INTRODUCTION:

Glipizide (GPZ) is chemically 1cyclohexyl 3[[p[2(5 methylpyrazin ecarboxamido) ethyl] phenyl] sulfonyl]urea^1-2.

Fig. No. 1 Structure for GPZ

GPZ is an oral medium-to-long acting anti-diabetic drug. It is classified as a second generation sulfonylurea, which means that it undergoes entero hepatic circulation. It acts by blocking potassium channels in the beta cells of the islets of Langerhans³. Literature survey reveals for estimation of GPZ by HPTLC⁴ method, HPLC⁵ method, RP-HPLC⁶ in plasma, and RP-HPLC⁷ in dosage form has been reported. In combination with other drugs LC⁸ method, ion pair RP-LC⁹, HPLC¹⁰,¹¹ and HPTLC¹² method has been reported for estimation of GPZ.

METHOD DEVELOPMENT:

Solubility Test:

Solubility test for the drug GPZ was performed by using various solvents. The solvents include Ethanol, Dist. water, Acetic Acid, 0.1 N Hydrochloric Acid, 0.1 N Sodium Hydroxide and Chloroform. However, 0.1 N Sodium Hydroxide was chosen as a solvent for developing the method.

Preparation of Stock Solution:

The standard stock solution of 100 μg/mL of GPZ was prepared by weighing 100 mg of the drug, taken in 100 mL volumetric flask and was dissolved in 0.1 N NaOH up to the mark. Further 1 ml of above solution was pipette out and diluted up to 10 ml with 0.1 N NaOH to produce 1000 μg/mL.

Preparation of Working Standard Solution:

5 ml of stock solution was taken in 10 ml volumetric flask and was diluted with 0.1 N NaOH Further 1 ml above resulting solution was taken and diluted up to 10 ml with 0.1 N NaOH to produce final concentration of 5μg/mL. Further dilutions were made with 0.1 N NaOH to obtain concentrations ranging from 10-50 μg/mL.

Determination of λ max:

By appropriate dilution of standard solutions with distilled water, solutions containing 50 μg/mL of GPZ were scanned in the range of 200-800 nm to determine the wavelength of maximum absorbance for the drugs. GPZ showed absorbance maxima at 227 nm.

Fig. No.2 λmax of GPZ at 227 nm

Assay of GPZ:

Accurately weigh and powder the 20 tablets. Take a drug which is equivalent to 50 mg of GPZ transfer to a 50 mL of volumetric flask; adjust the final volume up to 50 mL with solvent. Further take 5 ml of above solution and dilute to 45 ml with solvent. Finally check the absorbance of this resulting solution at 227 nm.

Table No 1: Assay of GPZ

Brand Name	Label Claim	Assay
Glynase	5 mg	97.04%

METHOD VALIDATION:

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its predetermined specifications and quality characteristics^13,14. The method was validated for different parameters like Linearity, Accuracy, Precision, LOD, LOQ, Robustness, and Ruggedness.

Linearity and Range:

Various aliquots were prepared form the stock solution ranging from 10-50 μg/ml. The samples were scanned in UV-VIS Spectrophotometer using 0.1N NaOH as a blank. It was found that the selected drug shows linearity between the range 10-50 μg/ml (Table 2).

Accuracy:

The accuracy of the method was determined by preparing solutions of different concentrations that is 80%, 100% and 120%. Samples were prepared in triplicates and the accuracy were indicated by % RSD (Table 3).

Precision:

Precision of the method was demonstrated by intraday and interday variation studies. In intraday variation study, 6 different solutions of same concentration that is 20μg/ml were prepared and analyzed in a day and the absorbance were noted. The result was indicated by % RSD (table no.6, and table no.7). In the interday variation study, solutions of same concentration 40μg/ml were prepared and analyzed for three consecutive days and the absorbance's were noted. The result was indicated by % RSD (Table 2, 4 and 5).

Ruggedness:

Ruggedness of the method was determined by analyzing same sample (different batches) by different analysts at different conditions and the respective absorbance’s were noted (Table 6)

Robustness:

Robustness of the method was determined by carrying out the analysis at two different wavelengths i.e. at (+2nm and -2nm). (Table 2, 7)

Limit of Detection (LOD):

The limit of detection (LOD) was determined by preparing solutions of different concentrations ranging from 10-50 μg/ml. The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantized as an exact value (Table 2).

Limit of Quantification (LOQ):

The LOQ is the concentration that can be quantized reliably with a specified level of accuracy and precision. The LOQ was calculated using the formula involving standard deviation of response and slope of calibration curve (Table 2).

RESULTS AND DISCUSSION:

The developed method was found to be precise as the %RSD values for intra-day and inter-day were found to be less than 2%. Good recoveries (99% to 102%) of the drug were obtained at each added concentration, indicating that the method was accurate. The method was also found to be robust and rugged as indicated by the % RSD values which are less than 2%. The results of Assay showed that the amount of drug was in good agreement with the label claim of the formulation as indicated by % recovery. Summary of validation parameters of proposed spectro photometric method is shown in table 2.

Table No. 2 Validation Summery

Parameters	Results
Beers Limit	10-50 μg/ml
Linearity indicated by correlation coefficient	0.999
Linear regression equation	y = 0.027x
Intraday Precision indicated by %RSD	0.441
Interday Precision indicated by Avg. %RSD	0.225
Robustness indicated by Avg. %RSD	0.21
Assay indicated by % Recovery	97.04%

RESULTS AND DISCUSSION:

Linearity

Fig. No. 3 Linearity curve for GPZ

Accuracy

Table No.3 Accuracy of GPZ

No. of Prepⁿ	Cocⁿ(μg/ml)		Avg % Recovery	%RSD
No. of Prepⁿ	Formulation	Pure Drug	Avg % Recovery	%RSD
S1-S3 80%	10	8	102.25	0.77
S4-S6 100%	10	10	100.10	0.061
S7-S9 120%	10	12	99.87	0.89

Precision

Table No.4 Intraday Precision

Avg.	0.555
SD	0.002
%RSD	0.44%

Table No.5 Interday Precision

	Day –I	Day-II	Day III
Mean	1.113	1.112	0.118
SD	0.002	0.003	0.001
%RSD	0.0019	0.0031	0.0016

Ruggedness

Table No.6 Ruggedness of GPZ

Sr. No.	Parameter	Set I	Set II
1	System	Bioera	Schimadzu 1700
2	Sample	Batch No-X	Batch.No- Y
3	Sample	10 μg/ml	10 μg/ml
4	Assay	99.47 %	99.62%

Robustness

Table No.7 Robustness of GPZ

Sr. No.	Cocⁿ(μg/ml)	Wavelength(nm)	Calculations
1	50	229	Mean = 1.205 S. D. = 0.004 %RSD = 0.331


2	50	225	Mean = 1.222 S. D. = 0.0012 %RSD = 0.10

Limit of Detection (LOD):

The LOD for GPZ was found to be 0.52μg/ml.

Limit of Quantification (LOQ):

The LOQ for GPZ was found to be 0.61μg/ml.

CONCLUSION:

The proposed method development and validation of UV-Vis Spectrophotometrically method was to determine Glipizide. The developed method was validated in 0.1 N NaOH according to ICH guideline and shown to be accurate, precise and cost effective. It do not require expensive or sophisticated and chemicals in contrast with chromatographic method. It can be used for the routine Q. C. analysis and quantification of the drug in the formulations.

ACKNOWLEDGEMENT:

The authors are wish to thanks Principal and Management of Shivnagar Vidya Prasarak Mandals College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune and Mr. Prabhatkumar Jain, Scan Research Bioanalytical Laboratories, Bhopal, for providing required lab facilities with enthusiastic environment.

REFERENCES:

1. United States Pharmacopeia and National Formulary, Asian edition 2005, United States Pharmacopeial Convention. Rockville, MD, 2005, p.903-904

2. Wilson and Gisvolds, Text book of organic medicinal and pharmaceutical chemistry, eleventh edition, edited by John H. Block, John M. Beale, Jr. Lippincott Willians and Wilkins , Wolters Kluwer, London UK: 2004; p 670.

3. Goodman and Gilman’s, The Pharmacological Basis of Therapeutics,11th edition, edited by Laurence L. Brunton, John S. Lazo, Keith L. Parkar, McGraw-Hill, Medical Publishing Division, 2006; p 1635-1638.

4. Shelesh Jain and Swarnlata Saraf, Development and validation of high performance thin layer chromatographic (HPTLC) technique for quantification of glipizide in tablet dosage forms, Biosciences Biotechnology Research Asia, 05(1).

5. S Dhawan, A K Singla, High Performance liquid chromatographic analysis of glipizide: application to in vitro and in vivo studies. J Chromatogr Sci. 2003 ;41 (6):295-300

6. Swaroop R. Lahoti, Prashant K. Puranik, Ashish A. Heda, Rajesh B. Navale. Development and Validation of RP-HPLC Method for Analysis of Glipizide in Guinea Pig Plasma and its Application to Pharmacokinetic Study, International Journal of Pharm Tech Research, 2010 ;Vol.2, No.3, : 1649-1654.

7. Shaikh Rahila, Karigar Asif, Reverse phase high performance liquid chromatographic method for the analysis of glipizide in pharmaceutical dosage forms, International Journal Of Research In Ayurveda and Pharmacy, 2010;1(2), : 455-458

8. S. Abu Ruza, J. Millershipb and J. McElnayb, The development Medical and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma, J of Chromatography B, 2005; 817 (2): 277-286.

9. B. Udaykumar Rao and Anna Pratima Nikalje, Determination of Glipizide, Glibenlamide and Glimeperide in a Tablet Dosage Form in the Presence of Metformin Hydrochloride by Ion Pair –Reversed Phase Liquid Chromatographic Technique, J Anal Bioanal Techniques, 2010; 1(2):1000105.

10. Mousumi Kar, P. K. Choudhury, HPLC Method for Estimation of Metformin Hydrochloride in Formulated Microspheres and Tablet Dosage Form, Indian J Pharm Sci. 2009; 71(3):318–320.

11. Anna Gumieniczek and Anna Berecka, Quantitative analysis of gliclazide and glipizide in Tablets by a new validated and stability-indicating RPTLC method, J of Planar Chromatography, 2010;23(2):129-133

12. Darshana K. Modi and Bhavesh H. Patel, Simultaneous determination of metformin hydrochloride and glipizide in tablet formulation by HPTLC, J. of Liquid Chromatography and Related Technologies, 2012; 35(1):28-39

13. Baokar Shrikrishna, Ranpise Nisharani, Development and validation of RP-HPLC method for Simultaneous determination of Dicyclomine and Mefanamic acid, Journal of Pharmaceutical Research Vol 13 (1), pp 16-19, (2014).

14. Shrikrishna B. Baokar, Kiran Khapake, Patil R.N., New Dissolution Method for the Evaluation of Acyclo vir using pH 7.4 Phosphate Buffer in-Vitro and Determination of its content by Validated UV Spectrophotometric Method, Research Journal of Pharmaceutical Dosage Forms and Technology 5 (1), 46-48, (2013).

Received on 21.11.2015 Modified on 15.12.2015

Res. J. Pharm. Dosage Form. & Tech. 8(1): Jan.-Mar. 2016; Page 05-08

DOI: 10.5958/0975-4377.2016.00010.0