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Sample preparation for the analysis of MEGX, Xylidine and GX involved addition of an 75 μl internal standard solution (EMGX 5 μg/ml in aqua dest.) and 150 μl aqua dest. to a 150 μl sample. Analysis of the much higher lidocaine concentrations required a 20-fold dilution of the sample in supplemented Williams' E medium. The isolation of lidocaine and its metabolites was performed by extraction. For this, 150 μl sodium carbonate (0.1 M) and 600 μl chloroform were added. After 1 min. vortexing and 4 min. centrifugation at 8000 rpm the aqueous supernatant was removed and 150 μl aqua dest. and 350 μl HCl (0.1 M) were added to the organic phase. The vortexing and centrifugation procedure was repeated and the supernatant removed. A cooled sample storage compartment kept the residues at 4° C. prior to analysis. The mobile phase (0.5 M phosphate buffer, pH 4.5) was pumped at a flow rate of 1.7 ml/min. (Perkin Elmer 250) and pretreated by a Quard-column (Superspher 60 RP 8, length 10 cm, 4 μm particles, Bischoff Chromatography, Germany). An auto sampler (Gilson Sample Injector model 231, France) injected 50 μl aliquots onto a temperature regulated (55° C., Chrompac Column Thermostat, The Netherlands) HPLC column (Superspher 60 RP 8, length 20 cm, i.d. 4.6 mm, 4 μm particles, Bischoff Chromatography, Germany). Detection was at 198 nm (Schoeffel SF 770 UV-spectrophotometer, Germany) and peak areas were calculated with the aid of an Olivetti M250 computer utilizing integration software (Chrompac PCI, version 5.12, The Netherlands). The samples were quantified by comparing the peak area ratio of the component of interest to that of the internal standard. Standard curves were obtained for lidocaine (5-80 μg/ml), MEGX (0.5-16 μg/ml), Xylidine (5-80 μg/ml) and GX (1-32 μg/ml) and showed linearity (r=0.996, n=6). The detection limit was 0.4 μ/ml for GX, 0.3 μg/ml for Xylidine, 0.2 μg/ml for MEGX, 0.4 μg/ml for EMGX and 0.5 μg/ml for lidocaine and the retention times were 2.2 min., 2.4 min., 3.2 min., 4.1 min., and 5.8 min., respectively. Column stabilization time was limited to 20 minutes by washing with a phosphate/acetonitrile/phosphoric acid buffer (50 mM, pH=1.7) and an acetonitrile solution (aqua dest.:ACN=1:1) to remove the chloroform peak.

1. concentrationAnalysis of the much higher lidocaine concentrations
2. extractionThe isolation of lidocaine and its metabolites was performed by extraction
3. additionFor this, 150 μl sodium carbonate (0.1 M) and 600 μl chloroform were added
4. customAfter 1 min. vortexing and 4 min. centrifugation at 8000 rpm the aqueous supernatant was removed
5. additionand 350 μl HCl (0.1 M) were added to the organic phase
6. customat 4° C.
7. custom55° C., Chrompac Column Thermostat