Salting-out assisted liquid–liquid extraction coupled with high-performance liquid chromatography for the determination of vitamin D3 in milk samples

Nur Hidayah Sazali,Moi Me Chong,Mazidatulakmam Miskam,Anas Alshishani,Bahruddin Saad,Ker Yin Chew

doi:10.1098/rsos.190952

Abstract

In this study, salting-out assisted liquid–liquid extraction (SALLE) as a simple and efficient extraction technique followed by high-performance liquid chromatography (HPLC) was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate were used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250x i.d 4.6 mm, 5 µm) HPLC column that was coupled with diode array detector. Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 min. Good linearity (r2 > 0.99) was obtained within the range of 25–600 ng g−1 with the limit of detection of 15 ng g−1 and limit of quantification of 25 ng g−1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 to 113.5%.

Highlights

Cholecalciferol, commonly known as vitamin D3, is a fat-soluble vitamin, which is of great nutritional interest, that supports metabolism processes and improves the efficiency of proteins and enzymes [1]
The results demonstrated that ammonium sulfate provided better phase separation and higher peak areas compared to sodium chloride
Significant advancement is sample pretreatment method based on salting-out assisted liquid–liquid extraction (SALLE) for the high-performance liquid chromatography (HPLC) determination of vitamin D3 in milk is demonstrated

Summary

Introduction

Cholecalciferol, commonly known as vitamin D3, is a fat-soluble vitamin, which is of great nutritional interest, that supports metabolism processes and improves the efficiency of proteins and enzymes [1]. It plays important roles within the body as it regulates blood calcium and phosphorus levels by promoting the absorption in intestines and reabsorption of calcium in the kidney that is key to the mineralization of the bones [2,3]. Among dairy products, processed milk products and infant formulas are fortified with vitamin D3. Vitamin D3 is sensitive to heat and light and is oxidized [7]

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Discussion

Conclusion