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A relatively new technique to reduce postoperative pain for total knee arthroplasty is to inject a mixture of 266 mg of liposomal bupivacaine and 125 mg of 0.25% bupivacaine HCl with epinephrine 1:300,000 around the knee joint at the time of surgery. Currently, no publications report serum bupivacaine concentrations over time after periarticular injection of liposomal mixed with free bupivacaine. This information is important to ensure safe serum bupivacaine concentrations are maintained especially when considering supplemental or rescue peripheral nerve blocks.A total of 40 subjects scheduled for primary unilateral total knee arthroplasty with intraoperative periarticular injection of the liposomal bupivacaine and bupivacaine HCl mixture were included. Total serum bupivacaine concentrations were measured after the last injection at selected time points and calculated by gas chromatography. Quantile regression techniques were used to analyze the data over time. This study is registered with ClinicalTrials.gov (ID NCT02626559).Peak serum concentration ranged from 0.17 to 1.2 μg/mL and occurred from 10 minutes to 48 hours. Across all time points, the 48-hour interval had the highest mean concentration of total serum bupivacaine at 0.55 μg/mL (SD, 0.27). Quantile regression showed total serum bupivacaine concentrations increased over the 48 hours measured. None of the participants demonstrated signs or symptoms of local anesthetic toxicity.Total serum concentrations of bupivacaine after periarticular administration of liposomal bupivacaine mixed with bupivacaine HCl remained below the described toxicity threshold (2.5 μg/mL) within the first 48 hours, and no patients demonstrated signs or symptoms of toxicity. However, peak serum concentration time was not achieved within the 48-hour interval. Additional studies are needed to describe the course of serum bupivacaine levels after 48 hours and to ascertain the risk of toxicity when combining this method of periarticular injection with peripheral nerve blocks.