Development of validated HPTLC and UHPLC methods for quantiﬁcation of Withanolide in extract and formulation

Sufiyan Ahmad; Mahendra Ganbase; Nitin Deshmukh; Khushabu Patil; Lokesh Barde; Mahesh Deshpande; Asmita Gaikwad; Pravin Tajane

doi:10.52756/ijerr.2023.v30.035

Sufiyan Ahmad Department of Pharmacognosy, Gangamai College of Pharmacy, Nagaon, Maharashtra, India https://orcid.org/0000-0003-4131-4025
Mahendra Ganbase Department of Pharmacognosy, Gangamai College of Pharmacy, Nagaon, Maharashtra, India https://orcid.org/0000-0001-6139-7898
Nitin Deshmukh Department of Science and Technology, Savitribai Phule Pune University, Pune, Maharashtra, India https://orcid.org/0009-0001-1379-0465
Khushabu Patil Department of Pharmaceutical Chemistry, Arunamai College of Pharmacy, Mamurabad, Maharashtra, India https://orcid.org/0009-0004-1037-2585
Lokesh Barde Department of Quality Assurance, S.N.D. College of Pharmacy, Yeola, Maharashtra, India https://orcid.org/0000-0002-2308-1478
Mahesh Deshpande Department of Pharmaceutical Quality Assurance, Amrutvahini College of Pharmacy, Sangamner, Maharashtra, India https://orcid.org/0000-0003-4367-2192
Asmita Gaikwad Department of Pharmaceutical Chemistry, Sharadchandra Pawar College of Pharmacy, Otur, Pune, Maharashtra, India https://orcid.org/0000-0003-3287-4397
Pravin Tajane Department of Pharmaceutical Chemistry, S.V.N.H.T.’s College of B. Pharmacy, Rahuri, Maharashtra, India https://orcid.org/0000-0001-5949-6918

DOI: https://doi.org/10.52756/ijerr.2023.v30.035

Keywords: HPTLC, ICH guidelines, TLC densitometric method, UHPLC, Withanolide

Abstract

High performance thin layer chromatography (HPTLC) and Ultra-High-Performance Liquid Chromatography (UHPLC) techniques were developed and validated to quantify Withanolide in extract and formulation. On Al-backed silica gel 60 F254 TLC plates (10 cm × 10 cm, layer thickness 0.2 mm), which had been prewashed with methanol, HPTLC separation was carried out. Dichloromethane: Methanol: Toluene: Acetone in various ratios produced good separation in mobile phase (5:1:1:0.5 v/v). Camag TLC scanner densitometric scanning at 365 nm determined and quantified. This approach produced compact Withanolide spots at Rf 0.48. ICH guidelines verified HPTLC's precision, reproducibility, and accuracy. Withanolide linearity was 500-3000 ng/spot with R²= 0.9994. LOD & LOQ were found to be 9.48 & 28.73 ng respectively. For UHPLC, Cosmosil C18 was used with acetonitrile: water (0.2 % OPA) (70:30, v/v) mobile phase. Flow rate was 1.5 mL/min. Under optimal chromatographic conditions, Withanolide was retained for 5.9 min and detected at 254 nm. ICH guidelines verified UHPLC's precision, repeatability, and accuracy. Withanolide linearity was 10-60 μg/mL with R²= 0.9994. LOD along with LOQ were 0.411 and 1.245 μg. HPTLC and UHPLC procedures utilized for regular quality control and quick screening of active components from plant extracts.

References

Abdelwahed, M. T., Hegazy, M. A., & Mohamed, E. H. (2023). Major biochemical constituents of Withania somnifera (ashwagandha) extract: A review of chemical analysis. Reviews in Analytical Chemistry, 42(1), 20220055. https://doi.org/10.1515/revac-2022-0055

Afewerky, H. K., Ayodeji, A. E., Tiamiyu, B. B., Orege, J. I., Okeke, E. S., Oyejobi, A. O., & Adeyemi, S. B. (2021). Critical review of the Withania somnifera (L.) Dunal: ethnobotany, pharmacological efficacy, and commercialization significance in Africa. Bulletin of the National Research Centre, 45, 1-16. https://doi.org/10.1186/s42269-021-00635-6

Ahmad, M., Saleem, S., Ahmad, A. S., Ansari, M. A., Yousuf, S., Hoda, M. N., & Islam, F. (2005). Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Human & Experimental Toxicology, 24(3), 137-147. https://doi.org/10.1191/0960327105ht509oa

Alanazi, H. H., & Elfaki, E. (2023).The immunomodulatory role of Withania somnifera (L.) dunal in inflammatory diseases. Frontiers in Pharmacology, 14, 430. https://doi.org/10.3389/fphar.2023.1084757

Anwar, F., Muhammad, G., Hussain, M. A., Zengin, G., Alkharfy, K. M., Ashraf, M., & Gilani, A. H. (2016).Capparisspinosa L.: A plant with high potential for development of functional foods and Nutraceuticals/pharmaceuticals. International Journal of Pharmacology, 12(3), 201-219. https://doi.org/10.3923/ijp.2016.201.219

Ashraf, M. U., Muhammad, G., Hussain, M. A., & Bukhari, S. N. (2016).Cydoniaoblonga M., a medicinal plant rich in phytonutrients for pharmaceuticals.Frontiers in Pharmacology, 7, 163. https://doi.org/10.3389/fphar.2016.00163

Bala, S., Govindarajan, R., Rawat, A. S., & Mehrotra, S. (2004). HPLC analysis of withaferinA in Withania somnifera (L.) Dunal.Indian Journal of Pharmaceutical Sciences, 66(2), 236.

Bhattacharya, A., Ramanathan, M., Ghosal, S., & Bhattacharya, S. K. (2000). Effect of Withania somnifera glycol Withanolides on iron‐induced hepatotoxicity in rats. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 14(7), 568-570. https://doi.org/10.1002/1099-1573(200011)14:7%3C568::AID-PTR663%3E3.0.CO;2-Q

Ganzera, M., Choudhary, M. I., & Khan, I. A. (2003).Quantitative HPLC analysis of Withanolides in Withania somnifera. Fitoterapia, 74(1-2), 68-76.

https://doi.org/10.1016/S0367-326X(02)00325-8

Hepper, F. N. (1991). Old World Withania (Solanaceae): A taxonomic review and key to the species. Solanaceae III: taxonomy, chemistry, evolution, pp. 211-227.

Hussain, M. S., Alqahtani, S. S., Ahmad, S., & Iqbal, M. R. (2023). A Comprehensive Scientific Claims on Ethnobotany, Phytochemical Pharmacological and Toxicology of Withania somnifera (L.) Dunal.Journal of Drug Delivery and Therapeutics, 13(3), 194-207.

https://doi.org/10.22270/jddt.v13i3.6003

Indhira, D., Aruna, A., Manikandan, K., Albeshr, M. F., Alrefaei, A. F., Vinayagam, R., & Srinivasan, R. (2023).Antimicrobial and Photocatalytic Activities of Selenium Nanoparticles Synthesized from Elaeagnusindica Leaf Extract. Processes, 11(4), 1107. https://doi.org/10.3390/pr11041107

International Conference on Harmonization. Q2A (1995).Text on validation of analytical