20 mg of lamotrigine in least amount of methanol and complete to 100 ml with water.

For method C (First Derivative Technique):
Standard solution of lamotrigine (1 mg ml -1 ) was prepared by dissolving 100 mg of the drug in 100 ml methanol.

Chemicals and Reagents:
All chemicals were of analytical grade and all solutions were freshly prepared.

Procedures: For method A (Charge Transfer Complex Technique):
Aliquots of stock drug solution containing ( 0.25 -2 mg ) of lamotrigine solution (1 mg ml -1 ) with CA or ( 0.06 -0.42 mg ) with DDQ were transferred into a series of 10 ml volumetric flasks, 2 ml of 0.1% CA or 3.5 ml of 0.3 % DDQ solution were added then, the volumes were adjusted with acetonitrile.The absorbance of the developed purple colored complexes was measured at 520 nm and 588 nm against the reagent blank for CA and DDQ, respectively.

For method B (Ion-Pair Technique): i-Using BTB:
Aliquots of standard lamotrigine solution (0.2 mg ml -1 ) containing (0.05 -0.3 mg ) were transferred into a series of 125 ml of separating funnels then 3 ml of phthalate buffer pH ( 2.6 ) were added followed by 5 ml of 0.1 % BTB solution.The aqueous layer was adjusted to 15 ml with water and extracted with chloroform by shaking for about half minute.The extracts were collected into 25 ml volumetric flasks, (double extraction 2 x 10 ml) and the volumes were adjusted to 25 ml with chloroform.The absorbances of the formed complexes were measured at 419 nm.

ii-Using BPB:
Aliquots of standard lamotrigine solution (0.2 mg ml -1 ) containing (0.05 -0.3 mg ) were transferred into a series of 125 ml separating funnels then 3 ml of phthalate buffer pH ( 2.8 ) were added followed by 5 ml of 0.1% BPB solution.The aqueous layer was adjusted to 15 ml with water and extracted with chloroform by shaking for about half minute.Then, the procedure was completed as maintained under -Using BTB‖.The absorbance of the formed complexes was measured at 417 nm.

iii-Using BCG:
Aliquots of stock lamotrigine solution ( 0.2 mg ml -1 ) containing (0.05 -0.3 mg) were transferred into a series of 125 ml separating funnels, followed by 3 ml of phthalate buffer pH ( 2.6 ) and 5 ml of 0.1% BCG solution.The aqueous phase was adjusted to 15 ml with water then, extracted with chloroform by shaking for about half minute.Then the procedure was completed as maintained under -Using BTB‖.The absorbance of the formed complexes was measured at 417 nm.

For method C (First -Derivative Technique):
Aliquots of lamotrigine standard solution (1mg ml -1 ) containing (0.1 -0.7 mg) were transferred into a series of 10 ml volumetric flasks and diluted to the mark with methanol.First-derivative ( 1 D) spectra of the drug were recorded against methanol as blank.The peak height at 291 nm was measured in mm for each drug concentration.Calibration curve relating peak height in mm to drug concentration in µg ml -1 was constructed.

Analysis of pharmaceutical preparations:
For method A, an accurately weighed quantity of the well-mixed powdered Lamotrine® 100 mg and Lamictal® 200 mg tablets equivalent to 100 mg of lamotrigine was shaken with 100 ml acetonitrile and filtered.The obtained solution of lamotrigine (1 mg ml -1 ) was subjected to colorimetric determination by the proposed method as mentioned under pure sample.
In the method B, an accurately weighed quantity of the well-mixed powdered tablets equivalent to 20 mg of lamotrigine were shaken with 30 ml of methanol then filtered into 100 ml volumetric flask and the volume was completed to 100 ml with water.The finally obtained solution of drug (0.2 mg ml -1 ) was subjected to colorimetric determination as detailed under pure sample.
For method C, an accurately weighed quantity of each Lamotrine ® 100 mg and Lamictal ® 200 mg very fine powdered tablets equivalent to 100 mg of lamotrigine was introduced into 100 ml volumetric flask, extracted by using 30 ml methanol by shaking for about 5 minutes, filtered into 100 ml volumetric flask, then, the volume was adjusted by methanol to the mark.The obtained solution (1 mg ml -1 ) was analyzed by adopting the 1 D procedure as detailed under pure sample.

In method A (Charge Transfer Complex Technique):
This method depends on the reaction of acceptors with basic nitrogenous compounds as ndonors to form charge transfer complexes.LTG acts as n-donor "D" forms a purple red colored charge transfer complex with both CA and DDQ as π -acceptors, exhibiting absorption maxima at 520 and 588 nm respectively, as shown in figures (1&2).The reaction can be expressed as following (20) .

D + A [ D A ] D . + + A - Donor Acceptor DA complex Radical anion
Acetonitrile affords maximum color intensity and good solvating power for the acceptors.So that acetonitrile was the solvent of choice.Different parameters involved in the reaction were studied such as concentration of the reagent and reaction time.The effect of reagent volume was studied; Figure (3) revealed that, 3 ml of 0.3 % DDQ were sufficient to give maximum absorbance.The maximum color intensity for both CA and DDQ was attained immediately at room temperature and remained stable for further one hour Figure (4).Molar ratio method and Continuous variation method (Job's Method) (19) were applied for the determination of the stoichiometry of LTG.The drug : reagent ratio was found to be 1:1 as shown in figures (5&6).Under the optimum conditions, beer s law is obeyed in the range of 25-200 and 6-42 µg ml -1 for CA and DDQ respectively figures (7&8).

Figure (2): Absorption Spectrum of lamotrigine (30 µg ml -1 ) with 0.3% DDQ (-)
and the Reagent Only (……).Lamotrigine is one of many pharmaceutical compounds which have been determined colorimetrically by ion-pair technique due to its basic nitrogen which reacts with acidic dyes at suitable pH.In this method, lamotrigine reacts with BTB, BPB and BCG to form ion-pair complexes.The formed yellow ion-pair complexes were quantitatively extracted by chloroform and their absorption spectra display absorption peaks at 419 for BTB and 417 nm for both BPB and BCG, respectively.Neither the cited drug nor acidic dyes alone exhibit any significant absorption at the described wavelength under the same conditions as shown in figure (9).The different factors affecting the reaction were studied to establish the optimum pH range; the extraction of the cited drug was carried out over pH range (1.2 -5).The results indicate that, the quantitative extraction of the cited drug was optimum at pH 2.6, 2.8 and 2.6 in presence of BTB, BPB and BCG respectively, figure (10).Phthalate buffer serves well in   maintaining the proper pH range of the buffer mentioned.The volume of buffer solution added was studied and complete color development was attained by adding 3 ml of buffer solution, figure (11).The amount of acidic dye should be sufficient enough and it was found that, 5 ml of reagent solution (0.1%) gives the maximum absorbance the as shown in figure (12).The effect of different extracting solvent was studied, it was found that chloroform (25 ml) is the ideal solvent for extraction of the formed complexes, as shown in figure (13).Also, shaking time for half minute produces reproducible results.The effect of time on the absorbance maxima at the suggested wavelengths was studied.The results obtained showed that full color development was attained after 5 minutes and the intensity of the color stayed constant for at least 24 hours as shown in figures (14&15).The composition of the cited drug ─ BTB, BPB and BCG ion-pair complexes were determined at optimum pH, by applying the molar ratio (19) and continuous variation ( Job's ) method (19) .The results indicate that, the cited drug reacts with the acidic dyes in ratio 1 : 2 as shown in figures (17&18).Under the optimum experimental conditions calibration curves were constructed at 419 nm for BTB and 417 nm for both BPB and BCG respectively.Then a series of standard solutions of lamotrigine revealed linearity over the range 2-12 µg ml -1 for all reagents as shown in figures (16).

For method C (First -Derivative Technique):
First derivative ( 1 D) spectrophotometry was developed for selective determination of lamotrigine in presence of its degradation product.

Degradation of Lamotrigine:
Accelerated degradation of lamotrigine was achieved upon heating under reflux with 1 N hydrochloric acid for 30 hours.This will cause the formation of the trisalt of lamotrigine through the two amino groups and one nitrogen atom of the triazine ring followed by cleavage of lamotrigine molecule upon neutralization with NaOH as illustrated in the following scheme:

Confirmation of degradation product:
For isolation of lamotrigine degradation product from the reaction medium the solution after reflux with 1 N hydrochloric acid for 30 hour was cooled, neutralized with 3 M sodium hydroxide, evaporated under vacuum till dryness and extracted with methanol.The obtained solution was tested by TLC on silica gel 60 GF254 plates.Separation of the intact drug and its corresponding degradate was achieved by using mobile phase consists of methanol -chloroform -acetone (1 : 9 : 1 by volume) and UV detection at 254 nm.Rf values of intact lamotrigine and its corresponding degradate were 0.54 and 0.73, respectively.Complete degradation of lamotrigine was confirmed by absence of spot in the region of the degradate corresponds to the spot of the intact drug.
IR spectrum of the intact drug showed strong bands at 3448, 3320 and 3212 cm-1 of the free two amino groups due to the stretching and bending vibration absorption of the amino group.Also, the presence of strong band at 1622 due to the (-N =N-) stretching vibration like the carbonyl absorption as shown in figure (17).While, IR of the degradate showed broad band of the hydrogen bonded amino and OH group at 3412 cm-1 and also, a band at 1602 cm-1 of the (-N=N-) of the triazine moiety which is shifted to a lower stretching frequency due to the attack of OH group onto the triazine ring as shown in figure (18).1HNMR spectrum of the intact drug showed singlet signal of NH2 group at 6.4 ppm due to the presence of four protons as shown in figure (19).However, 1HNMR of the degradate showed an increase in the number of the aromatic protons due to cleavage of the phenyl moiety from the triazine moiety in addition to a singlet signal at 5.39 ppm indicating the presence of an OH group on the triazine ring as shown in figure (20).
The Zero-order spectra of intact LTG and its degradation product show severe overlapping as shown in figure (21).However, this severe overlapping in zero-order spectra can be resolved by conversion of zero-order to higher first derivative spectra of LTG and its degradation product.Figure ( 22) showing that, the sever overlapping in zero-order spectra can be resolved at 291 nm, at this wavelength zero cross point of degradation product showing no interference to intact LTG.So that, the peak height at this wavelength was chosen for selective determination of the intact drug in presence of its degradate.At the described wavelength linear relationship was obtained between the peak height and the LTG concentration in the range (10 -70 µg ml -1 ).

Validation of the procedures: Linearity:
The linearity range of the absorbances and the corresponding concentrations for methods A was found in the range of 25 -200 and 6-42 µg ml -1 for CA and DDQ respectively.In method B the range was 2 -12 µg ml -1 for BTB, BPB and BCG.While in method C, under the optimized conditions, a linear relationship between the peak height and the corresponding drug concentrations was obtained over the range of 10-70 µg ml -1 .

LOD and LOQ:
The LOD and LOQ were assessed using the slope of the calibration curve and the standard deviation of the blank Table (1).

Accuracy and precision:
Intraday and interday of the proposed procedures were calculated, table (2) revealed the results of the developed methods.

Stability of solutions
The stability of LTG solution for methods A, B and C was evaluated by analysis of the stock solution for each procedure which was found to be stable for 4-6 days at room temperature and 10 days if stored in refrigerator.Also, the reagents were stable for 12 hours at room temperature and 3 days if stored in refrigerator in method A, while reagents of method B are stable for 2 days either stored at room temperature or in refrigerator.

Analysis of pharmaceutical preparation
The proposed methods were used for determination of the cited drug in its pharmaceutical preparations (Lamotrine ® 100 mg and Lamictal ® 200 mg tablets), without interference by excipients and additives.Recovery study was performed by adopting standard addition technique.Different concentrations of standard lamotrigine solution were added to previously analyzed tablets.Results are presented in table (3&4) and revealed that, the proposed method with high recovery.
The results obtained by the proposed procedures were statistically compared with those obtained by the reported one (4) depending on difference spectrophotometry (∆A) assay.
The calculated " t" and " F " values are less than tabulated ones confirming accuracy and precision at 95 % confidence limits table (5).

Specificity:
The specificity of the proposed first derivative method was assured by applying the laboratory prepared mixtures of the intact drug together with its degradation product.The proposed method was adopted for the specific determination of intact LTG in presence of up to 57.14 % of its degradate.The percentage recovery ± SD % was 100.28 + 1.189 %, table (6).

Conclusion
The proposed procedures are simpler, faster and more sensitive than the reported method.

Figure ( 3
Figure (3): Effect of 0.1% CA and 0.3 % DDQ Volume on the Absorbance of Their ReactionProduct with Lamotrigine.

Figure ( 4
Figure (4): Effect of Time on Colour Development of Lamotrigine with 0.1% CA and 0.3%DDQ.
is concentration in g ml -1 .