Reversed-phase chromatography is the mainly used in chromatographic mode, it is used to separate neutral molecules in solution based on their hydrophobicity. This technique is the reverse of normal-phase chromatography in the intelligence that it involves the employ of a polar mobile phase and a non-polar stationary phase. A sensitive, accurate, rapid, cost effective and robust HPLC method was developed for the quantification of Saxagliptin Hydrochloride (SGH) with UV detector. In this method, a reversed-phase Grace C18 (250mm x 4.6ID, Particle size: 5 micron) column with a mobile phase of methanol: water (80:20; v/v) at 0.8ml/min flow rate was used to separate SGH with a detection of 212nm.The volume injected was 20 µL. The retention time of SGH was obtained as 4.196 min. Hence it can be applied for routine analysis of Saxagliptin Hydrochloride (SGH) in bulk drug.
Keywords: Saxagliptin Hydrochloride (SGH), RP-HPLC, Assay, Method validation

Gaikwad, D D

Jadhav, S L

Patel, S G

Zinjad, Sanchay S

Journal of Drug Delivery and Therapeutics (JDDT)

Zinjad et al                                                                                                                Journal of Drug Delivery & Therapeutics. 2019; 9(4):274-278  ISSN: 2250-1177                                                                                  [274]                                                                                 CODEN (USA): JDDTAO Available online on 15.07.2019 at http://jddtonline.info Journal of Drug Delivery and Therapeutics Open Access to Pharmaceutical and Medical Research © 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted non-commercial use, provided the original work is properly cited Open  Access                                                                                                                                                     Research Article Analytical Method Development of Saxagliptin HCl by RP-HPLC Sanchay S Zinjad*, Patel S G, Dr. Gaikwad D D, Dr. Jadhav S L Vishal Institute of Pharmaceutical Education and Research, Ale, Tal- Junnar, Pune (412411) Maharashtra, India  ABSTRACT Reversed-phase chromatography is the mainly used in chromatographic mode, it is used to separate neutral molecules in solution based o n their hydrophobicity. This technique is the reverse of normal-phase chromatography in the intelligence that it involves the employ of a polar mobile phase and a non-polar stationary phase. A sensitive, accurate, rapid, cost effective and robust HPLC method was developed for the quantification of Saxagliptin Hydrochloride (SGH) with UV detector. In this method, a reversed-phase Grace C18 (250mm x 4.6ID, Particle size: 5 micron) column with a mobile phase of methanol: water (80:20; v/v) at 0.8ml/min flow rate was used to separate SGH with a detection of 212nm.The volume injected was 20 µL. The retention time of SGH was obtained as 4.196 min. Hence it can be applied for routine analysis of Saxagliptin Hydrochloride (SGH) in bulk drug.  Keywords: Saxagliptin Hydrochloride (SGH), RP-HPLC, Assay, Method validation.  Article Info: Received 05 May 2019;     Review Completed 25 June 2019;     Accepted 26 June 2019;     Available online 15 July 2019 Cite this article as: Zinjad SS, Patel SG, Gaikwad DD, Jadhav S L, Analytical Method Development of Saxagliptin HCl by RP-HPLC, Journal of Drug Delivery and Therapeutics. 2019; 9(4):274-278  http://dx.doi.org/10.22270/jddt.v9i4.3040                                                  *Address for Correspondence:   Sanchay S Zinjad, Vishal Institute of Pharmaceutical Education and Research, Ale, Tal- Junnar, Pune (412411) Maharashtra, India   1. INTRODUCTION Analytical chemistry termed as science of determining the components of materials in terms of the elements or compound contained. The techniques of this science are used to identify the substances which may be present in a material and to determine the exact amounts of the identified substances. Analytical chemistry is important in nearly all aspects of chemistry, e.g. clinical, agricultural, environmental, forensic, manufacturing metallurgical and pharmaceutical chemistry. Analytical techniques proved in assuring and maintaining the quality of substance and are critical components of QA and QC1, 2. Analytical method should be,  1. Most productive, economical and convenient, 2. As accurate and precise as required, 3. As simple as possible, 4. Most specific Should be fully optimized before transfer for validation of its characteristics such as precision, accuracy, sensitivity etc.2 According  to  USP,  system  suitability  tests  are  integral  part  of  chromatographic methods.  These  tests  are  used  to  verify  that  the  reproducibility  and  resolution  of  the system are adequate for the analysis to be performed. Parameters such as plate count, tailing  factor,  resolution  and  reproducibility  (%RSD  retention  time  and  area  for  6 repetitions)  are  determined  and  compared  against  the  specifications  set  for  the method.3, 4 Now a day reversed-phase chromatography is the most commonly used separation technique in HPLC due to its broad application range. It is estimated that over 65% (possibly up to 90%) of all HPLC separations are carried out in the reversed phase mode. The reasons for this include the simplicity, versatility, and scope of the reversed-phase method as it is able to handle compounds of a diverse polarity and molecular mass5-7. Reversed phase chromatography has found both analytical and preparative applications in the area of biochemical separation and purification. Molecules that possess some degree of hydrophobic character can be separated by reversed phase chromatography with excellent recovery and resolution8. The separation mechanism in reversed phase chromatography depends on the hydrophobic binding interaction between the solute molecule in the mobile phase and the immobilised hydrophobic ligand, i.e. the stationary phase. The actual nature of the hydrophobic binding interaction itself is a matter of heated debate9 but the conventional wisdom assumes the binding interaction to be the result of a favourable entropy effect. Zinjad et al                                                                                                                Journal of Drug Delivery & Therapeutics. 2019; 9(4):274-278  ISSN: 2250-1177                                                                                  [275]                                                                                 CODEN (USA): JDDTAO 2. AIM AND OBJECTIVE Aim:  Analytical method development and validation of Saxagliptin HCL by RP-HPLC method. Objectives: 1. To develop a simple validated stability indicating method for the estimation for Saxagliptin HCL by RP-HPLC. 2. To validate methods as per ICH guidelines. 3. MATERIALS AND METHOD 3.1. UV Spectroscopic Method Selection of Wavelength: Weighed accurately about 10mg of working standard of Saxagliptin HCl and transferred in 10mL of volumetric flask. Added about 5mL of diluent, Sonicated to dissolve and volume was made up the mark with diluent. Further diluted 0.2mL of above solution to 10mL and volume was made up to the mark with diluent. Determination of λmax 10-11 Initially the Ultra violet (U.V) spectrum of Saxagliptin HCl was produced using appropriate U.V spectrophotometer, so as to determine the absorbance maxima or Lambda max (λ max). This is essential since HPLC detection is basically UV based, thus a 20ppm solution of Saxagliptin HCl in water was used to get the following spectra.  3.2 High Performance Liquid Chromatography Method Development by RP-HPLC Preparation of Saxagliptin HCl Standard:12-17 Weighed accurately about 10mg of Saxagliptin HCl standard and transffered into 10mL of volumetric flask, added about 5mL of diluent, shaked to dissolved and volume was made up to the mark with diluent. ( concentration of Saxagliptin HCl 1000ppm). Further diluted 0.5mL of above stock solution to 10mL of volumetric flask and volume was made up to the mark with diluent (concentration of Saxagliptin HCl 50ppm).                                                                                       Trial 1  Table 3.1: Chromatographic conditions for Trial 1 Column  Grace C18 (250mm x 4.6ID, Particle size: 5 micron) Mobile Phase Methanol : Buffer (70:30) Pump mode Isocratic Flow rate 0.8ml/min Detector UV- Detector Injection volume 20µL Column Temperature 25°C Wavelength 212nm Run time 7.71min  Preparation of Buffer:  0.136gm of Potassium dihydrogen phosphate dissolved in 100mL of distilled water, gives 10mM solution. Trial 2  Table 3.2: Chromatographic conditions for Trial 2 Column  Grace C18 (250mm x 4.6ID, Particle size: 5 micron) Mobile Phase Methanol : Buffer (80:20) Pump mode Isocratic Flow rate 0.8ml/min Detector UV- Detector Injection volume 20µL Column Temperature 25°C Wavelength 212nm Run time 6.88min     Zinjad et al                                                                                                                Journal of Drug Delivery & Therapeutics. 2019; 9(4):274-278  ISSN: 2250-1177                                                                                  [276]                                                                                 CODEN (USA): JDDTAO Trial 3 Table 3.3: Chromatographic conditions for Trial 3 Column  Grace C18 (250mm x 4.6ID, Particle size: 5 micron) Mobile Phase Methanol : Water (80:20) Pump mode Isocratic Flow rate 0.8ml/min Detector UV- Detector Injection volume 20µL Column Temperature 25°C Wavelength 212nm Run time 9.21min  Trial 4 Table 3.4: Chromatographic conditions for Trial 4 Column  Grace C18 (250mm x 4.6ID, Particle size: 5 micron) Mobile Phase Methanol : Water (80:20) Pump mode Isocratic Flow rate 0.8ml/min Detector UV- Detector Injection volume 20µL Column Temperature 25°C Wavelength 212nm Run time 7.15min  TABLE 4.1: FINAL METHOD FOR ASSAY PARAMETER CONDITIONS Stationary Phase (Column)  Grace C18 (250mm x 4.6ID, Particle size: 5 micron) Mobile Phase Methanol: Water (80:20) Diluent Methanol : Water (200:100) Flow rate 0.8ml/min Injection volume 20 µL Pump mode Isocratic  Detector PDA Wavelength 212nm Column Temperature 25°C Run Time 7.15 min Retention Time 4.195  Conclusion: The peak obtained in Trail 4 is sharper. Shape is better. The retention time of the Trial 4 was less i.e. 4.165’    Zinjad et al                                                                                                                Journal of Drug Delivery & Therapeutics. 2019; 9(4):274-278  ISSN: 2250-1177                                                                                  [277]                                                                                 CODEN (USA): JDDTAO 4. RESULT AND DISCUSSION  Figure 1.2: UV spectra of Saxagliptin HCl  Figure 3.2: Observed chromatogram of Trial 1  Figure 3.3: Observed chromatogram of Trial 2 5.455'- 2 0020406080100120140160180mV2 4 6 8 10 12 14 16 18 min4.413'03 06090120150180210240270300mV2 4 6 8 10 12 14 16 18 minZinjad et al                                                                                                                Journal of Drug Delivery & Therapeutics. 2019; 9(4):274-278  ISSN: 2250-1177                                                                                  [278]                                                                                 CODEN (USA): JDDTAO  Figure 3.4: Observed Chromatogram of Trial 3  Figure 3.5: Observed Chromatogram of Trial 4  5. CONCLUSION The present investigation is an attempt to develop method for estimation of saxagliptin HCl in bulk by RP-HPLC. The developed method is less costly with lower retention time than the method reported so far. The developed methods will be useful in the estimation of Saxagliptin HCl in bulk in future. 6. REFERENCES 1. Skoog DA, Holler FJ, Timothy A, Nieman NW. Principle of Instrumental Analysis.  5th edition. Bangalore: Eastern press, 2004, 678-688, 695-696.  2. Christian G, Analytical Chemistry, 5thedition, New York, John Wiley and Sons, 2003, 1-3 3. Willard HH, Merritt LL, Dean JA, Settle FA, Instrumental methods of analysis. 7th edition. New Delhi: CBS Publishers and Distributors; 2001, 465-506, 513-522, 530-534. 4. Backett H, Stanlake JB. Practical Pharmaceutical Chemistry part II. 4th edition. Delhi: CBS Publisher and Distributors; 2001, 255-280. 5.         Willard HH, Dean AJ. Instrumental Methods of Analysis. CBS Publishers and distributors, 7th edition 1986, 513-515, 580-604.  6.         Connors AK. A Text Book of Pharmaceutical Analysis. A Wiley Interscience publication, 3rd edition, 2005, 373-400. 7.         Ahuja S, Ahuja S. High Pressure Liquid Chromatography. Comprehensive Analytical Chemistry. Elsevier, 2006.  8.         Amesham Biosciences. Reversed Phase Chromatography. Principles and Methods. 6-8.  9.         Dorsey JG, Cooper WT. Retention mechanisms of bonded-phase liquid chromatography. Anal. Chem. 66th edition 1994, 857A-867A. 10. ICH Q2 (R1),    Harmonized   tripartite   guideline,   validation   of   analytical procedure methodology, IFPMA, proceedings of the international conference on harmonization, Geneva, 2005. 11. ICH (Q2A), Harmonized   tripartite   guideline, validation   of   analytical procedure methodology, IFPMA, proceedings of the international conference on harmonization, Geneva, 1994. 12. ICH-Q2A, (R1) Text on Validation of Analytical Procedures, U.S. Department of Health and Human Services. USFDA. 1995. A1- A13. 13.  Pallavi et al. / SGVU Journal of Pharmaceutical Research & Education, 2017, 1(2), 243-260 14. Md. Saiful Islam, et al, (2016), Development and validation of RP-HPLC method for determination of saxagliptin hcl in bulk and tablet dosage form, World journal of pharmacy and pharmaceutical sciences, Volume 5, Issue 5, 107-119. 15. P.B.N. Prasad, et al, (2015), Development and validation of a method for simultaneous determination of metformin and saxagliptin in a formulation by RP-HPLC, American journal of analytical chemistry, 2015, 6, 841-850. 16. Hanan A. Merey, et al, (2017), Chromatographic methods for the simultaneous determination of binary mixture of saxagliptin hcl and metformin hcl, Bulletin of faculty of pharmacy, Cairo university, 55 (2017) 311-317. 17. Dr. Advaita B. Patel, et al, (2017), Development and validation of stability indicating method for the for the simultaneous estimation of saxagliptin hcl and dapagliflozin using RP-HPLC method in tablet dosage form, World journal of pharmacy and pharmaceutical sciences, Volume 6, Issue 10, 444-458. 4.196'02 0406080100120140160mV2 4 6 8 10 12 14 16 18 min4.195'09 0180270360450540630720810mV2 4 6 8 10 12 14 16 18 min

Analytical Method Development of Saxagliptin HCl by RP-HPLC

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Analytical Method Development of Saxagliptin HCl by RP-HPLC

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