INTRODUCTION:

Glipizide is an oral hypoglycemic agent in the second-generation sulfonylurea drug class that is used to control blood sugar levels in patients with type 2 diabetes mellitus.¹According to the 2018 Clinical Practice Guidelines by Diabetes Canada, sulfonylurea drugs are considered a second-line glucose-lowering therapy following metformin.² Because sulfonylureas require functional pancreatic beta cells for their therapeutic effectiveness, sulfonylureas are more commonly used for early-stage type 2 diabetes when there is no progressed pancreatic failure.³ Compared to other members of the sulfonylurea drug group, glipizide displays rapid absorption and onset of action with the shortest half-life and duration of action, reducing the risk for long-lasting hypoglycemia that is often observed with blood glucose-lowering agents.⁴ Glipizide is a blood glucose-lowering agent. The initial onset of blood glucose-lowering effect occurs around 30 minutes post-administration with the duration of action lasting for about 12 to 24 hours.⁵

The main therapeutic actions of glipizide primarily occur at the pancreas where the insulin release is stimulated, but glipizide also mediates some extra pancreatic effects, such as the promotion of insulin signaling effects on the muscles, fat, or liver cells.⁶Due to its action on the endogenous cells, sulfonylureas including glipizide is associated with a risk for developing hypoglycemia and weight gain in patients receiving the drug.^7,3Chronic administration of glipizide may result in down-regulation of the sulfonylurea receptors on pancreatic beta cells, which are molecular targets of the drug, leading to a reduced effect on insulin secretion.⁸

Fig. 1: Structure of Glipizide

UPLC:

Ultra-Performance Liquid Chromatography (UPLC) is specially designed to withstand higher system pressures during chromatographic analysis so that it enables significant decrease in separation time and solvent consumption. The UPLC columns packed with 1.7 μm sized particles provides not only increased efficiency but also the ability to work at increased linear velocity without loss of efficiency, providing both resolution and speed. Using advantages of UPLC, a number of applications in different fields including pharmacy,^9-11 clinical analysis, pesticide analysis¹² and tetracyclines in human urine ¹³ have been reported. The UPLC is based on the principle of use of stationary phase consisting of particles less than 2μm, while UPLC columns are typically filled with particles of 3 to 5μm. The underlying principles of this evolution are governed by the Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency).¹ The Van Deemter curve, governed by an equation with three components shows that the usable flow range for a good efficiency with a small diameter particles is much greater than for larger diameters.^8,7

METHOD DEVELOPMENT:

Based on drug solubility and P^kaValue following conditions has been used to develop the method estimation of Glipizide.

Optimized Chromatographic Conditions:

Column: BEH C18 (100mm x 2.1 mm, 1.8m)

Mobile phase: 0.01N KH₂PO₄ (4.8): Acetonitrile (50:50)

Flow rate: 1.0ml/min

Detector: Acquity TUV 234nm

Temperature: 30⁰C

Injection Volume: 0.30µL

Fig.2: Optimized chromatogram

Observation:

Glipizide eluted with good peak shape and retention time and tailing was passed.

MATERIALS AND METHODS:

Diluent: Based up on the solubility of the drug, diluent was selected, acetonitrile and buffer are taken in the ratio of 50:50.

Preparation of Standard stock solution:

Accurately weighed 5mg of Glipizide was transferred to 50ml volumetric flasks, 3/4^th of diluents were added and sonicated for 10 minutes. Volumetric flasks was made up with diluents and labeled as standard stock solution (100µg/ml of Glipizide).

Preparation of Standard working solution (100% solution):

1ml of Glipizide from each stock solution was pipetted out and taken into 10ml volumetric flask which was made up with diluent. (10µg/ml of Glipizide).

Preparation of Sample stock solution:

Five tablets were accurately weighed and the average weight of each tablet was calculated, then the weight which is equivalent to 1 tablet was transferred to 50ml volumetric flask. 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (100µg/ml of Glipizide).

Preparation of Sample working solution (100% solution):

1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (10µg/ml of Glipizide)

Preparation of buffer:

0.1% Ortho Phosphoric acid Buffer:

1ml of Perchloric acid was diluted to 1000ml with HPLC grade water.

Buffer: 0.01N Potassium dihyrogen orthophosphate: Accurately weighed 1.36gm of Potassium dihydrogen Orthophosphate was taken into 1000ml volumetric flask. Add about 900ml of milli-Q water and degas to sonicate, finally make up the volume with water and add 1ml of triethylamine, so pH is adjusted to 3.0 with dilute Orthophosphoric acid solution.

Preparation of Standard stock solution:

Accurately weighed 5mg of Glipizide were transferred to 50ml volumetric flask and 3/4^th of diluents were added and sonicated for 10 minutes. Volumetric flasks were made up with diluents and labeled as Standard stock solution (100µg/ml of Glipizide).

25% Standard solution:

0.25ml each from two standard stock solutions was pipetted out and made up to 10ml (2.5µg/ml of Glipizide)

50% Standard solution:

0.5ml each from two standard stock solutions was pipetted out and made up to 10ml (5µg/ml of Glipizide)

75% Standard solution:

0.75ml each from two standard stock solutions was pipetted out and made up to 10ml (7.5µg/ml of Glipizide)

100% Standard solution:

1.0ml each from two standard stock solutions was pipetted out and made up to 10ml (10µg/ml of Glipizide)

125% Standard solution:

1.25ml each from two standard stock solutions was pipetted out and made up to 10ml (12.5µg/ml of Glipizide)

150% Standard solution:

1.5ml each from two standard stock solutions was pipetted out and made up to 10ml (15µg/ml of Glipizide)

ACCURACY:

Preparation of Standard stock solution:

Five tablets were accurately weighed and average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into 50ml volumetric flask, 5ml of diluent was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters. (100µg/ml of Glipizide).

Preparation of 50% Spiked Solution:

0.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out and made up to the mark with diluent.

Preparation of 100% Spiked Solution:

1.0ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 150% Spiked Solution:

1.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Acceptance criteria:

The percentage recovery of each level should lie between 98.0 to 102%.

Robustness:

Small deliberate changes in the method like flow rate, mobile phase ratio and temperature were made but there was no change in the result and lies within the range of ICH guidelines. Robustness conditions like flow minus (0.27ml/min), flow plus (0.33ml/min), mobile phase minus, mobile phase plus, temperature minus (25°C) and temperature plus (35°C) were maintained and the samples were injected in duplicate manner. System suitability parameters was not much effected and %RSD also lies within the limit.

LOD sample Preparation:

0.25ml of Standard stock solution were pipetted out and transferred to 10ml volumetric flask which was made up with diluents. From the above solution, 0.1ml of Glipizide were transferred to 10ml volumetric flasks and made up with the same diluents.

LOQ sample Preparation:

0.25ml of Standard stock solution were pipetted out and transferred to 10ml volumetric flasks which was made up with diluents. From the above solution, 0.3ml of Glipizide were transferred to 10ml volumetric flasks and made up with the same diluents.

Assay Methodology:

Assay of the marketed formulation was carried out by injecting sample corresponding to equivalent weight into HPLC system and percent purity was found out.

RESULTS AND DISCUSSIONS:

System suitability:

A Standard solution of Glipizide working standard was prepared as per procedure and injected five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms obtained by calculating the % RSD of retention time, tailing factor, theoretical plates and peak areas from five replicate injections are within range.

Table 1: Peak Name - Glipizide

Sl.no.	Peak name	RT	Area	USP Plate Count	USP Tailing
1	Glipizide	1.248	467517	3308	1.83
2	Glipizide	1.248	472303	3316	1.87
3	Glipizide	1.250	475521	3297	1.78
4	Glipizide	1.251	472259	3532	1.80
5	Glipizide	1.252	473651	3424	1.81
6	Glipizide	1.254	470290	3397	1.72
Mean			471924
Std. Dev.			2766.5
% RSD			0.6

Fig. 3: System suitability Chromatogram

PRECISION:

Repeatability:

Six working sample solutions of 10ppm are injected and the percentage amount was calculated and %RSD was found to be 0.5 and chromatogram was shown .

Repeatability Chromatogram:

Intermediate precision:

Five working sample solutions of 10ppm are injected on the next day of the preparation of samples and the % amount found was calculated and %RSD was found to be 0.3.

Fig. 4: Intermediate precision Chromatogram

LINEARITY:

To demonstrate the linearity of assay method, inject 6 standard solutions with concentrations of about 2.5 ppm to 15 ppm of Glipizide. Plot a graph of concentration versus peak area. Slope obtained was 47139, Y-Intercept was 834.9 and Correlation Co-efficient was found to be 0.999 and linearity plot was shown.

Table 2: Linearity Concentration and Responses

Linearity Level (%)	Concentration (ppm)	Area
0	0	0
25	2.5	115226
50	5	233660
75	7.5	363573
100	10	475736
125	12.5	589272
150	15	703198

Accuracy:

Three Concentrations of 50%, 100%, 150% are injected in a triplicate manner and % recovery was calculated as 99.99.

Table 3: Accuracy data

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	5	5.06	101.16	99.99%
	5	5.03	100.57
	5	5.00	100.07
100%	10	9.99	99.90
	10	9.97	99.72
	10	9.94	99.44
150%	15	15.09	100.58
	15	14.85	99.02
	15	14.92	99.48

LOD:

Detection limit of the Glipizide in this method was found to be 0.05µg/ml.

Fig. 5 (a): LOD Chromatogram of Glipizide

Fig. 5 (b): LOQ Chromatogram of Glipizide

LOQ:

Quantification limit of the Glipizide in this method was found to be 0.14µg/ml.

Robustness:

Small deliberate changes in this method is made like flow minus, flow plus, mobile phase minus, mobile phase plus, temperature minus, temperature Plus and %RSD of the above conditions is calculated.

Table 4: Robustness Data

Parameter	%RSD
Flow Minus(0.27ml/min)	0.7
Flow Plus(0.33ml/min)	1.3
Mobile phase Minus(55:45)	1.4
Mobile phase Plus(45:55)	1.4
Temperature minus (25⁰C)	1.5
Temperature plus (35⁰C)	1.6

ASSAY OF MARKETED FORMULATION:

Standard solution and sample solution were injected separately into the system and chromatograms were recorded and drug present in sample was calculated using before mentioned formula.

Table 5: Assay of Formulation

Sample No	Standard	Sample	%Assay
1	467517	470229	99.24
2	472303	472163	99.65
3.	475521	476639	100.60
4.	472259	474619	100.17
5.	473651	474079	100.05
6.	470290	476068	100.47
AVG	471924	473966	100.03
STDEV	2766.5	2417.4	0.51
%RSD	0.6	0.5	0.5

CONCLUSION:

UPLC method was developed for estimation of drug glipizide in pharmaceutical dosage form. UPLC method was optimized by studying different mediums and conditions. The Sample recoveries in all the formulations were in good agreement with their respective labeled claims and the proposed method was validated as per ICH guidelines in all suitable parameters. This method can be used in quality control testing process of anti-diabetic drug (Glipizide) in pharmaceutical dosage forms.

RFERENCES:

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Received on 04.04.2020 Modified on 24.05.2020

Research J. Pharm. and Tech 2021; 14(3):1370-1374.

DOI: 10.5958/0974-360X.2021.00244.4