Chinese Journal of Chromatography ›› 2020, Vol. 38 ›› Issue (8): 929-936.DOI: 10.3724/SP.J.1123.2019.09026

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Combination of dispersive liquid-liquid microextraction using multifunctional ionic liquids with high performance chromatography for determination of phthalate ester metabolites in human urine sample

SUN Qian1,2, DAI Haoqiang2, CHEN Peipei2, SHE Hui2, WU Jia2,*()   

  1. 1 Clinical Laboratory, Lihuili Hospital, Ningbo Municipal Medical Center, Ningbo 315040, China
    2 Wenzhou Medical University, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou 325035, China
  • Received:2019-10-14 Online:2020-08-08 Published:2020-12-11
  • Contact: WU Jia
  • Supported by:
    Zhejiang Provincial Natural Science Foundation(LQ17H260005)


A novel sensitive method for the determination of the five primary metabolites of phthalate esters (PAEs) in urine was developed by combining dispersive liquid-liquid microextraction (DLLME) using ionic liquids with high performance liquid chromatography (HPLC). The factors affecting the efficiency of DLLME were optimized. The types and proportions of extraction solvent and dispersants, as well the ultrasonic extraction time, cooling time, and centrifugal time, were determined. The optimal conditions were as follows:extraction solvent[C8MIM]PF6] 35 μL; dispersants[BSO3HMIm]OTf] 30 μL, [C4MIM]BF6] 120 μL; NH4PF6 0.1 g, extraction at 35℃, ultrasonic dispersion for 5 min, cooling in ice water for 5 min, centrifugation at 4000 r/min for 5 min. After optimization, the five primary metabolites of PAEs were determined. The method showed a good linear relationship within the concentration range of 0.5-1000 μg/L. The determination coefficients (R2) were greater than 0.9955. The detection limit was in the range of 0.16-0.19 μg/L. Under the optimized conditions, the extraction recoveries for the PAEs were 92.9%-105.0%, and the relative standard deviations (RSDs) of the intra- and inter-day precisions were < 5.96%. Urine samples collected from 10 diabetic patients were tested, and the exposure level of the population to the PAE metabolites was evaluated. All the PAE metabolites were detected in these samples, and the detection rate of 2-ethylhexyl hydrogen phthalate (MEHP) was 100%. In conclusion, no toxic organic reagents were added during the extraction process in this method, and multifunctional ionic liquids were used as the extraction agent, dispersant, and salting-out agent. In other words, the extraction process was demonstrated to be green, simple, and efficient. The developed method has high sensitivity and stability, and it is suitable for the determination of trace PAE metabolites in human urine.

Key words: dispersive liquid-liquid microextraction (DLLME), high performance liquid chromatography (HPLC), phthalate metabolites, urine, multiple functional ionic liquids