Ecl. Quím., São Paulo, 35(1): 47 - 54, 201054 Artigo Article www.scielo.br/eq Volume 35, número 1, 2010 55 odic stripping voltammetry using a carbon paste electrode modified with ion-exchange resin Amberlite IR120. Ecl Quim 2006;31:45-52. [12] Ferrari RA, Oliveira VS, Scabio A. Biodiesel from soy- bean: characterization and consumption in an energy genera- tor. Quim Nova 2005;28:19-23. [13] Silva FC, Brandão KSR. Optimization of methyl and eth- yl biodiesel from babassu. Proceedings of the First Congress of the Brazilian Biodiesel Technology Network, v. 2, p.119- 125. Brasília, Brazil, 2006. [14] IUPAC - Standard methods for the analysis of oils, fats and derivatives. 7th ed. London: Blackwell Scientific Publi- cations; 1987. Artigo/Article SenSitiVe SPeCtroPHotometriC DeterminAtion oF LAmotrigine in BuLK Drug AnD PHArmACeutiCAL FormuLAtionS uSing BromoCreSoL green N. Rajendraprasad, K. Basavaiah* and K. B. Vinay Department of Chemistry, university of Mysore, Manasagangothri, Mysore-570 006, india *Corresponding e-mail: basavaiahk@yahoo.co.in. Mobile: +91-9448939105 Fax: 0091-821-2421263, 2516133. Abstract: Two new, simple, rapid and reproducible spectrophotometric methods have been de- veloped for the determination of lamotrigine (LMT) both in pure form and in its tablets. The first method (method A) is based on the formation of a colored ion-pair complex (1:1 drug/dye) of LMT with bromocresol green (BCG) at pH 5.02±0.01 and extraction of the complex into di- chloromethane followed by the measurement of the yellow ion-pair complex at 410 nm. In the second (method B), the drug-dye ion-pair complex was dissolved in ethanolic potassium hydro- xide and the resulting base form of the dye was measured at 620 nm. Beer’s law was obeyed in the concentration range of 1.5-15 µg mL-1 and 0.5-5.0 µg mL-1 for method A and method B, respectively, and the corresponding molar absorptivity values are 1.6932 x 104 and 3.748 x 104 L mol-1cm-1. The Sandell sensitivity values are 0.0151 and 0.0068 µg cm-2 for method A and method B, respectively. The stoichiometry of the ion-pair complex formed between the dug and dye (1:1) was determined by Job’s continuous variations method and the stability constant of the complex was also calculated. The proposed methods were applied successfully for the determination of drug in commercial tablets. Keywords: lamotrigine; spectrophotometry; ion-pair complex; bromocresol green; pharmaceuticals introduction Lamotrigine, (LMT), [6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diami- ne], is an anticonvulsant drug. As an antiepileptic, it has been used successfully to treat epilepsy and bipolar disorder as monotherapy and as an adjunct with other antiepileptics for treatment of partial and generalized toxic-chronic seizures. It is also used to treat neurological lesions and as a tranqui- lizer [1, 2]. Lamotrigine is not official in any phar- macopoeia. The analysis of LMT in biological samples is abundantly described in the literature. Chromatographic techniques have been wide- ly employed since they are powerful separation techniques. The methods based on the high-per- formance liquid chromatography (HPLC) [3-10], high-performance thin layer chromatography (HPTLC) [11] and gas-chromatography (GC) [12] have been described. There is an extensive literature on the determination of lamotrigine in pharmaceuticals include planar chromatography [13], TLC and HPLC [14], HPLC and GC [15], capillary electrophoresis [16, 17]. The immuno- assay techniques [18, 19] have been developed for Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 Ecl. Quím., São Paulo, 35(1): 55 - 66, 201056 Artigo Article Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 57 Artigo Article the determination of this drug in biological sam- ples. UV-spectrophotometric method [20] was used for determination of LMT in tablets, where the tablet extract in 0.1 M NaOH was measured at 305 nm. Youssef and Taha [14] have reported the application of the technique for the determination of LMT using chloranilic acid as a chromogen. The reported method is less sensitive with a linear range 10-200 µg mL-1 and the molar absorptivity of 1.28 x 103 L mol-1cm-1. Though the method is claimed to be selective, any N-containing basic moiety would definitely interfere with the assay. Many of the other reported methods [13- 17] are sensitive and selective but they are time consuming, require expensive instrumental setup, and some require preliminary sample treatment. Adsorptive stripping voltammetric method [21] is highly complicated and is reported to be less pre- cise (RSD ~10 %). Considering these drawbacks, there was a need to develop more advantageous spectrophotometric method for its determination in bulk powder and commercial dosage forms. Al- though many analytical methods were reported to analyze LMT in biological samples and pharma- ceutical samples, none of these methods was sui- table for routine analysis of LMT in pharmaceu- tical preparation. Extractive spectrophotometric procedures are popular for their sensitivity in the assay of drugs and, therefore, ion-pair extractive spectrophotometry has received considerable at- tention for the quantitative determination of many pharmaceutical compounds [22-25] and different alkaloids [26, 27]. We, therefore, developed two higly sensiti- ve, selective, reproducible and economical spec- trophotometric methods for the determination of LMT in bulk powder and in tablets by exploiting its basic nature and its ability to form ion-pair complex with an anionic dye bromocresol green. The first method (method A) is based on the for- mation of an ion-pair complex between drug and dye at pH 5.02±0.01 followed by extraction of the complex into dichloromethane (DCM), and the yellow drug-dye complex was measured at 410 nm. In the second method (method B), the drug- dye ion-pair was broken in ethanolic alkali and the blue color of base form of the dye was measu- red at 620 nm. The method B is a highly sensitive approach for determination of LMT in bulk drug and in tablets. experimental Apparatus A Systronic model 106 digital spectropho- tometer equipped with 1 cm quartz cells was used for absorbance measurements. A digital pH meter Model Elico L1 120 was used for pH measure- ments. Reagents All chemicals used were of analytical gra- de. Solvents used were of the spectroscopic grade. Distilled water was used through out the investi- gation. Sulphuric acid (0.1 M): Concentrated acid (S.D. Fine Chem, Mumbai, India, sp. gr. 1.84) was appropriately diluted with water to get 0.1 M acid. Bromocresol green (0.4%): Dissolved 400 mg of the dye (S.D.Fine Chem Ltd, Mumbai, In- dia) in 10 ml of ethanol and diluted to 100 ml with water. Sodium acetate (1 M): Prepared by dissol- ving 13.61 g of the pure sodium acetate (Merck Specialities Pvt Ltd, Mumbai, India) in 100 ml water. Buffer solution (pH 5.02): Mixed 50 ml of 1 M sodium acetate and 15 ml of 1 M hydrochlo- ric acid (Merck Specialities Pvt Ltd, Mumbai, In- dia, Sp, gr. 1.18) and volume was made upto 250 ml, and pH was adjusted to 5.02 by using dilute NaOAc/HCl solution. Ethanolic KOH (1%): One gram of the pure KOH (S.D.Fine Chem Ltd, Mumbai, India) was dissolved in and diluted to 100 ml with etha- nol. Standard drug solution (30 µg mL-1): LMT (pharmaceutical grade, 99.88 % pure) was procu- red from Cipla India Ltd, Mumbai, India, as a gift and was used as received. A stock standard solu- tion of lamotrigine (300 µg mL-1) was first prepa- red by dissolving 30 mg LMT in 0.1 M H2SO4 and diluting to 100 ml in calibrated flask with the same acid. Stock solution was diluted with the same acid to get a working solution of 30 µg mL-1. Lamosyn 100 and lamosyn 25 (both from Sun Pharmaceuticals Ltd, Mumbai, India) and La- metec 50-DT (Cipla India Ltd, Mumbai, India)- all tablets were purchased from local market and were used in the investigation. recommended Procedures Method A Into a series of 125 ml separating funnels, aliquots of lamotrigine standard solution (30 µg mL-1) equivalent to 1.5 – 15.0 µg mL-1 LMT were accurately transferred and the total volume was brought to 5 ml by adding 0.1 M H2SO4 to each funnel. To each funnel were added 20 ml of wa- ter, 4 ml of 1 M NaOAc and 5 ml buffer of pH 5.02 followed by 5 ml dye (0.4 %), and the con- tent was mixed well. The funnels were shaken vi- gorously with 10 ml of dichloromethane (added from microburette) for 30 seconds, and then allo- wed to stand for clear separation of the two pha- ses. The separated organic layer was passed over anhydrous sodium sulphate and absorbance of the yellow ion-pair complex was measured at 410 nm against a reagent blank similarly prepared. Method B Varying aliquots of LMT-BCG ion-pair complex (10 µg mL-1 in LMT; prepared by follo- wing the procedure described in method A) equi- valent to 0.5 – 5.0 µg mL-1 with respect to LMT were transferred into a series of 10 mL standard flasks and the total volume was brought to 5 mL by adding dichloromethane. To each flask, 1 mL of 1 % alcoholic KOH was added, the content mixed and kept aside for 5 min. Finally, the volume was made upto mark with ethanol and the absorbance measured at 620 nm against the reagent blank. In both the methods, the standard calibra- tion curve was prepared or regression equation derived to calculate the amount of analyte drug in unknown samples. Procedure for the Dosage Forms Twenty tablets were weighed and ground into a fine powder using a pestle and mortar. An accurately weighed portion of the powder equiva- lent to 30 mg LMT was transferred into a 100 ml calibrated flask, 50 ml of 0.1 M H2SO4 was added and the flask was shaken for 20 min. The volume was made upto mark with 0.1 M H2SO4. After 5 min, the solution was filtered through Whatman No 42 filter paper. First 10 ml portion of the filter was discarded and a suitable aliquot was diluted to get a working concentration of 30 µg mL-1 and used for assay by method A. The ion-pair complex of this tablet LMT-BCG (10 µg mL-1) was prepa- red for assay by applying the procedure described in method B. results and Discussion Spectral Characteristics Absorption spectrum of the yellow colored LMT-BCG ion-pair complex is shown in Fig. 1 which has a maximum absorbance (λmax) at 410 nm. This drug dye ion-pair complex was broken in ethanolic base to yield the base form of the blue dye which had maximum absorbance at 620 nm. 16 A B C D Fig. 1. Absorption spectra of A: ion-pair complex (9.5 µg mL-1 LMT); B: blank in method A; C: base form of dye (1.5 µg mL-1 LMT) and D: blank in method B. A B Fig. 2. Effect of water on the absorbance of A: ion-pair complex (9.0 µg mL-1 LMT) and B: blank. Fig. 1. Absorption spectra of A: ion-pair complex (9.5 µg mL-1 LMT); B: blank in method A; C: base form of dye (1.5 µg mL-1 LMT) and D: blank in method B. In both the cases, the blanks had negli- gible absorbance. Ecl. Quím., São Paulo, 35(1): 55 - 66, 201058 Artigo Article Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 59 Artigo Article Reaction Mechanisms Anionic dyes such as BCG forms ion-pair complex with positively charged drugs. The drug-dye stoichiometric ratio as calculated by the Job’s continuous variations method [28] is found to be 1:1. Each drug-dye complex molecule, with two oppositely charged ions, behaves as a single unit held toge- ther by an electrostatic force of attraction (Scheme 1). 15 N N N Cl Cl NH2NH2 H + N N N Cl Cl NH3 + NH2 N N N Cl Cl NH3 + NH2 N N N Cl Cl NH3 + NH2 O Br Br Br Br OH S O O O O Br Br Br Br OH S O O O + LMT LMT-protonated LMT-protonated BCG LMT-BCG ion-pair complex measured at 410 nm Method A Scheme 1. Reaction pathway for method A. N N N Cl Cl NH3 + NH2 O Br Br Br Br OH S O O O N N N Cl Cl NH2NH2 OH O Br Br Br Br OH S O O O + Method B LMT-BCG ion-pair complex Blue colour of the basic form of dye measured at 620 nm LMT Scheme 2. Reaction pathway for method B. Scheme 1. Reaction pathway for method A. In alcoholic alkaline medium, this ion-pair complex gets disturbed and it breaks to form a blue colored basic dye and the drug. The mechanism of this breaking is shown in scheme 2. 15 N N N Cl Cl NH2NH2 H + N N N Cl Cl NH3 + NH2 N N N Cl Cl NH3 + NH2 N N N Cl Cl NH3 + NH2 O Br Br Br Br OH S O O O O Br Br Br Br OH S O O O + LMT LMT-protonated LMT-protonated BCG LMT-BCG ion-pair complex measured at 410 nm Method A Scheme 1. Reaction pathway for method A. N N N Cl Cl NH3 + NH2 O Br Br Br Br OH S O O O N N N Cl Cl NH2NH2 OH O Br Br Br Br OH S O O O + Method B LMT-BCG ion-pair complex Blue colour of the basic form of dye measured at 620 nm LMT Scheme 2. Reaction pathway for method B. Scheme 2. Reaction pathway for method B. optimisation of Variables and method Development A number of preliminary experiments established optimum conditions necessary for rapid and quantitative formation of colored ion-pair complex to achieve the maximum stability and sensitivity. Optimum condition was fixed by varying one pa- rameter at a time while keeping other parameters constant and observing its effect on the absorban- ce either at 410 nm in method A or at 620 nm in method B. Method A Effect of pH In order to establish the optimum pH range, 2 ml of LMT solution in 0.1 M H2SO4 and 3 ml 0.1 M H2SO4 were taken and diluted with 5, 10, 15, 20 and 25 ml of water. Five ml of the dye solution was added after the addition of 4 ml of 1 M NaO- Ac and 5 ml buffer of pH 5.02. The effective pH value of above resulting aqueous solutions was 5.02±0.01. Fig. 2 shows that at 20 ml of water, the absorbance of the ion-pair complex was maxi- mum and the respective blank had shown negligi- ble absorbance. The pH of the aqueous phase was constant at 5.02±0.01 for water volumes ranging from 5-25 ml, at a volume of 20 ml water, blank had a lower absorbance. At water volumes greater than 20 ml, the sensitivity was less. It was also found that a ratio of ~4:1 of aqueous to organic phases was required for the efficient extraction of the colored species. Hence, an aqueous phase of 39 ml which includes 20 ml of H2O was used in all subsequent work. 16 A B C D Fig. 1. Absorption spectra of A: ion-pair complex (9.5 µg mL-1 LMT); B: blank in method A; C: base form of dye (1.5 µg mL-1 LMT) and D: blank in method B. A B Fig. 2. Effect of water on the absorbance of A: ion-pair complex (9.0 µg mL-1 LMT) and B: blank. Fig. 2. Effect of water on the absorbance of A: ion- pair complex (9.0 µg mL-1 LMT) and B: blank. Effect of sodium acetate, BCG and buffer solution Various amounts of NaOAc were added to the acidic solution of lamotrigine to bring the pH to the optimum value and measurements were carried out as recommended. Maximum absor- bance for sample and negligible absorbance for blank was observed when the volume of NaOAc was 4 mL (1 M) in a total volume of 39 ml. The effect of BCG concentration was investigated by varying the volume of dye solution, and using a fixed amount of drug. The complex formation and its extraction were unaffected in the range of 3.0 to 8.0 ml of 0.4 %BCG solution. Hence, 5 ml of 0.4 % BCG solution was fixed in a total volume of 39 ml of aqueous phase. Various amounts of buffer solution were used in the investigation to establish its effect on absorbance. There was almost no influence on the absorbance up to 10 ml, but an amount less than 4 ml resulted in unsatisfactory separation of the organic phase during the extraction. So, 5 ml of buffer was used through out the investigation. Reaction time After the addition of dye, the effect of standing time was studied in the time range 5-30 min before extraction. After a contact time of 5 min, measured absorbance of the complex after extraction into dichloromethane, showed almost constant absorbance values from 5-30 min. So a contact time of 5 min was adequate to form the complex. Effect of shaking time Shaking times ranging from 30 to 60 sec produced no change in absorbance, by maintai- ning all other parameters constant. So a 30 sec shaking time was fixed. Selecting of the Extracting Solvents The effect of the extracting solvent on the ion-pair complex was examined. Dichloromethane was preferred to other solvents (carbon tetrachlo- Ecl. Quím., São Paulo, 35(1): 55 - 66, 201060 Artigo Article Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 61 Artigo Article ride, benzene, cyclohexane, hexane, chloroform, 1, 2-dichloroethane and ether) because of its sli- ghtly higher efficiency on color intensity, selecti- ve extraction of the LMT-BCG complex from the aqueous phase and obtained highest absorbance with dichloromethane. Effect of number of extractions Under optimum conditions, the drug-dye complex in the aqueous phase was extracted with three 10 ml portions of DCM and absorbance was measured each time. After the second extraction, The absorbance of the organic layer was negligi- bly small. Hence, a single extraction with 10 ml DCM was selected for the extraction because of complete recovery of the complex. Equilibration time and stability of the coloured complexes The organic and aqueous phases were cle- arly separated in less than 1 min. The drug-dye ion-pair complex was stable for more than 15 h at laboratory temperatute (30±20 C). Effect of order of addition of reactants The sequence of order of addition of the reactants prior to extraction had small change in the absorbance values. So the order of addition of reactants should be in the described manner. Composition of ion-pair Complexes The composition of the ion-pair complex was established by Job’s method of continuous variations [28] using equimolar concentrations of the drug and the dye (1.955 x 10-4 M). The results indicated that 1:1 (drug:dye) ion-pair is formed through the electrostatic attraction between the positive protonated drug and the anion of dye. Six solutions containing LMT and BCG in various molar ratios, with a total volume of 5 ml, in addi- tion to 20 ml H2O, 4 ml of 1 M NaOAc and 5 ml buffer solution were prepared. The extraction was performed using 10 ml of dichloromethane and the absorbance was subsequently measured at 410 nm. The graph of the results obtained (Fig. 3) 17 Fig. 3. Job’s method of continuous variations plot for ion-pair complex of LMT-BCG in dichloromethane at 410 nm. Fig. 3. Job’s method of continuous variations plot for ion-pair complex of LMT-BCG in dichloromethane at 410 nm. gave a maximum at a molar ratio of Xmax = 0.5 which indicated the formatiuon of a 1:1 LMT:BCG complex. The conditional stability constant (Kf) of the ion-association complex was calculated from the continuous variation data using the following equation [29]: 8 Equilibration time and stability of the coloured complexes The organic and aqueous phases were clearly separated in less than 1 min. The drug-dye ion-pair complex was stable for more than 15 h at laboratory temperatute (30±20 C). Effect of order of addition of reactants The sequence of order of addition of the reactants prior to extraction had small change in the absorbance values. So the order of addition of reactants should be in the described manner. Composition of ion-pair Complexes The composition of the ion-pair complex was established by Job’s method of continuous variations [28] using equimolar concentrations of the drug and the dye (1.955 x 10-4 M). The results indicated that 1:1 (drug:dye) ion-pair is formed through the electrostatic attraction between the positive protonated drug and the anion of dye. Six solutions containing LMT and BCG in various molar ratios, with a total volume of 5 ml, in addition to 20 ml H2O, 4 ml of 1 M NaOAc and 5 ml buffer solution were prepared. The extraction was performed using 10 ml of dichloromethane and the absorbance was subsequently measured at 410 nm. The graph of the results obtained (Fig. 3) gave a maximum at a molar ratio of Xmax = 0.5 which indicated the formatiuon of a 1:1 LMT:BCG complex. The conditional stability constant (Kf) of the ion-association complex was calculated from the continuous variation data using the following equation [29]: [ ] n M n m m f nCAA AA K )(/1 / 2+− = where A and Am are the observed maximum absorbance and the absorbance value when all the drug present is associated, respectively. CM is the mole concentration of drug at the maximum absorbance and n is the stoichiometry which BCG ion associates with drug. The log Kf value was found to be 5.60. where A and Am are the observed maxi- mum absorbance and the absorbance value when all the drug present is associated, respectively. CM is the mole concentration of drug at the maximum absorbance and n is the stoichiometry which BCG ion associates with drug. The log Kf value was found to be 5.60. Method B Studies on the effect of alkali concentration required to break the complex into its components revealed that 1 mL of 1 % alcoholic KOH with a standing time of 5 min was sufficient to yield ma- ximum absorbance at 620 nm. The stability of the resulted blue coloured dye was stable for more than 20 h at laboratory temperatute (30±20 C). method Validation Linearity, sensitivity, limits of detection and quantification Calibration graphs were constructed from ten points covering the concentration ranges 1.5-15 µg mL-1 and 0.5-5.0 µg mL-1 for method A and method B, respectively. Regression analysis of the Beer’s law data indicated a linear relationship between absorbance and concentration, table 1. Sensitivity and regression parameters Parameter Method A Method B λmax, nm 410 620 Color stability > 24 h > 20 h Linear range, µg mL-1 1.5-15 0.5-5.0 Molar absorptivity(ε), l mol-1 cm-1 1.6932 x 104 3.748 x 104 Sandell sensitivity*, µg cm-2 0.0151 0.0068 Limit of detection (LOD), µg ml-1 0.43 0.19 Limit of quantification (LOQ), µg ml-1 1.30 0.59 Regression equation, Y** Intercept (a) -0.0007 0.0007 Slope (b) 0.0661 0.1463 Standard deviation of a (Sa) 0.0998 0.0998 ± tSa/√n 0.124 0.124 Standard deviation of b (Sb) 0.0073 0.022 ± tSb/√n 0.0091 0.027 Variance (Sa2) 0.0096 0.0096 Regression coefficient (r) 0.9993 0.9996 *Limit of determination as the weight in µg per ml of solution, which corresponds to an ab- sorbance of A = 0.001 measured in a cuvette of cross-sectional area 1 cm2 and l = 1 cm. **Y=a+bX, Where Y is the absorbance, X is concentration in µg mL-1, a is intercept, b is slope, ± tSa/√n = confidence limit for intercept, ± tSb/√n = confidence limit for slope. Ecl. Quím., São Paulo, 35(1): 55 - 66, 201062 Artigo Article Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 63 Artigo Article which is corroborated by high values (close to unity) of the correlation coefficients. A plot of log absorbance and log concentration, yielded straight lines with slope equal to 0.996 and 1.009 for method A and method B, respectively, further establishing the linear relation between the two variables. The calculated molar absorptivity and Sandell sensitivity [30] values are summarized in Table 1. The limits of detection (LOD) and quan- tification (LOQ), calculated according to the ICH guidelines [31] using the formulae: LOD = 3.3 S/b and LOQ = 10 S/b, (where S is the standard deviation of blank absorbance va- lues, and b is the slope of the calibration plot) are also summarized in Table 1. The high values of ε and low values of Sandell sensitivity and LOD indicate the high sensitivity of the proposed me- thods. Precision and accuracy The assays described under “general proce- dures” were repeated seven times within the day to determine the repeatability (intra-day preci- sion) and five times on different days to determine the intermediate precision (inter-day precision) of the methods. These assays were performed for three levels of analyte. The results of this study are summarized in Table 2. table 2. Evaluation of intra-day and inter-day accuracy and precision Method LMT taken, µg mL-1 Intra-day accuracy and precision (n=7) Inter-day accuracy and precision (n=5) LMT found ±CL, µg mL-1 %RE %RSD LMT found±CL, µg mL-1 %RE %RSD A 6.0 9.0 12.0 5.90±0.08 8.92±0.11 12.01±0.22 1.67 0.89 0.08 1.52 1.36 1.96 5.96±0.14 8.99±0.11 11.98±0.30 0.67 0.11 0.17 1.82 1.02 2.01 B 1.0 3.0 5.0 1.01±0.01 3.04±0.02 5.01±0.02 1.00 1.33 0.20 0.884 0.603 0.450 1.02±0.02 3.02±0.04 4.98±0.05 2.00 0.67 0.40 1.16 0.92 0.80 %RE. Percent relative error, %RSD. relative standard deviation and CL. Confidence limits were calcu- lated from: CL = ± tS/√n. (The tabulated value of t is 2.45 and 2.77 for six and four degrees of freedom respectively, at the 95% confidence level; S = standard deviation and n = number of measurements). The percentage relative standard devia- tion (%RSD) values were < 2 % (intra-day) and ≤ 2.01 % (inter-day) indicating high precision of the methods. The accuracy of the methods was determined by the percent mean deviation from known concentration, bias % = [(Concentration found - known concentration) x 100 / known concentration]. Bias was calculated at each con- centration and these results are also presented in Table 2. Percent relative error (%RE) values ≤ 2.0 % demonstrate the high accuracy of the proposed methods. Selectivity A systematic study was performed to deter- mine the effect of matrix by analyzing the place- bo blank and synthetic mixture containing LMT. A placebo blank of the composition: starch (10 mg), acacia (15 mg), hydroxyl cellulose ( 10 mg), sodium citrate (10 mg), talc (20 mg), magnesium stearate (15 mg) and sodium alginate (10 mg) was made and its solution was prepared as described under ‘tablets’, and then subjected to analysis. The absorbance of the placebo solution in each case was almost equal to the absorbance of the blank which revealed no interference. To assess the role of the inactive ingredients on the assay of LMT, a synthetic mixture was separately prepared by adding 10 mg of LMT to the placebo mentio- ned above. The drug was extracted and solution prepared as described under the general procedure for tablets. The solution after appropriate dilu- tion were analysed following the recommended procedures. The absorbance resulting from 12 µg mL-1 (method A) and 4.0 µg mL-1 (method B) were nearly the same as those obtained for pure LMT solutions of identical concentrations. This unequi- vocally demonstrated the non-interference of the inactive ingredients in the assay of LMT. Further, the slopes of the calibration plots prepared from the synthetic mixture solutions were about the same as those prepared from pure drug solutions. Robustness and ruggedness The robustness of the methods was eva- luated by making small incremental changes in volume of H2O/ethanolic KOH and contact time and the effect of the changes was studied on the absorbance of the colored systems. The changes had negligible influence on the results as revealed by small intermediate precision values expressed as % RSD (< 1.53 %). Method ruggedness was demonstrated having the analysis done by four analysts, and also by a single analyst performing analysis on four different instruments in the same laboratory. Intermediate precision values (%RSD) in both instances were in the range 0.88 – 1.65 % indicating acceptable ruggedness. The results are presented in Table 3. table 3. Method robustness and ruggedness expressed as intermediate precision (% RSD) Method LMT taken, µg mL-1 Robustness Ruggedness Parameters altered Inter-analysts (%RSD), (n=4) Inter-instruments (%RSD), (n=4) Volume of H2O/ Ethanolic KOH* Reaction/Breaking timeΨ A 6.0 1.02 1.23 0.88 1.65 B 3.0 0.96 1.53 0.99 1.58 *In method A, the volume of H2O was 17, 20 and 23 mL, and in method B the volumes of ethanolic KOH added were 0.8, 1.0 and 1.20 mL. ΨIn method A, the reaction times were 3, 5 and 7 min and in method B breaking times were 3, 5 and 7 min. Application The proposed methods were applied for the quantification of LMT in commercial tablets. The results were compared with these obtained using a published method [20]. The method consisted of the measurement of the absorbance of the ta- blet extract in 0.1 M NaOH at 305 nm. Statistical analysis of the results did not detect any signifi- cant difference between the performance of the proposed methods and reference method with res- pect to accuracy and precision as revealed by the Student’s t-value and variance ratio F-value [32]. The results of assay are given in Table 4. Ecl. Quím., São Paulo, 35(1): 55 - 66, 201064 Artigo Article Ecl. Quím., São Paulo, 35(1): 55 - 66, 2010 65 Artigo Article table 4. Results of analysis of tablets by the proposed methods and statistical comparison of the results with the reference method Tablet brand nameψ Nominal amount, (mg/tablet) Found* (Percent of label claim ± SD) Reference method Method A Method B Lamosyn-100a 100 98.56±0.76 98.14±1.12 t=0.71 F=2.17 99.04±1.06 t=0.83 F=1.95 Lamosyn-25a 25 101.3±0.62 100.6±0.86 t=1.49 F=1.92 101.1±0.90 t=0.42 F=2.11 Lametec-50 DTb 50 102.5±0.86 101.2±1.05 t=2.15 F=1.49 101.8±0.72 t=1.40 F=1.43 *Mean value of 5 determinations. (Tabulated t-value at the 95 % confidence level and for four degrees of freedom is 2.77). (Tabulated F-value at the 95 % confidence level and for four degrees of freedom is 6.39). ψMarketed by : aSun pharmaceuticals. bCipla India Ltd, Mumbai. Recovery study To further assess the accuracy of the me- thods, recovery experiments were performed by applying the standard-addition technique. The recovery was assessed by determining the agre- ement between the measured standard concentra- tion and added known concentration to the sam- ple. The test was done by spiking the pre-analysed tablet powder with pure LMT at three different le- vels (50, 100 and 150 % of the content present in the tablet powder (taken) and the total was found by the proposed methods. Each test was repeated three times. In all the cases, the recovery percen- tage values ranged between 99.22 and 104.3 % with standard deviation in the range 0.85 – 1.25 %. Closeness of the results to 100 % showed the fairly good accuracy of the methods. The results are shown in Table 5. table 5. Results of recovery study via standard-addition method Tablet studied Method A Method B LMT in tablet, µg mL-1 Pure LMT added, µg mL-1 Total found, µg mL-1 Pure LMT recovered (Percent±SD*) LMT in tablet, µg mL-1 Pure LMT added, µg mL-1 Total found, µg mL-1 Pure LMT recovered (Percent±SD*) Lamosyn-100 4.91 4.91 4.91 2.5 5.0 7.5 7.44 9.87 12.73 101.2±0.96 99.22±0.85 104.3±1.25 1.98 1.98 1.98 1.0 2.0 3.0 2.98 4.04 4.96 100.2±1.06 103.2±1.25 99.23±0.98 *Mean value of three determinations. Conclusions In the present study, the maximum co- lor development of LMT-BCG ion-pair complex (method A) and basic dye (method B) was ins- tantaneous. No heating or standing was needed. These methods do not involve procedural steps, do not take more operator time and expertise like HPLC and other methods. In terms of simplici- ty, rapidity, sensitivity and expense, the methods could be considered superior in comparison with the previously reported methods, especially with those based on chromatography [3-15]. The rea- gents utilized in the proposed methods are che- aper, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. The method is unaffected by slight variations in experimental conditions such as time, reagent concentration or temperature. The methods are highly sensitive compared to the only spectrophotometric method [14]. Since there was no interference from the tablet excipients, the methods are highly selective in comparison with the chloranilic acid and method based on charge- transfer complexation method [14]. The proposed methods gave results with good accuracy to permit determination of low concentration even down to 0.5 µg mL-1. The wide applicability of the described procedure for routi- ne quality control is well established by the assay of LMT in pure form, as well as in pharmaceutical preparations Aknowledgement Authors thank M/S. Cipla India Ltd, for gifting pure lamotrigine, two of the authors (NRP & KBV) are gratefully thanks the University of Mysore, Mysore, permission and facilities and on of the authors (NRP) gratefully acknowledges the UGC, New Delhi, for providing Meritorious Re- search Fellowship. references A.G. Gilman, J.G. Hardman, L.E. Limbird, “Goodman [1] and Gilman’s the Pharmacological Basis of Therapeutics.” 10th ed. McGraw Hill, New York, U.S.A. (2001) pp. 539. C. Sean, Sweetman, Martindale: The Complete Drug [2] Reference 34th ed. Pharmaceutical Press, London. (2005) pp. 363. M. Cociglio, R. Alric, O. Bouvier, J. Chromatogr. Biomed. [3] Appl. 110: 1-2, J.Chromatogr. 572 (1991) 269. G.L. Lensmeyer, B.E. Gidal, D.A. Wiebe, Ther. Drug Mo-Drug Mo-[4] nit. 19 (1997) 292. P. 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Artigo/Article ComPLeXeS oF 4-CHLoroPHenoXYACetAteS oF nD(iii), gD(iii) AnD Ho(iii) W.Ferenc1*, M.Bernat1, J.Sarzyński2 and H.Głuchowska1 1Faculty of Chemistry, Maria Curie-Sklodowska University, PL 20-031 Lublin, Poland 2Faculty of Physics, Maria Curie-Sklodowska University, PL 20-031 Lublin, Poland * Author for correspondence: e-mail: wetafer@poczta.umcs.lublin.pl Abstract: The complexes of 4-chlorophenoxyacetates of Nd(III), Gd(III) and Ho(III) have been synthesized as polycrystalline hydrated solids, and characterized by elemental analysis, spec- troscopy, magnetic studies and also by X-ray diffraction and thermogravimetric measurements. The analysed complexes have the following colours: violet for Nd(III), white for Gd(III) and cream for Ho(III) compounds. The carboxylate groups bind as bidentate chelating (Ho) or bridging ligands (Nd, Gd). On heating to 1173K in air the complexes decompose in several steps. At first, they dehydrate in one step to form anhydrous salts, that next decompose to the oxides of respective metals. The gaseous products of their thermal decomposition in nitrogen were also determined and the magnetic susceptibilites were measured over the temperature range of 76-303K and the magnetic moments were calculated. The results show that 4-chlo- rophenoxyacetates of Nd(III), Gd(III) and Ho(III) are high-spin complexes with weak ligand fields. The solubility value in water at 293K for analysed 4-chlorophenoxyacetates is in the order of 10-4mol/dm3. Keywords: 4-chlorophenoxyacetates, thermal stability, magnetic properties of Nd(III), Ho(III) and Gd(III). introduction The carboxylates play an important role in inorganic and bioinorganic chemistry. Many me- tal cations are a component of several vitamins and drugs [1,2]. The carboxylates of d- and 4f- ion elements may be used as electric materials in the modern branches of techniques and technology. They may have also applications as precursors in superconducting ceramic and magnetic field pro- ductions and may be used as catalysts, pigments, solvents, food preservatives and plastics produc- tions. Metal carboxylates are applied for the pro- ductions of high degree purity of metal oxides and polycarboxylic acids are used for supermolecular compound synthesis, which in many cases, form with metal ions the molecular polymers containing in their structures, pores and channels owning to them they appear catalytic and adsorption proper- ties. Therefore they may be used for the adsorp- tion of inorganic gases such as: argon, nitrogen and hydrocarbons or small molecules of another inorganic compounds. Polycarboxylic acids may also form the molecules with two- and three–di- mentional structures, yielding special magnetic and luminescence properties which let them be used in optical and electronic industries [3-10]. 4-Chlorophenoxyacetic acid is a white sol- id hardly soluble in water (K=7,9 ·10-4) and easily soluble in ether and ethanol [10,11]. It is used as growth hormone for plants [12]. In some papers the details of its molecular structure was pre- sented [13-15]. Literature survey informs that its complexes with Li(I), Cu(II), Co(II), Mn(II) and Ecl. Quím., São Paulo, 35(1): 67 - 75, 2010