1. INTRODUCTION:

Gliclazide, chemically known as 1-[(4- methylbenzene) sulfonyl] -3-octahydrocyclopenta [c] pyrrol-2-yl} urea¹, acts as oral anti-hyperglycaemic agent used for treating non-insulin-dependent diabetes mellitus (NIDDM)². Many studies that were carried out revealed that this drug possesses good general tolerability among individuals, almost reported few cases of hypoglycaemia, having a much decreased rate of secondary failure². To be an effective drug with anti-diabetic property it should show rapid absorption of the drug from the GI tract. It is observed that Gliclazide absorption is slow from the GI tract and it shows variation in individuals.

In various research works carried out on patients of diabetes and volunteers who are healthy it was observed that, time required to achieve the peak in concentration of plasma (t-max) ranges between 2-8 hours when a single dose of 80mg oral Gliclazide tabletwas administered². This slow absorption can be correlated with the hydrophobic nature of the drug or its poor ability of the drug to cross Gastrointestinal membrane. In a view of overcoming this problem, a controlled released bioadhesive formulation was developed. Bio adhesion allows the proper gastric residence of the formulation thereby enabling the proper release of the drug. To study the bioavailability, pharmacology, bioequivalence and pharmacokinetics, methods of quantitative analysis which are sensitive and selective are important. Survey of literature brings out that there is no method which is specific for the estimation of Gliclazide from this type of microsphere dosage form. In this current study, a simple and fast method of UHPLC was developed to estimate Gliclazide from microsphere formulation. Validation of the developed method was done for linearity, accuracy, sensitivity, system suitability, precision, robustness, and ruggedness according to ICH guidelines^3,4. Linearity is the ability of a developed analytical method to produce a result in a linear range which is directly proportional to the drug concentration in a given sample which is observed by plotting a calibration curve. Accuracy is the ability of method to produce close measurement results from multiple samples of a homogenous sample under specific conditions which is understood by %RSD calculation with different equipment, analyst and at different time.

2. MATERIAL AND METHODS:

2.1. Chemicals:

The chemicals used during developing the analytical method were HPLC grade acetonitrile, HPLC grade methanol and analytical grade Glacial acetic acid procured from Rankem, India.

Figure 1: Chemical Structure of Gliclazide

HPLC grade Distilled water was procured from Rankem, India. Gliclazide API was obtained from Legen Healthcare, India. The Bioadhesive microsphere of Gliclazide was prepared by the ionic gelation technique⁵ by using starch which was previously isolated from Banana (Musa assamica species).

2.2. Equipment:

The equipments used during developing the analytical method were Thermo Scientific Dionex Ultra 3000plus UHPLC integrated with a UV detector and assembled with an Autosampler monitored by Chromeleon 2 software, Sigma 200 electronic balance pH Unilab pH meter and PCi, 3.5 L sonicator.

2.3. Preparation of Microsphere:

Ionic gelation technique was used in the preparation of microsphere. Required amount of polymer was dispersed in a specific volume of 5% acetic acid solution, allowed to swell for 2 hours and transferred to distilled water. In another beaker, suitable amount of sodium alginate and drug was mixed well with 100ml of water. The previously prepared polymer solution was finally transferred to another beaker containing specified volume of calcium chloride with continuous stirring on a magnetic stirrer. The drug and the sodium alginate solution were added dropwise⁶.

2.3.1. FT-IR Study:

In order to find out the drug excipient compatibility studies, FTIR study was carried out initially with the pure drug and excipient individually and finally by mixing all the drug and excipients together. The FTIR study was carried out using Fourier Transform Infrared Spectroscopy (ATR) Bruker, ALPHA. About 2-3 mg of samples were mixed with dried KBr of equal weight and compressed to form KBr disks. The samples were scanned from 400 to 4000 cm^-1

Figure 2: FT-IR Study of Drug and excipients combined form

2.3.2. Estimation of Particle size:

The particle size of the prepared microsphere was estimated by using optical microscopy method⁷. A calibrated optical microscope fitted with a stage micro meter was used for this purpose. Approximately 100 microspheres were counted for particle size determination by using this technique. All the experiments were done in triplicate (n=3)⁸

2.3.3.Content Uniformity:

50 mg of microspheres were dispersed in 10 ml of 0.1 N HCl for 30 min with occasional shaking. The suspension was then centrifuged at 2000 rpm for 5 min and the supernatant was kept aside. The drug concentration was determined spectrophotometrically at 230 nm (Dionex Ultimate 3000, Thermo Fischer). The entrapment efficiency (n=3) was calculated by using following formula⁹.

2.3.4. Flow Properties of Microspheres:

Accurately weighed microspheres were gently poured using a glass funnel into a graduated cylinder exactly to 10 ml mark. Initial volume was noted. Bulk density and tapped density were noted using tapping method using measuring cylinder (of 10 ml). Angle of repose of prepared microspheres (n=3) was determined by fixed funnel standing method. The granules were allowed to flow through funnel orifice on a plane paper kept on the horizontal surface to form a pile of granules. Angle of repose (θ), Hausner’s ratio (H) and Carr’s index (% C) were calculated to study the flow properties of microspheres by using following formulas⁹

Tan Ø = h/r, where, h is height and r is radius of the pile, respectively

H = Dt/Db, %C = Dt-Db/Dt *100, where, Dt is tapped and Db is bulk density, respectively

2.3.5. Percentage Entrapment efficiency:

50 mg of microspheres were dispersed in 10ml of 0.1 N HCl for 30 min with occasional shaking. The suspension was then centrifuged at 2000 rpm for 5 min and the supernatant was kept aside. The drug concentration was determined spectrophotometrically at 230nm (Dionex Ultimate 3000, Thermo Fischer). The entrapment efficiency (n=3) was calculated by using following formula¹⁰.

%EE =Dcal/Dth *100

where, Dcal is the calculated drug content and Dth is the theoretical drug content, respectively.

Table 1. Entrapment efficiency of Microspheres

Formulation code	Theoritical drug content (%)	Practical drug content (%)	Entrappment efficincy (%)
F1	6.02	5.38	64.45±2.186
F2	8.15	6.73	72.44±2.38
F3	8.15	6.54	77.14±1.171
F4	9.45	7.29	80.24±1.436
F5	12.92	9.36	82.57±1.325
F6	15.25	9.83	89.36±2.428
F7	15.32	9.90	89.16±2.124
F8	18.36	12.57	87.06±2.018
F9	18.90	14.55	90.36±1.345

2.4. Chromatographic conditions:

A stainless steel YMC C8 column (25cm × 4mm) packed with end-capped octylsilane bonded to porous silica (4µm was used for the separation. The mobile phase prepared for this was phosphate buffer pH 3.4 and HPLC grade acetonitrile in 20:80 (v/v) ratio and the flow rate was kept at 1ml/min. The detector wavelength was kept at 230nm whereas the sample was injected at a volume of 20µl. Ambient temperature was kept during the analysis which had a run time of 5 minutes. Using 0.45µm membrane filter, filtration of mobile phase was done and was set to preconditioning for about 20minutes.

Table 2. Optimized Chromatographic Conditions for Gliclazide

Parameters Conditions

Stationary phase (column) C8 (25 cm×4mm, 4 μm)

Mobile phase Phosphate Buffer: Acetonitrile (20:80, v/v),

pH adjusted to 3.4

Flow rate (mL/min) 1.0

Run time (min) 5.0

Column temperature Ambient temperature

Volume of injection loop (μL) 20

Detection wavelength (nm) 230

Retention time (min) 1.915

2.5. Method Development:

2.5.1. Mobile phase selection and its preparation:

Different mobile phases consisting of acetonitrile, water, methanol, and phosphate buffer were used in different ratios and different flow rates. A sharp and symmetrical peak was found with when a mobile phase containing Phosphate Buffer: Acetonitrile in the ratio of 20:80 (v/v) (pH adjusted to 3.4 using glacial acetic acid) was used for the trial run. Preparation of the mobile phase was done by mixing 200ml of pH 3.4 Phosphate buffer and 800mL of HPLC grade acetonitrile which was kept 20 minutes for sonication and then filtered using a 0.45μm membrane filter.

2.5.2. Standard stock solution preparation:

The stock solution of Gliclazide (1000µg/ml) was prepared by taking 10mg of Gliclazide into a 10ml volumetric flask using HPLC grade methanol as the solvent. From this standard mother stock solution, aliquots of dilution were done in the range of 2µg/ml to 10µg/ml using methanol as the solvent. A steady baseline was recorded using with the chromatographic conditions which was optimized. The prepared standard stock solution was injected thrice in a volume of 20 µl. The retention time of Gliclazide was observed at 1.9 min. Construction of calibration curve by plotting the average peak areas against the respective concentration found against it.

2.6. Method Validation:

In accordance with ICH guidelines the developed analytical RP-UHPLC method for the estimation of Gliclazide was validated. Validation of method was done concerning accuracy, precision, linearity, range, robustness and sensitivity. As per guidelines of United States Pharmacopoeia system suitability was checked¹¹.

2.6.1. Determination of Linearity and Range:

Accurately weighed amount of Gliclazide was taken and dissolved in 10ml HPLC grade methanol for preparation of stock solution of 1mg/ml. Further from this stock, dilutions were made to yield a concentration of 2,4,6,8, and 10µg/ml respectively. To find the linearity and range, different concentrations of Gliclazide working standard solutions were injected and the readings were collected in triplicate. Construction of concentration versus peak area was done¹².

2.6.2. Determination of Accuracy:

Determination of accuracy was achieved by estimation of the percentage recovery of Gliclazide at samples of low (LQC), middle (MQC), and high (HQC). Gliclazide samples of concentration 2µg/ml, 4µg/ml, and 6µg/ml were prepared in triplicate and calculation of %RSD was done^13,14.

2.6.3. Determination of Precision:

Estimation of precision on intra-day and inter-day basis for the developed method was done by calculating the %RSD of triplicate reading of three different concentrations. The average Data was calculated¹⁵.

2.6.4. Determination of Robustness:

Determination of robustness for the developed method was done by producing deliberate variations in flow rate, ratio of the mobile phase, temperature, etc. The composition of the mobile phase varied at ±2% whereas variation in flow rate was at ±0.2% ^16,17.

2.6.5. Determination of Sensitivity:

To understand how sensitive the developed analytical method is Limit of detection (LOD) and Limit of Quantification (LOQ) was determined at 3times of LOD^18,19.

2.5.6. Determination of System Suitability:

To understand how suitable is the developed analytical method for analysis tests for System suitability was done by taking three replicate injections of a standard sample of Gliclazide solution of 50µg/ml and estimation of peak resolution, retention time, theoretical plate height etc were done²⁰.

2.6.7. Application of the validated RP-UHPLC method:

The validated analytical method was used to estimate Drug entrapment efficiency of Gliclazide loaded microsphere formulation. The %DEE of the prepared formulation was estimated²¹.

2.6.8. Determination of Drug Entrapment Efficiency:

In the core of the matrix amount of drug which is entrapped can be determined by performing Drug entrapment efficiency test. For the determination of %DEE, the drug-loaded microsphere was collected and dissolved in a suitable solvent. It was further filtered and centrifuged. For estimation of %DEE the validated analytical method was used.

3. RESULTS:

3.1. Method development:

The sample for developing the method was prepared in appropriate conditions by using HPLC grade methanol as the solvent. The sample was weighed accurately and was prepared in an amber-colored volumetric flask. The absorption maxima of Gliclazide was found at 230nm. RP-UHPLC method development was carried out using Phosphate buffer pH3.4 and acetonitrile as the mobile phase in the ratio of 20:80. 1.915min was found as the retention time of Gliclazide. Other parameters like theoretical plate height, peak resolution tailing factor, separation factor, etc. were in accepted limit.

Figure 3: UHPLC Chromatogram of Gliclazide standard

3.2. Validation of Method:

3.2.1. Specificity:

At 1.915min other than peak of Gliclazide no other peak was observed in the chromatogram. Hence it can be said that the analytical method developed is specific for Gliclazide estimation only.

Figure 4: UHPLC Chromatogram for Estimation of Specificity

3.2.2. Linearity and Range:

The calibration curve obtained for Gliclazide was linear in the concentration range between 2-10µg/ml. The determination of Regression coefficients (R²) for Gliclazide was 0.998. The equation obtained from the calibration curve was Y=11.515x-1.75.

Figure 5: Calibration Curve of Gliclazide

Table 3. Inter-Day precision Values of the RP-UHPLC method

Nominal Conc. Level (µg/ml)	Day 1				Day 2				Day 3
Nominal Conc. Level (µg/ml)	Found Conc. (µg/ml)	% Accuracy Day 1	Avg. % Bias	% RSD	Found Conc. (µg/ml)	% Accuracy Day 2	Avg. % Bias	% RSD	Found Conc. (µg/ml)	% Accuracy Day 3	avg. % Bias
2	1.95 (±0.02)	97.5 (±0.20)	2.50	1.4	2.10 (±0.30)	105.00	-5	1.8	2.50 (±0.40)	125	-25
4	4.05 (±0.50)	101.25 (±0.50)	-1.25	0.80	4.45 (±0.20)	111.25	-11.5	1.6	4.70 (±0.55)	117.5	-17.5
6	5.98 (±0.45)	99.66 (±0.40)	0.40	0.60	6.50 (±0.50)	108.33	-8.33	0.90	6.88 (±0.70)	114.6	-14.6

Data Expressed as Mean (±SD), n=3

Table 4. Accuracy values of Gliclazide

Analytes	Nominal Concentration Level (µg/ml)	Found Concentration(µg/ml)	% Accuracy	Avg.% Bias	%RSD
Gliclazide	2	1.89(±0.20)	94.5(±0.50)	5.5	1.2
	4	3.90(±0.40)	97.5(±0.22)	2.5	0.90
	6	5.80(±0.60)	96.6(±0.40)	3.4	0.60

Data Expressed as Mean (±SD), n=3

Table 5. Robustness data of RP-UHPLC method

Sl no.			Parameters	Analytes			Sample conc. (µg/ml)		Tailing Factor T	Theoretical Plates N
A. Change in composition of Mobile Phase
1		Buffer: Acetonitrile (30:70)			Gliclazide			2	1.83(±0.01)	3602(19)
								4	1.78(±0.04)	2809(05)
								6	1.55(±0.06)	2897(22)
								8	1.41(±0.08)	3833(10)
								10	1.48(±0.01)	2321(14)
2		Buffer: Acetonitrile (50:50)			Gliclazide			2	1.80(±0.01)	3482(45)
								4	1.60(±0.02)	3883(14)
								6	1.48(±0.06)	4120(26)
								8	1.35(±0.08)	4139(16)
								10	1.25(±0.01)	3670(28)
B. Change in flow rate of Mobile Phase
1	0.8ml/min					Gliclazide	2		1.80(±0.02)	2986(26)
							4		1.69(±0.05)	2982(12)
							6		1.55(±0.04)	2990(11)
							8		1.70(±0.01)	4076(30)
							10		1.90(±0.08)	2451(22)
	2ml/min					Gliclazide	2		1.65	2886(12)
							4		1.45	3562(11)
							6		1.17	4106(12)
							8		1.31	3562(11)
							10		1.71	2654(18)

3.2.3. Precision:

On the repeatability of the method, effect of random errors was expressed in terms of %RSD which was lesser than 2%.

3.2.4. Accuracy/ Recovery:

The % recovery of samples for the LQC, MQC, and HQC were found in acceptable range of 94%-98%.

3.2.5. Robustness:

Determination of robustness for the developed analytical method was done by deliberately producing variations in the mobile phase concentration and also a slight alteration in the mobile phase flow rate. The effect of this change on the tailing factor, resolution, and theoretical plates were considered for samples of LQC, MQC, and HQC. It was seen that a slight modification in the mobile phase composition and flow rate did not produce any significant variation in the above parameters which reflects the robustness of the developed method.

3.2.6. System suitability:

Retention time, theoretical plate height, resolution, tailing factor and separation factor were analyzed for the given drug molecule. The sample concentration was maintained at 50µg/ml and was injected to estimate the parameters of system suitability. Resolution was found as 16.94(±0.08) which gives a clear indication of well-resolved peaks. All the other parameters were under acceptable limits. It can be said that the developed analytical method fulfilled the criteria of system suitability.

3.2.7. Drug Entrapment Efficiency:

The extraction of encapsulated Gliclazide microsphere was done by the liquid-liquid extraction method with the help of chloroform and acetone in a ratio of 1:1. The sample was vortexed and centrifuged for about 5min at 1000rpm. Finally, the organic layer was collected and was subjected to the estimation of Gliclazide by the developed analytical method. The % DEE was calculated and was found to be 93.72 %.

4. DISCUSSION:

Linearity for the developed RP–UHPLC analytical method was in the range of 2–10μg/mL, In optimized conditions validation of the developed analytical method was performed successfully. The results obtained for different validation parameters in accordance with ICH guidelines were found in acceptable range and limits. In this chromatographic method, the % recovery of Gliclazide samples of LQC, MQC, and HQC were found in the acceptable range of 94%-98%. The % Drug entrapment Efficiency of the Gliclazide loaded microsphere was found to be 93.72 % which was satisfactory.

5. CONCLUSION:

The analytical Reversed-phase UHPLC method developed for estimation of Anti Diabetic Drug Gliclazide was found to be fast rapid and accurate. All the parameters were carried out as per the standard guidelines of ICH. Validation of the developed analytical method for different parameters like precision, accuracy, linearity, robustness, and system suitability were performed. Furthermore, the validated method was applied in terms of estimation of %DEE from the prepared microsphere formulation and the results found were within well acceptable limits. It can be concluded that this validated method can be further used in the estimation of Gliclazide from other sustained release dosage forms.

6. ACKNOWLEDGEMENT:

Authors Acknowledge Late. Dr. Partha Jyoti Gogoi, Regional Director, Regional Drugs Testing Laboratory (RDTL), Guwahati for providing the necessary instruments and facilities for carrying out this work.

7. CONFLICT OF INTEREST:

None.

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Received on 16.11.2020 Modified on 25.03.2021

Research J. Pharm.and Tech 2022; 15(2):773-778.

DOI: 10.52711/0974-360X.2022.00129