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)₆ ^3--Aliquat 336₃ ³⁺ 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.

Keywords: ET‐AAS; colorimetric; complexing agent; iron; serum; spectrophotometric; suspended droplet microextraction.

FIGURE 1

The schematic images showing the steps involved in the suspended droplet microextraction (SDME) of serum iron; (I) serum + HCl, (II) serum + HCl + NH₄SCN, (III) serum + HCl + NH₄SCN + Aliquat 336 + octanol and (IV) scarlet red coloured suspended droplet.

FIGURE 2

The scheme indicating the underlying mechanism involved in the suspended droplet microextraction (SDME) method used for separation and preconcentration of Fe³⁺ ions from the serum samples.

FIGURE 3

The UV–vis absorption spectrum of the methanol solubilised suspended droplet of ferric thiocyanate complex indicating an absorption peak at 505 nm. Inset: corresponding solution colour.

FIGURE 4

The effect of different parameters on the suspended droplet microextraction of serum iron at variable concentrations of (a) HCl (conditions: 400 ng/mL Fe³⁺, 1% NH₄SCN, 0.1% Aliquat 336, 1000 µL octanol), (b) NH₄SCN (conditions: 400 ng/mL Fe³⁺, 1.5% HCl, 0.1% Aliquat 336, 1000 µL octanol), (c) Aliquat 336 (conditions: 400 ng/mL Fe³⁺, 1.5% HCl, 0.7% NH₄SCN, 1000 µL octanol) and (d) octanol (conditions: 400 ng/mL Fe³⁺, 1.5% HCl, 0.7% NH₄SCN, 0.2% Aliquat 336).

FIGURE 5

(a) The stability of dissolved ferric thiocyanate complex, in terms of absorbance at 505 nm with time and (b) the effect of different quaternary ammonium salts (0.2%) on the suspended droplet microextraction of serum iron at 300 ng/mL Fe³⁺, 1.5% HCl, 0.7% NH₄SCN and 500 µL octanol.

FIGURE 6

(a) The calibration plot in SDME method showing the absorbance at 505 nm vs. Fe³⁺ concentration in serum and (b) corresponding absorption spectra. SDME, suspended droplet microextraction.