©2013 MFMER | slide-1

High-Throughput Analysis of Five Therapeutic Drug Monitoring Assays using Ultra Fast SPE-MS/MSEric Korman

American Society for Mass SpectrometryJune 8, 2013

©2013 MFMER | slide-2

Disclosures• Relevant Financial Relationship(s):

None

• Off Label Usage:None

©2013 MFMER | slide-3

Outline• Introduction• Extraction method using the RapidFire• Validation Data• Conclusions

©2013 MFMER | slide-4

Why the RapidFire vs. Turboflow?

• Non-endogenous compounds• High volume test• High drug concentrations

Why Therapeutic Drug Monitoring?

• Get patients their results as fast as possible• Free up instrumentation• Cost savings

©2013 MFMER | slide-5

High Volume TDM Assays in the Toxicology Lab• Lamotrigine - Anticonvulsant

• ~ 40,000 samples annually (110 daily)

• Levetiracetam - Anticonvulsant• ~ 60,000 samples annually (160 daily)

• Oxcarbazepine Metabolite (OMHC) - Anticonvulsant• ~ 18,000 samples annually (50 daily)

• Felbamate - Anticonvulsant• ~ 2,000 samples annually (6 daily)

• Voriconazole - Antifungal• ~ 10,000 samples annually (25 daily)

©2013 MFMER | slide-6

Extraction Method• 50 µL serum• Crash with 200 µL acetonitrile containing deuterium

labeled IS• Vortex/Centrifuge• Remove 30 µL of supernatant and dilute with

600 µL of mobile phase A.• Done on a Hamilton Starlet – 96 well plate format

©2013 MFMER | slide-7

Agilent RapidFire 300 and 6490 QQQ

©2013 MFMER | slide-8

©2013 MFMER | slide-9

©2013 MFMER | slide-10

©2013 MFMER | slide-11

©2013 MFMER | slide-12

RapidFire Methods

Step Lamotrigine Levetiracetam OMHC Felbamate Voriconazole

Inject 600 ms 600 ms 600 ms 600 ms 600 ms

Load 3500 ms 3500 ms 3500 ms 3500 ms 3500 ms

Wash 0 ms 0 ms 0 ms 0 ms 0 ms

Elute 3500 ms 4000 ms 4000 ms 4000 ms 3500 ms

Equil 500 ms 1000 ms 1000 ms 500 ms 1000 ms

Total (sec): 8.0 9.0 9.0 8.5 8.5 (x2) = 17

©2013 MFMER | slide-13

Standard Curve Responses

©2013 MFMER | slide-14

Validation Data

©2013 MFMER | slide-15

Lamotrigine LOQ (0.20 µg/mL)Blank

LOD

LOQ

©2013 MFMER | slide-16

Lamotrigine (n=100)

• Linear Range: 0.20 µg/mL – 25.0 µg/mL

©2013 MFMER | slide-17

Levetiracetam (n=100)

• Linear Range: 1.0 µg/mL – 100.0 µg/mL

©2013 MFMER | slide-18

OMHC (n=100)

• Linear Range: 1.0 µg/mL – 50.0 µg/mL

©2013 MFMER | slide-19

Felbamate (n=40)

• Linear Range: 5.0 µg/mL – 150.0 µg/mL

©2013 MFMER | slide-20

Voriconazole (n=100)

• Linear Range: 0.10 µg/mL – 20.0 µg/mL

©2013 MFMER | slide-21

Within Run Precision (n=20)

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

Lamotrigine Levetiracetam OMHC Felbamate Voriconazole

3.5%

2.5%2.0%

2.6%

3.3%3.8% 3.7%

1.5%

3.3% 3.3%

5.0%

2.2% 2.2%

5.3%

4.1%%C

V

Low QC Medium QC High QC

©2013 MFMER | slide-22

Between Run Precision (n=20)

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

10%

Lamotrigine Levetiracetam OMHC Felbamate Voriconazole

5.1%4.9%

3.5%

4.0%

4.6%

6.0% 5.8%

2.6%

5.0%

3.6%

6.0%

4.5%

2.0%

6.1%

4.6%

%C

V

Low QC Medium QC High QC

©2013 MFMER | slide-23

Compounds shown not to interfereDrugs Tested at 100 µg/mL for Interference AMPHS INCLUDE: BENZOS INCLUDE:

1 ACETOMINOPHEN 34 AZITHROMYCIN MDMA Oxazepam2 ATORVASTIN 35 ROSUVASTATIN MDA Nordiazepam3 AMOXICILLIN 36 CLOPIDOGREL Amphetamine Lorazepam4 LISINOPRIL 37 SIMVASTATIN Methamphetamine Hydroxyethyl-Flurazepam5 HYDROCHLOROTHIAZIDE 38 METOPROOL Pseudoephedrine a-Hydroxy-Alprazolam6 FUROSIMIDE 39 ARIPIPRAZOLE Phentermine a-Hydroxy-Triazolam7 ALPRAZOLAM 40 LEVOTHYROXINE Temazepam8 AMLODIPINE 41 COCAINE* BARBS INCLUDE: 7-Aminoclonazepam9 METFORMIN 42 LSD OXO LSD Pentobarbital 7-Aminoflunitrazepam

10 SERTRALINE 43 CATHINONE ANALOGS* Phenobarbital Clonazepam11 IBUPROFEN 44 FELBAMATE Secobarbital Diazepam12 CEPHALEXIN 45 CARBAMAZEPINE Butalbital Nordiazepam13 ZOLPIDEM 46 CLONZAPAM Amobarbital Midazolam14 PREDNISONE 47 GABAPENTIN Mephobarbital Chlordiazepoxide15 HYDROCODONE 48 OMHC Flurazepam16 ESOMEPRAZOLE 49 PHENYTOIN COCAINE INCLUDES: Flunitrazepam17 LORAZEPAM 50 PRIMIDONE Cocaine18 CITALOPRAM 51 TIAGABINE Benzoylecgonine OPIATES INCLUDE:19 PROXPOXYPHENE 52 TOPIRAMATE Norcocaine Hydromorphone20 WARFARIN 53 VALPROIC ACID m-Hydroxybenzoylecgonine Oxymorphone21 BUPIVICAINE 54 ZONISAMIDE Levamisole Oxycodone22 OPIATES* 55 POSACONAZOLE Cocaethylene Codeine23 BENZODIAZAPINES* 56 FLUCONAZOLE Morphine24 BARBS* 57 ITRACONAZOLE CATHINONE ANALOGS: 6-Monoacetylmorphine25 AMPHS* 58 OH-ITRACONAZOLE Methylone26 THC 59 LAMOTRIGINE Mephedrone27 FENTANYL 60 CLONAZEPAM MDPV28 NORFEENTANYL 61 MIDAZOLAM Ethylone29 METHADONE 62 CHLORDIAZEPOXIDE30 EDDP 63 DIAZEPAM31 PCP 64 NORDIAZEPAM32 KETAMINE 65 C101133 NORKETAMINE 66 LEVETERICETAM

©2013 MFMER | slide-24

What benefits did we realize?

©2013 MFMER | slide-25

Mass Spec Time Savings: 97%

0

100

200

300

400

500

600

700

Turbo-flow Method Rapidfire Method

650

18

Hou

rs p

er W

eek

QQQ Run Time

©2013 MFMER | slide-26

Estimated consumables annual cost savings: $205,000

Turboflow and analytical columns$97,000 Filter plates and

cap mats $85,000

Mobile phase reagents$23,000

©2013 MFMER | slide-27

Turn Around Time Comparison:

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

Lamotrigine Levetiracetam OMHC Felbamate Voriconizole

0.80 0.84

1.36

0.95

1.22

0.470.43

1.11

0.82

0.75

Day

s

Average TAT Before Average TAT After

©2013 MFMER | slide-28

Projects in Progress…• Immunosuppressants:

• Tacrolimus• Cyclosporin• Sirolimus• Everolimus

• Busulfan• Methotrexate• Lacosamide• Pregabalin• Rufinamide

©2013 MFMER | slide-29

Special Thanks Mayo ClinicLoralie Langman Ph.D.Paul Jannetto Ph.D.Frank Crow Ph.D.Robert EngerMatt BjergumDarlington DansoAmy AdlerMary Dallman

Agilent TechnologiesVaughn Miller Ph.D.Kari Schlicht Ph.D.Michelle Romm Ph.D.William Lamarr Ph.D.Maxine Jonas Ph.D.Patty Sun Ph.D.

©2013 MFMER | slide-30

Questions & Discussion