Introduction: When treating periprosthetic joint infection with a two-stage procedure, antibiotic-impregnated spacers can be used in the interval between prosthetic removal and reimplantation. In our experience, cultures of sonicated spacers are most often negative. The objective of the study was to assess whether that sonication causes an elution of antibiotics, leading to elevated antibiotic concentrations in the sonication fluid inhibiting bacterial growth and thus causing false-negative cultures.Methods: A prospective monocentric study was performed from September 2014 to March 2016. Inclusion criteria were a two-stage procedure for prosthetic infection and agreement of the patient to participate in the study. Spacers were made of gentamicin-containing cement to which tobramycin and vancomycin were added. Antibiotic concentrations in the sonication fluid were determined by mass-spectometry (LC-MS).Results: 30 patients were identified (15 hip and 14 knee and 1 ankle arthroplasties). No cases of culture positive sonicated spacer fluid were observed in our serie. In the sonication fluid median concentrations of 13.2µg/ml, 392 µg/ml and 16.6 µg/ml were detected for vancomycin, tobramycin and gentamicin, respectively. According to the European Committee on antimicrobial susceptibility testing (EUCAST), these concentrations released from cement spacer during sonication are higher than the minimal inhibitory concentrations (MICs) for most bacteria relevant in prosthetic joint infections.Conclusion:Spacer sonication cultures remained sterile in all of our cases. Elevated concentrations of antibiotics released during sonication could explain partly negative-cultured sonicated spacers. Indeed, the absence of antibiotic free interval during the two-stages can also contribute to false-negative spacers sonicated cultures.