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Analysis of Benzodiazepines Trevor D. Gillis, M.S., D-ABC Criminalist Santa Clara County District Attorney’s Crime Laboratory Medical Indications • Anxiety (Anxiolytics) – associated with social/medical/personal problems • Insomnia (Sedative) – as a result of anxiety/age • Chronic pain – muscular, spasm, headaches, menopause/menses • • • • • • Skin conditions Dementia Anesthesia Muscle relaxant Withdrawal treatment Anticonvulsant Pharmacological Action • GABA receptor complex – Major inhibitory pathway – Composed of various subunits (/////) – Different brain regions have different subunit structures • Drug actions differ based on subunit affinity http://web.lemoyne.edu Medical Classification (t½) • Ultra-Short Acting (<6 hrs – Sedatives) – E.g. midazolam, triazolam • Short Acting (<12 hrs – Sedatives) – E.g. oxazepam, temazepam, lorazepam • Intermediate Acting (12-24 hrs - Anxiolytics) – E.g. clonazepam, flunitrazepam, alprazolam • Long Acting (>24 hrs - Anxiolytics) – E.g. chlordiazepoxide, diazepam, flurazepam, nitrazepam, medazepam Effects & Side Effects • • • • • • • • • • sedation anterograde amnesia ataxia low blood pressure poor balance cognitive impairment respiratory problems dependency drug interactions withdrawal Common Forensic Encounters • Implicated in drug facilitated sexual assaults • Can impair performance and behavior • Abuse is increasing • Additive/Synergistic with many sedatives Possible Analytical Schemes • EIA – Not sensitive to every benzodiazepine • GC or LC – Possible option (qualitative issues) • GCMS – Possible option (sensitivity issues) • LCMS (or LCMS2) – of course! Analytical Choice: LC/MSD • Easy sample prep. • Great selectivity – Screening – Confirmation • Great sensitivity – Low LODs – Small sample volume (1 mL) Instrument • Agilent Technologies • 1100 LC • Single Quadrupole – SL series Instrument Design • LC – In-line solvent degasser • Binary Pump with solvent selection • 96-wellplate autosampler with needlewash • Thermostated column compartment with column selection • In-line DAD Instrument Design MSD • API (ESI) or APCI • 2 modes (positive & negative) • Single quadrupole • 4 data channels • Chemstation Software Atmospheric Pressure Ionization Spray Chamber Design • API (ESI) • Nebulizing Needle • Hot N2 •Ionization Aid •Instrument Potential The Analytical Approach • SPE Extraction • Screening (Slow Gradient) - SIM – Low fragmentation voltage • Confirmation – (Fast Gradient) – SIM and Full Scan – High fragmentation voltage Specifications • 2mm SB-C8 guard • 150 x 2.1m Zorbax SB-C18 Column • Varian Certify SPE Cartridges • glass vials with 300L inserts Static Instrument Settings • • • • 2 sec. Needlewash Pump flow 0.200 mL/min. Isothermal 50°C column Spray chamber settings (API) – Drying gas 350°C @ 10.0 L/min. – Nebulizer pressure 25 psig – Capillary Voltage 2500 V • MS in Positive Mode Sample Preparation • 1 mL blood or urine sample • 30 ng Prazepam (300 L of 1.0 g/mL) • 2 hour, 37°C urine hydrolysis (2000 units glucuronidase Type L-II e. coli pH 6.8) • 4 mL of 0.1 M Phosphate buffer pH 6.0 • Sonicate 15 min. • Centrifuge 10 min. (5000 rpm) SPE Extraction • Bond Elut Certify – 130 mg mixed-mode sorbent bed: octyl & benzene sulfonic acid • • • • • • Column Prep (Methanol then pH 6 buffer) Sample Added Wash and dry column Elution with 98:2 Ethyl Acetate: NH3 Dry at 40°C Reconstitute 300L 1:2 Acetonitrile Screening Analysis • • 10 l injected Gradual Gradient (0.200 mL/min.) – 30% Acetonitrile (0.1% formic acid) for 14 min. to 100% at 19 min. – Total time 27 min. • QC procedures: – Standard mix first & last in run – Cutoff mix first & last in run – Blanks first and last in run Screening – Single Ion (M+H+) • SIM windows • Optimal Ionization Settings • Greatest Signal • Extremely Sensitive Alprazolam MW=308 Compounds in the Procedure • • • • • • • • 7-aminoclonazepam (285/286) norchlordiazepoxide (285/286) 7-aminoflunitrazepam (283/284) chlordiazepoxide (299/300) desalkylflurazepam* (288/289) nitrazepam (281/282) oxazepam (286/287) lorazepam (321/321) * not tested in urine • • • • • • • • (MW/SIM Signal) clonazepam (315/ 316) nordiazepam (270/271) flurazepam (387/388) alprazolam (308/309) flunitrazepam (313/314) triazolam (343/343) temazepam (300/301) diazepam (284/285) Screening - Analytical Requirements • Integration is optimized for each compound based on cut-off standards • Screening is positive if: – Peak shape is similar to the standards – Integration is acceptable – Retention Time match (0.1 min.) – All blanks are negative – Cutoff standards contain results Why Confirm at all? • SIM M+H+ ion is not enough character, especially at low levels • The potential for coeluting compounds • Provides a greater level of certainty Confirmation Options Targeted Analysis 2 Options: – Fragmentation – SIM – Fragmentation – SCAN • Each drug group has its own method – Clonazepam/7-Aminoclonazepam – Diazepam/Nordiazepam/Oxazepam/ Temazepam – Etc. Confirmation Analysis • • 20 l injected Standard: – Only 1 drug class per standard – Concentration similar to sample (based on screening result) Example: – Screening: • • • – 89 ng/mL 7-aminoclonazepam 85 ng/mL clonazepam 25 ng/mL lorazepam Confirmation standards used: • • 100 ng/mL Clonazepam Mix 20 ng/mL Lorazepam Confirmation Analysis Gradients Group Gradient (0.1% Formic Acid in Acetonitrile) Total Alprazolam 30% for 3 min. to 100% by 10 min. 16 min. Clonazepam 20% for 3 min. to 100% by 10 min. 18 min. Chlordiazepoxide 20% for 6 min. to 100% by 8 min. 16 min. Diazepam 50% for 2 min. to 100% by 10 min. 12 min. Flunitrazepam 30% for 3 min. to 100% by 10 min. 16 min. Flurazepam 30% for 3 min. to 100% by 10 min. 16 min. Lorazepam 30% for 3 min. to 100% by 10 min. 16 min. Nitrazepam 30% for 3 min. to 100% by 10 min. 16 min. Oxazepam 40% for 2 min. to 100% by 8 min. 14 min. Triazolam 30% for 3 min. to 100% by 10 min. 16 min. Confirmation Mass Spectrometry Lorazepam Channel 1 SIM – 130V Channel 2 Scan – 250V Confirmation Analytical Requirements Detected if (SIM): – – – – All peaks are present Peak shape is similar to standard Retention times within ± 0.1 min. for all peaks Ion ratios for all qualifiers within ± 20% of standard – Acceptable integration Detected if (Scan): – Spectral Match is clear – Retention times within ± 0.1 min. Detection Limits (Blood) • 1 ng/mL – flurazepam, nitrazepam, oxazepam, lorazepam, clonazepam, nordiazepam, desalkylflurazepam, alprazolam, flunitrazepam, triazolam, temazepam, diazepam • 5 ng/mL – 7-aminoclonazepam, norchlordiazepoxide, chlordiazepoxide, 7-aminoflunitrazepam Detection Limits (Urine) • 5 ng/mL – chlordiazepoxide, norchlordiazepoxide, flunitrazepam, 7-aminoflunitrazepam, flurazepam, alprazolam, triazolam • 10 ng/mL – nitrazepam, lorazepam, diazepam, nordiazepam • 20 ng/mL – 7-aminoclonazepam, clonazepam, oxazepam, temazepam Interferences • Used NIST Compound Search • Search compounds with the same MW • Tested all compounds where a standard could be obtained • Tested 29 different compounds • No interferences detected Carry-Over • Carry-over exists in all methods where the same instrument is used multiple times • 0.025% was detected for flurazepam • None detected after 100 g/mL injection for remainder Extract Stability • stable for at least 1 week (instrument) • most are stable up to 4 weeks • chlordiazepoxide and clonazepam are known to be light sensitive • 80-95% loss of norchlordiazepoxide and 7-aminoflunitrazepam by 4 weeks • 30-65% loss of nitrazepam, oxazepam, nordiazepam, alprazolam, and temazepam by 4 weeks Summary • LCMSD Powerful analytical tool • Easy to maintain • Meets the analytical requirements for a forensic toxicology laboratory