Analytical and Bioanalytical Chemistry

, Volume 410, Issue 12, pp 2921–2935 | Cite as

Combination of in situ metathesis reaction with a novel “magnetic effervescent tablet-assisted ionic liquid dispersive microextraction” for the determination of endogenous steroids in human fluids

  • Jia Wu
  • Zilin Xu
  • Yixuan Pan
  • Yi Shi
  • Xiujie Bao
  • Jun Li
  • Yu Tong
  • Han Tang
  • Shuyan Ma
  • Xuedong WangEmail author
  • Jianxin LyuEmail author
Research Paper


Herein, a novel magnetic effervescence tablet-assisted microextraction coupled to in situ metathesis reaction of ionic liquid (IS-META-ILDM) is presented for the determination of four endogenous steroids in human urine, pregnant women’s blood, and fetal umbilical cord blood. The magnetic effervescent tablets, which were composed of Fe3O4 nanoparticles, sodium carbonate (alkaline source), and tartaric acid (acidic source), were used to disperse the extractant and for convenient magnetic separation. After the effervescent reaction, in situ reaction between NH4PF6 and [C6MIM]BF4 was adopted to change hydrophilic ionic liquid to hydrophobic liquid, which could be separated from the aqueous phase. The newly developed method has three obvious advantages: (1) combination of effervescent dispersion and magnetic nanoparticles’ retrieval is cost-effective and the dispersion and collection of the extractant can be completed almost simultaneously; (2) as compared to temperature-controlled ionic liquid dispersive microextraction and cold-induced solidified microextraction, this method avoids a heating and cooling process which significantly reduces the extraction time and energy cost; and (3) the combination of adsorption by magnetic nanoparticles with extraction by in situ metathesis reaction easily produces high recoveries for target analytes. The optimized composition of effervescent tablet and experimental parameters are as follows: 0.64 g mixture of sodium carbonate and tartaric acid, 7 mg of Fe3O4 (20 nm) as magnetic sorbents, 40 μL of [C6MIM]BF4 as the extraction solvent, 0.15 g NH4PF6, and 300 μL of elution solvent. Under the optimized conditions, the newly developed method provided high extraction recoveries (90.0–118.5%) and low LODs (0.14–0.17 μg L−1) in urine and blood samples. In total, this IS-META-ILDM method provided high extraction efficiency, fast and convenient separation, and underutilization of any organic solvent, and thus it has great potential for the determination of trace endogenous steroids in complex human fluids.

Graphical abstract

The newly developed method has three obvious advantages: combination of effervescent dispersion and magnetic nanoparticles’ retrieval is cost-effective and the dispersion and collection of the extractant can be completed almost simultaneously. It avoids a heating and cooling process which significantly reduces the extraction time and energy cost and easily produces high recoveries for target analytes


Endogenous steroid hormones Magnetic effervescent tablet In situ metathesis reaction Effervescence-assisted microextraction Magnetic nanoparticles Human urine and blood 



This work was jointly supported by the Natural Science Foundation of Zhejiang Provincial (LQ17H260005), the National Natural Science Foundation of China (21577107), and the Wenzhou Municipal Science and Technology Bureau (Y20160181).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

All individual participants received a complete description of the study and gave written informed consent before providing the urine (healthy male) and blood samples (pregnant women’s blood and fetal umbilical cord blood). The studies were approved by The Ethics Committee of the Wenzhou People’s Hospital and performed in accordance with the ethical standards.

Supplementary material

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ESM 1 (PDF 135 kb)
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  1. 1.
    Abdel-Khalik J, Bjorklund E, Hansen M. Simultaneous determination of endogenous steroid hormones in human and animal plasma and serum by liquid or gas chromatography coupled to tandem mass spectrometry. J Chromatogr B. 2013;928:58–77.CrossRefGoogle Scholar
  2. 2.
    Keevil B-G. LC-MS/MS analysis of steroids in the clinical laboratory. Clin Biochem. 2016;49:989–97.CrossRefGoogle Scholar
  3. 3.
    Gervasoni J, Schiattarella A, Primiano A, D'Addurno I, Cocci A, Zuppi C, et al. Simultaneous quantification of 17-hydroxyprogesterone, androstenedione, testosterone and cortisol in human serum by LC-MS/MS using TurboFlow online sample extraction. Clin Biochem. 2016;49:998–1003.CrossRefGoogle Scholar
  4. 4.
    Shibayama Y, Higashi T, Shimada K, Kashimada K, Onishi T, Ono M, et al. Liquid chromatography-tandem mass spectrometric method for determination of salivary 17α-hydroxyprogesterone: a noninvasive tool for evaluating efficacy of hormone replacement therapy in congenital adrenal hyperplasia. J Chromatogr B. 2008;867:49–56.CrossRefGoogle Scholar
  5. 5.
    Kushnir M-M, Rockwood A-L, Roberts W-L, Yue B, Bergquist J, Meikle A-W. Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories. Clin Biochem. 2011;44:77–88.CrossRefGoogle Scholar
  6. 6.
    Jones A-M, Honour J-W. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol. 2006;64:234–44.CrossRefGoogle Scholar
  7. 7.
    Gao W, Stalder T, Kirschbaum C. Quantitative analysis of estradiol and six other steroid hormones in human saliva using a high throughput liquid chromatography-tandem mass spectrometry assay. Talanta. 2015;143:353–8.CrossRefGoogle Scholar
  8. 8.
    Xu W, Li H, Guan Q, Shen Y, Cheng L. A rapid and simple liquid chromatography-tandem mass spectrometry method for the measurement of testosterone, androstenedione, and dehydroepiandrosterone in human serum. J Clin Lab Anal. 2016:1–7.Google Scholar
  9. 9.
    Aufartova J, Mahugo-Santana C, Sosa-Ferrera Z, Santana-Rodriguez J-J, Novakova L, Solich P. Determination of steroid hormones in biological and environmental samples using green microextraction techniques: an overview. Anal Chim Acta. 2011;704:33–46.CrossRefGoogle Scholar
  10. 10.
    Psychogios N, Hau D-D, Peng J, Guo A-C, Mandal R, Bouatra S, et al. The human serum metabolome. PLoS One. 2011;6(2):e16957.CrossRefGoogle Scholar
  11. 11.
    Bouatra S, Aziat F, Mandal R, Guo A-C, Wilson M-R, Knox C, et al. The human urine metabolome. PLoS One. 2013;8(9):e73076.CrossRefGoogle Scholar
  12. 12.
    Leong M-I, Fuh M-R, Huang S-D. Beyond dispersive liquid-liquid microextraction. J Chromatogr A. 2014;1335:2–14.CrossRefGoogle Scholar
  13. 13.
    Zhou Q, Bai H, Xie G, Xiao J. Temperature-controlled ionic liquid dispersive liquid phase micro-extraction. J Chromatogr A. 2008;1177:43–9.CrossRefGoogle Scholar
  14. 14.
    Lin P-C, Tseng M-C, Su A-K, Chen Y-J, Lin C-C. Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification. Anal Chem. 2007;79:3401–8.CrossRefGoogle Scholar
  15. 15.
    Wan J, Cai W, Meng X, Liu E. Monodisperse water-soluble magnetite nanoparticles prepared by polyol process for high-performance magnetic resonance imaging. Chem Commun. 2007;0:5004–5006.Google Scholar
  16. 16.
    Zhao G, Song S, Wang C, Wu Q, Wang Z. Determination of triazine herbicides in environmental water samples by high-performance liquid chromatography using graphene-coated magnetic nanoparticles as adsorbent. Anal Chim Acta. 2011;708:155–9.CrossRefGoogle Scholar
  17. 17.
    Roman I-P, Chisvert A, Canals A. Dispersive solid-phase extraction based on oleic acid-coated magnetic nanoparticles followed by gas chromatography-mass spectrometry for UV-filter determination in water samples. J Chromatogr A. 2011;1218:2467–75.CrossRefGoogle Scholar
  18. 18.
    Lasarte-Aragones G, Lucena R, Cardenas S, Valcarcel M. Effervescence-assisted carbon nanotubes dispersion for the micro-solid-phase extraction of triazine herbicides from environmental waters. Anal Bioanal Chem. 2013;405:3269–77.CrossRefGoogle Scholar
  19. 19.
    Jiang W, Chen X, Liu F, You X, Xue J. Effervescence-assisted dispersive liquid-liquid microextraction using a solid effervescent agent as a novel dispersion technique for the analysis of fungicides in apple juice. J Sep Sci. 2014;37:3157–63.CrossRefGoogle Scholar
  20. 20.
    Yang M, Wu X, Jia Y, Xi X, Yang X, Lu R, et al. Use of magnetic effervescent tablet-assisted ionic liquid dispersive liquid-liquid microextraction to extract fungicides from environmental waters with the aid of experimental design methodology. Anal Chim Acta. 2016;906:118–27.CrossRefGoogle Scholar
  21. 21.
    Lasarte-Aragones G, Lucena R, Cardenas S, Valcarcel M. Effervescence assisted dispersive liquid-liquid microextraction with extractant removal by magnetic nanoparticles. Anal Chim Acta. 2014;807:61–6.CrossRefGoogle Scholar
  22. 22.
    Sun J, Chen J, Shi Y. Multiple functional ionic liquids based dispersive liquid-liquid microextraction combined with high performance chromatography for the determination of phenolic compounds in water samples. Talanta. 2014;125:329–35.CrossRefGoogle Scholar
  23. 23.
    Liu Y, Li H, Lin J-M. Magnetic solid-phase extraction based on octadecyl functionalization of monodisperse magnetic ferrite microspheres for the determination of polycyclic aromatic hydrocarbons in aqueous samples coupled with gas chromatography-mass spectrometry. Talanta. 2009;77:1037–42.CrossRefGoogle Scholar
  24. 24.
    Gao Q, Luo D, Ding J, Feng Y. Rapid magnetic solid-phase extraction based on magnetite/silica/poly(methacrylic acid-co-ethylene glycol dimethacrylate) composite microspheres for the determination of sulfonamide in milk samples. J Chromatogr A. 2010;1217:5602–9.CrossRefGoogle Scholar
  25. 25.
    Li D, Teoh W-Y, Gooding J-J, Selomulya C, Amal R. Functionalization strategies for protease immobilization on magnetic nanoparticles. Adv Funct Mater. 2010;20:1767–77.CrossRefGoogle Scholar
  26. 26.
    Arain M-S, Arain S-A, Kazi T-G, Afridi H-I, Ali J, Naeemulllah, et al. Temperature controlled ionic liquid-based dispersive micro-extraction using two ligands, for determination of aluminium in scalp hair samples of Alzheimer's patients: a multivariate study. Spectrochim Acta A Mol Biomol Spectrosc. 2015;137:877–85.CrossRefGoogle Scholar
  27. 27.
    Fang H, Yang F, Sun J, Tian Y, Zeng Z, Xu Y. Centrifuge microextraction coupled with sweeping-MEKC to analyze trace steroid hormones in urine samples. Talanta. 2011;85:2148–53.CrossRefGoogle Scholar
  28. 28.
    Almeida C, Nogueira J-M. Determination of steroid sex hormones in real matrices by bar adsorptive microextraction (BAμE). Talanta. 2015;136:145–54.CrossRefGoogle Scholar
  29. 29.
    Stopforth A, Burger B-V, Crouch A-M, Sandra P. The analysis of estrone and 17β-estradiol by stir bar sorptive extraction-thermal desorption-gas chromatography/mass spectrometry: application to urine samples after oral administration of conjugated equine estrogens. J Chromatogr B. 2007;856:156–64.CrossRefGoogle Scholar
  30. 30.
    Guo T-D, Chan Y-M, Soldin S-J. Steroid profiles using liquid chromatography tandem mass spectrometry with atmospheric pressure photoionization source. Arch Pathol Lab Med. 2004;128:469–75.Google Scholar
  31. 31.
    Soldin O-P, Guo T-D, Weiderpass E, Tractenberg R-E, Hilakivi-Clarke L, Soldin S-J. Steroid hormone levels in pregnancy and 1 year postpartum using isotope dilution tandem mass spectrometry. Fertil Steril. 2005;84:701–10.CrossRefGoogle Scholar
  32. 32.
    Sippell W-G, Bidlingmaier F, Becker H, Brunig T, Dorr H, Hahn, et al. Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol and cortisone. J Steroid Biochem. 1978;9(1):63–74.CrossRefGoogle Scholar
  33. 33.
    Sippell W, Becker H, Versmold H, Bidlingnaier F, Knorr D. Longitudinal studies of plasma aldosterone, corticosterone, deoxycorticosterone, progesterone, 17-hydroxyprogesterone, cortisol, and cortisone determined simultaneously in mother and child at birth and during the early neonatal period. I. Spontaneous delivery. J Clin Endocrinol Metab. 1978;46:971–85.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jia Wu
    • 1
  • Zilin Xu
    • 1
  • Yixuan Pan
    • 1
  • Yi Shi
    • 1
  • Xiujie Bao
    • 1
  • Jun Li
    • 2
  • Yu Tong
    • 2
  • Han Tang
    • 1
  • Shuyan Ma
    • 1
  • Xuedong Wang
    • 3
    Email author
  • Jianxin Lyu
    • 1
    Email author
  1. 1.Key Laboratory for Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life ScienceWenzhou Medical UniversityWenzhouChina
  2. 2.Department of Clinical LaboratoryWenzhou People’s HosptialWenzhouChina
  3. 3.Key Laboratory of Watershed Science and Health of Zhejiang ProvinceWenzhouChina

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