Spectrophotometric Determination of Glimepiride in Pure and Pharmaceutical Formulations Using Bromophenol Blue and Thymol Blue
Abdul Aziz Ramadan^{1}*, Hasna Mandil^{2}, Souad Zeino^{3}
Department of Chemistry, Faculty of Sciences, University of Aleppo, Syria
*^{1}Corresponding Author Email: ^{1}dramadan@scsnet.org, ^{2}promandil955@gmail.com, ^{3}Souad198977@gmail.com
ABSTRACT:
Two simple, direct and accurate spectrophotometric methods have been developed for the determination of Glimepiride (GLM) in pure and pharmaceutical formulations by ionpair complexes with Bromophenol Blue (BPB) and Thymol Blue (TB). The methods involve the formation of yellow ionpair complexes between BPB and TB with GLM at pH<3.8; after reacting GLM with Na_{2}CO_{3} to give C_{24}H_{33}N_{4}H^{+}O_{5}NaS which is prepared in chloroform. The formed complexes [GLM]:[BPB] in chloroform and [GLM]:[TB]_{2} in acetonitrile were measured at ^{l}^{max} 424 and 392 nm, respectively. Molar absorptivity(e) for complexes were 23000 and 28000^{ L.mol1.cm1}, respectively. Beer’s law was obeyed in the concentration range of 1.126 – 46.608 mg.mL^{1 }with relative standard deviations (RSD)3.8%_{ }in presence of 5.0x10^{4} mol.L^{1} of BPB and 0.319 – 34.343 mg.mL^{1 }with RSD_{}4.0% in presence of 2.5x10^{4} mol.L^{1} of TB. The proposed method was validated for specificity, linearity, precision, accuracy, repeatability, sensitivity (LOD and LOQ) and robustness.
The results for the determination of GLM using TB were more sensitive and accurate than using BPB. The developed method is applicable for the determination of GLM in pure and different dosage forms with average assay of 99.0 to 101.0% and the results are in good agreement with those obtained by the RPHPLC reference method.
KEYWORDS: Direct spectrophotometric method Ionpair complexes, Glimepiride; Bromophenol Blue, Thymol Blue.
INTRODUCTION:
GLM belongs to sulfonylurea oral anti diabetic and GLM is an antidiabetic drug which is used for the treatment of diabetes. GLM is a white to yellowishwhite, odorless, crystalline powder insoluble in water. It is chemically described as 1[[p[2(3ethyl4methyl2oxo3pyrroline1carboxamido) ethyl] phenyl]sulfonyl]3(trans4methylcyclohexyl)urea (C_{24}H_{34}N_{4}O_{5}S) with a mol. mass of 490.62 g [1], see Scheme 1.
BPB (C_{19}H_{10}Br_{4}O_{5}S), acts as a weak acid in solution. It can thus be in deprotonated or protonated form, appearing blue to yellow. It is bluish green in neutral solution. The deprotonating of the neutral form results in a highly conjugated structure, accounting for the difference in color. An intermediate of the deprotonating mechanism is responsible for the greenish color in neutral solution, mol. mass 669.96 g [1]. Bromophenol blue has been used as a reagent to form ion pair complex with many drugs [2, 3], see Scheme 2.
TB (C_{27}H_{30}O_{5}S) belongs to thymolsulfonphthalein is a brownishgreen or reddishbrown crystalline powder that is used as a pH indicator. It transmits from red to yellow respectively. It is orange in neutral solution, mol. mass 466.59 g [3], see Scheme 3.



Scheme 1: Chemical structure of Glimepiride (GLM). 
Scheme 2: Chemical structure of Bromophenol blue (BPB). 
Scheme 3: Chemical structure of Thymol blue (TB). 
A simple, accurate and sensitive UVVisible spectrophotometric method have been developed and validated for the quantitative determination of cefixime and glimepiride in pure form or in their dosage forms. The method is based on the formation of a ternary complex with copper (II) and eosin. The method does not involve solvent extraction. Appropriate conditions were examined for the reaction to obtain maximum absorptivity and sensitivity. The color of the produced complex is measured at 550 and 544 nm with apparent molar absorptivities of 1.49 X 10^{4} and 1.657 X 10^{4} L.mol^{1}.cm^{1}, respectively. The method is applicable over concentration range of 428 and 550 μg.mL^{1} for cefixime and glimepiride, respectively. The analytical performance of the developed method was fully validated and the results obtained revealed high accuracy (recovery values, 100 ± 1%) and precision (with relative standard deviation <1.50%). Furthermore, the developed methods hold their accuracy and precision well when applied to the determination of cefixime and glimepiride in their dosage forms, therefore, they could be used for routine analysis of the two drugs in their pharmaceutical dosage forms^{ }[4].
Two simple and sensitive visible spectrophotometric methods have been developed for the quantitative estimation of glimepiride from its tablet formulation. The developed methods are based on formation of chloroform extractable colored complex of drug with methylene blue and safranine. The chloroform extracted complex of drug with methylene blue showed absorbance maxima at 652 nm and linearity was observed in the concentration range of 1550 μg.mL^{1} (methodI), while with safranine showed absorbance maxima at 536 nm and linearity was observed in the concentration range of 1080 μg.mL^{1} (methodII). Results of analysis for both the developed methods were validated statistically and by recovery studies [5].
Various spectrophotometric methods^{ }[614] have been reported for the determination of Glimepiride in pure as well as in dosage forms. Most spectrophotometric methods employ extraction procedures. In this case, the extracted complexes were into an organic solvent, which is immiscible with water, and the concentration of the resulting complex in the organic phase is determined spectrophotometrically. The complex extraction technique has some difficulties and inaccuracies due to incomplete extraction or the formation of emulsions between the hydrocarbon solvent and the basic compoundcontaining solution. In response to the problems resulting from extraction of the complex, it is better to determine formed complex without extraction^{ }[15]. Also none of the methods reported in the literatures is based on the formation of complexes between BPB and TB with GLM.
In this study, extractionfree spectrophotometric method for determination of GLM through ionpair complexes formation with BPB and TB were developed.
MATERIALS AND METHODS:
Equipment and materials:
Spectrophotometric measurements were made in Spectro scan 80 DV UVVIS spectrophotometer with 1 cm quartz cells. An ultrasonic processor model Powersonic 405 was used to sonicate the sample solutions. The diluter pipette model DIP1 (Shimadzu), having 100 μl sample syringe and five continuously adjustable pipettes covering a volume range from 20 to 5000 μL (model Piptman P, GILSON).^{ Centrifuge (Centurion Scientific Ltd., Model: K2080Manufactured in the United Kingdom)} was used for preparation of the experimental solutions. SARTORIUS TE64 electronic balance was used for weighing the samples.
Glimepiride (99.98%) was supplied by Chempi fine chemicals (INDIA), Metformin HCl (99.0%) and Rosuvastatin Calcium (98.6%). Bromophenol blue (99%) of analytical grade, chloroform SigmaAldrich, BPB was prepared as 1×10^{2} mol. L^{1} in chloroform. Thymol blue (99%), acetonitrile of analytical grade, and Na_{2}CO_{3} extra pure were from Merck. TB was prepared as 1×10^{3} mol. L^{1} in acetonitrile. All solvents and reagents were analytical grade chemicals.
Tablet commercial formulations were used for the analysis of GLM. The pharmaceutical formulations subjected to the analytical procedure were:
(1) Amarium tablets, Racha Lab, Aleppo–Syria, each tablet contains 2 and 4 mg of GLM, (Mfg. 4/2017, Exp. 4/2020).
(2) Amapiride tablets, Avenzor Pharmaceutical Industries, Damascus–Syria, each tablet contains 2 and 4 mg of GLM, (Mfg. 12/2017, Exp. 12/2020).
Standard stock solutions:
1´10^{3} mol.L^{1} of pure GLM was prepared in chloroform. This solution was prepared by good mixing 12.27 mg of GLM with 0.05 g of Na_{2}CO_{3}, adding 0.1 mL methanol, after that it was dissolved in chloroform into a volumetric flask (25 mL) and diluted up to mark with chloroform. The solution was stored in dark bottles and kept in the refrigerator for not more than a week. The stock solution was further diluted daily just before the use to obtain working solutions of GLM in the concentration range 2.5 95 μM (1.126 – 46.608 μg.mL^{1} of GLM) and 0.6570 μM (0.319 – 34.343 μg.mL^{1} of GLM) for BPB and TB, respectively.
Recommended Procedure:
Aliquots of 1´10^{3} mol.L^{1} GLM solution (6.5, 7.5, 10, 25, 35, 50, 75, 85, 100, 200, 300, 400, 500, 600, 700, 850 and 950 μL) containing 0.65, 0.75, 1.0, 2.5, 3.5, 5.0, 7.5, 8.5, 10, 20, 30, 40, 50, 60, 70, 85 and 95 μM (0.319, 0.368, 0.491, 1.226, 1.717, 2.453, 3.680, 4.170, 4.906, 9.812, 14.719, 19.625, 24.531, 29.437, 34.343, 41.703 and 46.608 μg.mL^{1}) of GLM were transferred into a series of 10 mL calibrated volumetric flasks. Then 0.500 mL of BPB solution (1x10^{2} mol.L^{1}) and 2.500 mL of TB solution (1x10^{3} mol.L^{1}) was added respectively. The volume was diluted up to the mark with the solvent and the absorbance were measured at l_{max }424 nm in chloroform for BPB and l_{max }392 nm in acetonitrile for TB, against a similar reagent blank.
Procedure for pharmaceutical formulations:
Twenty tablets of each studied pharmaceutical formulation were weighed accurately, finely powdered and mixed well. An amount of the powder equivalent to the weight of one tablet was mixed well with 0.05 g of Na_{2}CO_{3}, adding 0.1 mL methanol and solved in chloroform using ultrasonic for 15 min, 10 ml of chloroform was added, filtered over a 10 ml flask and washed by the same solvent, then diluted to 10 ml with chloroform. This solution contains the following: 200 and 400 mg.mL^{1} of GLM for all studied pharmaceutical formulations contains 2 and 4 mg/tab, respectively.
Five solutions were prepared daily by diluting 1.0 mL from each stock solution of pharmaceutical formulations for contents: 2 or 4 mg/tab, then 0.4 ml from stock standard solution of BPB or 4.0 ml from stock standard solution of TB was added and adjusted the volume up to 10 ml with chloroform or acetonitrile (these solutions contain 20 or 40 mg.mL^{1} of GLM respectively and they contain 4x10^{4 }mol.L^{1} and 2.5x10^{4 }mol.L^{1} of BPB and TB, respectively; test solutions).
RESULTS AND DISCUSSION:
The effect of solvent:
The effect of the solvents (acetone, acetonitrile, dichloroethane, dichloromethane, chloroform and ethyl acetate) on absorbance of reagents BPB, TB, formed complexes [GLM]:[BPB]_{ }or [GLM]:[TB]_{2}_{.} It was found that chloroform and acetonitrile solvents were the best for complex [GLM]:[BPB] and complex [GLM]:[TB]_{2}, respectively.
Fig.1. (a) UVVis spectra in chloroform of: 1 0.8x10^{4 }mol.L^{1 }of GLM; 2 5.0x10^{4 }mol.L^{1} of BPB; 3 0.5x10^{4 }mol.L^{1 }ionpair complex (0.5x10^{4 }mol.L^{1} of GLM with 5.0x10^{4 }mol.L^{1}of BPB ); Blank is 5.0x10^{4} mol.L^{1 }of BPB, (b) UVVis spectra in acetonitrile of: 1 0.8x10^{4 }mol.L^{1 }of GLM; 2 1.0x10^{4 }mol.L^{1} of TB; 3 0.5x10^{4 }mol.L^{1 }ionpair complex [GLM]:[ TB] (0.5x10^{4 }mol.L^{1} of GLM with 0.5x10^{4 } mol.L^{1}of TB); Blank is acetonitrile, 4 0.5x10^{4 }mol.L^{1 }ionpair complex [GLM]:[ TB]_{2} (0.5x10^{4 }mol.L^{1} of GLM with 4.0x10^{4 }mol.L^{1 }of TB ); Blank is 4.0x10^{4} mol.L^{1 }of TB, ℓ =1 cm.
Absorption spectra:
UVVis spectra of GLM, BPB and the formed complex [GLM]:[BPB] solutions in chloroform was obtained and spectra of GLM, TB and the formed complexes [GLM]:[TB] and [GLM]:[TB]_{2} solutions in acetonitrile was obtained. GLM solutions do not absorb in the range 300600 nm. BPB and TB solutions have small absorption at l_{max} 424 ^{nm and }392 nm^{ }for BPB and TB, respectively^{ (}^{e }^{≈ 245}^{ L mol1 cm1 in }^{chloroform}^{ and}^{ }^{e }^{≈ 637 }^{L mol1 cm1 in }acetonitrile for BPB and TB^{)}. [GLM]:[BPB] and [GLM]:[TB]_{2} complexes solutions have maximum absorption at l_{max }424 nm and^{ }392 nm, e for the complexes were 23000^{ L mol1 cm1 }and 28000^{ L.mol1.cm1}, see (Fig. 1) as an example.
Optimization of variables:
The different experimental parameters affecting the spectrophotometric determination of GLM through ionpair complexes [GLM]:[BPB] and [GLM]:[TB]_{2} formation with BPB and TB were studied in order to determine the optimal conditions for the determination of GLM.
The effect of time and temperature:
The effect of time and temperature on the complexes [GLM]:[BPB] and [GLM]:[TB]_{2} formation were studied within the ranges 5120 min and ^{1545oC.} It was found that the formed complexes weren't affected by time or temperature at those ranges.
The effect of BPB and TB concentration:
The effect of BPB and TB concentration on complexes [GLM]:[BPB] and [GLM]:[TB]_{2} formations were investigated. It was observed that the ^{absorbance of the formed complexes increased coinciding with increasing the ratio of C}_{BPB}:C_{GLM} and^{ C}_{TB}:C_{GLM} until the ratio (5:1) and (4:1), respectively, then slowly increased until the absorbance became a quasistatic at ratio more than 10 and 6, respectively.
Stoichiometric Relationship:
The molar ratio method:
The composition of GLM: TB complexes were determined by the molar ratio method and Job's method of continuous variation^{ }[14, 15]. The stoichiometry of GLM:TB complex was studied by molar ratio method according to the following equation: A_{max}= f ([TB]/ [GLM]) at l_{max} 392 nm. It confirmed that the binding ratio of GLM:TB complexes are equal to (1:1 and 1:2); where the concentration of GLM was constant (50 mM) and the concentrations of TB changed from 0 to 250 mM, see (Fig. 2) in acetonitrile as example. The formation constant of the ion pair complexes [GLM]:[TB] and [GLM]:[TB]_{2} are 45.7 x10^{5} and 28.75x10^{5}, respectively.
Fig.2. Molar ratio method to calculate binding ratio of GLM:TB complexes at l_{ max} 392 nm in acetonitrile ([GLM]= 50 mM, blank is acetonitrile, ℓ =1 cm).
The Job's method:
Continuous variation was utilized to check the composition of GLM: TB complexes at l_{max }392 nm in acetonitrile. The absorbance of the complexes in used solvent were plotted against the mole fraction [TB]/([GLM]+[TB]), where [GLM]+[TB]=100 mM. The plot reached maximum values at a mole fraction of 0.5 and 0.67, see (Fig. 3) as example. This indicated complexes formation (GLM: TB) in the ratio of (1:1 and 1:2). The formation constant of the ion pair complexes [GLM]:[TB] and [GLM]:[TB]_{2} are 18x10^{5} and 78.5x10^{5} in acetonitrile.
Fig.3. Job's method of continuous variation to calculate binding ratio of GLM: TB complex at l_{max} 392 nm in acetonitrile ([GLM]+[TB]=100 mM, blank is acetonitrile, ℓ =1 cm).
Mechanism of Reaction:
Anionic dyes such as TB form ionpair complexes with the positively charged nitrogencontaining molecule. The color of such dyes is due to the opening of lactoid ring and subsequent formation of quinoid group (deprotonated). GLM(C_{24}H_{34}N_{4}O_{5}S) is reacted with Na_{2}CO_{3} to give (C_{24}H_{33}N_{4}H^{+}O_{5}NaS), protonated and forms yellow ionpair complexes [GLM]:[TB] and [GLM]:[TB]_{2} with the dye. Each drugdye complex with two oppositely charged ions (positive on the drug and negative on the dye) behaves as a single unit held together by an electrostatic binding [14]. The suggested mechanism of GLM:TB ionpair complexes formation in in acetonitrile is shown in Scheme 4.
i First step:
iiSecond step:
iii Third step (formation of [GLM]:[TB]):
iv Fourth step:
v Fifth step (formation of [GLM]:[TB]_{2}):
Scheme 4: The possible reaction mechanism of [GLM]:[TB] and [GLM]:[TB]_{2} complexes formation.
Calibration curve:
The calibration curves of GLM in pure form through complications with BPB and TB showed an excellent linearity over concentration range of 1.226 – 46.608 μg.mL^{1} in presence of 5.0´10^{4} mol.L^{1 }of BPB in chloroform and the concentration range of 0.319 – 34.343 μg.mL^{1} in presence of 2.5´10^{4} mol.L^{1 }of TB in acetonitrile. Regression^{ equation at }^{l}^{max was as the follows: }y=0.0455x+0.0091(R^{2}=0.9997) and y=0.0571x+0.0003 (R^{2}=0.9998) for the BPB and TB respectively, (Figs. 4 and 5).
The spectra characteristics of the method ^{such as} the molar absorptivity^{ (}^{e}^{), Beer's law, }regression equation at ^{l}^{max (y=a.x+b); where y=absorbance, a=slope, x=concentration of GLM }^{by }^{μg.mL1, b=intercept, the correlation coefficient, }limit of detection (LOD) and limit of quantification (LOQ) and the optimum conditions for spectrophotometric determination of GLM through ionpair complex formation using BPB in chloroform^{ is summarized in Table }1^{.}
Fig.4. Spectra and Calibration curve of [GLM]:[BPB] complex in presence of 5.0´10^{4} M of BPB; where C_{GLM} as the follows: 0.491, 1.226, 1.717, 2.453, 3.680, 4.170, 4.906, 9.812, 14.719, 19.625, 24.531, 29.437, 34.343, 36.797, 41.703 and 46.608 μg.mL^{1} {Blank is BPB solution in chloroform 5.0´10^{4} M; ℓ = 1cm}.
Fig.4. Spectra and Calibration curve of [GLM]:[BPB] complex in presence of 5.0´10^{4} M of BPB; where C_{GLM} as the follows: 0.491, 1.226, 1.717, 2.453, 3.680, 4.170, 4.906, 9.812, 14.719, 19.625, 24.531, 29.437, 34.343, 36.797, 41.703 and 46.608 μg.mL^{1} {Blank is BPB solution in chloroform 5.0´10^{4} M; ℓ = 1cm}.
ANALYTICAL RESULTS:
Spectrophotometric determination of GLM through complexation with BPB and TB within optimal conditions using calibration curve was applied. The results, summarized in Tables 2 and 3, showed that the determined concentration of GLM was rectilinear over the range of 1.126 – 46.608 μg.mL^{1} and 0.319 – 34.343 μg.mL^{1} for BPB and TB, respectively. With relative standard deviation (RSD) not more than 3.8% and 4.0%, respectively. The results obtained from the developed method have been compared with the official RPHPLC method^{ }[16] and a good agreement was observed between them.
APPLICATIONS:
The developed spectrophotometric method was applied to determine GLM in some pharmaceutical preparations through complexes formation by BPB and TB according to the optimal conditions. The results of quantitative analysis for GLM in pharmaceutical preparations were summarized in Table 3. The proposed method was simple, direct, specific and successfully applied to the determination of GLM in pharmaceuticals without any interference from excipients. Average recovery ranged between 99.0 to 101.0%. The results obtained by this method agree well with the contents stated on the labels and were validated by RPHPLC method [16].
^{ }
Table 1. The parameters established for spectrophotometric determination of GLM by complexes formation with BPB and TB.
parameters 
Operating values 
^{ }^{BPB} 

^{Solvent} 
Chloroform 
^{l}^{max of GLM: BPB }complex in chloroform, nm 
^{424} 
^{Beer’s Law Limit, }μ^{M } 
^{2.595} 
^{Beer’s Law Limit, }μg.mL^{1} 
1.126 ^{– 46.608} 
Molar absorptivity of [GLM]:[BPB] complex (e ), L.mol^{1}.cm^{1 } 
23000 
^{Regression equation for }[GLM]:[BPB] ^{at }^{l}^{max 424 }nm^{:} 

^{Slope} 
^{0.0455} 
^{Intercept} 
^{0.00}^{91} 
^{Correlation coefficient (R2)} 
^{0.999}^{7} 
^{LOD, }μg.mL^{1 } 
^{0.}^{1}^{4} 
^{LOQ, }μg.mL^{1 } 
^{0}^{.}^{43} 
^{RSD%} 
^{3.8} 
^{CBPB}:C_{GLM}, M 
^{≥5} 
Stability 
^{24 h} 
^{Temperature of solution} 
^{25±5oC} 
^{ }^{TB} 

^{Solvent } 
^{acetonitrile} 
^{l}^{max of GLM:TB complexes in acetonitrile, nm} 
^{392} 
^{Beer’s Law Limit, }μ^{M } 
^{0.6570} 
^{Beer’s Law Limit, }μg.mL^{1} 
^{0.319 – 34.343} 
Molar absorptivity of [GLM]:[TB] complex (e_{1}), L.mol^{1}.cm^{1 } 
^{20200} 
Molar absorptivity of [GLM]:[TB]_{2} complex (e_{2}), L.mol^{1}.cm^{1 } 
^{28000} 
^{Regression equation for }[GLM]:[TB]_{2 }^{at }^{l}^{max 392 nm:} 

^{Slope} 
^{0.0570} 
^{Intercept} 
^{0.0024} 
^{Correlation coefficient (R2)} 
^{0.9998} 
^{LOD, }μg.mL^{1 } 
^{0.}^{04} 
^{LOQ, }μg.mL^{1 } 
^{0.13} 
^{RSD% } 
^{4.0} 
^{CTB}:C^{ GLM}, M^{ } 
^{≥4} 
Stability 
^{36 h} 
^{Temperature of solution} 
^{25±5oC} 
^{ }
^{T}^{able 2: Spectrophotometric determination of GLM through complexes formation with BPB (in chloroform) and TB (in acetonitrile) }^{with}^{in optimal conditions using calibration curve}_{ }^{(n=5, t=2.776).}
_{}±SD, mg.mL^{1} RPHPLC[16] 
BPB 
TB 
X_{i}, mg.mL^{1} (Taken) 

RSD % 
_{}, mg.mL^{1} 
_{}±SD^{, } mg.mL^{1} (Found) 
RSD% 
_{}, mg.mL^{1} 
_{}±SD^{, } mg.mL^{1} (Found) 

Not determine 
 
 
Not determine 
4.0 
0.310±0.0154 
0.310±0.0124 
0.319 
Not determine 
 
 
Not determine 
3.8 
0.366± 0.0173 
0.366±0.0140 
0.368 
0.496±0.0280 
 
 
Not determine 
3.5 
0.503±0.0218 
0.503±0.0176 
0.491 
1.221±0.0465 
3.8 
1.120±0.0528 
1.120±0.0426 
3.4 
1.256±0.0535 
1.256±0.0431 
1.226 
1.708±0.0586 
3.5 
1.691±0.0735 
1.691±0.0592 
3.2 
1.728±0.0686 
1.728±0.0553 
1.717 
2.430±0.0712 
3.3 
2.426±0.0994 
2.426±0.0800 
3.0 
2.420±0.0901 
2.420±0.0726 
2.453 
3.718±0.1015 
3.2 
3.738±0.1485 
3.738±0.1196 
2.8 
3.762±0.1308 
3.762±0.1053 
3.680 
4.100±0.1122 
3.0 
4.169±0.1553 
4.169±0.1251 
2.8 
4.161±0.1446 
4.161±0.1165 
4.170 
4.920±0.1288 
3.0 
4.837±0.1801 
4.837±0.1451 
2.6 
5.065±0.1635 
5.065±0.1317 
4.906 
9.934±0.2156 
2.8 
9.426±0.3276 
9.426±0.2639 
2.5 
9.315±0.2891 
9.315±0.2329 
9.812 
14.890±0.3244 
2.7 
15.002±0.5029 
15.002±0.4050 
2.4 
14.914±0.4444 
14.914±0.3579 
14.719 
18.965±0.4052 
2.6 
19.675±0.6351 
19.675±0.5115 
2.2 
19.345±0.5283 
19.345±0.4256 
19.625 
25.112±0.4435 
2.5 
24.932±0.7738 
24.932±0.6233 
2.0 
24.714±0.6136 
24.714±0.4943 
24.531 
29.885±0.5524 
2.4 
29.721±0.8855 
29.721±0.7133 
2.0 
29.480±0.7320 
29.480±0.5896 
29.437 
34.325±0.6947 
2.3 
34.745±0.9921 
34.745±0.7991 
2.2 
34.285±0.9379 
34.285±0.7555 
34.343 
41.562±0.7352 
2.2 
41.778±1.1410 
41.778±0.9191 
 
 
 
41.703 
46.892±0.7682 
2.4 
45.912±1.368 
45.912±1.102 
 
 
 
46.608 
Table 3: Determination of GLM, in some Syrian pharmaceutical preparations using spectrophotometric method through complexes formation with BPB and TB ^{(n=5).}
Tablet dosage form 
Label Claim^{ } ^{of }GLM, ^{mg/tab.} 
Mean ±SD (GLM)^{, } ^{mg/tab.} 
^{RSD%} 
^{Assay %} 
Mean ±SD (GLM)^{, mg/tab. } by RPHPLC[ 16] 
^{Assay} %, by RPHPLC^{ }[ 16] 
^{GLM:BPB, }l_{max }424 nm 

Amarium 
^{2} 
^{1.984±0.060} 
^{3.0} 
^{99.2} 
^{2.008±0.040} 
^{100.4} 
^{4} 
^{3.976±0.113} 
^{2. 8} 
^{99.4} 
^{3.980±0.062} 
^{99.5} 

Amapiride 
^{2} 
^{2.020±0.063} 
^{3.1} 
^{101.0} 
^{2.026±0.043} 
^{101.3} 
^{4} 
^{3.964±0.111} 
^{2.8} 
^{99.1} 
^{3.956±0.062} 
^{98.9} 

[GLM]:[TB]_{2}^{, }l_{max }392 nm 

Amarium 
^{2} 
^{1.980±0.055} 
^{2.8} 
^{99.0} 
^{2.008±0.040} 
^{100.4} 
^{4} 
^{3.992±0.104} 
^{2.6} 
^{99.8} 
^{3.980±0.062} 
^{99.5} 

Amapiride 
^{2} 
^{2.000±0.058} 
^{2.9} 
^{100.0} 
^{2.026±0.043} 
^{101.3} 
^{4} 
^{4.036±0.109} 
^{2.7} 
^{100.9} 
^{4.040±0.062} 
^{101.0} 
METHOD VALIDATION:
The developed method for simultaneous estimation of GLM has been validated in accordance with the International Conference on Harmonization guidelines (ICH) [17].
Selectivity and Specificity:
Selectivity test determines the effect of excipients on the assay result. To determine the selectivity of the method, standard solution of GLM, commercial product solution and blank solutions were analyzed. The results of the tests proved that the components other than the drug did not produce any interfere. The specificity of the method was ascertained by analyzing standard GLM in presence of excipients. There was no interference from most of the common excipients.
Linearity:
Several aliquots of standard stock solution of GLM were taken in different 10 ml volumetric flasks and diluted up to the mark with chloroform such that their final concentrations were 1.126 – 46.608 μg.mL^{1} of GLM for complex GLM:BPB and with acetonitrile such that their final concentrations were 0.319 – 34.343 μg.mL^{1} of GLM for complex GLM:(TB)_{2}. Absorbance was plotted against the corresponding concentrations to obtain the calibration graph, see (Figs. 5, 6) and Table 2. Linearity equations obtained were y=0.0455x+0.0091(R^{2}=0.9997) and y=0.0571x+0.0003 (R^{2}=0.9998) for the BPB and TB, respectively.
Precision and Accuracy:
The precision and accuracy of proposed method was checked by recovery study by addition of standard drug solution to preanalyzed sample solution at three different concentration levels (80%, 100% and 120%) within the range of GLM linearity for complex GLM:(TB)_{2}. The basic concentration level of sample solution selected for spiking of the GLM standard solution was 4.170 μg.mL^{1}. The proposed method was validated statistically and through recovery studies, and was successfully applied for the determination of GLM in pure and dosage forms with percent recoveries ranged from 99.8% to 101.8%, see Table 4.
Table 4: Results of recovery studies (n=5).
Level 
% Recovery 
80% 
100.4 
100% 
99.8 
120% 
101.8 
Repeatability and robustness:
The repeatability was evaluated by performing 10 repeat measurements for 3.680 μg.mL^{1} of GLM using the studied spectrophotometric method under the optimum _{conditions. The found amount of GLM (}_{}_{± SD) }3.722±0.1042 μg.mL^{1} _{and the} percentage recovery was found to be 101.1 ± 2.83. These values indicate that the proposed method has high repeatability for GLM analysis. The robustness of the method adopted is demonstrated by the constancy of the absorbance with the deliberated minor change in the experimental parameters such as the change in the concentration of excipients, C_{TB}:C_{GLM} (6±5%), temperature (25±5^{o}C), stability (36±0.5 h) and reaction time (5±1 min), see Table 5 which indicates the robustness of the proposed method. The absorbance was measured and assay was calculated for five times.
Sensitivity (LOD and LOQ):
The sensitivity of the method by TB was evaluated by determining the LOD and LOQ. The values of LOD and LOQ for GLM are 0.04 and 0.13 μg.mL^{1}, respectively.
The homogenization of tablets:
The homogenization of tablets in terms of the weight and the amount of drug was studied. It was found that the mean weight and amount drug in the tablets was 0.1794 ± 0.0040 g (i.e. ±2.22%), 0.1795 ± 0.0030 g (i.e. ±1.67%) for Amarium tablets (2 and 4 mg/tab), respectively, and 0.1000 ± 0.0010g (i.e. ±1.00%) and 0.1012 ± 0.0012 g (i.e. ±1.86%) for Amapiride tablets (2 and 4 mg/tab), respectively. While the mean amount drug in the tablets was ^{1.980±0.055} ^{mg/tab} (i.e. ±2.78%) and ^{3.992±0.104} ^{mg/tab} (i.e. ±2.61%) for Amarium tablets (2 and 4 mg/tab), respectively, and ^{2.000±0.058} ^{mg/tab} (i.e. ±2.90%) and^{ 4.036±0.109} ^{mg/tab} (i.e. ±2.70%) for Amapiride tablets (2 and 4 mg/tab), respectively; which shows that homogeneity of tablets is good.
Interferences:
Metformin HCL up to 1000 mg with 2 mg of glimepiride does not interfere, but Pioglitazone HCL and Rosuvastatin Calcium interfere.
Table 5: Robustness of the proposed spectrophotometric method(n=10).
Experimental parameter variation 
Average recovery (%) 

C_{GLM} 

3.680 g.mL^{1} 
29.437g.mL^{1} 

Temperature 15^{o}C 25^{o}C 
99.8 101.1 
100.0 100.2 
Stability 35.5 h 36.5 h 
101.4 101.8 
99.9 100.5 
Reaction time 4.0 min 6.0 min 
99.9 101.6 
99.6 101.0 
CONCLUSION:
The developed spectrophotometric methods for determination of GLM using BPB in chloroform and TB in acetonitrile were studied. Liner calibration graphs A=f(C_{GLM}), were obtained in the concentration ranges of 1.126 – 46.608 μg.mL^{1} for complex GLM:BPB with RSD3.8% and 0.319 – 34.343 μg.mL^{1} with RSD4.0% for complex GLM:(TB)_{2}. This method showed sensitive results for the determination of GLM than that obtained using BPB or TB. The results for the determination of GLM using TB were more sensitive and accurate than the results obtained by using BPB (the sensitivity was increased about 3.8 times). The developed method is applied for the determination of GLM in pure and its commercial tablets without any interference from excipients with average assay of 99.0 to 101.0%.
REFERENCES:
1. Meyer D, Thierry, 2014 Substituent effects on absorption spectra of pH indicators: An experimental and computational study of sulfonphthaleins dyes, Dyes Pigments, 102:241–250.
2. Rahman N, Najmul S, Azmi H. Extractive spectrophotometric methods for determination of diltiazem HCl in pharmaceutical formulations using bromothymol blue, bromophenol blue and bromocresol green. J. Pharm. Biomed. Anal. 2000; 24: 3341.
3. Ashour S, Shehna MF, Bayram R. Spectrophotometric determination of alfuzosin HCl in pharmaceutical formulations with some sulphonephthalein dyes. Int. J. Biomed. Sci. 2006; 2: 273278.
4. Almasri IM, AlLaham MK. Development and validation of spectrophotometric method for determination of cefixime and glimepiride by ternary complex formation with eosin and Cu(II). IAJPR. 2014; 12: 56705677.
5. Ravindra N, Singhvi I. Spectrophotometric Estimation of Glimepiride from Pharmaceutical Dosage Forms. Asian J. Chem. 2008; 6: 43794382.
6. Rahman N, Azmi SNH. Extractive spectrophotometric methods for determination of diltiazem HCl in pharmaceutical formulations using bromothymol blue, bromophenol blue and bromocresol green. J. Pharm. Biomed. Anal. 2000; 24: 3341.
7. Rahman N, Khan NA, Azmi SNH. Extractive spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations using bromocresol green, bromophenol blue, bromothymol blue and eriochrome black T. Il Farmaco. 2004; 59: 4754.
8. Altinöz S, Tekeli D. Analysis of glimepiride by using derivative UV spectrophotometric method. J. pharm. Biomed. Anal. 2001; 24: 507522.
9. Sevgi TU. Spectrophotometric Determination of Glimepiride in Pharmaceutical Preparations Based on the Formation of Charge Transfer and Ion_Pair Complexes. J. Analyt. Chem. 2013; 68: 606610.
10. Bonfilio de Rudy B, Araujo Magali, Salgado Hérida RN. Development and Validation of an UVDerivative Spectrophotometric Method for Determination of Glimepiride in Tablets. J. Braz. Chem. Soc. 2011; 22: 292301.
11. Siddiqui MR, AlOthman ZA, Rahman N. Analytical techniques in pharmaceutical analysis: A review, Arabian J. Chem. 2013; 10: 14091421.
12. AL Othman ZA, Rahman N, Siddiqui MR. Review on pharmaceutical impurities, stability studies and degradation products: an analytical approach, Rev. Adv. Sci. Eng. 2013; 2: 155221.
13. Rahman N, Azmi SNH, Wu HF. The importance of impurity analysis in pharmaceutical products: an integrated approach. Accred. Qual. Assur. 2006; 11: 6974.
14. Ramadan AA, Zeino S. Direct Spectrophotometric Determination of Glimepiride in Pure Form and Pharmaceutical Formulations Using Bromocresol Purple. J. Adv. Chem. 2018; 15: 61866198.
15. Ashour S, Chehna MF, Bayram R. Spectrophotometric Determination of Alfuzosin HCl in Pharmaceutical Formulations with some Sulphonephthalein Dyes. Int. J. Biomed Sci. 2006; 2: 273281.
16. Karthik A, Subramanian G, Mallikarjuna Rao C, Krishnamurthy Bhat, Ranjithkumar A, Musmade P, Surulivelrajan M, Karthikeyan K, Udupa N. Simultaneous determination of pioglitazone and glimepiride in bulk drug and pharmaceutical dosage form by RPHPLC method. Pak J Pharm Sci. 2008; 21(4): 421426.
17. ICH: Proceedings of the International Conference on Harmonization of Technical Requirement of Registration of Pharmaceuticals for Human Use (ICH Harmonized Tripartite Guidelines) 2000.
Received on 25.10.2018 Modified on 23.12.2018
Accepted on 12.01.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(3): 11691177.
DOI: 10.5958/0974360X.2019.00193.8