59-05-032 Proceeding

69 Proceedings of the Princess Maha Chakri Sirindhorn Congress VALIDATION OF HPLC METHOD FOR THE DETERMINATION OF BENZODIAZEPINES IN PLASMA Phonpan Prasongthum 1 , Supawadee Daodee 2 , Akhom Kesorn 3 and Wanna Sirisangtrakul 4 Abstract : This studyaimed todevelopanalyticalmethodandmethodvalidation forBenzodiazepines (BZDs); lorazepam, alprazolam, diazepam and midazolam, which were the most frequently observed compounds in case of drug-facilitated sexual assaults (DFSA).The isolation of drugs from plasma by liquid-liquid extraction with ethyl acetate, and reverse phase-high performance liquid chromatographic (HPLC) with UV detection at 210 nm was performed. The chromatographic separation was generated using Hypersil ODS, 5 µm (4.6 × 250 mm) column and a mobile phase containing phosphate buffer, pH 5.8: acetonitrile: methanol (58:20:22, v/v/v). The linearity range for 4 Benzodiazepines ranged from0.0101-52.10 µg/mL (r2 ≥ 0.999).The limit of detection (LOD) and the limit of quantification (LOQ) for 4 benzodiazepines ranged from 0.004-0.082 and 0.112- 0.210 µg/mL, respectively. Within-day and between-day precision of 4 benzodiazepines were less than 1.874 and 2.202 respectively, and accuracy as % recovery was from 90.106-97.333%. This validated analytical method gave high accuracy, precision and could be applied to analyze some benzodiazepines in plasma sample of DFSA cases. Keywords : benzodiazepines, drug facilitated sexual assault, plasma sample, HPLC I. Introduction Benzodiazepines (BZDs) are class of psychoactive drugs that have been used as sedative, hypnotic, anxiolytic, antidepressant, muscle relaxant, and anticonvulsant drug worldwide. All BZDs share the same mechanism of action through interaction with the benzodiazepine site on GABA A receptor, where they potentiate the action on the inhibitory neurotransmitter γ -aminobutyric acid (GABA). The benzodiazepines are notably safe in use regarding to lower toxicity than their antecedent barbiturates ([1], [11]). However, they can still cause severe side effect such as drowsiness, paradoxical anxiety, cognitive impairment and amnesia and moreover, possess a potential for abuse, subjected to concomitant administration of other central nervous system such as ethanol and other related stimulant drug [1], [4], [5], [9]). Furthermore, toward to sedative effect and ability to cause amnesia, the BZDs have also been found to be present in a suspected drug facilitated sexual assault (DFSA) cases. InThailand, the BZDs; alprazolam, diazepam lorazepamandmidazolamare themost frequently observed compounds in case of drug-facilitated crime [13]. Regard to unconscious and amnesia caused by drug, panic or embarrassment for notice 1 Forensic Science Program, Faculty of Science, Khon Kaen University 2 Faculty of Pharmaceutical Sciences, Khon Kaen University 3 Scientific Crime Detection Division 4. 4 Forensic Science Program, Faculty of Science, Khon Kaen University