INTRODUCTION:

Diabetes mellitus is a foremost health problem and a vital cause of prolonged ill health and early death. Many anti-diabetic drugs with different mechanisms of action are now available for treatment of type 2 diabetes mellitus. Sulfonylureas have been extensively used for treatment of type 2 diabetes¹. Gliclazide, glipizide, glibenclamide and glimepiride are second-generation sulfonylureas, currently used, while first-generation drugs (such as tolbutamide and chlorpropamide) are no longer used. Second-generation drugs are equally effective in lowering blood glucose concentrations¹. Gliclazide is a potent hypoglycaemic agent which compares favourably with others of its type. It has a low incidence of side effects, few problems with hypoglycaemia, and retains its efficacy longer than other sulphonylureas. Gliclazide may therefore be considered a first choice for the therapy of type II diabetes mellitus².

It also selectively inhit pancreatic K+ ATP channels, antioxidant activity and other beneficial haemobiological effects³. Figure 1 shows that gliclazide is chemically [1-(3-azabicyclo(3,3,0)oct-3-yl)-3-ptolylsulfonylurea]. It is mainly used in the non-insulin dependent diabetes mellitus (NIDDM) treatment. It is second generation hypoglycemic sulfonylurea⁴. It activates the beta cells of the Islet of Lengerhans in the pancreas to release insulin. It increases peripheral insulin sensitivity. Overall it potentiates insulin release and improves insulin dynamics⁵.

Literature survey revealed that gliclazide has been estimated by analytical methods such as spectrophotometry⁶, HPTLC and HPLC⁷–¹⁰. HPLC has also been used for combination containing gliclazide. The application of design of experiments (DoE) in robustness testing as a part of extensive method validation has been scantly reported.

Robustness testing has attracted immense attention by analysts due to strict requirements by regulators and high probability of variability due to men and machine. The validation data are also of great importance during method transfer from R&D centre to the plant. Hence, in the present work chromatographic method along with Quality by Design (QbD) approaches been reported to study robustness of the developed method.

Now-a-days QbD is widely used in the pharmaceutical field especially in the formulation and development of different dosage form. It leads to the use of Qbd in development of analytical methods also. Pharmaceutical regulatory agencies also insist to use of QbD principles at research and development level¹¹. Implementation of QbD helps to develop robust method facilitating continuous improvement throughout the life-cycle of the method.

DoE is a good alternative to the classical approach (one variable at a time (OVAT) tool) to evaluate robustness. DoE offers maximum information about how the factors influence the response while requiring minimum time that further can facilitate process of method transfer protocol from transferring to receiving site¹²–¹⁵. Thus, in the present study, experimental design is integrated to study influence of the simultaneous variation of different variables for a response to understand the robustness of the method.

In above context, the objective of the present research was systematic development and validation of accurate, precise and robust RP-HPLC method for the determination of gliclazide.

MATERIAL AND METHODS:

Standards and reagents:

Standard drug sample of gliclazide was procured from Alembic Pharmaceutical Ltd; Vadodara. Methanol, potassium dihydrogen ortho phosphate and ortho phenylene di-amine (HPLC grade) was obtained from Merck, Mumbai, India. Mili Q water was purchased from SICART laboratories, Vallabh Vidhyanagar, Anand.

Instrumentation:

High-performance liquid chromatography system, with LC solutions data handling system (Shimadzu LC 2010-CHT) with PDA detector (ShimadzuSPD-M20A) was used for the analysis. The packed column used was PHEMOMENEX, C₁₈ (internal diameter 5μm, 250× 4.6mm). LC 2010 software is used for data collection and record. Weighing was performed on an analytical balance (Shimadzu Corporation ELB 300).

The data were recorded using LC 2010 solutions software-1.25. An analytical balance (Shimadzu Corporation ELB 300) was used in the study.

Preparation of Standard Solution:

Standard stock solution was prepared by weighing accurately 10mg of gliclazide and dissolved in 5ml methanol in 10ml-capacity volumetric flask. The solution was sonicated using bath sonicator to ensure complete solubilisation of drug and final volume was made up to 10ml using methanol to get the stock solution with the strength of 1000μg/ml¹⁶. Then the solution was diluted 10 times using methanol to make the strength of 100μg/ml. Further, five ml of the drug solution was withdrawn and made up volume with mobile phase up to 10 ml to obtain a solution containing 50μg/ml concentration of the drug.

Preparation of Mobile phase:

Potassium dihydrogen orthophosphate (0.27218 g) was dissolved in 100 ml of Mili Q water. The pH of final solution was adjusted with OPA- orthophosphoric acid to 3.5, followed by degassing. Mix 700 volumes of methanol and 300 volumes of 0.02M phosphate buffer completely. Add above solution into 500 volumes of mobile phase reservoir bottle and mixed it properly. Sonication was performed to remove dissolved gas for 10 min. using batch sonicator.

METHOD:

The HPLC analysis was carried out on Shimadzu LC-2010C HT and UV visible detector. The separation was achieved using LINOMAX C₁₈ (250 × 4.6mm, packed with 5μm particle size) column with a constant flow rate of 1.2ml/min. The isocratic mobile phase consisted of an organic phase methanol (70%) and 0.02 M potassium dihydrogen orthophosphate (30%), with pH adjusted to 3.5. The mobile phase was filtered through a 0.22-μm membrane and degassed prior to use. UV detection was performed at 210 nm. e. The injection volume was 20μL for standards and samples. All analyses were done at ambient temperature. The samples were analysed at maximum wavelengths of 210 nm¹⁷.

METHOD VALIDATION:

The objective of the validation of method is to demonstrate that weather the method is suitable for intended purpose or not as per the ICH guideline¹⁵. The developed method is validated for different validation parameters like linearity, precision, accuracy, specificity, limit of detection, limit of quantification and robustness. System suitability test was performed prior to validation.¹⁸

System suitability study:

The performance of system was verifies by measuring the system suitability parameters. System suitability was determined by taking the percent relative standard deviation RSD of the six standard injections to assess system suit parameters such as area, capacity factor, resolution, theoretical plates and tailing. The system precision was determined on six replicate injections of standard preparations containing the drug and solvents. Tailing factor and theoretical number of plate were determined using LC 2010 solution software and percentage relative standard deviation was calculated. In all cases, the % RSD should be <2.0 % for the five consecutive injections of analytes peak¹⁹.

Linearity and Range:

The linearity of analytical method is its ability to elicit test results that are directly proportional to concentration of analyte in sample within given range. The linearity range of analytical method is interval between upper and lower level of analyte including level that have been demonstrated to be determining with precision and accuracy using method. The linearity is express in term of correlation co-efficient of linear regression analysis²⁰. Linearity was evaluated by injecting a series of solution ranging from 5-100μg/ml. Each concentration were prepared and injected six times. Regression analysis was performed and linearity was observed between injected concentration and its respective peak area.

Limit of quantitation (LOQ) and limit of detection (LOD)

The parameters LOD and LOQ were determined on the basis of signal to noise ratio, LOD and LOQ were calculated by the method based on the standard deviation (SD) of the response and the slope (S) of the calibration curve at levels approximating the LOD and LOQ. The values of LOD and LOQ were determined as follows²¹²²:

Standard deviation of y intercept

Limit of detection (LOD) = 3.3 * -------------------------------------------------- Equation 1

Slop of calibration curve

Standard deviation of y intercept

Limit of Quantification (LOQ) = 10*--------------------------- Equation 2

Slop of calibration curve

Precision:

The precisions of the proposed HPLC method were determined by injecting three different concentrations at low (LOQ), mid and high end of the calibration curve²³. For intra-day variation, sets of six replicates of the above three concentrations were analysed on the same day; for inter-day variation, six replicates were analyzed on different days. Percentage RSD of the all assays is also reported²³.

Accuracy:

Accuracy of an analytical method is the closeness of test results to the true value (100%). It was determined by the application of analytical procedure to recovery studies, where a known amount of standard is spiked into pre-analysed sample solutions²⁰. Three different concentration 50%, 100% and 150% were selected for the study. The solutions were analysed by the method described above. Each experiment was performed in triplicate and % RSD was calculated. ²².

Specificity:

The specificity of the analytical method is the ability of the method to estimate the analyte response in the presence of additional components such as impurities, degradation products and matrix²⁴. Chromatographic peaks of gliclazide were evaluated for spectral purity using the recorded UV spectra by UV detector in HPLC system. The peak purity of gliclazide were assessed by comparing the retention time of standard and sample of gliclazide ²⁴,²⁵.

Robustness:

The robustness of an analytical procedure refers to its capability to remain unaffected by small but deliberate variations in the analytical method, as per the ICH guideline¹⁵. The conditions studied were pH 3.5±0.5, concentration of phosphate buffer in the mobile phase composition 30±5% and flow rate of mobile phase 1.2±0.2ml/min.

Simultaneous variations of the factors can be effectively studied using DOE approach which was applied efficiently to test method robustness. Box-Behnken design was employed to obtain predictive model describing the changes in the responses within the experimental domain²⁶. Box-Behnken design is balanced incomplete block designs. Box-Behnken design three factors were studied at three levels. In BBD, the number of experiments (N) required for k number of factors are 2k (k−1) runs and at least one centre point (Co) run.

Statistical validity of the parameters was established on the basis of ANOVA in Design Expert software. Also, three dimensional response surface graphs and two dimensional contour plot were constructed using Design Expert software.

Application of method to analyse Gliclazide formulations:

The developed and validated method was used to analyse gliclazide content in the marketed formulation Reclide 80mg. Six replicates of each batch was separately prepared and analysed using validated method. The tablets were weighed, powdered and equivalent to 50mg of gliclazide powder was taken. Then, the powder was transferred to 25ml methanol containing 100ml volumetric flask. The solution was sonicated in bath sonicator for complete solubilisation of drug. The solution was filtered to remove un-dissolved particles using whatman filter paper no. 41. Then the solution volume was adjusted to 100ml using mobile phase. Further dilution were made to require 50μg/ml of gliclazide solution²⁷.

RESULTS AND DISCUSSION:

METHOD VALIDATION:

System Suitability Parameter:

The system suitability parameters like repeatability of peak area for Gliclazide (720811±0.0112), theoretical plate (5634.75±0.578) and tailing factor (asymmetric factor, 0.91±0.321) were as per the specifications discussed before. The results of system suitability parameter are shown in Table 1. The method applicability was found to be suitable.

Table 1: System Suitability parameters of Gliclazide

Concentration	Sample number	Area	R_t (min)
50 µg/ml	1	720914	6.26
	2	720891	6.19
	3	720797	6.32
	4	720773	6.29
	5	720800	6.28
	6	720694	6.26
Statistical analysis	Mean	720811.5	6.266
	SD	80.56985	0.0004
	%RSD	0.0112	0.009

Linearity and Range:

Linearity was measured by preparing different dilution from the stock solution. Concentration range of gliclazide was ranging from 5, 10, 25, 50, 75, and 100μg/ml. Each solution was injected in same quantity at 1.2ml/min. flow rate. The solution absorbance was measured at 210nm wavelength using UV detector in HPLC system. Calibration curve was plotted between the concentration of solution and peak area. Linearity factor (r²) was found to be 0.999. The results are shown in Figure 2 and Figure 3.

Detection and quantitation limits:

The LOD and LOQ for GLZ were found to be 5.51 and 16.70 ng/ml, respectively. The low value of LOD and LOQ indicated adequate sensitivity of the analytical method.

Precision:

Inter-day precision was measured by injecting 50μg/ml of the solution for three times on the same day. Intraday precision was measured by injecting the same volume at three different days and peak area was determined. The % RSD was found to be 0.0076 and 0.0086 for intra-day and inter-day respectively as shown in Table 2.

Table 2: Precision for Gliclazide

	Precision
Injections (50 µg/ml)	Peak area
Injections (50 µg/ml)	Intra-day	Inter-day
1	720773	720914
2	720800	720891
3	720694	720797
Mean	720756	720867
SD	55.08	61.98
% RSD	0.0076	0.0086

Accuracy:

Gliclazide reference standards were precisely weighed and all other excipients of tablet were added into the drug, at three different concentration levels (25, 50and 75μg/ml). At each level, samples were formulated in triplicate and the percentage recovery was determined. The results are shown in Table 3.

Robustness:

Robust analytical method can be developed only if external variables that can influence the process are identified with the concept of QBD and DOE. Robustness of the analytical method can be fulfilled if the claim of robustness is developed by sound statistical technique. Many of the analyst’s uses mean standard deviation and % RSD to define robustness. This method can be augmented by using appropriate statistical design such as placket- Barman design, factorial design, fractional factorial design and three variable designs such as Box-Behnken design. In this present work BBD was chosen assure robustness of method. Three level designs are useful to elaborate non linearity in the response. Furthermore, design was successfully used to test the robustness of the developed HPLC method. Box-Behnken experimental design was employed successfully for evaluation of robustness. From results of ANOVA and analysis of response surfaces plots; it can be concluded that responses peak area and retention time are robust for all the three factors mobile phase composition, flow rate and pH of the buffer within selected range. Perturbation plots were constructed (Figure 4) to evaluate the effect of factors on the response. From the perturbation plot of retention time, it can be concluded that factor B and C. Also, factor A in medial value has no effect but low level of factor A retention time was found to be highest. Same effect can be seen from perturbation plot of Peak area. The level C was kept constant because of higher p value (0.84 for Peak area and 0.79 for retention time).

Table 4: Experimental domain of BBD for robustness

Experimental runs	A: pH	B: Flow rate (ml/min)	C: Buffer composition	R₁: Retention time (min)	R₂: Peak area
1	4.0	1.2	35	3.03	764553
2	3.5	1.2	30	7.88	542290
3	3.0	1.0	30	4.39	239105
4	3.0	1.4	30	4.56	256926
5	4.0	1.0	30	6.25	489807
6	3.0	1.2	35	9.76	317099
7	3.5	1.2	30	4.56	251311
8	3.5	1.2	30	4.55	255705
9	3.5	1.0	35	3.83	608078
10	3.5	1.2	30	2.55	749662
11	3.5	1.4	25	4.56	258146
12	4.0	1.4	30	7.72	448918
13	3.5	1.4	35	5.26	777247
14	3.5	1.0	25	5.23	787778
15	4.0	1.2	25	5.20	776427
16	3.5	1.2	30	5.25	786586
17	3.0	1.2	25	5.22	776667

Table 5: ANOVA Summary

Source	Retention time (Y₁)			Peak area (Y₂)
Source	Coded coefficient	F Value	p value	Coded Coefficient	F Value	p value
Intercept	4.96	0.42	0.88	1.112E+005	1.07	0.47
X₁	-0.22	0.07	0.78	-47941.50	1.91	0.20
X₂	0.30	0.15	0.70	-16505.25	0.35	0.57
X₃	0.21	0.07	0.79	-14677.50	0.04	0.84
X₁X₂	0.32	0.08	0.77	1.119E+005	0.01	0.90
X₁X₃	-1.68	2.36	0.16	1.747E+005	0.97	0.35
X₂X₃	0.52	0.23	0.64	-53773.65	2.36	0.16
X₁²	0.93	0.76	0.41	-1.046E+005	0.23	0.64
X₂²	-0.16	0.02	0.88	1.953E+005	0.89	0.37
X₃²	-0.083	6.054-003	0.94	1.112E+005	3.10	0.12

Figure 4: Perturbation effect chart of Retention time and Peak area

Figure 5: 3D plot of Retention time and Peak area

Analysis of marketed dosage formulation:

Different batches of marketed formulation were evaluated by validated method. All the three analysed batches of the first tablets (Reclide 80 mg) showed results very close to the labelled amount. The content in the tablets samples varied from 99.55± 0.25 to 100.80± 0.45% indicating applicability of the method in the routine quality control testing of bulk drugs and marketed formulation.

CONCLUSION:

A robust method is developed and validated integrating the principles of quality by design approach for the estimation of gliclazide in bulk or in any pharmaceutical product. The method is also sensitive, simple and economical which is also applicable at industrial level. Validation of the method supported excellent accuracy, system suitability, linearity, precision, specificity. Box behnken design was successfully applied to evaluate method robustness. Statistical analysis showed that the model represents the phenomenon quite well and the variations in responses were correctly co-related to the variations of the factors. From results of ANOVA and analysis of response surfaces plots; it can be concluded that responses, R₁ retention time; R₂, peak area; are robust for X₁ and X₃ within selected range.

ACKNOWLEDGEMENT:

The authors are thankful to the authorities of Anand Pharmacy College, Anand for providing the facilities and Alembic Pharmaceutical Ltd; Vadodara, India for providing gift sample of Gliclazide.

CONFLICT OF INTEREST:

The authors do not have any conflict of interest.

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Received on 28.08.2018 Modified on 29.09.2018

Research J. Pharm. and Tech 2019; 12(1): 135-141.

DOI: 10.5958/0974-360X.2019.00026.X

	Concentration (µg/ml)	Peak area			Mean	% Recovery	SD	% RSD
	Concentration (µg/ml)	1	2	3	Mean	% Recovery	SD	% RSD
50%	25	346583	346601	346577	346587	99.84	12.49	0.36
100%	50	720800	720797	720773	720790	100.001	14.79	0.20
150%	75	1029818	1029793	1029813	1029808	99.97	13.22	0.12