Spectrophotometric Determination of Gliclazide in Bulk and Pharmaceutical Formulation using 1, 2-naphthoquinone-4-sulfonic acid Sodium salt
Batoul Jeha*, Maysam Salami
Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.
*Corresponding Author E-mail: batul.pharmacy@gmail.com
ABSTRACT:
This article describes the development of a new, selective and extraction-free spectrophotometric method for the determination of gliclazide (GLZ) in both pure form and pharmaceutical formulation preparations. The proposed method is based on nucleophilic substitution reaction of GLZ with 1, 2-naphthoquinone-4-sulfonic acid sodium salt (NQS) in methanol solvent to form reddish orange product peaking at 454 nm. The stoichiometry of the reaction was studied by Job’s method of continuous variation. The parameters that affect the reaction were carefully optimized. Under the optimized reaction conditions, Beer’s law was obeyed in the concentration range of 30-110 µg/ml with correlation coefficient (R2=0.9978). Intra-day and inter-day precision and accuracy of the method were established by following the International Conference of Harmonization (ICH) guidelines, the limit of detection(LOD) and quantification(LOQ) were also reported. The method is successfully applied to the estimation of GLZ in its pharmaceutical tablets without interference from excipients and additives.
KEYWORDS: Gliclazide, 1, 2-naphthoquinone-4-sulfonic acid sodium salt (NQS), Nucleophilic Substitution, Spectrophotometric Method, Pharmaceutical Formulation
INTRODUCTION:
Gliclazide which is chemically known as 1-(Hexahydrocyclopenta[c]pyrrol-2(1H)-yl)-3-[(4-methylphenyl) sulphonyl] urea (Fig.1)1 is an oral antihyperglycemic agent used for the treatment of non-insulin dependent diabetes mellitus (NIDDM). It belongs to the sulfonylurea class of insulin secretagogues, which act by stimulating β cells of the pancreas to release insulin2, increase peripheral glucose utilization, decrease hepatic gluconeogenesis, and may increase the number and sensitivity of insulin receptors3, it also has anti-platelet adhesive activity4.
Fig.1: Chemical structure of Gliclazide
The drug assay is listed in the monograph of the British Pharmacopoeia (BP)1 which recommends a potentiometric titration of GLZ with 0.1 M perchloric acid in a anhydrous acetic acid medium. Several methods have been reported for the determination of GLZ in pharmaceutical formulations, including high-performance liquid chromatography (HPLC)5,6,7 spectrophotometry8,9,10, Capillary Gas Chromatography11 although those methods are sensitive, some of them are time-consuming, complicated, and require expensive instrumentation. In addition the estimation of GLZ in its dosage forms based on the direct measurement of the absorption for ultraviolet is susceptible to potential interferences from the co-extracted common excipients and/or impurities. Therefore, derivatization is more selective for the determination. The present work describes accurate and simple visible spectrophotometric method for the quantification of GLZ in bulk and tablets by using NQS as derivatizing agent which react with secondary amine of the drug to produce reddish orange colored complex having a maximum absorption at 454 nm.
MATERIALS AND METHODS:
Instrumentation:
HITACHI U-1800 ratio beam UV-Visible Spectrophotometer, Sartorius-Germany analytical balance, water bath, micropipette.
Materials and Reagents:
Analytical grade Gliclazide, its purity was 99.25%.
Methanol (99.9%, SIGMA – ALDRICH).
A solution of 0.2% (w/v) of 1,2-Naphthoquinone-4 -sulfonic acid sodium salt (NQS) (British Drug Houses B.D.H – England) was prepared by dissolving 0.1g in 50 ml methanol with good shaking, the solution was freshly prepared.
Tablets of GLZ (Diamicron MR® produced by Servier - France, Unicron® produced by Unipharma- Syria).
Preparation of Gliclazide Standard Solution:
A stock solution (1000µg/ml) was prepared by dissolving 50mg of GLZ in 50 ml of methanol.
Analytical procedure:
Aliquot volumes of GLZ standard solution were transferred into a series of 10ml volumetric flasks, to give final concentrations of 30-110µg/ml. 1.5ml of NQS 0.2% w/v was added to each flask then closed and heated at 90℃ for 20min in a water bath, the flasks were cooled and made up to the volume with methanol. The absorbance of the reddish orange colored product was measured at 454nm against the reagent blank treated similarly. The drug concentrations were calculated from the corresponding regression equation of the calibration curve.
Preparation of Tablets Sample Solution:
Twenty tablets of Gliclazide were accurately weighed and powdered. A portion of the powder equivalent to 25 mg of GLZ was accurately weighed and transferred into a 25ml volumetric flask.15ml of methanol was added to the flask, and the container was shaken thoroughly for 15–20min to extract the drug into the liquid phase then the solution was made up to the mark with same solvent. The contents were mixed well and filtrate. An aliquot of the filtrate (1000µg/ml GLZ) was further diluted with methanol to obtain working concentrations of 500µg/ml. The solution was suitably diluted and analyzed as given under the assay procedure for bulk sample. Then the concentration of GLZ in tablets was calculated using calibration curve.
RESULTS AND DISCUSSION:
Absorption Spectra:
The standard solution of Gliclazide (80µg/ml) was scanned in the range of 400-800nm which shows maximum absorbance at 454nm (Fig.2)
Fig.2: Absorption spectrum of the derivative product against reagent blank (80µg/ml GLZ)
Optimization of the experimental conditions:
Effect of diluting solvent:
Different solvents such as water, methanol, and acetonitrile were tested as potential diluting media. Methanol was found to be the optimum solvent and the highest absorbance values were obtained.
Effect of NQS concentration:
It was studied over the range 0.08-0.5% w/v. It was found that absorbance increases with increasing NQS concentration and reaches its maximum value by using 0.2% w/v of reagent, after that, it decreases as shown in (Fig.3)
Effect of NQS volume:
A different volumes of reagent (0.2% w/v) was studied in the range 0.5-3 ml. the highest absorption intensity was attained at NQS volume of 1.5 ml then it decreased (Fig.3)
Fig.3: Effect of NQS concentration (
) and volume ( ) on the reaction of GLZ with NQS.
Effect of temperature:
It was studied by carrying out the reaction at different temperatures (25-90℃). About 90℃ was found to be optimal for maximum color development (Fig.4)
Effect of heating time:
It was investigated in the range (10-60 min). The experimental results show that the optimal absorbance was attained in heating about 20 min at 90℃ and longer heating time up to 20min did not affect the reaction (Fig.4)
Fig.4: Effect of
temperature ( ) and time (
) on the reaction of GLZ with NQS.
Stability of the reaction product:
Under the overe mentioned optimum conditions. The formed colour was stable for 2 h at an ambient temperature.
Stoichiometry of the reaction:12
In order to establish the stoichiometry between GLZ and NQS, Job’s method of continuous variations was applied. The plot reached a maximum value at a mole fraction of 0.5 which indicated the formation of 1:1 (GLZ: NQS) complex (Fig.5).
Fig.5: Job’s plot for determination of stoichiometry of the reaction between GLZ and NQS.
[GLZ]: 2×10−3 M; [NQS]: 2×10−3 M; [GLZ] + [NQS]: 4 mL
Method validation:
Linearity and Sensitivity:
A series of six concentrations of GLZ standard solution prepared in triplicate were derivatized as previously described. The absorbance responses were linear in relation to the concentration of GLZ over the ranges of 30-110µg/ml (Fig.6), Regression parameters of the developed assay, limit of detection (LOD), limit of quantification (LOQ) are calculated as per the current ICH guidelines13 and are summarized in (Table.1)
Fig.6: Calibration curve for GlZ after derivatization with NQS
Table.1: Sensitivity and regression parameters.
|
Parameter |
Value |
|
λmax, nm |
454 |
|
Beer’s law limit, µg/mL |
30-110 |
|
Molar absorptivity (ε), L mol−1 cm−1 |
1290 |
|
Regression equation Slope, a Intercept, b |
y=b+ac 0.0038 0.0122 |
|
Correlation Coefficient, R2 |
0.9978 |
|
Limit of detection (LOD), µg/ml |
5.9 |
|
Limit of quantification (LOQ), µg/ml |
17.9 |
Accuracy and Precision:
The repeatability of proposed method was estimated by measuring six replicate samples of one concentration (60 µg/ml) of GLZ prepared in the same day. The precision expressed as a relative standard deviation (RSD%).
In addition, the accuracy and the intermediate precision of the method were evaluated by preparing and analyzing three replicate solutions of pure drug at three different concentration levels during the same day (intra-day) and over three consecutive days (inter-day), and the results are summarized in (Table.2). The recovery and RSD % values indicate the good accuracy and precision of the method.
Table.2: Accuracy and precision of the proposed method for the determination of GLZ.
|
Precision |
Concentration µg/ml |
Recovery % |
RSD |
|
Repeatability* (inter-day) |
60 |
100.28 |
1.483 |
|
Intermediate precision** (intra-day) |
40 60 80 |
99.92 100.82 99.1 |
1.571 1.299 0.529 |
|
Accuracy |
Concentration µg/ml |
Recovery % |
Mean ± SD |
|
(inter-day)** |
40 60 80 |
99.65 99.47 99.39 |
99.5 ± 0.133 |
*Recovery Value is mean of 6 determinations; RSD: Relative standard Deviation
**Recovery Values are mean of 3 determinations; SD: Standard Deviation
Specificity:
The specificity of the method was evaluated by investigating the interference liabilities from the common excipients that might be added during pharmaceutical formulation. Sample was prepared by mixing known amount (30mg GLZ) with various amounts of the common excipients: maize starch (30 mg), PVP30 (5mg), HPMC (60mg), lactose (35mg), stearic acid (6mg) and magnesium stearate (4mg). These laboratory prepared samples were analyzed by the proposed method applying the general recommended procedure previously mentioned. Good recovery 99.75% with RSD 1.33% were obtained. This confirmed the absence of interference from any of the common excipients with the determination of GLZ by the proposed method.
Robustness:
The robustness of the developed method was examined by evaluating the influence of small but deliberate variations of the reaction conditions. One parameter was changed, whereas all others were kept constant. Variation of the reagent concentration by ±0.02 % w/v, reagent volume by ±0.1 mL, heating time by 1 min and measurement wavelength by ±2 nm. These variations did not significantly affect the developed method. This provided an indication for the reliability of the proposed methods during their routine application for the analysis of GLZ.
Assay in tablets:
Commercial products were analysed using the proposed spectrophotometric method. (Table.3) showed that the results agreed well with the label claim, and within the required limits.
Table.3: Application of the proposed method to the determination of GLZ in tablets.
|
Brand name |
Label claim (mg/tablet) |
Amount obtained (mg/tablet) |
Recovery % ± SD* |
RSD |
|
Diamicron MR® |
60 |
59.6 |
100.06 ± 1.65 |
1.65 |
|
61.18 |
||||
|
59.35 |
||||
|
Unicron® |
80 |
82.71 |
104.8 ± 1.26 |
1.20 |
|
84.14 |
||||
|
84.49 |
* n = 3
CONCLUSION:
The present study describes the successful evaluation of NQS as analytical reagent in the development of simple, economical and sensitive spectrophotometric method for the determination of GLZ in bulk and tablets.
Statistical analysis of the results showed that the proposed procedure has good precision and accuracy.
Compared with most of the existing methods for the determination of GLZ, the recommended method is very simple, it does not need expensive sophisticated apparatus, free from a complicated extraction procedure nor strict pH control, and using materials are available in any analytical laboratory. Moreover, this method has excellent shelf life and showed no interference from common excipients, therefore, it is suited for the assay of drug in quality control laboratories.
CONFLICT OF INTEREST:
The authors declare that there is not any conflict of interest related to this work.
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Received on 02.05.2019 Modified on 28.05.2019
Accepted on 29.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11):5310-5314.
DOI: 10.5958/0974-360X.2019.00920.X