Agilent Technologies, Inc.

1. S QQQ Applications:A Sensitive and Selective Technique for the Quantitation and Confirmation of Benzodiazepines in Biological Matrices Agilent Technologies, Inc.

2. Who are the customers? • What is their need? • Why do they need a solution? • How do they reach their goal? What are the needs for analyzing benzodiazepines? • Who are the customers? • Forensics / Clinical Toxicology Labs – private and government • What is their need? • - Need to analyze illegally used, or low-dose prescription amounts in biological matrices including urine, blood, oral fluid, and hair • Why do they need a solution? • - Polar and non-volatile nature makes them difficult to analyze • by GC and GC/MS • - Matrices contribute large amounts of background • - Newer benzos are more potent and require lower limits of detection • How do they reach their goal? • - Sensitive and fast LC/MS/MS technique for quantitation and confirmation

3. Agilent 1200 Series RRLC with 6410 QQQ What is the solution?

4. Analysis of > 20 benzodiazepines in biological matrices Need quantitation and confirmation ions for each compound along with associated internal standards Need good reproducibility inter- and intra-day < 20% RSD at LOQ Quantitation ranges Reference LOQs

5. Agilent 1200 Series RRLC with 6410 QQQ What is the solution? – What we offer now

6. Quad Mass Filter (Q3) Quad Mass Filter (Q1) Collision Cell Q1 lets only target ion 210 pass through Collision cell breaks ion 210 apart Q3 monitors only characteristic fragments 158 and 191 from ion 210 for quant and qual. 210 210 222 158 210 158 268 191 280 165 191 190 160 150 170 190 210 170 210 250 290 190 210 MS Solution: Sensitivity and confirmation QQQ - MRM (Multiple Reaction Monitoring) Spectrum with backgroundions (from ESI)

7. LC Solution: Rapid Resolution High Throughput e.g. Bisphenol A, particle size comparison 4.6x50mm

8. Instrument Actuals/Status (configurable) System Diagram Real-Time Spec/Chro Display Method Editor LC + MS ! Worklist (Sequence) Agilent MassHunter Acquisition Software Method development – where to start ‘Flat’ User Interface All Info At-A-Glance

9. Autotune

10. Autotune and Calibration SystemBuilt-in calibrant delivery system is unique to Agilent Built-in calibrant delivery system used for fast auto-calibration calibrant solutions

11. Triple (QQQ) Reporting (Excel) –tune

12. Method development – MS only (no column) Flow injection analysis (FIA) in LC solvent mix of 50:50 water/methanol with 20 mM ammonium formate Use APCI with typical settings for 500 µL/min flow: Vaporizer = 400 C Nebulizer = 50 psi Dry gas flow = 5 L/min Dry gas temp = 350 C Optimize for only 2 parameters: Fragmentor (Frag) and Collision Energy (CE) voltages

13. 120 120 240 240 200 200 160 160 80 80 40 40 0 V 0 V EIC 287.1 Oxazepam Optimal Frag = 120 V [M + H]+ = 287.1 Method development – MS only (no column) FIA optimization of Fragmentor voltage for Oxazepam

14. Quant: 321.1 > 275.1 (CE = 5V) Qual: 321.1 > 229.1 (CE = 15 V) Method development – MS only (no column) 15 20 25 5 0 V 30 FIA optimization of Collision Energy for Lorazepam 10 Lorazepam

15. A = 20 mM ammonium formate in H2O B = 20 mM ammonium formate in ACN Time (min) %B 0 – 4.5 50 5.0 80 6.0 100 Stop time = 8.2 Post time = 4.5 Method development – LC (establish RTs) Use APCI with typical settings for 800 µL/min flow: Vaporizer = 400 C Nebulizer = 50 psi Dry gas flow = 5 L/min Dry gas temp = 350 C Optimize gradient for good chromatography (Same as 500 µL/min used for FIA) Zorbax 4.6 x 50 mm, 1.8-µm particle size column (922975-902) Create segments for MRM experiments

16. Chromatogram of 21 benzos and 6 IStds 6 segments, total run time of 8 minutes

17. Filter by Sample Type View Results from Single or Multiple compounds Outlier Flagging + Filtering Compounds Compounds Compounds Samples Samples MassHunter Quant Analysis – Batch-At-A-Glance Batch Table Sample- or Compound-Centric Orthogonal Navigation Context- located Tool strips Compound Information (Chroms + Specs) Calibration Curve + Curve Fit Assistant

18. Quantitative View - Method Editor

19. Quantitative Analysis – CurveFit Assistant Alternate choice Try other curve types for better fits

20. a-OH-triazolam in urine 10 – 100 ng/mL R2 > 0.999 Alprazolam in blood 1 – 100 ng/mL R2 > 0.999 Nitrazepam in hair 50 – 1000 pg/mg R2 > 0.9999 Midazolam in oral fluid 0.5 – 40 ng/mL R2 > 0.9999 Quantitation results - linearity

21. a-OH-triazolam in urine – 10 ng/mL Midazolam in oral fluid – .5 ng/mL Nitrazepam in hair – 50 pg/mg Quantitation results - confirmation Alprazolam in blood – 1 ng/mL

22. Quantitation Summary Report

23. Quantitation results - reproducibility Immunalysis Inter- and intra-day variability

24. Quantitation results – transferability and reproducibility

25. Summary • Fast and sensitive LC/MS/MS technique using RRLC and QQQ • to meet customer needs for analyzing benzodiazepines • Easy method development optimizing only two parameters • using FIA. • Acquisition software easy to use for creating methods • Methods transferable to other customers • Processing of data in Quantitative Analysis is fast and detailed • - Recall fast method development, ion ratios for • confirmation, and CurveFit Assistant • Reporting capability though industry standard Excel