59-05-032 Proceeding

70 Proceedings of the Princess Maha Chakri Sirindhorn Congress in DFSA victim, there will be a 24-72 hr. or longer delay between victim’s report and the drug’s administration. In addition, drug used is difficult to detect becausemetabolic clearance or the active metabolite is chemically unstable. Therefore, in such accompaniment, to prolong the window of detection drug’s presence in blood is particularly important. This study aims to develop a simple and sensitive analytical method for BZDs determination in plasma using high performance liquid chromatography (HPLC) and UV-visible spectrophotometer detector. The results of the analysis will be useful to judge the case. II. Materials and Methods A. Chemicals and reagents Acetonitrile and methanol were purchased from Honey well Burdick & Jackson ® , Germany, NaOHwas purchased fromFisher ® , England and internal standard: 5-(4-methylphenyl)- 5-phenylhydantoin was a product of Aldrich Co ® , Germany. Lorazepam, alprazolam, diazepam, midazolam and nitrazepam (USP) were kindly provided by Scientific Crime Detection Division 4, Thailand. Plasma sample was kindly obtained from Blood bank, faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. B. Preparation of standard solution Stock solutions of lorazepam, alprazolam, diazepam and midazolam (1 mg/mL) and internal standard, 5-(4-methylphenyl)-5-phenylhydantoinwere prepared by separately dissolving suitable amounts of each standard drug inmethanol. For working solution prepared by diluting the stock standard solution of 4 BZDs in methanol to the concentration 100 µg/mL prior to injection. C. Extraction Procedure Plasma samples (250 µL) were placed in 1 mLmicrocentrifuge tube and stock standard solution of lorazepam, alprazolam, diazepam, midazolamand internal standard were added.Then sample solutionwas incubated in ultrasonic bath for 1 hr. and extractedwith 750 µL of ethyl acetate. The samples were vortex-mixed for 5 minutes and centrifuged at 10,000 rpm/10 minutes for the protein precipitation. The upper layer was collected and dried under N 2 then reconstituted with 100 µL of mobile phase. The sample solutions were vortex-mixed and centrifuged as described above again. Supernatant was separated and injected into HPLC. D. HPLC analysis The assay was conducted by Agilent 1100 series HPLC and UV-VWD at 210 nm (Agilent®, Japan), using isocratic solvent containing 10 mMphosphate buffer, pH 5.8, acetonitrile andmethanol (58:20:22) as mobile phase. Separationwas performed onODSHYPERSYL column; 5 µm, 4.6 × 250 mm, (Agilent, Germany). Flow rate was 1 mL/min and injection volume was 20 µL.