DEVELOPMENT AND VALIDATION OF AN RP-HPLC METHOD FOR THE ESTIMATION OF SILDENAFIL, FLUOXETINE, AND LOVASTATIN

INDU MELKANI; BIMLESH KUMAR; SHASHI; NARENDRA KUMAR PANDEY

doi:10.22159/ajpcr.2025v18i5.54054

Authors

INDU MELKANI Department of Pharmacology, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India. Department of Pharmacology, Amity Institute of Pharmacy, Amity University Uttar Pradesh, Lucknow Campus, Lucknow, Uttar Pradesh, India.
BIMLESH KUMAR Department of Pharmacology, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India. https://orcid.org/0000-0001-8072-5172
SHASHI Department of Pharmaceutics, Chitkara University, School of Pharmacy, Chitkara University, Himachal Pradesh, India
NARENDRA KUMAR PANDEY Department of Pharmacology, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India.

DOI:

https://doi.org/10.22159/ajpcr.2025v18i5.54054

Keywords:

RP-HPLC, Neuropathic pain, Elution, Analytical method, Validation

Abstract

Objective: Neuropathic pain (NP) arises from trauma to the somatosensory nervous system and can be managed using selective serotonin reuptake inhibitors, such as fluoxetine (FLX) and phosphodiesterase inhibitors, such as sildenafil (SD), and cholesterol-lowering agents such as lovastatin (LOVA). The present study aimed to develop and validate an analytical method for the simultaneous estimation of these drugs (SD, FLX, and LOVA [SFL]) using reverse-phase high-performance liquid chromatography (RP-HPLC).

Methods: An RP-HPLC method was developed and validated for the quantification of SFL. Chromatographic separation was achieved using a C-18 reverse-phase ODS column with a mobile phase consisting of acetonitrile and 0.2 M ammonium acetate buffer (55:45) in gradient elution mode. The flow rate was maintained at 1 mL/min, and detection was carried out at 228 nm. The method was validated following the ICH Q2 (R2) guidelines, assessing parameters such as linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ).

Results: The developed method exhibited linearity within the concentration range of 20–100 μg/mL, with a regression coefficient (r2) of 0.9992. Retention times for FLX, SD, and LOVA were recorded at 6.481, 4.238, and 19.778 min, respectively. Recovery studies demonstrated an accuracy range of 94.61–110.44%, with a relative standard deviation of 0.06–2.00%, confirming the precision of the method. The LOD values for FLX, SD, and LOVA were found to be 12.77 μg/mL, 14.81 μg/mL, and 13.28 μg/mL, respectively, while the LOQ values were 45.16 μg/mL, 42.33 μg/mL, and 38.71 μg/mL.

Conclusion: The validated RP-HPLC method met all required validation criteria and demonstrated suitability for the accurate quantification of FLX, SD, and LOVA in pharmaceutical formulations. These findings support the potential use of these drugs as an alternative therapeutic strategy for NP.

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References

Barbas R, Llinas A, Prohens R. The solid state landscape of the sildenafil drug. J Pharm Sci. 2022;111(4):1104-9.

Hezave AZ, Rajaei H, Lashkarbolooki M, Esmaeilzadeh F. Analyzing the solubility of fluoxetine hydrochloride in supercritical carbon dioxide. J Supercrit Fluids. 2013;73:57-62.

Goswami S, Vidyarthi AS, Bhunia B, Mandal T. A review on lovastatin and its production. J Biochem Technol. 2013;4(1):581-7.

Sheu MT, Hsieh CM, Chen RN, Chou PY, Ho HO. Rapid-onset sildenafil sublingual drug delivery systems: In vitro evaluation and in vivo pharmacokinetic studies in rabbits. J Pharm Sci. 2016;105(9):2774-81. doi: 10.1016/j.xphs.2016.01.015

Yoon MH, Park KD, Lee HG, Kim WM, An TH, Kim YO, et al. Additive antinociception between intrathecal sildenafil and morphine in the rat formalin test. J Korean Med Sci. 2008;23(6):1033-8. doi: 10.3346/jkms.2008.23.6.1033

Melkani I, Kumar B, Panchal S, Singh SK, Singh A, Gulati M, et al. Comparison of sildenafil, fluoxetine and its co-administration against chronic constriction injury induced neuropathic pain in rats: An influential additive effect. Neurol Res. 2019;41(10):875-82. doi: 10.1080/01616412.2019.1630091

Dupuis A, Wattiez AS, Pinguet J, Richard D, Libert F, Chalus M, et al. Increasing spinal 5-HT2A receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy in neuropathic rats. Evidence for GABA release. Pharmacol Res. 2017;118:93-103. doi: 10.1016/j.phrs.2016.09.021

Abdel-Salam OM. Anti-inflammatory, antinociceptive, and gastric effects of Hypericum perforatum in rats. Sci World J. 2005;5:586-95. doi: 10.1100/tsw.2005.78

Ciurleo R, Bramanti P, Marino S. Role of statins in the treatment of multiple sclerosis. Pharmacol Res. 2014;87:133-43.

Wolozin B, Wang SW, Li NC, Lee A, Lee TA, Kazis LE. Simvastatin is associated with a reduced incidence of dementia and Parkinson’s disease. BMC Med. 2007;5:20.

Zamrini E, McGwin G, Roseman JM. Association between statin use and Alzheimer’s disease. Neuroepidemiology. 2004;23(1-2):94-8.

Pannu R, Barbosa E, Singh AK, Singh I. Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats. J Neurosci Res. 2005;79(3):340-50.

Chen SF, Hung TH, Chen CC, Lin KH, Huang YN, Tsai HC, et al. Lovastatin improves histological and functional outcomes and reduces inflammation after experimental traumatic brain injury. Life Sci. 2007;81(4):288-98.

Xu L, Zhang Y, Huang Y. Advances in the treatment of neuropathic pain. Adv Exp Med Biol. 2016;904:117-29. doi: 10.1007/978-94-017- 7537-3_9

Finnerup NB, Kuner R, Jensen TS. Neuropathic pain: From mechanisms to treatment. Physiol Rev. 2021;101(1):259-301. doi: 10.1152/ physrev.00045.2019

Mangus LM, Dorsey JL, Laast VA, Ringkamp M, Ebenezer GJ, Hauer P, et al. Unraveling the pathogenesis of HIV peripheral neuropathy: Insights from a simian immunodeficiency virus macaque model. ILAR J. 2014;54(3):296-303. doi: 10.1093/ilar/ilt047

Moore RA, Derry S, Moore M, McQuay HJ. Single dose oral tiaprofenic acid for acute postoperative pain in adults. Cochrane Database Syst Rev. 2009 Jan;2009:CD007542. doi: 10.1002/14651858.CD007542. pub2

Flatters SJ, Bennett GJ. Studies of peripheral sensory nerves in paclitaxel-induced painful peripheral neuropathy: Evidence for mitochondrial dysfunction. Pain. 2006;122(3):245-57. doi: 10.1016/j. pain.2006.01.037

Mitsikostas DD, Moka E, Orrillo E, Aurilio C, Vadalouca A, Paladini A, et al. Neuropathic pain in neurologic disorders: A narrative review. Cureus. 2022;14(2):e22419. doi: 10.7759/cureus.22419

Song XJ, Huang ZJ, Song WB, Song XS, Fuhr AF, Rosner AL, et al. Attenuation effect of spinal manipulation on neuropathic and postoperative pain through activating endogenous anti-inflammatory cytokine interleukin 10 in rat spinal cord. J Manipulative Physiol Ther. 2016;39(1):42-53. doi: 10.1016/j.jmpt.2015.12.004

Millan MJ. The induction of pain: An integrative review. Prog Neurobiol. 1999;57(1):1-164. doi: 10.1016/S0301-0082(98)00048-3

Attal N, Bouhassira D. Mechanisms of pain in peripheral neuropathy. Acta Neurol Scand Suppl. 1999;173:12-24; discussion 48-52.

Jarvis MF, Boyce-Rustay JM. Neuropathic pain: Models and mechanisms. Curr Pharm Des. 2009;15(15):1711-6. doi: 10.2174/138161209788186272

Schumacher MA. Transient receptor potential channels in pain and inflammation: Therapeutic opportunities. Pain Pract. 2010;10(3):185- 200. doi: 10.1111/j.1533-2500.2010.00358.x.TRP

Devan BD, Bowker JL, Duffy KB, Bharati IS, Jimenez M, Sierra- Mercado D Jr., et al. Phosphodiesterase inhibition by sildenafil citrate attenuates a maze learning impairment in rats induced by nitric oxide synthase inhibition. Psychopharmacology (Berl). 2006;183(4):439-45. doi: 10.1007/s00213-005-0232-z

Anjaneyulu M, Chopra K. Possible involvement of cholinergic and opioid receptor mechanisms in fluoxetine mediated antinociception response in streptozotocin-induced diabetic mice. Eur J Pharmacol. 2006;538(1-3):80-4. doi: 10.1016/j.ejphar.2006.03.067

Clark AK, Old EA, Malcangio M. Neuropathic pain and cytokines: Current perspectives. J Pain Res. 2013;6:803-14.

Chen M, Hoshino H, Saito S, Yang Y, Obata H. Spinal dopaminergic involvement in the antihyperalgesic effect of antidepressants in a rat model of neuropathic pain. Neurosci Lett. 2017;649:116-23. doi: 10.1016/j.neulet.2017.04.017

Navarro X, Vivó M, Valero-Cabré A. Neural plasticity after peripheral nerve injury and regeneration. Prog Neurobiol. 2007;82:163-201. doi: 10.1016/j.pneurobio.2007.06.005

Melkani I, Kaur G, Kaur S, Rana R, Kumar B, Kumar S, et al. Neuropathic pain and diabetes: A complicated clinical condition. AIP Conf Proc. 2024;2986(1):030091.

Kaur M, Singh A, Kumar B, Singh SK, Bhatia A, Gulati M, et al. Protective effect of co-administration of curcumin and sildenafil in alcohol induced neuropathy in rats. Eur J Pharmacol. 2017Aug;805:58- 66. doi: 10.1016/j.ejphar.2017.03.012

Ahlawat A, Sharma S. A new promising simultaneous approach for attenuating type II diabetes mellitus induced neuropathic pain in rats: INOS inhibition and neuroregeneration. Eur J Pharmacol. 2018;818:419-28.

Davidson EP, Holmes A, Coppey LJ, Yorek MA. Effect of combination therapy consisting of enalapril, α-lipoic acid, and menhaden oil on diabetic neuropathy in a high fat/low dose streptozotocin treated rat. Eur J Pharmacol. 2015;765:258-67. doi: 10.1016/j.ejphar.2015.08.015

Pottabathini R, Kumar A, Bhatnagar A, Garg S, Ekavali E. Ameliorative potential of pioglitazone and ceftriaxone alone and in combination in rat model of neuropathic pain: Targeting PPARγ and GLT-1 pathways. Pharmacol Rep. 2016;68(1):85-94. doi: 10.1016/j.pharep.2015.06.010

Pedersen LH, Nielsen AN, Blackburn-Munro G. Anti-nociception is selectively enhanced by parallel inhibition of multiple subtypes of monoamine transporters in rat models of persistent and neuropathic pain. Psychopharmacology (Berl). 2005;182(4):551-61. doi: 10.1007/ s00213-005-0120-6

Abdelshakour MA, Salam RA, Hadad GM, Abo-ElMatty DM, Hameed EA. HPLC-UV and UPLC-MS/MS methods for the simultaneous analysis of sildenafil, vardenafil, and tadalafil and their counterfeits dapoxetine, paroxetine, citalopram, tramadol, and yohimbine in aphrodisiac products. RSC Adv. 2021;11(14):8055-64.

Aboul-Enein HY, Hefnawy MM. Rapid determination of sildenafil citrate in pharmaceutical preparations using monolithic silica HPLC column. J Liquid Chromatogr Relat Technol. 2003;26(17):2897-908.

Yang YJ, Song DM, Jiang WM, Xiang BR. Rapid resolution RP-HPLC-DAD method for simultaneous determination of sildenafil, vardenafil, and tadalafil in pharmaceutical preparations and counterfeit drugs. Anal Lett. 2010;43(3):373-80.

Dural E. Investigation of the presence of sildenafil in herbal dietary supplements by validated HPLC method. Turk J Pharm Sci. 2020;17(1):56-62.

Baokar SB, Shirke B, Sivanand V, Pratheesh GK. Analytical method development and validation for estimation of sildenafil citrate from tablet dosage form by using RP-HPLC. Int J Res Pharm Sci. 2011;2(2):130.

Sheu MT, Wu AB, Yeh GC, Hsia A, Ho HO. Development of a liquid chromatographic method for bioanalytical applications with sildenafil. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;791(1-2):255-62.

Katakam LN, Ettaboina SK, Dongala T. A simple and rapid HPLC method for determination of parabens and their degradation products in pharmaceutical dosage forms. Biomed Chromatogr. 2021;35(10):e5152.

Tuchilă C, Baconi D. Bioanalytical methods for determination of fluoxetine, an antidepressant, in biological fluids. Rom J Cogn BehavTher Hypn. 2015;2(4):6.

Perrone PA. Mobile phase control. In: HPLC in Nucleic Acid Research. Boca Raton: CRC Press; 2020. p. 161-78.

El-dawy MA, Mabrouk MM, El-Barbary FA. Liquid chromatographic determination of fluoxetine. J Pharm Biomed Anal. 2002;30(3):561-71.

Shashi, Pandey NK, Kumar B, Singh SK, Baghel SD, Sudhakar K, et al. Original article validated reversed-phase high-performance liquid chromatography method for the estimation of tetrabenazine in self-nano emulsifying drug delivery systems. Int J Appl Pharm. 2024;16(5):388-94.

Patel S, Patel NJ. Simultaneous RP-HPLC and HPTLC estimation of fluoxetine hydrochloride and olanzapine in tablet dosage forms. Indian J Pharm Sci. 2009;71(4):477-80.

Silva TD, Oliveira MA, De Oliveira RB, Vianna-Soares CD. Development and validation of a simple and fast HPLC method for determination of lovastatin, pravastatin and simvastatin. J Chromatogr Sci. 2012;50:831-8.

Wassel AA, El-agezy H. Development and validation of (HPLC) method for simultaneous determination of atomoxetine HCl & fluoxetine HCl in their pharmaceutical dosage forms. Biomed J Sci Technical Res. 2021;34(4):26943-50.

Marais S, Du Preez JL, Du Plessis LH, Du Plessis J, Gerber M. Determination of lovastatin, mevastatin, rosuvastatin and simvastatin with HPLC by means of gradient elution. Pharmazie. 2019;74(11):658-60.

Bayat F, Bozorgi AH, Hollow fiber-based liquid-phase microextraction and HPLC-UV determination of lovastatin in biological fluids. J Rep Pharm Sci. 2020;9(2):203-8.

Ayothiraman S, Murugesan N, Sethi G. Critical analysis of analytical techniques developed for statins in biological fluids, environmental and fermentation samples. Crit Rev Biotechnol. 2024;21:1-31.

Seenivasan A, Gummadi SN, Panda T. Comparison of the elution characteristics of individual forms of lovastatin in both isocratic and gradient modes and HPLC-PDA method development for pure and fermentation-derived lovastatin. Prep Biochem Biotechnol. 2017;47(9):901-8.

Göktaş EF, Kabi E, Yeşilçimen ES, Dirikolu L. Screening analysis of doping agents in horse urine and plasma with dilute and shoot using liquid chromatography high resolution mass spectrometry. Analyst. 2025;150:773-92.

Mabry SJ, Cao X, Zhu Y, Rowe C, Patel S, Gonzales-Arancibia C, et al. Fusobacterium nucleatum Determines the Expression of Amphetamine- Induced Behavioral Responses Through an Epigenetic Phenomenon. bioRxiv [Preprint]; 2005. p. 2001-25.

Iudicello A, Genovese F, Strusi V, Dominici M, Ruozi B. Development and validation of a new storage procedure to extend the in-use stability of azacitidine in pharmaceutical formulations. Pharmaceuticals (Basel). 2021;14(9):943.

Hardeep, Pandey NK, Singh SK, Kumar B, Corrie L, Goutam U, et al. Development and validation of reverse-phase high-performance liquid chromatography based bioanalytical method for estimation of simvastatin in rat’s plasma. Assay Drug Dev Technol. 2022;20(8):349-58.

Gandla Y, Pabba P, Akula G. RP-HPLC Method development and validation for estimation of glycopyrrolate in bulk and tablet dosage forms. Asian J Pharm Clin Res. 2011;4(4):37-40.

Awasthi A, Kumar A, Kumar R, Vishwas S, Khursheed R, Kaur J, et al. RP-HPLC method development and validation for simultaneous estimation of mesalamine and curcumin in bulk form as well as nanostructured lipid carriers. South Afr J Bot. 2022;151:529-37. doi: 10.1016/j.sajb.2022.05.044

Marri S. The Development and Validation of the HPLC Method for Determination of Artesunate and Amodiaquine in Novel Antimalarial Formulations. Hatfield: University of Hertfordshire; 2024.

Lambarki LZ, Jhilal F, Slimani L, El Hajji R, Bakkali F, Iskandar S, et al. Comparison of approaches for assessing detection and quantitation limits in bioanalytical methods using HPLC for sotalol in plasma. Sci Rep. 2025;15(1):5472.

Sunkara B, Tummalapalli Naga Venkata GK. Development of novel gradient RP-HPLC method for separation of dapagliflozin and its process-related impurities: Insight into stability profile and degradation pathway, identification of degradants using LCMS. Future J Pharm Sci. 2023;9(1):107.

Ravisankar P, Rao GD. Development and validation of RP-HPLC method for determination of levamisole in bulk and dosage form. Asian J Pharm Clin Res. 2013;6(3):169-73.

Kumar R, Kumar R, Khursheed R, Awasthi A, Khurana N, Singh SK, et al. Development and validation of RP-HPLC method for estimation of fisetin in rat plasma. South Afr J Bot. 2021;140:284-9.

Pandey SN, Kumar B, Singh SK, Baghel DS, Sudhakar K, Singh S. Validated reversed-phase high-performance liquid chromatography method for the estimation of tetrabenazine in self-nano emulsifying drug delivery systems. Int J Appl Pharm. 2024;16(5):388-94.

Guiard BP, Gotti G. The high-precision liquid chromatography with electrochemical detection (HPLC-ECD) for monoamines neurotransmitters and their metabolites: A review. Molecules. 2024;29(2):496.

Thaakur SR, Pokkula S. Ameliorative effects of vanda testacea in sciatic nerve transection-induced neuropathy in rats. Int J Pharma Biosci. 2013;4(1):271-84.

Moscato S, Cortelli P, Chiari L. Physiological responses to pain in cancer patients: A systematic review. Comput Methods Programs Biomed. 2022;217:106682.