High-Performance Liquid Chromatography Method Validation for the Determination of Cyproheptadine Hydrochloride in Tablets
Article Sidebar
Main Article Content
Page: 3082-3094
Abstract
Cyproheptadine hydrochloride is an antihistamine drug widely available in tablet dosage forms; therefore, a validated analytical method is required to ensure product quality. This study aimed to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for the determination of cyproheptadine hydrochloride 4 mg in tablets according to ICH Q2(R2) and USP 2024 guidelines. The analysis was performed using an XBridge C18 column (2.1 mm × 50 mm; 3.5 µm) with an isocratic mobile phase of acetonitrile:0.1% acetic acid (60:40), a flow rate of 1.0 mL/min, an injection volume of 10 µL, and PDA detection at 285 nm. Validation parameters included specificity, linearity, accuracy, precision, robustness, LOD and LOQ. The method showed excellent linearity with R² of 0.997, recovery of 100.26%, precision with % RSD of 1.42%, LOD 0.0022% and LOQ of 0.0066%. Specificity testing showed no interfering peaks, while robustness was confirmed for a ±5% change in mobile phase composition (%RSD 1.26% for retention time and 1.97% for peak area) but not for a ±5% change in flow rate, where retention time gave %RSD of 2.14%. The procedure is fit for routine quality control assay of cyproheptadine hydrochloride tablets, with the stated limitation on flow-rate robustness.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
References
Harrison ME, Norris ML, Robinson A, Spettigue W, Morrissey M, Isserlin L. Use of cyproheptadine to stimulate appetite and body weight gain: A systematic review. Appetite 2019;137:62–72. https://doi.org/10.1016/j.appet.2019.02.012. DOI: https://doi.org/10.1016/j.appet.2019.02.012
Josefsson M, Rydberg I. Determination of methylphenidate and ritalinic acid in blood, plasma and oral fluid from adolescents and adults using protein precipitation and liquid chromatography tandem mass spectrometry-A method applied on clinical and forensic investigations. J Pharm Biomed Anal 2011;55:1050–9. https://doi.org/10.1016/j.jpba.2011.03.009. DOI: https://doi.org/10.1016/j.jpba.2011.03.009
Ermer J. Method Validation in Pharmaceutical Analysis Edited by Related Titles from Wiley-VCH : LC / MS Applications in Drug Development Reference Materials for Chemical Analysis. 2005.
Tuani Y, Amo-koi S, Pharmacopoeia US, Mingle D, Asor A. Stability-Indicating HPLC Methods for the Quantification of Cyproheptadine Hydrochloride, Vitamins B1, B5 And B6 in Tablets using RP-HPLC with UV Detection. Chem Mater Res 2020. https://doi.org/10.7176/cmr/12-6-06. DOI: https://doi.org/10.7176/CMR/12-6-06
Abdelrahman MM, Abdelaleem EA, Ali NW, Emam RA. Development and validation of stability indicating high-performance liquid chromatographic method for determination of cyproheptadine hydrochloride, its impurity and degradation product. J Chromatogr Sci 2021;59:128–33. https://doi.org/10.1093/chromsci/bmaa085. DOI: https://doi.org/10.1093/chromsci/bmaa085
Mendes GD, Arruda A, Chen LS, de Almeida Magalhães JC, Alkharfy KM, De Nucci G. Quantification of cyproheptadine in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study. Biomed Chromatogr 2012;26:129–36. https://doi.org/10.1002/bmc.1618. DOI: https://doi.org/10.1002/bmc.1618
United States Pharmacopeia. Cyproheptadine Hydrochloride. USP 47-NF 42 2024;1000:1–2.
Kementerian Kesehatan Republik Indonesia. Farmakope Indonesia Edisi VI. 2020.
United States Pharmacopeia. Validation Of Compendial Procedures. USP 47-NF 42 2024:1–6.
Wu AB, Huang MC, Ho HO, Yeh GC, Sheu MT. Investigation on liquid chromatographic separation of basic compounds using silica column with aqueous/organic mobile phase containing triethylamine and acetic acid. Biomed Chromatogr 2004;18:443–9. https://doi.org/10.1002/bmc.334. DOI: https://doi.org/10.1002/bmc.334
Andi S, Ayusari Ramly E. Validasi Metode Analisis Kapsul Rifampisin Dengan HPLC-PDA. Farmasains J Ilm Ilmu Kefarmasian 2021;8:31–6. https://doi.org/10.22236/farmasains.v8i1.5602. DOI: https://doi.org/10.22236/farmasains.v8i1.5602
Aryani A. Validation of Analytical Method of Testing Cefotaxime Sodium in Injection Powder Preparation by High-Performance Liquid Chromatography. Berk Ilm Kim Farm 2023;10:28–33. https://doi.org/10.20473/bikfar.v10i2.47179. DOI: https://doi.org/10.20473/bikfar.v10i2.47179
Sanchez JM. Estimating detection limits in chromatography from calibration data: Ordinary least squares regression vs. weighted least squares. Separations 2018;5. https://doi.org/10.3390/separations5040049. DOI: https://doi.org/10.3390/separations5040049
Zhaoxia WH ，CHEN. Assay of cyproheptadine hydrochloride and the related substances with HPLC 2017:60–9. https://doi.org/10.3969/j.issn.1006-0111.2017.01.014.
Gritti F. Thermodynamic interpretation of the drift and noise of gradient baselines in reversed-phase liquid chromatography using mobile phase additives. J Chromatogr A 2020;1633. https://doi.org/10.1016/j.chroma.2020.461605. DOI: https://doi.org/10.1016/j.chroma.2020.461605
Moosavi SM, Ghassabian S. Linearity of Calibration Curves for Analytical Methods: A Review of Criteria for Assessment of Method Reliability. Calibration Valid Anal Methods - A Sampl Curr Approaches 2018. https://doi.org/10.5772/intechopen.72932. DOI: https://doi.org/10.5772/intechopen.72932
Chowdary GL, , Ravisankar P, Akhil Kumar G, Mounika K SBP. Analytical Method Validation Parameters: An Updated Review. Int J Pharm Sci Rev Res 2020;61:1–7.
Elder D. Validation of analytical procedures – ICH Q2(R2). Eur Pharm Rev 2024;29:5.
More SS, Kadam SP, K.Redasani DV. A Review on Analytical Method Development And Validation. Int J Pharm Res Appl 2025;10:1177–95. https://doi.org/10.35629/4494-100111771195. DOI: https://doi.org/10.35629/4494-100111771195
Sridhar S. Analytical Method Development and Validation Strategies for Quantitative Determination of Novel Drugs Using High Performance Liquid Chromatography. Int J Pharm Sci 2026;4:2304–10. https://doi.org/10.5281/zenodo.18640178.
Sharma S, Goyal S, Chauhan K. A review on analytical method development and validation. Int J Appl Pharm 2021;10:8–15. https://doi.org/10.22159/ijap.2018v10i6.28279. DOI: https://doi.org/10.22159/ijap.2018v10i6.28279
Kazusaki M, Ueda S, Takeuchi N, Ohgami Y. Validation of analytical procedures by high−performance liquid chromatography for pharmaceutical analysis. Chromatography 2012;33:65–73. https://doi.org/10.15583/jpchrom.2012.005. DOI: https://doi.org/10.15583/jpchrom.2012.005
Kocic D, Shalliker RA. Using active flow technology columns for high through-put and efficient analyses : The drive towards ultra-high through-put high-performance liquid chromatography with mass spectral detection. J Chromatogr A 2015;1421:60–7. https://doi.org/10.1016/j.chroma.2015.09.002. DOI: https://doi.org/10.1016/j.chroma.2015.09.002
Gupta BP, Thakur N, Jain NP, Banweer J, Jain S. Osmotically controlled drug delivery system with associated drugs. J Pharm Pharm Sci 2010;13:571–88. https://doi.org/10.18433/j38w25. DOI: https://doi.org/10.18433/J38W25
Nelson DJ, Marbury GD. A risk-based approach to validation of ion chromatography methods using suppressed conductivity. AAPS Open 2021;7. https://doi.org/10.1186/s41120-021-00044-z. DOI: https://doi.org/10.1186/s41120-021-00044-z
Mishra AS, Vasanthan M. Design and validation of a robust stability-indicating reversed-phase HPLC method for quantification of mesalamine in formulated drug products. BMC Chem 2025;19. https://doi.org/10.1186/s13065-025-01666-0. DOI: https://doi.org/10.1186/s13065-025-01666-0
César IDC, Pianetti GA. Robustness evaluation of the chromatographic method for the quantitation of lumefantrine using Youden’s test. Brazilian J Pharm Sci 2009;45:235–40. https://doi.org/10.1590/S1984-82502009000200007. DOI: https://doi.org/10.1590/S1984-82502009000200007
Sharaf El-Din MK, Belal F, Tolba MM, Elmansi H. Stability indicating HPLC Method Coupled with Fluorescence Detection for the Determination of Cyproheptadine Hydro-chloride in Its Tablets. Studies on Degradation Kinetics. Anal Chem Lett 2018;8:565–77. https://doi.org/10.1080/22297928.2018.1451366. DOI: https://doi.org/10.1080/22297928.2018.1451366