Salting‐out homogeneous liquid‐liquid microextraction for the determination of azole drugs in human urine: Validation using total error concept

A salting‐out homogeneous liquid‐liquid microextraction was proposed for the quantification of four azole drugs in human urine prior to high‐performance liquid chromatography analysis. The procedure involved the mixing of the sample with acetonitrile in appropriate volumes followed by the addition of sodium sulfate solution in order to facilitate phase separation. The parameters influencing the extraction performance were studied and optimized using a two‐step experimental design. The analytical procedure was thoroughly validated using the accuracy profiles as a graphical decision‐making tool. The β‐expectation tolerance intervals did not exceed the acceptance criteria of ±15% meaning that 95% of future results will be included in the defined bias limits. The limits of detection of the procedure were satisfactory, ranging between 0.01 and 0.03 μg/mL. The mean analytical bias in the spiking levels was satisfactory and ranged between –10.3 and 4.2% while the relative standard deviation was lower than 5.6%. Monte‐Carlo simulations followed by capability analysis were employed to investigate the ruggedness of the sample preparation protocol. The developed method offers advantages compared to previously reported approaches for the same type of analysis including extraction efficiency and scaling down of the sample volume and extraction time.