| Objective: Systematic toxicological analysis (STA) is defined as the logical chemical-analytical search for potentially harmful substances whose presence is uncertain and whose identity is unknown. We established a general sample preparation method and a chromatographic screening method for a broad range of drugs from plasma, and set up a database including UV spectra and relative retention time (RRT) of 61 central nervous system drugs which iucluded analgesics, antipsychotics, anesthetics, benzodiazepines. The identification is based on the comparison of the UV absorption spectrum and RRT with corresponding data stored in the database. The study is very significant for diagnosis and treatment of patients with drug poisoning in emergency.Methods and results1 To establish a general chromatographic screening method and set up a database including UV spectra and RRTThe following acetonitrile- phosphate buffer elution gradient was applied: the proportion of acetonitrile linearly increased from 5 % to 50 % at a rate of 1.5 % min-1 in 30 min, then increased linearly to 80 % in 5 min at a rate of 6 % min-1. The equilibration time between two consecutive sample analyzed in series was set at 15 min. The flow rate of mobile phase was 1.5 mL min-1 and the injection volume was 30μL. The injection loop volume was 100μL .The temperature of column was set at 35℃. The UV detection wavelength was set at 210 nm and 254 nm, and full spectra were recorded from 200 nm to 364 nm. 1-nitrobutane was chosen as internal standard.Durg standard solution with optimal concentration was injected into the HPLC system respectively. Their RRT and UV spectra were recorded, and a database including UV spectra and the RRT was established. The identification is based on the comparison of the UV absorption spectrum and RRT with corresponding data stored in the database.2 To establish a general sample preparation from plasma2.1 Protein precipitation procedure 1 mL plasma was added with 1.5 mL acetonitrile, vortex mixed, centrifuged, and the supernate was filtrated and then injected into the HPLC system. The absolute recoveries of 61 drugs tested exceeded 80 % with RSD ranging from 0.94 % to 11.23 %.2.2 The solid-phase extraction preparation by WCX columnThe column was preconditioned with 3 mL of acetonitrile, 1 mL of water, 2 mL of buffer solution subsequently. The pretreated plasma was transferred to the column. The column was washed with 2mL of water. Then 2mL of acetonitrile was added into the column and the elution was collected as the neutral and acid fraction. 3mL of trifluoroacetic acid–acetonitrile (2+98) was added into the column and the elution was collected as basic fraction. The two fractions were evaporated respectively at 40℃in a water bath under a nitrogen stream until about 100μL of solvent remained, then make it the volumn of 1 mL with 5 % of acetonitrile. The absolute recoveries of 53 drugs tested exceeded 50 %, 37 drugs tested exceeded 80 % with RSD ranging from 0.08 % to 14.03 %.3 To evaluate the selection of the UV spectra and RRT in the identification of unknown substancesDiscrimination power (DP) and Mean list length (MLL) were used to evaluate the selection of the UV spectra and RRT in identification of unknown substances. If UV spectra and RRT were both taken into account, DP was 0.9995 and MLL was 1.0328, which signified that, on average, every substance was indistinguishable only from 0.03 others.4 Case studyThis method has been used in our laboratory to screen about 47 plasma specimens,in which 37 plsama is successfully detected. The detect rate is 77.1%.Conclusion1,Protein Precipitation by acetonitrile is suitable for STA.2,The solid-phase extraction preparation using WCX may be appropriate for STA, however, other methods need to be supplemented. 3,HPLC-DAD has been proven to be a reliable, promising method for substance identification in STA. |
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