Analytical and Bioanalytical Chemistry

, Volume 408, Issue 19, pp 5109–5124 | Cite as

Hydrodynamic chromatography coupled to single-particle ICP-MS for the simultaneous characterization of AgNPs and determination of dissolved Ag in plasma and blood of burn patients

  • Marco RomanEmail author
  • Chiara Rigo
  • Hiram Castillo-Michel
  • Ivan Munivrana
  • Vincenzo Vindigni
  • Ivan Mičetić
  • Federico Benetti
  • Laura Manodori
  • Warren R. L. Cairns
Research Paper
Part of the following topical collections:
  1. Single-particle-ICP-MS Advances


Silver nanoparticles (AgNPs) are increasingly used in medical devices as innovative antibacterial agents, but no data are currently available on their chemical transformations and fate in vivo in the human body, particularly on their potential to reach the circulatory system. To study the processes involving AgNPs in human plasma and blood, we developed an analytical method based on hydrodynamic chromatography (HDC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) in single-particle detection mode. An innovative algorithm was implemented to deconvolute the signals of dissolved Ag and AgNPs and to extrapolate a multiparametric characterization of the particles in the same chromatogram. From a single injection, the method provides the concentration of dissolved Ag and the distribution of AgNPs in terms of hydrodynamic diameter, mass-derived diameter, number and mass concentration. This analytical approach is robust and suitable to study quantitatively the dynamics and kinetics of AgNPs in complex biological fluids, including processes such as agglomeration, dissolution and formation of protein coronas. The method was applied to study the transformations of AgNP standards and an AgNP-coated dressing in human plasma, supported by micro X-ray fluorescence (μXRF) and micro X-ray absorption near-edge spectroscopy (μXANES) speciation analysis and imaging, and to investigate, for the first time, the possible presence of AgNPs in the blood of three burn patients treated with the same dressing. Together with our previous studies, the results strongly support the hypothesis that the systemic mobilization of the metal after topical administration of AgNPs is driven by their dissolution in situ.

Graphical Abstract

Simplified scheme of the combined analytical approach adopted for studying the chemical dynamics of AgNPs in human plasma/blood


Silver nanoparticles Hydrodynamic chromatography Single-particle ICP-MS Synchrotron radiation Burns Wound dressings 



The authors are grateful to the Italian Ministry of Education, University and Research for financial support through the project MIUR-FIRB number RBFR08M6W8. The European Synchrotron Radiation Facility is acknowledged for provision of beamtime at ID21. ELGA LabWater is acknowledged for providing the PURELAB Option-Q and Ultra Analytic systems, which produced the ultra-pure water used in these experiments. Francesca Benetello and Bruno Pavoni from Ca’ Foscari University of Venice are acknowledged for the lyophilization of standards and samples.

Conflict of interest

The authors declare that they have no competing interests.


  1. 1.
    Rigo C, Ferroni L, Tocco I, Roman M, Munivrana I, Gardin C, Cairns WRL, Vindigni V, Azzena B, Barbante C, Zavan B (2013) Active silver nanoparticles for wound healing. Int J Mol Sci 14(3):4817–4840CrossRefGoogle Scholar
  2. 2.
    Wilkinson LJ, White RJ, Chipman JK (2011) Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety. J Wound Care 20(11):543–549CrossRefGoogle Scholar
  3. 3.
    Liu J, Wang Z, Liu FD, Kane AB, Hurt RH (2012) Chemical transformations of nanosilver in biological environments. ACS Nano 6(11):9887–9899CrossRefGoogle Scholar
  4. 4.
    Reidy B, Haase A, Luch A, Dawson KA, Lynch I (2013) Mechanisms of silver nanoparticle release, transformation and toxicity: a critical review of current knowledge and recommendations for future studies and applications. Materials 6(6):2295–2350CrossRefGoogle Scholar
  5. 5.
    Gnanadhas DP, Ben Thomas M, Thomas R, Raichur AM, Chakravortty D (2013) Interaction of silver nanoparticles with serum proteins affects their antimicrobial activity in vivo. Antimicrob Agents Chemother 57(10):4945–4955CrossRefGoogle Scholar
  6. 6.
    Liu J, Sonshine DA, Shervani S, Hurt RH (2010) Controlled release of biologically active silver from nanosilver surfaces. ACS Nano 4(11):6903–6913CrossRefGoogle Scholar
  7. 7.
    You CG, Han CM, Wang XG, Zheng YR, Li QY, Hu XL, Sun HF (2012) The progress of silver nanoparticles in the antibacterial mechanism, clinical application and cytotoxicity. Mol Biol Rep 39(9):9193–9201CrossRefGoogle Scholar
  8. 8.
    Pyrz WD, Buttrey DJ (2008) Particle size determination using TEM: a discussion of image acquisition and analysis for the novice microscopist. Langmuir 24(20):11350–11360CrossRefGoogle Scholar
  9. 9.
    Luo P, Morrison I, Dudkiewicz A, Tiede K, Boyes E, O’Toole P, Park S, Boxall AB (2013) Visualization and characterization of engineered nanoparticles in complex environmental and food matrices using atmospheric scanning electron microscopy. J Microsc 250(1):32–41CrossRefGoogle Scholar
  10. 10.
    Grobelny J, DelRio F, Pradeep N, Kim D-I, Hackley V, Cook R (2011) Size measurement of nanoparticles using atomic force microscopy. In: McNeil SE (ed) Characterization of nanoparticles intended for drug delivery, vol 697. Methods in molecular biology. Humana, pp. 71–82. doi:  10.1007/978-1-60327-198-1_7
  11. 11.
    Hagendorfer H, Kaegi R, Parlinska M, Sinnet B, Ludwig C, Ulrich A (2012) Characterization of silver nanoparticle products using asymmetric flow field flow fractionation with a multidetector approach—a comparison to transmission electron microscopy and batch dynamic light scattering. Anal Chem 84(6):2678–2685CrossRefGoogle Scholar
  12. 12.
    Proulx K, Wilkinson KJ (2014) Separation, detection and characterisation of engineered nanoparticles in natural waters using hydrodynamic chromatography and multi-method detection (light scattering, analytical ultracentrifugation and single particle ICP-MS). Environ Chem 11(4):392–401CrossRefGoogle Scholar
  13. 13.
    Mitrano DM, Barber A, Bednar A, Westerhoff P, Higgins CP, Ranville JF (2012) Silver nanoparticle characterization using single particle ICP-MS (SP-ICP-MS) and asymmetrical flow field flow fractionation ICP-MS (AF4-ICP-MS). J Anal At Spectrom 27(7):1131–1142CrossRefGoogle Scholar
  14. 14.
    Wimuktiwan P, Shiowatana J, Siripinyanond A (2015) Investigation of silver nanoparticles and plasma protein association using flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (FlFFF-ICP-MS). J Anal At Spectrom 30(1):245–253CrossRefGoogle Scholar
  15. 15.
    Ramos K, Ramos L, Camara C, Gomez-Gomez MM (2014) Characterization and quantification of silver nanoparticles in nutraceuticals and beverages by asymmetric flow field flow fractionation coupled with inductively coupled plasma mass spectrometry. J Chromatogr 1371:227–236CrossRefGoogle Scholar
  16. 16.
    Philippe A, Gangloff M, Rakcheev D, Schaumann GE (2014) Evaluation of hydrodynamic chromatography coupled with inductively coupled plasma mass spectrometry detector for analysis of colloids in environmental media—effects of colloid composition, coating and shape. Anal Methods 6(21):8722–8728CrossRefGoogle Scholar
  17. 17.
    Lewis DJ (2015) Hydrodynamic chromatography-inductively coupled plasma mass spectrometry, with post-column injection capability for simultaneous determination of nanoparticle size, mass concentration and particle number concentration (HDC-PCi-ICP-MS). Analyst 140(5):1624–1628CrossRefGoogle Scholar
  18. 18.
    Soto-Alvaredo J, Montes-Bayon M, Bettmer J (2013) Speciation of silver nanoparticles and silver(I) by reversed-phase liquid chromatography coupled to ICPMS. Anal Chem 85(3):1316–1321CrossRefGoogle Scholar
  19. 19.
    Franze B, Engelhard C (2014) Fast separation, characterization, and speciation of gold and silver nanoparticles and their ionic counterparts with micellar electrokinetic chromatography coupled to ICP-MS. Anal Chem 86(12):5713–5720CrossRefGoogle Scholar
  20. 20.
    Laborda F, Bolea E, Jimenez-Lamana J (2014) Single particle inductively coupled plasma mass spectrometry: a powerful tool for nanoanalysis. Anal Chem 86(5):2270–2278CrossRefGoogle Scholar
  21. 21.
    Yang Y, Long CL, Yang ZG, Li HP, Wang Q (2014) Characterization and determination of silver nanoparticle using single particle-inductively coupled plasma-mass spectrometry. Chin J Anal Chem 42(11):1553–1559CrossRefGoogle Scholar
  22. 22.
    Lee S, Bi XY, Reed RB, Ranville JF, Herckes P, Westerhoff P (2014) Nanoparticle size detection limits by single particle ICP-MS for 40 elements. Environ Sci Technol 48(17):10291–10300CrossRefGoogle Scholar
  23. 23.
    Mitrano DM, Ranville JF, Bednar A, Kazor K, Hering AS, Higgins CP (2014) Tracking dissolution of silver nanoparticles at environmentally relevant concentrations in laboratory, natural, and processed waters using single particle ICP-MS (spICP-MS). Environ Sci Nano 1(3):248–259CrossRefGoogle Scholar
  24. 24.
    Furtado LM, Hoque ME, Mitrano DF, Ranville JF, Cheever B, Frost PC, Xenopoulos MA, Hintelmann H, Metcalfe CD (2014) The persistence and transformation of silver nanoparticles in littoral lake mesocosms monitored using various analytical techniques. Environ Chem 11(4):419–430CrossRefGoogle Scholar
  25. 25.
    Mitrano DM, Lesher EK, Bednar A, Monserud J, Higgins CP, Ranville JF (2012) Detecting nanoparticulate silver using single-particle inductively coupled plasma-mass spectrometry. Environ Toxicol Chem 31(1):115–121CrossRefGoogle Scholar
  26. 26.
    Peters RJB, Rivera ZH, van Bemmel G, Marvin HJP, Weigel S, Bouwmeester H (2014) Development and validation of single particle ICP-MS for sizing and quantitative determination of nano-silver in chicken meat. Anal Bioanal Chem 406(16):3875–3885Google Scholar
  27. 27.
    Loeschner K, Navratilova J, Kobler C, Molhave K, Wagner S, von der Kammer F, Larsen EH (2013) Detection and characterization of silver nanoparticles in chicken meat by asymmetric flow field flow fractionation with detection by conventional or single particle ICP-MS. Anal Bioanal Chem 405(25):8185–8195CrossRefGoogle Scholar
  28. 28.
    Pergantis SA, Jones-Lepp TL, Heithmar EM (2012) Hydrodynamic chromatography online with single particle-inductively coupled plasma mass spectrometry for ultratrace detection of metal-containing nanoparticles. Anal Chem 84(15):6454–6462CrossRefGoogle Scholar
  29. 29.
    Grombe R, Allmaier G, Charoud-Got J, Dudkiewicz A, Emteborg H, Hofmann T, Larsen EH, Lehner A, Llinas M, Loeschner K, Molhave K, Peters RJ, Seghers J, Solans C, von der Kammer F, Wagner S, Weigel S, Linsinger TPJ (2015) Feasibility of the development of reference materials for the detection of Ag nanoparticles in food: neat dispersions and spiked chicken meat. Accred Qual Assur 20(1):3–16CrossRefGoogle Scholar
  30. 30.
    Liu JY, Murphy KE, MacCuspie RI, Winchester MR (2014) Capabilities of single particle inductively coupled plasma mass spectrometry for the size measurement of nanoparticles: a case study on gold nanoparticles. Anal Chem 86(7):3405–3414CrossRefGoogle Scholar
  31. 31.
    Cornelis G, Hassellov M (2014) A signal deconvolution method to discriminate smaller nanoparticles in single particle ICP-MS. J Anal At Spectrom 29(1):134–144CrossRefGoogle Scholar
  32. 32.
    Roman M, Rigo C, Munivrana I, Vindigni V, Azzena B, Barbante C, Fenzi F, Guerriero P, Cairns WRL (2013) Development and application of methods for the determination of silver in polymeric dressings used for the care of burns. Talanta 115:94–103CrossRefGoogle Scholar
  33. 33.
    Solé VA, Papillon E, Cotte M, Walter P, Susini J (2007) A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra. Spectrochim Acta B 62(1):63–68CrossRefGoogle Scholar
  34. 34.
    Ravel B, Newville M (2005) ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J Synchrotron Radiat 12(4):537–541CrossRefGoogle Scholar
  35. 35.
    Tuoriniemi J, Cornelis G, Hassellov M (2014) Improving the accuracy of single particle ICPMS for measurement of size distributions and number concentrations of nanoparticles by determining analyte partitioning during nebulisation. J Anal At Spectrom 29(4):743–752CrossRefGoogle Scholar
  36. 36.
    Montano MD, Badiei HR, Bazargan S, Ranville JF (2014) Improvements in the detection and characterization of engineered nanoparticles using spICP-MS with microsecond dwell times. Environ Sci Nano 1(4):338–346CrossRefGoogle Scholar
  37. 37.
    Hineman A, Stephan C (2014) Effect of dwell time on single particle inductively coupled plasma mass spectrometry data acquisition quality. J Anal At Spectrom 29(7):1252–1257CrossRefGoogle Scholar
  38. 38.
    Pace HE, Rogers NJ, Jarolimek C, Coleman VA, Higgins CP, Ranville JF (2011) Determining transport efficiency for the purpose of counting and sizing nanoparticles via single particle inductively coupled plasma mass spectrometry. Anal Chem 83(24):9361–9369CrossRefGoogle Scholar
  39. 39.
    Cedervall T, Lynch I, Lindman S, Berggard T, Thulin E, Nilsson H, Dawson KA, Linse S (2007) Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci U S A 104(7):2050–2055CrossRefGoogle Scholar
  40. 40.
    Shannahan JH, Lai XY, Ke PC, Podila R, Brown JM, Witzmann FA (2013) Silver nanoparticle protein corona composition in cell culture media. PLoS One 8(9)Google Scholar
  41. 41.
    Rigo C, Roman M, Munivrana I, Vindigni V, Azzena B, Barbante C, Cairns WRL (2012) Characterization and evaluation of silver release from four different dressings used in burns care. Burns 38(8):1131–1142CrossRefGoogle Scholar
  42. 42.
    Adams NWH, Kramer JR (1999) Silver speciation in wastewater effluent, surface waters, and pore waters. Environ Toxicol Chem 18(12):2667–2673CrossRefGoogle Scholar
  43. 43.
    Larese FF, D’Agostin F, Crosera M, Adami G, Renzi N, Bovenzi M, Maina G (2009) Human skin penetration of silver nanoparticles through intact and damaged skin. Toxicology 255(1–2):33–37CrossRefGoogle Scholar
  44. 44.
    Armitage SA, White MA, Wilson HK (1996) The determination of silver in whole blood and its application to biological monitoring of occupationally exposed groups. Ann Occup Hyg 40(3):331–338CrossRefGoogle Scholar
  45. 45.
    Wang XQ, Kempf M, Mott J, Chang HE, Francis R, Liu PY, Cuttle L, Olszowy H, Kravchuk O, Mill J, Kimble RM (2009) Silver absorption on burns after the application of Acticoat(TM): data from pediatric patients and a porcine burn model. J Burn Care Res 30(2):341–348CrossRefGoogle Scholar
  46. 46.
    Schneider L, Korber A, Grabbe S, Dissemond J (2007) Influence of pH on wound-healing: a new perspective for wound-therapy? Arch Dermatol Res 298(9):413–420CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Marco Roman
    • 1
    • 2
    Email author
  • Chiara Rigo
    • 1
  • Hiram Castillo-Michel
    • 3
  • Ivan Munivrana
    • 4
  • Vincenzo Vindigni
    • 4
  • Ivan Mičetić
    • 5
  • Federico Benetti
    • 5
  • Laura Manodori
    • 5
  • Warren R. L. Cairns
    • 2
  1. 1.Department of Environmental Sciences, Informatics and Statistics (DAIS)Ca’ Foscari University of VeniceVenezia MestreItaly
  2. 2.Institute for the Dynamics of Environmental Processes (IDPA-CNR)Venezia MestreItaly
  3. 3.European Synchrotron Radiation Facility (ESRF)GrenobleFrance
  4. 4.Burns Center, Division of Plastic SurgeryUniversity Hospital of PaduaPaduaItaly
  5. 5.European Center for the Sustainable Impact of Nanotechnology (ECSIN)RovigoItaly

Personalised recommendations