LCMS APPLICATIONS TCFS LAB. Applications of Triple Quadrupole LC/MS/MS in a Forensic Toxicology Laboratory CURRENT LCMS ANALYTES
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1 Applications of Triple Quadrupole LC/MS/MS in a Forensic Toxicology Laboratory Jinee D. Rizzo, Ph.D. Travis County Forensic Center Austin, Texas jinee.rizzo@co.travis.tx.us LCMS APPLICATIONS TCFS LAB Simplify or enhance GC methods: Omit derivatization - Amphetamines Improve detection limit Zolpidem Short extract/analyze time Benzodiazepines Targeted analysis - Qualitative screens DFSA drugs zolpidem, eszopiclone, etc. ED drugs sildenafil, vardenafil, tadalafil CURRENT LCMS ANALYTES Benzodiazepines Amphetamines Quetiapine Olanzapine Trazodone Paroxetine Fluoxet/norflux Verapamil Propoxy/norprop Buproprion Zolpidem Warfarin Topiramate THC/THCCOOH Eszopiclone Aripiprazole Sildenafil, etc
2 General LC Chromatographic Conditions LC Columns - Polar RP, 5 x 2mm, 4um - Luna C18(2), 5 x 2mm, 5um - Zorbax XDB-C8,15 x 4.6mm,5um Mobile Phase: - MeOH or MeCN (3 85%) -Buffers NH 4 Formate or NH 4 Acetate (ph ) General LCMS Conditions Flow rates: 2 4 ul/min, isocratic Injection volumes: 5 2 ul Column Temp : 35 o C Electrospray Ionization(ESI) (+) Polarity THC - Atmospheric Pressure Chemical Ionization (APCI) General MS Parameters Detection: MRM mode - 2 transitions Quant/Qualifier ion ratios +/- 2% of closest standard Curtain/Nebulizer/CAD gas Nitrogen Ion Spray Voltage 5. kv Dwell times 2 to 5 msecs Collision Energy (CE) Cell Exit Potential (CXP) 3. LCMS EXTRACTION METHOD 1 ml Blood - Standard Curves, QC sample 1 ml PM Blood, diluted (2 1X) 1 ml ph1, Borate buffer + 5mL n-butyl-c Evaporate/Reconst 6 ul MPH (~ 17x conc) Hexane wash (if recovery good) Typical calibration curves from 1 2 ng/ml with control at 4 ng/ml.
3 FLURAZEPAM MIDAZOLAM 7-AmFLUNITRAZ 7AmCLON Αl-OH-ALPRAZ-d5 CLONAZ ALPRAZ TRIAZOLAM TEMAZEPAM desalkfluraz Y=.248x (r 2 =.9998) XIC of +MRM (11 pairs): 15./119. amu from Sample 2 (Pos Ctrl Mix) of Data26Mar2 Am... Max. 7. cps. AMPHETAMINE EXTRACTION METHOD 1.4e4 1.3e4 1.2e4 EPHEDRINE/PSEUDO 1 ml PM Blood (diluted) +.2 ml IS(s).2 ml of.5n NaOH with vortex 5 ml n-butyl-cl, rotate, spin and remove organic layer Back-extract with.15 ml.1n Formic H + 1.1e4 1.e METHAMPHET 1.97 PHENTERMINE MDA Inject 5 ul AMPHET MDMA
4 ZOLPIDEM EXTRACTION METHOD ZOLPIDEM LCMS CHROMO.5 ml Blood Standards, QC.5 ml PM Blood, diluted appropriately Add 1 ul IS (zolp-d6).1 ml aliquot to 2 ml PPC tube.1 ml ph1, Borate buff +.6 ml n-butyl Evaporate/Reconst 1 ul MPH Inject 5 ul XIC of +MRM(3 pairs): 38.4/235. amu fromsample 7 (blk blood) of Data26Sep2 Zolpidem Max cps XICof +MRM(3 pairs): 314./236. amu fromsample 2 (1) of Data26Sep2 ZolpidemQua... Max cps pg on column THC EXTRACTION PROCEDURE 1mL blood (2uL IS) + 2 ml MeCN Add 5 ml.1m, ph 6 NH 4 Acetate Buffer Load Utak- 2mg/1mL ZSTHC SPE THC EXTRACTION PROCEDURE Elute THC-COOH with Hexane:ETOAc:HOAc (75:25:1), Dry N 2 Reconstituted 5 ul MPh, Inject 2 ul Wash SPE with buffer, Dry 1 min vacuum Elute THC with Hexane/EtOAc (95:5) Wash SPE - 4 ml MeOH/H 2 (1:1), Dry 1 min vacuum MPhase - MeOH: 4 mm, ph4 NH 4 Formate (85:15) Column Phenomenex Luna C18(2) APCI ion source
5 Sample Name: "2/5" Sample ID: "" File: "Data26Aug31 THC Quant.wiff" Sample Index: 2 Sample Type: Standard Concentration: 5. ng/ml Calculated Conc: 5.27 ng/ml Acq. Date: 8/31/6 Acq. Time: 3:34:57 PM Modified: Yes Bunching Factor: 1 Noise Threshold: 1.53 cps Area Threshold: 7.63 cps Num. Smooths: 1 RT Window: 3. sec Expected RT: 1.27 min Sep. Width:.2 Sep. Height:.1 Exp. Peak Ratio: 5. Exp. Adj. Ratio: 4. Exp. Val. Ratio: 3. Use Relative RT: No Int. Type: Base To Base Retention Time: 1.21 min Area: 9.93e+2 counts Height: 1.16e+2 cps Start Time: 1.3 min End Time: 1.47 min Sample Index: 2 Sample Type: Standard Concentration:. ng/ml Calculated Conc:. ng/ml Acq. Date: 8/31/6 Acq. Time: 3:34:57 PM Modified: Yes Bunching Factor: 1 Noise Threshold: 2.17 cps Area Threshold: 1.84 cps Num. Smooths: 1 RT Window: 3. sec Expected RT: 2.49 min Sep. Width:.2 Sep. Height:.1 Exp. Peak Ratio: 5. Exp. Adj. Ratio: 4. Exp. Val. Ratio: 3. Use Relative RT: No Int. Type: Manual Retention Time: 2.32 min Area: 1.36e+3 counts Height: 1.16e+2 cps Start Time: 2.1 min End Time: 2.54 min Sample Index: 2 Sample Type: Standard Concentration: 1. ng/ml Calculated Conc: N/A Acq. Date: 8/31/6 Acq. Time: 3:34:57 PM Modified: Yes Bunching Factor: 1 Noise Threshold: 1. cps Area Threshold: 9.48 cps Num. Smooths: 1 RT Window: 3. sec Expected RT: 1.23 min Sep. Width:.2 Sep. Height:.1 Exp. Peak Ratio: 5. Exp. Adj. Ratio: 4. Exp. Val. Ratio: 3. Use Relative RT: No Int. Type: Manual Retention Time: 1.18 min Area: 1.7e+3 counts Height: 1.95e+2 cps Start Time:.977 min End Time: 1.47 min Sample Index: 2 Sample Type: Standard Concentration: 1. ng/ml Calculated Conc: N/A Acq. Date: 8/31/6 Acq. Time: 3:34:57 PM Modified: Yes Bunching Factor: 1 Noise Threshold: 1.38 cps Area Threshold: cps Num. Smooths: 1 RT Window: 3. sec Expected RT: 2.44 min Sep. Width:.2 Sep. Height:.1 Exp. Peak Ratio: 5. Exp. Adj. Ratio: 4. Exp. Val. Ratio: 3. Use Relative RT: No Int. Type: Base To Base Retention Time: 2.27 min Area: 3.29e+4 counts Height: 2.83e+3 cps Start Time: 2. min End Time: 2.83 min Peak Name: "THCCOOH299.1" Mass(es): "345.1/299.1 amu" Comment: "" Annotation: "" THC-COOH Sample Name: "2/5" Sample ID: "" File: "Data26Aug31 THC Quant.wiff" Peak Name: "THC 259.1" Mass(es): "315.1/259.1 amu" Comment: "" Annotation: "" THC Sample Name: "2/5" Sample ID: "" File: "Data26Aug31 THC Quant.wiff" Peak Name: "THCCOOH-D9" Mass(es): "354.1/38.1 amu" Comment: "" Annotation: "" Sample Name: "2/5" Sample ID: "" File: "Data26Aug31 THC Quant.wiff" Peak Name: "THC-D3" Mass(es): "318.1/196.1 amu" Comment: "" Annotation: "" THCCOOH-D THC-D THC Standard Curve Stats Analyte DoA slope r^2 THC 6/2/ /2/ /29/ /27/ Mean:.74 StdDev:.3 RSD(%): 4.5 Analyte DoA slope r^2 THC-COOH 6/2/ /2/ /29/ /27/ Mean:.229 StdDev:.24 RSD(%): XIC of +MRM (3 pairs): 172./137.1 amu from Sample 5 (5Gaba) of DataGabapentin8_14_3.w... Max cps. GABAPENTIN EXTRACTION METHOD 1.29e4 1.2e4 Baclofen (IS) MRM 214/15 5 μl sample PPC tube 1.1e4 1.e4 9. Gabapentin 5mg/L Add 4 μl acetonitrile containing 5 mg/l baclofen (IS). 8. MRM Centrifuge - transfer supernatant Dry under nitrogen at 5 C / /137 Reconstitute in 1 μl mobile phase -- inject 5 μl * Protein precipitation methods may cause ion suppression
6 XIC of +MRM (7 pairs): 3.1/215.1 amu from Sample 12 (3-18UrineOpiatesBackExt) of Dat... Max cps. LCMS Opiate Prelim. Experiments Heroin 6MAM Morphine Codeine Lengthy extraction, derivitize with TMS 2.8e4 2.6e4 2.4e4 2.2e4 2.e4 1.8e4 1.6e4 Case urine opiate liquid-liquid extraction with back extract Morphine 6-acetylmorphine CYP 2D6 Codeine Morphine 1.4e4 1.2e4 1.e4 Codeine D6 Polymorphism SubTher vs Toxic XIC of +MRM (3 pairs): 286.1/165.1 amu from Sample 6 (3-1111UrineSPE) of DataStdTestsE... Max cps. XIC of +MRM (3 pairs): 286.1/165.1 amu from Sample 7 (3-1111Blood3uL) of DataStdTests... Max cps Morphine-3- glucuronide Morphine-6- glucuronide 5.23 Case urine SPE for morphine and morphine glucuronides ~ 85:15 water:acetonitrile Agilent Zorbax Eclipse XDB- C8 15 mm x 4.6 mm 5 μm particle size Morphine Morphine-3- glucuronide Morphine-6- glucuronide 5.23 Case blood for morphine and morphine glucuronides Zorbax Eclipse XDB-C8 15 mm x 4.6 mm; 5 μm Morphine
7 LCMS OPIATES TIC: from Sample 7 (5) of DataBlood Opiate parallel 6166.wiff (Turbo Spray) 1.6e5 1.5e5 Timothy D. Dahn,Technical Director American Institute of Toxicology Max. 1.6e5 cps. 4.1Hydrocodone GCMS vs LCMS CORRELATION Blood Opiate Correlation 1.4e5 1.3e5 1.2e5 1.1e5 1.e5 9.e4 8.e4 7.e4 6.e4 5.e4 4.e4 3.e4 2.e4 1.e4 Codeine 3.68 Oxycodone MAM Oxymorphone 3.13 Morphine 1.91 Hydromorphone 2.53 GC/MS (ng/ml) y =.842x R 2 = LC/MSMS (ng/ml) Method Validation Parameters Establish the: Linear range including LLOQ and ULOQ Calibration curves 4-5 calibr points DAY 1 6 replicates of each conc.,1 blkblk,1-blkis DAY 2 and 3 duplicates of each conc. Inter/Intraday Accuracy and Precision (+/- 2%) 3 non-consecutive days Stability of Drug (literature refer useful) Method Validation Parameters Interference Studies Matrix effects, ion suppression (6 PM bloods,? anticoags) Specificity with most common analytes Autosampler stability Single reinjection of curve Quality Control Samples 3 per day Outside source In house prepared
8 LCMS TARGETED SCREENS ED Drug - Herbal Analogs High probability drug interactions, P45 inhibitors Antibiotics Erythromycin,Ciprofloxin Antifungals Keto and Fluconazole Antiarrythmia Verapamil, Diltiazem Narrow Therapeutic Index Digoxin, Theophylline ED drugs Silendifil, Tadalafil, Vardenafil DFSA Zolpidem, Eszopiclone, Summary of LCMS in Forensics Good for polar, nonvolatile cpds No derivitization, adequate sensitivity Benzodiapzepines Amphetamines THC, Opitaes Simplify extraction procedures One solvent extract or Protein ppt Method Validation Targeted and NonTargeted Screens Acknowledgements THC SPE method Adapted from Dallas County Forensic Sciences THC and Benz LC conditions, APCI transitions Pascal Mireault, Pierre Picotte, Laboratoire de Sciences judiciaires, Montreal, Canada
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