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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