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LC/MS/MS Analysis of in Urine, Oral Fluid and Blood Julie Cichelli, Rory M Doyle MSACL 2015 Agilent Technologies, Inc., Wilmington, DE Poster #32

Introduction Experimental Results and Discussion

Barbiturates are still analyzed in forensic laboratories today. Therefore in this case, we Method Precision/ Specificity developed an LC/MS/MS analytical method and evaluated various columns and HPLC Conditions- The intra–assay precision (%CV) of the barbiturates in each matrix were determined combinations in order to demonstrate the chromatography separation, Agilent 1260 Infinity HPLC series binary pump, well plate, thermostatted column by extracting and quantifying five replicates of the Quad-level QC material from UTAK detection and quantification of barbiturates in urine, oral fluid and blood for compartment resulting in a mean of 14.5 ng/mL, 305 ng/mL, 758 ng/mL and 1525 ng/mL for urine and blood. QC Level 1 was not observed in the oral fluid. The inter-assay precision , , , hexobarbital, , , Column: Agilent Technologies Zorbax Eclipse Plus C18, and . The sample preparation choices were kept simple and was determined over 5 consecutive days and was found to have a %CV <10% for 2.1 x 50 mm, 1.8 µm each barbiturate for Level 1, 2, 3 and 4 of 15, 300, 750 and 1500 ng/mL respectively included dilute and shoot for urine and oral fluid and protein crash for blood and the o methodologies were developed on an Agilent 1260 Infinity HPLC and 6460 Triple Column Temperature: 55 C in urine and blood but Level 1 was not observed in oral fluid. Quadrupole LC/MS with a 6 minute analytical gradient method with 1.5 min post- Injection Volume: 20 µL (urine, oral fluid), 10 µL (blood) o Accuracy equilibration time in negative ionization mode. Autosampler Temperature: 4 C The accuracy was determined by the analysis of the Quad-level UTAK QC material as Needle Wash: Flush port (50%:50%) 10 seconds the percentage deviation from the targeted mean and the results were <10% for all Mobile Phase A: 5mM Ammonium Acetate in Water levels in each matrix. Therefore, the analytical method in negative mode can achieve Mobile Phase B: Acetonitrile the required levels for the analysis of barbiturates particularly in urine and blood Flow Rate: 0.65 mL/min while oral fluid may need a better sample preparation cleanup. Gradient: 0 min- 82%A:18%B Table 3- Chromatograms 4.2 min- 82%A:18%B 4.3 min- 5%A:95%B 5.5 min- 5%A:95%B 5.6 min- 82%A:18%B Run/Stop time: 6 min/1.5 min

MS Conditions- Urine 100 ng/mL Agilent 6460 Triple Quadruple LC/MS- Dynamic MRM Amobarbital Butabarbital Butalbital Ion mode: Agilent JetStream Negative Mode Gas Temperature: 120oC Gas Flow: 8 L/min Nebulizer: 20 psi Sheath Gas Temperature: 400oC Sheath Gas Flow: 12 L/min Capillary Voltage: 400 V Oral Fluid 100 ng/mL Nozzle Voltage: 5 V Q1/Q2 Resolution: Wide/Unit Delta EMV: -600 V Cell Accelerator Voltage: 2 Methohexital Pentobarbital Phenobarbital RMTable 1: M Acquisition Table- * Quantifier Ion

Compound Rt MRM Fragmentation Collision min Transitions Voltage Energy Blood 100 ng/mL Amobarbital 4.37 225.1 - 182.1/42.2* 77 8/16 Amobarbital-D5 4.29 230.2 - 42.2* 77 20 Secobarbital Hexobarbital Butabarbital 1.8 211.1 – 168/42.2* 77 8/16 Cpd 1: Phenobarbital: -ESI MRM Frag=72.0V CF=0.000 DF=0.000 [email protected] (231.1 -> 42.2) 500 ngml-U.d Smooth x10 5 Butabital 2.34 223.1 – 180/42.2* 77 8/16 1 1 1.551 473097 Butalbital-D5 2.31 228.1 – 42.2 77 16 0 Cpd 3: Butabarbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (211.1 -> 42.2) 500 ngml-U.d Smooth Experimental x10 5 Hexobarbital 3.64 235.1 – 42.2* 77 16 1 1

1 1.789 428793 Methohexital 5.16 261.1 42.2* 77 16 Reagents, Standards, Calibrators and Controls 0 Cpd 5: Butalbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (223.1 -> 42.2) 500 ngml-U.d Smooth Standard/Calibrators- Cerilliant Methohexital-D5 5.16 266.2 – 42.2 82 12 x10 5 µ 1 1 Amobarbital: 1 mg/mL Amobarbital-D5: 100 g/mL 1 2.334 588640 Butabarbital: 1 mg/mL Butalbital-D5: 100 µg/mL Pentobarbital 4.09 225.1 – 182/42.2* 77 8/16 0 µ 5 Cpd 6: Hexobarbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (235.1 -> 42.2) 500 ngml-U.d Smooth Butalbital: 1 mg/mL Methohextial-D5: 100 g/mL Pentobarbital-D5 3.97 230.2 – 42.2 77 20 x10 Methohexital: 1 mg/mL Phenobarbital-D5: 100 µg/mL 1 1 1 Pentobarbital: 1 mg/mL Pentobarbital-D5: 100 µg/mL Phenobarbital 1.56 231.1 – 188/42.2* 72 4/16 3.629 544273 0 Phenobarbital: 1 mg/mL Secobarbital-D5: 100 µg/mL Cpd 8: Pentobarbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (225.1 -> 42.2) 500 ngml-U.d Smooth Phenobarbital-D5 1.53 236.1 – 42.2 77 12 x10 4 Hexobarbital: 1 mg/mL 1 1 Secobarbital: 1 mg/mL Secobarbital 5.09 237.1 – 194/42.2* 77 8/16 4.083 417271 4.349 533844

0 Secobarbital-D5 5.09 242.2 – 42.2 77 16 4 Cpd 10: Amobarbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (225.1 -> 42.2) 500 ngml-U.d Smooth Reagents x10 Acetonitrile: Honeywell Ammonium Acetate: Sigma-Aldrich 1 1 4.077 402648 4.347 517682 Isopropanol: Honeywell Methanol: Honeywell Results and Discussion 0 Cpd 12: Secobarbital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (237.1 -> 42.2) 500 ngml-U.d Smooth x10 5

Sample Preparation- Urine 1 1 • f100 µL o urine sample, calibrators, controls was taken and 10 µL of ISTD at 1000 2.5 5.093 418023 Table 2- Sensitivity ng/mL were added to each and vortexed briefly 0 Cpd 14: Methohexital: -ESI MRM Frag=77.0V CF=0.000 DF=0.000 [email protected] (261.1 -> 42.2) 500 ngml-U.d Smooth • f890 µL o HPLC grade water was added to each tube and vortexed for 1 min prior x10 5 1 1 2 to centrifugation for 10 minutes at 13000 rpm Compound LOD LOD LOD LOQ LOQ LOQ 5.165 292386 • The supernatant was transferred to an MS vial (ng/mL) Urine Oral Fluid Blood Urine Oral Fluid Blood 0 • All in-house calibrators were prepared in drug-free urine (Golden West Biological, 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 Inc) Amobarbital 10 20 10 25 40 25 Counts vs. Acquisition Time (min) Sample Preparation- Oral Fluid Butabarbital 5 20 5 10 20 10 • 100 µL of oral fluid sample solution (25 µL of oral fluid and 75 µL of buffer), Butalbital 5 20 5 10 40 10 calibrators, controls was taken and 2.5 µL of ISTD at 1000 ng/mL were added to Conclusions each and vortexed briefly Hexobarbital 5 20 5 10 20 10 µ • 397.5 L of HPLC grade water was added to each tube and vortexed for 1 min prior Methohexital 2.5 5 2.5 5 10 5 to centrifugation for 10 minutes at 13000 rpm • Baseline separation of the barbiturates particularly amobarbital and pentobarbital in • The supernatant was transferred to an MS vial Pentobarbital 10 20 10 25 40 25 under 6 minutes with good LOD/LOQ in negative mode was achieved in three • All in-house calibrators were prepared in negative oral fluid (Immunalysis, Inc) Phenobarbital 5 20 10 10 20 10 different matrix types • Simple sample preparation in three matrixes (urine, oral fluid and blood) achieved Sample Preparation- Blood Secobarbital 2.5 5 10 5 10 10 • f100 µL o blood sample, calibrators, controls was taken and 10 µL of ISTD at 1000 desirable LOD/LOQ but oral fluid requires better sample cleanup to achieve lower ng/mL were added to each and vortexed briefly sensitivity. • 100f µL o HPLC grade Acetonitrile was added to each tube and vortexed for 1 min Linearity/Sensitivity • Excellent linearity of calibration curves with acceptable accuracy, precision and prior to centrifugation for 10 minutes at 13000 rpm The linear range of the barbiturates in each matrix was from 10 to 5000 ng/mL for reproducibility in negative mode was achieved in all matrices <10% for %CV. • The supernatant was transferred to an MS vial and 400 µL of HPLC grade water urine and blood and from 25 to 5000 ng/mL for oral fluid. The linearity of each matrix • Further work to improve the peak shape of the barbiturates while still maintaining was added was determined in triplicate over 5 days and the results are shown with LOD and the baseline separation using sub-2 µm core shell columns • All in-house calibrators were prepared in Drug-free blood (Golden West Biological, LOQ being determined as 3:1 and 10:1 of signal to noise respectively where possible • Further evaluate different sample preparation techniques to determine which gives Inc) and the mean coefficient of determination (R2) > 0.99 for each matrix and the %CV the best results while maintaining low cost and ease of use. for each calibration point were all <10%. For Forensic Use.