Evaluation and Application of a High Performance Liquid Chromatographic Method for Prilocaine Analysis in Human Plasma

Background: Prilocaine, a local anesthetic of the amide type, is frequently applied in substantial doses during tumescent liposuction. Although it cannot be excluded that the subcutaneously infiltrated narcotic may enter the circulation and trigger adverse systemic reactions, prilocaine plasma levels have rarely been measured during routine tumescent surgery. We established and evaluated a high performance liquid chromatography (HPLC) method for analysis of this narcotic and used it to measure the drug in plasma samples drawn in the course of tumescent liposuction with prilocaine local anesthesia. Methods: After approval by the local ethics committee and written informed consent, 283 heparin plasma samples were collected from 132 patients during and about 6, 12, and 24 hours after tumescent liposuction with prilocaine infused at doses of 19 +/- 5 mg/kg body weight. Calibrators and controls were prepared by spiking blank plasma with prilocaine. Following addition of internal standard and sodium hydroxide, plasma was extracted with diisopropyl ether. For HPLC analysis, dried extracts were dissolved in methanol - 4.35 mmol/L ammonium phosphate, pH7.0, (60:40 v/v) and applied to a Synergy 4 mu m Fusion-RP column (250 x 4.6 mm) rinsed with the same buffer. Analytes were detected by absorption at 237 nm. For liquid chromatography mass spectrometry (LC-MS), extracts were dissolved in acetonitrile - 2 mmol/L ammonium acetate - formic acid (5:95:0.2 v/v/v), applied to a Synergy 4 mu m Polar-RP column (75 x 2 mm), and eluted with a gradient of acetonitrile in 2 mmol/L ammonium acetate - formic acid. Analytes were detected by an ion trap mass spectrometer with electrospray ionization run in a MS/MS mode. Results: In the HPLC assay established, prilocaine and the internal standard lidocaine eluted at about 14 and 25 minutes, respectively. The limit of detection of prilocaine was 0.002 mg/L, the measurable range extended to 30 mg/L. At prilocaine concentrations between 0.08 and 10.0 mg/L, inter-assay coefficients of variation of 6.2 to 9.9% were obtained. Analyses of plasma pools spiked with variable amounts of prilocaine showed recoveries of 91-101%. Results measured in 20 plasma samples by both HPLC and an independent LC-MS assay agreed acceptably (y(HPLC) = 0.07 + 1.19x(LC-MS), R 0.98). Prilocaine plasma concentrations measured by HPLC in 132 plasma samples drawn in the late phase of liposuction ranged between 0.01 and 32.0 mg/L, roughly one third of all samples exhibiting levels above 5 mg/L. About 6 hours later, prilocaine levels measured in 46 plasma samples were lower (0.13 - 1.56 mg/L) and decreased further in the evening of the operative day (n = 49, 0.10 - 0.62 mg/L) and on the morning of the first postoperative day (n = 55, 0.03 - 0.25 mg/L). Conclusions: An HPLC method for determination of prilocaine was established and successfully applied to analysis of this drug in human plasma. Our results clearly indicate that during tumescent liposuction a significant portion of the subcutaneously infiltrated prilocaine enters the circulation, resulting in potentially harmful blood levels in about one third of the patients studied. 6 hours after liposuction, however, all samples exhibited prilocaine plasma levels far below a critical concentration and these levels further decreased in the evening of the day of treatment and on the next morning.