Novel Analytical Method Development and Validation for Estimation of Clinical Important Simvastatin in Bulk and Pharmaceutical Dosage Form by UV Spectrometric Method Using Phosphate Buffer Solubility

Suvarna G. Bhokare1* and R. P. Marathe2

1 Government College of Pharmacy (BAMU), Aurangabad - 431001, INDIA

2 Yash Institute of Pharmacy, Aurangabad - 431001, INDIA

* Correspondence: E-mail:

(Received 01 July, 2018; Accepted 28 Sept, 2018; Published 22 Nov, 2018 )

ABSTRACT: According to World Health Organization statistics, more than 16 million people die of cardiovascular disease each year, and 7.2 million deaths in 2001 were caused by heart disease. By the year 2020, approximately 25 million deaths annually worldwide are expected from cardiovascular disease, and almost half of those deaths (11.1 million) will be from coronary heart disease. Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is administered in the form of lactone prodrug. Simvastatin lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase and found most effective for the treatment of cardiovascular disease. The present study was undertaken to develop and validate a simple, accurate, precise, reproducible and cost effective UV-Visible spectrophotometric method for the estimation of simvastatin in bulk and pharmaceutical formulation. The solvent used throughout the experiment was methanol and water. Absorption maximum of the drug was found to be 238nm in phosphate buffer pH 7.4. Beer’s law was obeyed in the range of 02-200µg/ml. The method was shown linear in the mentioned concentrations having line equation y = 0.021x + 0.063 with correlation coefficient R2 of 0.977. The recovery values for simvastatin ranged from 99.95%-100.21%.The percent relative standard deviation (%RSD) of interday and intraday precision range was found below 2%. The percent relative standard deviation of robustness and ruggedness of the method was found within the prescribed limit as per recommended guideline of ICH. Hence, proposed method was precise, accurate and cost effective. This method could be applicable for quantitative determination of the bulk drug as well as dosage formulation.

Keywords: UV Spectroscopy; Method Development; Validation; Simvastatin and Regulatory Requirements for Drug Development.

INTRODUCTION: Hyperlipidemia is commonly diagnosed each year. There is a strong correlation between hypercholesterolemia and coronary heart disease. 1-4 Statin is a group of drugs that used primarily in lowering blood cholesterol. Statins were discovered in 1976 when Endo et al. found that a product of mould Penicillium citricum was able to inhibit the activity of one of the enzymes in the cascade of cholesterol synthesis, 3-hydroxy-3 methylglutaryl coenzyme A reductase (HMG CoA reductase). This substance was used for further name statins (vastatins). Two decades of research after discovery were sufficient for the conclusion that statins were considered a very important group of drugs (professor Roberts stated that statins were for atherosclerosis the same as was penicillin for infectious diseases).6-9 Statins have become one of the basic pillars in the secondary and primary prevention of atherosclerosis. Their role is documented in preventing the progression of atherosclerosis, and they are even able to cause regression of the disease. The decrease of cardiovascular endpoints, but also total mortality found in clinical studies could not be explained only by the decrease of atherogenic lipids. 10-16 It was greater than benefit from lipid lowering. Speculation about so-called pleiotropic effects (non-lipid-modifiable) appeared and was confirmed by further research activity. Statin is generally capable of lowering cholesterol by 20 to 60 percent. The discovery of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme-A which is act as inhibitors called statin that was a breakthrough in the prevention of hypercholesterolemia and related diseases such as cardiovascular diseases related to high levels of cholesterol are among the main causes of death in our societies, there is a high incentive for developing processes for the production of statins, an FDA approved drug.17-18

All natural statins have a common molecular structure, a hexahydro-naphthalene system and a -hydroxy-lactone, but they differ from each other due to side chains and a methyl group around the ring. Statins also are fungal secondary metabolites and were the first enzyme in cholesterol biosynthesis. Statins are available either in Tablet or capsule form; statins are usually taken with dinner or bedtime.19 The results are typically evident after a period of four to six weeks of use. Medications in this group are usually easy to tolerate and cause few side effects. The mechanism that involved in controlling the production of plasma cholesterol 14 levels is the reversible inhibition of HMG-CoA reductase by the statins that are related to the structural. The statins differ with respect to their ring structure and substituents. These differences in structure affect the pharmacological properties of the statins.20-21 Sometimes, statins have been grouped into two groups of statins according to their structure. Statins that belong to type-1 are pravastatin and simvastatin. Statins that are fully synthetic and have larger groups linked to the HMG-like moiety is often referred to as type 2 statins. Statins that belong to this group are atorvastatin and rosuvastatin. Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is administered in the form of lactone prodrug. Simvastatin lowers plasma cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoAreductase.22-23

The HMG-CoA reductase inhibitors, or statins, have a beneficial effect on the primary and secondary prevention of cardiovascular morbidity and mortality, primarily by lowering the concentration of circulating LDL. Statins exert their effect by inhibition of HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthesis pathway. Therefore, the HMGCR gene is a good candidate for studies on genetic variation influencing the cholesterol-lowering effect of statin therapy. Studies have shown that polymorphisms in the HMGCR gene are associated with a lower reduction in levels of total and LDL-cholesterol, within different populations and settings. Another important candidate gene is the LDL receptor (LDLR) gene, since statins increase LDLR expression. Studies on genetic variation in this gene showed a decreased response to statin therapy. Furthermore, cytochrome P450 3A4 (CYP3A4) metabolizes simvastatin, atorvastatin, and lovastatin, and in a recent study, the CYP3A4 intron 6 C>T SNP was associated with an increased total and LDL-cholesterol lowering response to simvastatin therapy. Nevertheless, there is a considerable variability between individuals in response to statins, in terms of both cholesterol lowering and clinical outcomes, of which the origins are poorly understood. Simvastatin belongs to another class of cholesterol-lowering agents, the statins. Statins work by inhibiting the first step involved in cholesterol synthesis.24-25 They competitively block HMG-CoA reductase and lower the amount of cholesterol that is synthesized by the body. This is the rate-limiting enzyme in the liver necessary for cholesterol production.26-28

Pharmaceutical analyses is one of the most challenging field of analytical chemistry.29-34 Pharmaceutical analysts carry out the qualitative and quantitative control of APIs and drug products and also develop and validate appropriate methods. These methods are routinely used by manufacturing companies in-process testing and by authorities for the quality control of drug products. In the vast majority of pharmaceutical analyses, instrumental analytical methods are applied. It is the subject of science, which deals with the interaction of radiation and matter. 35-46 All atoms and molecules are capable of absorbing energy in accordance with certain restrictions, these limitations depending upon the structure of the substance.47-49 Spectroscopic analytical methods are based on measuring the amount of radiation produced and absorbed by molecular or atomic species. The kind and amount of radiation absorbed depends upon the number of molecules interacting with the radiation. The study of these dependencies is called absorption spectroscopy.50-54 Absorption spectroscopy is one of the most valuable analytical techniques; its advantages include simplicity, speed, specificity and sensitivity. Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. The analytical procedure refers to the way of performing the analysis. It should describe in detail the steps necessary to perform each analytical test. This may include but is not limited to: the sample, the reference standard and the reagents preparations, use of the apparatus, generation of the calibration curve, use of the formula for the calculation, etc.55-59 A suitable and validated method has to be available for the analysis of drug in the bulk, in drug delivery systems, from release dissolution studies and in biological samples. If a suitable method, for specific need, is not available then it becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples. The efficient analytical method development and its validation are critical elements in the development of pharmaceuticals. An analytical method is selected on the basis of criteria such as accuracy, precision, sensitivity, selectivity, robustness, ruggedness, and the amount of available sample, the amount of analyte in the sample, time, cost, and availability of equipment.60-72 Thus, present study was undertaken to develop and validate a simple sensitive, accurate, precise and reproducible UV method for simvastatin.


Instruments: The analysis was performed by using the analytical balance (Rodwag Wagi Electronics Switzerland model AS22201xs), pH meter (Cyber scan), UV spectrophotometer (UV-1800 Shimadzu,Japan. equipped with variable wavelength detector and data integration software).

Reagents and solutions: Simvastatin IP grade and reagents for phosphate buffer were used as analytical grade.

Preparation of solvent system:

Phosphate buffer of pH 7.4: 50ml of 0.2M potassium dihydrogen phosphate was placed in 200 ml volumetric flask, 39.1 ml of 0.2M sodium hydroxide was added, water was added to volume and pH adjusted to 7.4 accordingly.

Standard stock solution of simvastatin : 100 mg of simvastatin weighted accurately and transferred into a 100 ml volumetric flask containing phosphate buffer. Solution sonicated to dissolve simvastatin and cooled at room temperature and mix well. Pipette out 10ml solution and transferred into 100 ml volumetric flask resulting solution contained 100µg/ml solution (Stock solution A). Pipette out 10, 20, 30, 40, 50ml solution respectively of standard stock solution A and transferred into a 100 ml volumetric flask, mixed well and diluted up to volume with buffer solution. The resulting solution contains 10 to 50µg/ml of simvastatin.

Spectral study: The final stock solution scanned in UV spectrophotometer over the range 200-400nm (Figure 1).

Validation of the Method :

Linearity and Range: A series of dilutions were prepared in the concentration range of 10-50mg/ml. Separate calibration curve was plotted between concentration Vs response and slope, intercept and correlation coefficient value (r 2) was determined.

Accuracy: To test accuracy, recovery studies were performed. To a preanalyzed sample solution, a definite quantity of known concentration of standard drug solution was added and then its recovery was studied. Different concentration of pure drug was added to preanalyzed sample, and then the solution was analyzed to determined recovery study.

Precision: A standard stock solution of drug was prepared in same manner. The tests were repeated thrice for all concentration. The intermediate precision was performed by doing repeatability, day-to-day variation, and analyst-to-analyst variation.

RESULTS AND DISCUSSION: The methods discuss in the present work provide a convenient, precise and accurate way for simvastatin pharmaceutical dosage form. An absorption maximum of simvastatin was selected at 238nm for the analysis. Regression analysis shows linearity over the concentration range of 02-200µg/ml for correlation coefficients of 0.977 (Figure 2). The % RSD for repeatability (n=6) precision was found to be less than 2% indicating the precision of method. Accuracy of proposed methods was ascertained by recovery studies and the results are expressed as percentage recovery. Percentage recovery for simvastatin was found within the range of 99.15 % and 101.21%. The assay for simvastatin was found to be 101.00 ± 0.22. The % RSD value for simvastatin was found to be less than 2%. In this study the simvastatin was carried out by UV Spectroscopy method satisfactorily. The result of developed method and validation was given in table 1.


Figure 1: UV Spectra of Simvastatin.

Table 1 : Result of method development and validation.







Slope and


0.021x + 0.063






% Recovered

99.15 to 101.21





Method was found repeatable

(Validated three times as per ICH guideline)



Analyst to analyst

Method was found precise when performed by different analyst

(Validated three times as per ICH guideline)



Day to day

Method was found precise when performed three consecutive days

(Validated three times as per ICH guideline)



Ruggedness & Robustness


(Validated three times as per ICH guideline)



*Each value is mean of three replicates


Figure 2: Calibration curve of Simvastatin.

CONCLUSION: The analytical method for determination of simvastatin has been validated according to validation protocol of ICH guidelines. All parameters mentioned in the protocol were tested and they fulfilled the requirement of ICH analytical method validation for the drug. The results obtained are well within the set limit; indicates that the described analytical method is suitable for determination of simvastatin in bulk as well as tablet formulation.


1. Goldstein, J. L., Schrott, H. G., Hazzard, W. R., Bierman, E. L., & Motulsky, A. G. (1973). Hyperlipidemia in coronary heart disease II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. The Journal of clinical investigation, 52(7), 1544-1568.

2. Ross, R. and Harker, L., (1976). Hyperlipidemia and atherosclerosis. Science, 193 (4258), pp.1094-1100.

3. Ikramuddin, S., Korner, J., Lee, W. J., Connett, J. E., Inabnet, W. B., Billington, C. J., & Ahmed, L. (2013). Roux-en-Y gastric bypass vs intensive medical management for the control of type 2 diabetes, hypertension, and hyperlipidemia: the Diabetes Surgery Study randomized clinical trial. Jama, 309(21), 2240-2249.

4. Assmann, G., & Schulte, H. (1988). The Prospective Cardiovascular Munster (PROCAM) study: prevalence of hyperlipidemia in persons with hypertension and/or diabetes mellitus and the relationship to coronary heart disease. American heart journal, 116(6), 1713-1724.

5. Thielmann, M., Neuhauser, M., Marr, A., Jaeger, B. R., Wendt, D., Schuetze, B. & Jakob, H. (2007). Lipid-lowering effect of preoperative statin therapy on postoperative major adverse cardiac events after coronary artery bypass surgery. The Journal of thoracic and cardiovascular surgery, 134(5), 1143-1149.

6. Gupta, A. (2018). Systematic approach to novel drug delivery system. A & V Publication India. Ist edition, 58-64.

7. Bruckert, E., Hayem, G., Dejager, S., Yau, C., & Begaud, B. (2005). Mild to moderate muscular symptoms with high-dosage statin therapy in hyperlipidemic patients-the PRIMO study. Cardiovascular Drugs and Therapy, 19(6), 403-414.

8. Chalasani, N., Aljadhey, H., Kesterson, J., Murray, M. D., & Hall, S. D. (2004). Patients with elevated liver enzymes are not at higher risk for statin hepatotoxicity. Gastroenterology, 126(5), 1287-1292.

9. Farnier, M., & Davignon, J. (1998). Current and future treatment of hyperlipidemia: the role of statins. The American journal of cardiology, 82(4), 3J-10J.

10. Ellen BSc, R. L., & McPherson, M. D. (1998). Long-term efficacy and safety of fenofibrate and a statin in the treatment of combined hyperlipidemia. The American journal of cardiology, 81(4), 60B-65B.

11. Athyros, V. G., Papageorgiou, A. A., Hatzikonstandinou, H. A., Didangelos, T. P., Carina, M. V., Kranitsas, D. F., & Kontopoulos, A. G. (1997). Safety and efficacy of long-term statin-fibrate combinations in patients with refractory familial combined hyperlipidemia. The American journal of cardiology, 80(5), 608-613.

12. Chalasani, N. (2005). Statins and hepatotoxicity: focus on patients with fatty liver. Hepatology, 41(4), 690-695.

13. Horne, B. D., Muhlestein, J. B., Carlquist, J. F., Bair, T. L., Madsen, T. E., Hart, N. I., & Anderson, J. L. (2000). Statin therapy, lipid levels, C-reactive protein and the survival of patients with angiographically severe coronary artery disease. Journal of the American College of Cardiology, 36(6), 1774-1780.

14. Schachter, M. (2005). Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundamental & clinical pharmacology, 19(1), 117-125.

15. Mason, R. P., Walter, M. F., Day, C. A., & Jacob, R. F. (2005). Intermolecular differences of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors contribute to distinct pharmacologic and pleiotropic actions. The American journal of cardiology, 96(5), 11-23.

16. Arnaboldi, L., & Corsini, A. (2010). Do structural differences in statins correlate with clinical efficacy?. Current opinion in lipidology, 21(4), 298-304.

17. Wilson, S. H., Herrmann, J., Lerman, L. O., Holmes Jr, D. R., Napoli, C., Ritman, E. L., & Lerman, A. (2002). Simvastatin preserves the structure of coronary adventitial vasa vasorum in experimental hypercholesterolemia independent of lipid lowering. Circulation, 105(4), 415-418.

18. Noel, B. (2007). Lupus erythematosus and other autoimmune diseases related to statin therapy: a systematic review. Journal of the European Academy of Dermatology and Venereology, 21(1), 17-24.

19. Gupta, A. (2018). Basic concepts in clinical pharmacy practice. A & V Publication India. Ist edition, 78-85.

20. Gupta, A. (2018). Insight of solubility. A & V Publication India. Ist edition, 12-26.

21. Sirtori, C. R. (2014). The pharmacology of statins. Pharmacological research, 88, 3-11.

22. Jones, P., Kafonek, S., & Hunninghake, D. (1998). Comparative Dose Efficacy Study of Atorvastatin Versus Simvastatin, Pravastatin, Lovastatin, and Fluvastatin in Patients With Hypercholesterolemia (The CURVES Study) fn1. The American journal of cardiology, 81(5), 582-587.

23. Davidson, M. H., McGarry, T., Bettis, R., Melani, L., Lipka, L. J., LeBeaut, A. P. & Ezetimibe Study Group. (2002). Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. Journal of the American College of Cardiology, 40(12), 2125-2134.

24. Notarbartolo, A., Davi`, G., Averna, M., Barbagallo, C. M., Ganci, A., Giammarresi, C., ... & Patrono, C. (1995). Inhibition of thromboxane biosynthesis and platelet function by simvastatin in type IIa hypercholesterolemia. Arteriosclerosis, Thrombosis, and Vascular Biology, 15(2), 247-251.

25. Wilson, S. H., Herrmann, J., Lerman, L. O., Holmes Jr, D. R., Napoli, C., Ritman, E. L., & Lerman, A. (2002). Simvastatin preserves the structure of coronary adventitial vasa vasorum in experimental hypercholesterolemia independent of lipid lowering. Circulation, 105(4), 415-418.

26. Ballantyne, C. M., Abate, N., Yuan, Z., King, T. R., & Palmisano, J. (2005). Dose-comparison study of the combination of ezetimibe and simvastatin (Vytorin) versus atorvastatin in patients with hypercholesterolemia: the Vytorin Versus Atorvastatin (VYVA) study. American heart journal, 149(3), 464-473.

27. Sen, K., Misra, A., Kumar, A., & Pandey, R. M. (2002). Simvastatin retards progression of retinopathy in diabetic patients with hypercholesterolemia. Diabetes research and clinical practice, 56(1), 1-11.

28. Tomás, M., Sentí, M., García-Faria, F., Vila, J., Torrents, A., Covas, M., & Marrugat, J. (2000). Effect of simvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholesterolemic patients. Arteriosclerosis, thrombosis, and vascular biology, 20(9), 2113-2119.

29. Ermer, J., & Miller, J. H. M. (Eds.). (2006). Method validation in pharmaceutical analysis: A guide to best practice. John Wiley & Sons.

30. Beckett, A. H., & Stenlake, J. B. (Eds.). (1988). Practical Pharmaceutical Chemistry: Part II Fourth Edition (Vol. 2). A&C Black.

31. Ahuja, S., & Scypinski, S. (Eds.). (2010). Handbook of modern pharmaceutical analysis (Vol. 10). Academic press.

32. Gupta, A., Yadav, V., & Rawat, S. (2011). An analytical approach for the determination of amoxicillin and potassium clavulanate in pharmaceutical dosage form. Drug Invention Today, 3(4).

33. Rawat, S., Sangali, S., & Gupta, A. (2018). Formulation and Evaluation of Floating Matrix Tablets of Acyclovir using 32 Factorial Design. Research Journal of Pharmaceutical Dosage Forms and Technology, 10(1), 01-09.

34. Connors, K. A. (2007). A textbook of pharmaceutical analysis. John Wiley & Sons.

35. Watson, D. G. (2015). Pharmaceutical Analysis E-Book: A Textbook for Pharmacy Students and Pharmaceutical Chemists. Elsevier Health Sciences.

36. Ermer, J. (2001). Validation in pharmaceutical analysis. Part I: An integrated approach. Journal of pharmaceutical and biomedical analysis, 24(5-6), 755-767.

37. Bakeev, K. A. (Ed.). (2010). Process analytical technology: spectroscopic tools and implementation strategies for the chemical and pharmaceutical industries. John Wiley & Sons.

38. Khan, T. A., Peh, K. K., & Ch’ng, H. S. (2002). Reporting degree of deacetylation values of chitosan: the influence of analytical methods. J Pharm Pharmaceut Sci, 5(3), 205-212.

39. Gupta, A., Rawat, S., Gandhi, M., & Namdeo, R. (2011). Simultaneous Estimation of Amoxycillin and Potassium Clavulanate in Injection Formulation by Simultaneous Equation Method. Journal of Pharmacy Research, 4(4), 1244-1245.

40. Shrivastava, A., & Gupta, V. B. (2011). Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles of Young Scientists, 2(1), 21.

41. Mach, H., Volkin, D. B., Burke, C. J., & Middaugh, C. R. (1995). Ultraviolet absorption spectroscopy. In Protein stability and folding (pp. 91-114). Humana Press.

42. O’Connor, R. T. (1955). Ultraviolet absorption spectroscopy. Journal of the American Oil Chemists' Society, 32(11), 616-624.

43. Costello, J. T., Mosnier, J. P., Kennedy, E. T., Carroll, P. K., & O'Sullivan, G. (1991). X-UV absorption spectroscopy with laser-produced plasmas: a review. Physica Scripta, 1991(T34), 77.

44. Platt, U., & Stutz, J. (2008). Differential absorption spectroscopy. In Differential Optical Absorption Spectroscopy (pp. 135-174). Springer, Berlin, Heidelberg.

45. Green, J. M. (1996). Peer reviewed: a practical guide to analytical method validation. Analytical chemistry, 68(9), 305A-309A.

46. Swartz, M. E., & Krull, I. S. (1997). Analytical method development and validation. CRC Press.

47. Ermer, J., & Miller, J. H. M. (Eds.). (2006). Method validation in pharmaceutical analysis: A guide to best practice. John Wiley & Sons.

48. Taverniers, I., Van Bockstaele, E., & De Loose, M. (2010). Analytical method validation and quality assurance. Pharmaceutical Sciences Encyclopedia: Drug Discovery, Development, and Manufacturing, 1-48.

49. Vander Heyden, Y., Nijhuis, A., Smeyers-Verbeke, J., Vandeginste, B. G. M., & Massart, D. L. (2001). Guidance for robustness/ruggedness tests in method validation. Journal of pharmaceutical and biomedical analysis, 24(5-6), 723-753.

50. Chan, C. C., Lee, Y. C., Lam, H., & Zhang, X. M. (Eds.). (2004). Analytical method validation and instrument performance verification. John Wiley & Sons.

51. González, A. G., & Herrador, M. Á. (2007). A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles. TrAC Trends in Analytical Chemistry, 26(3), 227-238.

52. Davidson AG (2002) Ultraviolet-visible absorption spectrophotometry. In BeckettAH, Stenlake JB, (4thedn), Practical Pharmaceutical chemistry. CBS Publishers and distributors, New Delhi, 275-278.

53. Ali, H., & Nazzal, S. (2009). Development and validation of a reversed-phase HPLC method for the simultaneous analysis of simvastatin and tocotrienols in combined dosage forms. Journal of pharmaceutical and biomedical analysis, 49(4), 950-956.

54. ICH, Q2A Validation of Analytical Procedure: Methodology International Conference on Harmonization, Geneva, October 1994, pg 1-8.

55. Gupta, A., Yadav, V., Yadav, J. S., & Rawat, S. (2011). An analytical approach of doxofylline: a review. Asian journal of pharmaceutical analysis, 1(4), 67-70.

56. Trivedi, R. K., Kallem, R. R., Mullangi, R., & Srinivas, N. R. (2005). Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC–MS/MS with electrospray ionization: assay development, validation and application to a clinical study. Journal of pharmaceutical and biomedical analysis, 39(3-4), 661-669.

57. Shah, B. B., Patel, B. B., Gohil, K. N., & Patel, P. M. (2012). Difference Spectrophotometric Method Development and Validation For Simultaneous Estimation of Rosuvastatin Calcium and Telmisartan in Bulk and Combined Dosage Form. International Journal of Research in Pharmacy & Science, 2(2)15-21.

58. Gupta, A., Rawat, S., & Pandey, A. (2011). Method Development and Photolytic Degradation Study of Doxofylline by RP-HPLC and LC-MS/MS. Asian J Pharm Anal, 1, 29-33.

59. Harvey D., Modern Analytical Chemistry, 1st edition, McGraw-Hill Publication, Kingsport-USA, 2000, pg 1-2.

60. Gupta, A., Rajkumar, V. Y., & Rawat, S. (2011). Method development and alkali degradation study of doxofylline by RP-HPLC and LC-MS/MS. Drug Invention Today, 3(4), 30-32.

61. Skoog D. A. et al; Principles of Instrumental Analysis, 5th edition, Thomson Books, Delhi, 2001, pg 11-16.

62. Rawat, S., & Gupta, A. (2011). Spectrophotometric method for simultaneous estimation of nimesulide and diclofenac sodium in pharmaceutical dosage forms. Asian Journal of Pharmaceutical Analysis, 1(4), 85-87.

63. Pratik P., et al. (2012) Spectrophotometric method Development and Validation for estimation of alpha lipoic acid in tablet dosage form, Intl J of Pharmaand Pharm sci., 5, 519-522.

64. Rawat, S., & Gupta, A. (2011). Regulatory requirements for drug development and approval in United States: a review. Asian J. Pharm. Res, 1(1), 01-06.

65. Reddy, G. V. R., Reddy, B. V., Haque, S. W., Gautam, H. D., Kumar, P., Kumar, A. P., & Park, J. H. (2011). Development and validation of a stability-indicating UPLC method for rosuvastatin and its related impurities in pharmaceutical dosage forms. Quimica Nova, 34(2), 250-255.

66. Trivedi, H. K., & Patel, M. C. (2012). Development and validation of a stability-indicating RP-UPLC method for determination of rosuvastatin and related substances in pharmaceutical dosage form. Scientia pharmaceutica, 80(2), 393.

67. Rawat, S., & Gupta, A. (2011). Development of novel HPTLC method for estimation of Qurcetine in Ocimum sanctum. Asian J. Pharm. Tech, 1(4), 149-151.

68. Chirag et al. (2014) Development and validation of UV spectrophotometric method for simultaneous estimation of metformin hydrochloride and alogliptin benzoate in bulk drugs and combined dosage forms, Der Pharma Chemica, 6, 303-311.

69. Gupta, A., Rajkumar, V. Y., & Rawat, S. (2011). Simultaneous estimation of amoxycillin and potassium clavulanate in injection formulation by dual wavelength spectrophotometry. Drug Invention Today, 3(4).

70. Sevda, R. R., Ravetkar, A. S., & Shirote, P. J. (2011). UV Spectrophotometric estimation of rosuvastatin calcium and fenofibrate in bulk drug and dosage form using simultaneous equation method. Int. J. Chem. Tech. Res, 3, 629-635.

71. Najma, S., Arayne, M. S., & Safila, N. (2011). Validated Method for the Simultaneous Determination of Lisinopril, Pravastatin, Atorvastatin and Rosuvastatin in API, Formulations and Human Serum by RP-HPLC. Chinese Journal of Chemistry, 29(6), 1216-1220.

72. Gupta, A., Yadav, V., Yadav, J. S., & Rawat, S. (2011). An analytical approach of doxofylline: a review. Asian journal of pharmaceutical analysis, 1(4), 67-70.