Development of validated HPTLC and UHPLC methods for quanti?cation 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

Authors

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 ?g/mL with R²= 0.9994. LOD along with LOQ were 0.411 and 1.245 ?g. HPTLC and UHPLC procedures utilized for regular quality control and quick screening of active components from plant extracts.

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