Development of Suspended Droplet Microextraction Method for Spectrophotometric Determination of Serum Iron

Abstract

A facile, selective and sensitive method was devised for the Fe³⁺ quantification in low volume samples, such as bovine serum based on suspended droplet microextraction (SDME). Various process parameters such as concentrations of acid (1.5% hydrochloric acid), complexing agent (0.7% ammonium thiocyanate), quaternary ammonium salt (0.2% Aliquat 336) and extracting solvent (500 µL octanol) were optimised. Ammonium thiocyanate forms water soluble, red coloured, anionic ferric thiocyanate complex [Fe(SCN)₆]³⁻ with Fe³⁺ ions released from the iron–protein complex under an acidic medium. Negatively charged [Fe(SCN)₆]³⁻ complex forms hydrophobic ion associate Fe(SCN)₆³⁻–Aliquat 336₃³⁺ with hydrophilic NH₄⁺ head groups of Aliquat 336 and drives out the formed micelle from aqueous solution along with the iron complex. After stirring, ion associate bonded micelles are separated into a hanging micro droplet of octanol. Red coloured ferric thiocyanate complex in a suspended droplet is solubilised in methanol and Fe³⁺ concentration in serum samples is obtained by recording the spectrophotometric absorbance at 505 nm. The recoveries ranged from 96.2%–98.8% with relative standard deviation (RSD) (%) values from 1.4% to 5.0% at 100–400 ng/mL confirming interference free quantification at optimised conditions. The developed method was linear over the range of 20–1000 ng/mL of Fe³⁺ with a limit of detection of 2.4 ng/mL for the serum matrix. The developed method is applied to various bovine serum samples and Fe³⁺ concentration values ranged from 62.7 to 1582.5 ng/mL. The obtained values were in accordance with the results obtained from the electrothermal atomic absorption spectrometry at 99% confidence level using t-test indicating the accuracy of the developed method. The proposed procedure offers various advantages such as enhanced sensitivity of the spectrophotometer towards iron determination, low-cost complexing agent, low sample volume, metal and biological interference free, simplicity and selectivity. Thus, the developed method can be an alternative to the routine spectrophotometric analysis of low volume samples such as serum and other biological fluids.