Major LC-MS Clinical Applications

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Neonatal developed with Chromsystems, Munich. • Immunosuppressant drugs developed with Recipe,. Munich. Please Note : The methods described in this ...
Agilent Clinical LC/MS:

Review of Established LC-MS QQQ Clinical Research Applications

dr. Jan Srbek, HPST Page 1

Major LC-MS Clinical Applications • Steroids/Endocrinology – Vitamin D – Testosterone, Estradiol and Cortisone – Thyroxin

• Neonatal/Metabolic Diseases – – – –

Amino Acids Fatty acid metabolites Glucose metabolites Organic compounds

• Therapeutic Monitoring – Immunosuppressant drugs – Pain Medications – Pharmacogenomic Profiling

• Drugs of Abuse/Toxicology – Opiates, Benzodiazepines, Amphetamines, Anabolic Steroids Page 2

Clinical applications developed so far… Today, it is possible able to implement 8 clinical applications at customer sites (including the sample preparation) : • Homocysteine in plasma • Drugs of Abuse in urine • THC and metabolites in whole blood • Methylmalonic Acid (MMA) in plasma • Cortisol in urine • Plasmatic Metanephrines (MN and NMN) • Neonatal developed with Chromsystems, Munich • Immunosuppressant drugs developed with Recipe, Munich Please Note : The methods described in this presentation are for research purposes only and not for diagnostic use.

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Clinical applications already available

PN 5990-5782EE

PN 5990-5785EE

PN 5990-5784EE

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Clinical applications already available – Sales Tools

PN 5990-5783EEE

PN 5990-5781EE

PN 5990-5426EE

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Therapeutic Drug Monitoring

2D LCMS/MS Method for the Analysis of Immunosuppressive Drugs in Blood : Tacrolimus, Sirolimus, Cyclosporin A and Everolimus

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Therapeutic Drug Monitoring TDM Immunosuppressants – 4 most commonly used substances • Cyclosporin A • Tacrolimus (also known as FK-506), • Sirolimus (Rapamycin) • Everolimus (RAD-001) • And others, not part of current method (e.g.Mycophenolic Acid (MPA) Current technologies used • Immunoassays • MEIA for SRL & TRL; FPIA & Innofluor® for RAD, CEDIA for SRL, Emit 2000 for TRL, ACMIA for CSA) • LCMS

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Why do customer adopt LC/MS/MS for immunosuppressant analysis?

Cost effectiveness (enormous cost savings vs Immunoassays) Specificity vs current immunoassays leading to less false positive results & more accurate quantitation Simultaneous detection and quantitation of 4 or more immunosuppressants, Fast turnaround time (~5 min per sample) New drugs require lower doses- thus higher sensitivity Easy to add new drugs to the list (vs. immunoassays) LC/MS/MS shows excellent reproducibility and dynamic range Very robust assay

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Method User Manual

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Method User Manual

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Components and Kit

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LC Equipment and Conditions LC Pumps Series

Pump A (Loading pump) - Isocratic pump 1200

Pump B (Elution pump) - Binary SL 1200 Series Column Switcher : 6 port switching valve Injection volume: 50 µL Autosampler temp: 4 ºC Column Temp:

60ºC (Analytical column)

Needle Wash:

Washing solution (MS1005)

Column:

Trapping Column Analytical Column

Starting Pump Flow:

SPE Column Analytical Column

0.1ml/min 0.5ml/min

Mobile Phase:

Trapping Column MP (MS1009) Analytical Column MP (MS1010)

Gradient:

Isocratic for both Stop time – 2 minutes

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Column Switching Setup Initial flow rates Pump A

0.1 ml/min

Pump B

0.5 ml/min

Column Switcher timetable

Flow rate Time (min)

0

Time (min)

Position

0

1

0.5

2

1.65

1

timetable Pump A

Pump B

Flowrate (ml/min)

Flowrate (ml/min)

0.5

0.01

0.1 2.5

0.5 1.3

0.5

1.35

1.0

1.5

2.5

1.55

1.0

1.65

0.5

1.8 2

0.1 0.5

0.1

NB – Diagram describes a 6 rather than a 10-port switching valve. The plumbing is similar with either system

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LC pump and column setup

Isocratic pump

Binary pump

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QQQ MRM Transitions

Analyte

Internal standard

Cyclosporine A

Cyclosporine D

Tacrolimus

Ascomycin

Everolimus

D4 - Everolimus

Sirolimus

D4 - Everolimus

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QQQ Conditions MS Conditions: Agilent 6460 QQQ QQQ MS conditions Ion Mode:

ESI Agilent JetStream, Positive

ESI conditions Drying Gas Temperature: 225 ºC Drying Gas Flow: 3 L/min Nebulizer Pressure: 35psi Sheath Gas Temperature: 325 ºC Sheath Gas Flow: 12 L/min Capillary: Nozzle Voltage:

4000 V 0V

QQQ EMV: Resolution:

500 V MS1 - Unit, MS2 – Unit

•The

selection of the spray chamber conditions for a multi-component method always involves a trade-off between the optimum settings of the individual compounds.

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Quantitative Results Calibration Levels Spiked into Whole Blood Sirolimus

Tacrolimus

Everolimus

Level

Conc. (ng/mL)

Level

Conc. (ng/mL)

Level

Conc. (ng/mL)

1

1.38

1

1.22

1

1.2

2

2.71

2

2.46

2

2.62

3

5.41

3

5.03

3

5.13

4

11.3

4

10.2

4

10.9

5

22.3

5

20.5

5

22.7

6

44.3

6

39.7

6

44.9

QC I

3.64

QC I

3.28

QC I

3.34

QC II

11.2

QC II

6.67

QC II

10.6

QC III

18.9

QC III

13.3

QC III

18.2

Cyclosporine A Level

Conc. (ng/ml)

1

24.3

2

48.4

3

92.4

4

187

5

472

6

1345

QC I

62.5

QC II

132

QC III

258

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Quantitative Results Sirolimus - Lowest Extracted Standard and Calibration Line Sirolimus

Lowest calibration standard – 1.38 ng/mL

I.S.

Linear fit with R2 = 0.9992

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Quantitative Results

Cyclosporine A

Tacrolimus

Sirolimus

Everolimus

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Opiates

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Propaxyphene

Amphetamine

EDDP

Benzoylecgonine Norfentnyl

Methaamphetamine

Fentanyl

Methadone

Norpropaxyphene

Tramadol

O-Desmethyltramadol

Oxycodone Hydrocodone

Codeine

Dihydrocodeine

Hydromorphone

Morphine Oxymorphone

19 Opiates 10 ng/mL Under 5 minutes

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47 Drugs of Abuse in 10 minutes 5

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List of 47 DOA Tested Compound Name Morphine Oxymorphone Hydromorphone Codeine Oxycodone 7-Aminonitrazepam 7-Aminoclonazepam Noroxycodone Hydrocodone 6-MAM Norhydrocodone Pregabalin Gabapentin 7-Aminoflunitrazepam O-Desmathyltremadol Bromazepam Norfentanyl Tramadol alpha-Hydroxytriazolam Hydroxyalprazolam Meperidine Normeperidine Desmethylflunitrazepa

RT Precursor Ion 1.60 286 1.74 302 1.91 286 2.51 300 2.71 316 2.77 252 2.78 286 2.84 302 2.85 300 2.93 328 2.96 286 3.16 160 3.20 172 3.27 284 3.27 250 4.08 316 4.09 233 4.28 264 4.53 359 4.54 325 4.66 248 4.79 234 4.80 300

Compound Name Oxazepam Estazolam Nitrazepam Lorazepam Alprazolam 2-hydroxyethylflurazepam Chlordiazepoxide Clonazepam Triazolam Norbuprenorphine Fentanyl Desalkylflurazepam Flunitrazepam EDDP Temazepam Meprobamate Nordiazepam Buprenorphine Midazolam Propoxyphane Diazepam Norpropoxyphane Methadone Prazepam

RT Precursor Ion 4.81 287 4.83 295 4.94 282 5.01 321 5.07 309 5.12 333 5.12 300 5.13 316 5.18 343 5.28 414 5.32 337 5.49 289 5.61 314 5.64 278 5.66 301 5.68 219 5.72 271 5.86 468 6.03 326 6.49 340 6.60 285 6.60 326 6.67 310 8.19 325

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Contact for applications [email protected]

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