Applications of Triple Quadrupole LC/MS/MS in a Forensic ...

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

General LC Chromatographic Conditions 

• LC Columns - Polar RP, 50 x 2mm, 4um 

- Luna C18(2), 50 x 2mm, 5um 

- Zorbax XDB-C8,150 x 4.6mm,5um 

• Mobile Phase: 

- MeOH or MeCN (30 – 85%) 

-Buffers–NH 4 Formate or NH 4 

Acetate (pH 3.0 – 7.4) 

General LCMS Conditions 

• Flow rates: 200 – 400 uL/min, isocratic 

• Injection volumes: 5 – 20 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 +/- 20% of 

closest standard 

• Curtain/Nebulizer/CAD gas – Nitrogen 

• Ion Spray Voltage – 5.0 kV 

• Dwell times – 200 to 500 msecs 

• Collision Energy (CE) – 25 - 35 

• Cell Exit Potential (CXP) – 3.0 

LCMS EXTRACTION METHOD 

• 1 mL Blood - Standard Curves, QC sample 

• 1 mL PM Blood, diluted (2 – 100X) 

• 1 mL pH10, Borate buffer + 5mL n-butyl-C 

• Evaporate/Reconst 60 uL MPH (~ 17x conc) 

• Hexane wash (if recovery good) 

• Typical calibration curves from 10 – 200 

ng/mL with control at 40 ng/mL.

FLURAZEPAM 

MIDAZOLAM 

7-AmFLUNITRAZ 

7AmCLON 

Αl-OH-ALPRAZ-d5 

CLONAZ 

ALPRAZ 

TRIAZOLAM 

TEMAZEPAM 

desalkFLURAZ 

Y=0.0248x + 0.00817 (r 2 = 0.9998) 

XIC of +MRM (11 pairs): 150.0/119.0 amu from Sample 2 (Pos Ctrl Mix) of Data2006Mar20 Am... 

Max. 7000.0 cps. 

AMPHETAMINE EXTRACTION METHOD 

1.4e4 

1.3e4 

1.2e4 

EPHEDRINE/PSEUDO 

• 1 mL PM Blood (diluted) + 0.020 mL IS(s) 

• 0.20 mL of 0.50N NaOH with vortex 

• 5 mL n-butyl-CL, rotate, spin and remove 

organic layer 

• Back-extract with 0.150 mL 0.10N Formic H + 

Intensity, cps 

1.1e4 

1.0e4 

9000.0 

8000.0 

7000.0 

6000.0 

5000.0 

4000.0 

3000.0 

METHAMPHET 

1.97 

PHENTERMINE 

MDA 

• Inject 5 uL 

2000.0 

1000.0 

AMPHET 

MDMA 

0.0 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 

Time, min

ZOLPIDEM EXTRACTION METHOD 

ZOLPIDEM LCMS CHROMO 

• 0.50 mL Blood – Standards, QC 

• 0.50 mL PM Blood, diluted appropriately 

• Add 10 uL IS (zolp-d6) 

• 0.100 mL aliquot to 2 mL PPC tube 

• 0.100 mL pH10, Borate buff + 0.600 mL n-butyl 

• Evaporate/Reconst 100 uL MPH 

• Inject 5 uL 

XIC of +MRM(3 pairs): 308.4/235.0 amu fromSample 7 (blk blood) of Data2006Sep20 Zolpidem... 

53 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0.11 

308 235 

308 263 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 

Time, min 

5.04 

Max. 53.3 cps. 

1.62 2.61 

0.24 4.82 

0.79 0.88 0.49 2.54 

4.57 4.71 

4.02 5.40 

1.23 6.10 

1.53 5.89 

3.39 3.64 4.50 

5.77 

3.02 1.832.21 6.36 

2.05 

3.34 

1.40 3.84 5.56 

XICof +MRM(3 pairs): 314.0/236.0 amu fromSample 2 (10) of Data2006Sep20 ZolpidemQua... 

Max. 3320.0 cps. 

3320 

3200 

3000 

2800 

2600 

2400 

2200 

50 pg on column 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 


5.17 

THC EXTRACTION PROCEDURE 

• 1mL blood (20uL IS) + 2 mL MeCN 

• Add 5 mL 0.1M, pH 6 NH 4 Acetate Buffer 

• Load Utak- 200mg/10mL ZSTHC SPE 

THC EXTRACTION PROCEDURE 

• Elute THC-COOH with Hexane:ETOAc:HOAc 

(75:25:1), Dry N 2 

• Reconstituted 50 uL MPh, Inject 20 uL 

• Wash SPE with buffer, Dry 10 min vacuum 

• Elute THC with Hexane/EtOAc (95:5) 

• Wash SPE - 4 mL MeOH/H 2 0 (1:1), Dry 10 

min vacuum 

• MPhase - MeOH: 4 mM, pH4 NH 4 Formate 

(85:15) 

• Column – Phenomenex Luna C18(2) 

• APCI ion source

Sample Name: "2/5" Sample ID: "" File: "Data2006Aug31 THC Quant.wiff" 

Sample Index: 2 

Sample Type: Standard 

Concentration: 5.00 ng/mL 

Calculated Conc: 5.27 ng/mL 

Acq. Date: 08/31/06 

Acq. Time: 03:34:57 PM 

Modified: Yes 

Bunching Factor: 1 

Noise Threshold: 1.53 cps 

Area Threshold: 7.63 cps 

Num. Smooths: 1 

RT Window: 30.0 sec 

Expected RT: 1.27 min 

Sep. Width: 0.20 

Sep. Height: 0.01 

Exp. Peak Ratio: 5.00 

Exp. Adj. Ratio: 4.00 

Exp. Val. Ratio: 3.00 

Use Relative RT: No 

Int. Type: Base To Base 

Retention Time: 1.21 min 

Area: 9.93e+002 counts 

Height: 1.16e+002 cps 

Start Time: 1.03 min 

End Time: 1.47 min 




Calculated Conc: 0.00 ng/mL 

Acq. Date: 08/31/06 

Acq. Time: 03:34:57 PM 

Modified: Yes 













Int. Type: Manual 









Calculated Conc: N/A 

Acq. Date: 08/31/06 

Acq. Time: 03:34:57 PM 

Modified: Yes 













Int. Type: Manual 









Calculated Conc: N/A 

Acq. Date: 08/31/06 

Acq. Time: 03:34:57 PM 

Modified: Yes 













Int. Type: Base To Base 







Peak Name: "THCCOOH299.1" Mass(es): "345.1/299.1 amu" 

Peak Name: "THCCOOH-D9" Mass(es): "354.1/308.1 amu" 

Comment: "" Annotation: "" 


1.22 

1.17 

125 

199 

120 

190 

115 

180 

110 

170 

105 

160 

100 

THC-COOH 

THCCOOH-D9 

95 

150 

90 

140 

85 

130 

80 

75 

345 299 

120 354 308 

70 

110 

65 

100 

60 

345 327 

90 

55 

50 

80 

45 

70 

40 

60 

35 

50 

30 

25 

40 

0.68 

20 

1.51 

30 

15 

20 

3.57 

10 

0.39 0.59 

10 

1.71 2.15 

5 

2.44 3.27 3.81 

0 

0 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 





Peak Name: "THC 259.1" Mass(es): "315.1/259.1 amu" 

Peak Name: "THC-D3" Mass(es): "318.1/196.1 amu" 



2.30 

2.25 

124 

2700 

120 

2600 

115 

2500 

110 

THC 

2400 

THC-D3 

105 

2300 

100 

2200 

95 

2100 

90 

2000 

315 193 

1900 

85 

318 196 

1800 

80 

1700 

75 

315 259 

1600 

70 

1500 

65 

1400 

60 

1300 

55 

1200 

50 

1100 

45 

1000 

40 

900 

800 

35 

1.51 

700 

30 

600 

25 

0.73 

0.39 

1.17 3.03 4.25 

3.81 4.64 

500 

20 

3.27 

4.10 

1.76 

400 

15 

300 



10 



200 

THC Standard Curve Stats 

Analyte DoA slope r^2 

THC 6/20/06 0.073 0.0992 

8/2/06 0.079 0.9995 

8/29/06 0.072 0.9997 

9/27/06 0.072 0.9902 

Mean: 0.0740 

StdDev: 0.003 

RSD(%): 4.5 

Analyte DoA slope r^2 

THC-COOH 6/20/06 0.207 0.0992 

8/2/06 0.244 0.9995 

8/29/06 0.256 0.9997 

9/27/06 0.210 0.9902 

Mean: 0.229 

StdDev: 0.024 

RSD(%): 10.7 

5 

0 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 


100 

0 

0.93 1.61 

1.86 

3.47 

4.05 4.93 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 


XIC of +MRM (3 pairs): 172.0/137.1 amu from Sample 5 (5Gaba) of DataGabapentin8_14_03.w... 

Max. 4027.0 cps. 

GABAPENTIN EXTRACTION METHOD 

1.29e4 

1.20e4 

Baclofen (IS) MRM 214/150 

• 50 μL sample PPC tube 

1.10e4 

1.00e4 

9000.00 

Gabapentin 

5mg/L 

•Add 400 μL acetonitrile containing 5 mg/L baclofen (IS). 

8000.00 

MRM 

•Centrifuge - transfer supernatant 

•Dry under nitrogen at 50 °C 

7000.00 

6000.00 

5000.00 

172/154 

172/137 

•Reconstitute in 100 μL mobile phase -- inject 5 μL 

4000.00 

1.41 

3000.00 

2000.00 

* Protein precipitation methods may cause ion suppression 

1000.00 

0.00 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 


XIC of +MRM (7 pairs): 300.1/215.1 amu from Sample 12 (03-1080UrineOpiatesBackExt) of Dat... 

Max. 7620.0 cps. 

LCMS Opiate Prelim. Experiments 

• Heroin 6MAM Morphine Codeine 

• Lengthy extraction, derivitize with TMS 

2.8e4 

2.6e4 

2.4e4 

2.2e4 

2.0e4 

1.8e4 

1.6e4 

Case urine opiate 

liquid-liquid 

extraction with back 

extract 

Morphine 

6-acetylmorphine 

CYP 2D6 

• Codeine Morphine 

1.4e4 

1.2e4 

1.0e4 

Codeine 

8000.0 

7.06 

• 2D6 Polymorphism – SubTher vs Toxic 

6000.0 

4000.0 

2000.0 

0.0 

1 2 3 4 5 6 7 8 9 10 11 


XIC of +MRM (3 pairs): 286.1/165.1 amu from Sample 6 (03-1111UrineSPE) of DataStdTestsE... 

Max. 2372.0 cps. 

XIC of +MRM (3 pairs): 286.1/165.1 amu from Sample 7 (03-1111Blood300uL) of DataStdTests... 

Max. 3158.0 cps. 

9830 

9500 

9000 

8500 

8000 

7500 

7000 

6500 

6000 

5500 

5000 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

Morphine-3- 

glucuronide 


glucuronide 

5.23 

Case urine SPE for morphine 

and morphine glucuronides 

~ 85:15 water:acetonitrile 

• Agilent Zorbax Eclipse XDB- 

C8 150 mm x 4.6 mm 5 μm 

particle size 


1 2 3 4 5 6 7 8 9 10 11 


3158 

3000 

2800 

2600 

2400 

2200 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 

0 


glucuronide 


glucuronide 

5.23 

Case blood for morphine and 

morphine glucuronides 

•Zorbax Eclipse XDB-C8 

150 mm x 4.6 mm; 5 μm 


1 2 3 4 5 6 7 8 9 10 11 


LCMS OPIATES 

TIC: from Sample 7 (50) of DataBlood Opiate parallel 061606.wiff (Turbo Spray) 

1.6e5 

1.5e5 

Timothy D. Dahn,Technical Director 

American Institute of Toxicology 

Max. 1.6e5 cps. 

4.01Hydrocodone 

GCMS vs LCMS CORRELATION 

Blood Opiate Correlation 


1.4e5 

1.3e5 

1.2e5 

1.1e5 

1.0e5 

9.0e4 

8.0e4 

7.0e4 

6.0e4 

5.0e4 

4.0e4 

3.0e4 

2.0e4 

1.0e4 

Codeine 3.68 

Oxycodone 3.86 

6-MAM 

Oxymorphone 

3.13 


1.91 

Hydromorphone 

2.53 

GC/MS (ng/ml) 

350 

300 

250 

200 

150 

100 

50 

0 

y = 0.8402x + 10.598 

R 2 = 0.915 

0 100 200 300 400 

LC/MSMS (ng/ml) 

0.0 

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 


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 

• (+/- 20%) 

• 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

LCMS TARGETED SCREENS 

ED Drug - Herbal Analogs 

• High probability drug interactions, P450 

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

Applications of Triple Quadrupole LC/MS/MS in a Forensic ...

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