Agilent Clinical LC/MS: Review of Established LC-MS QQQ Clinical Research Applications. dr. Jan Srbek, HPST. Page 1

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1 Agilent Clinical LC/MS: Review of Established LC-MS QQQ Clinical Research Applications dr. Jan Srbek, HPST Page 1

2 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

3 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. Page 3

4 Clinical applications already available PN EE PN EE PN EE Page 4

5 Clinical applications already available Sales Tools PN EEE PN EE PN EE Page 5

6 Therapeutic Drug Monitoring 2D LCMS/MS Method for the Analysis of Immunosuppressive Drugs in Blood : Tacrolimus, Sirolimus, Cyclosporin A and Everolimus Page 6

7 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 Page 7

8 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 Page 8

9 Method User Manual Page 9

10 Method User Manual Page 10

11 Components and Kit Page 11

LC Equipment and Conditions LC Pumps Pump A (Loading pump) - Isocratic pump 1200 Series Pump B (Elution pump) - Binary SL 1200 Series Column Switcher : 6 port switching valve Injection volume: 50 µl

12 LC Equipment and Conditions LC Pumps Pump A (Loading pump) - Isocratic pump 1200 Series Pump B (Elution pump) - Binary SL 1200 Series Column Switcher : 6 port switching valve Injection volume: 50 µl Autosampler temp: 4 ºC Column Temp: Needle Wash: 60ºC (Analytical column) Washing solution (MS1005) Column: Trapping Column Analytical Column Starting Pump Flow: SPE Column 0.1ml/min Analytical Column 0.5ml/min Mobile Phase: Gradient: Trapping Column MP (MS1009) Analytical Column MP (MS1010) Isocratic for both Stop time 2 minutes Page 12

Column Switching Setup Initial flow rates Pump A Pump B 0.1 ml/min 0.5 ml/min Column Switcher timetable Time (min) Position 0 1 0.5 2 1.

13 Column Switching Setup Initial flow rates Pump A Pump B 0.1 ml/min 0.5 ml/min Column Switcher timetable Time (min) Position Flow rate timetable Time (min) Pump A Flowrate(ml/min) Pump B Flowrate(ml/min) NB Diagram describes a 6 rather than a 10-port switching valve. The plumbing is similar with either system Page 13

14 LC pump and column setup Isocratic pump Binary pump Page 14

15 QQQ MRM Transitions Analyte Cyclosporine A Tacrolimus Everolimus Sirolimus Internal standard Cyclosporine D Ascomycin D4 - Everolimus D4 - Everolimus Page 15

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:

16 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: QQQ EMV: Resolution: 4000 V 0 V 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. Page 16

17 Quantitative Results Calibration Levels Spiked into Whole Blood Sirolimus Tacrolimus Everolimus Level Conc. (ng/ml) QC I 3.64 QC II 11.2 QC III 18.9 Level Conc. (ng/ml) QC I 3.28 QC II 6.67 QC III 13.3 Cyclosporine A Level Conc. (ng/ml) QC I 3.34 QC II 10.6 QC III 18.2 Level Conc. (ng/ml) QC I 62.5 QC II 132 QC III 258 Page 17

18 Quantitative Results Sirolimus - Lowest Extracted Standard and Calibration Line Sirolimus Lowest calibration standard 1.38 ng/ml I.S. Linear fit with R2 = Page 18

19 Quantitative Results Cyclosporine A Sirolimus Tacrolimus Everolimus Page 19

20 Opiates Page 20

21 19 Opiates 10 ng/ml Under 5 minutes Page 21 Morphine Oxymorphone Dihydrocodeine Codeine Hydromorphone Oxycodone Hydrocodone O-Desmethyltramadol Tramadol Norpropaxyphene Amphetamine Methaamphetamine Benzoylecgonine Norfentnyl Fentanyl EDDP Propaxyphene Methadone

22 47 Drugs of Abuse in 10 minutes 5 Page 22

23 List of 47 DOA Tested Compound Name RT Precursor Ion Morphine Oxymorphone Hydromorphone Codeine Oxycodone Aminonitrazepam Aminoclonazepam Noroxycodone Hydrocodone MAM Norhydrocodone Pregabalin Gabapentin Aminoflunitrazepam O-Desmathyltremadol Bromazepam Norfentanyl Tramadol alpha-hydroxytriazolam Hydroxyalprazolam Meperidine Normeperidine Desmethylflunitrazepa Compound Name RT Precursor Ion Oxazepam Estazolam Nitrazepam Lorazepam Alprazolam hydroxyethylflurazepam Chlordiazepoxide Clonazepam Triazolam Norbuprenorphine Fentanyl Desalkylflurazepam Flunitrazepam EDDP Temazepam Meprobamate Nordiazepam Buprenorphine Midazolam Propoxyphane Diazepam Norpropoxyphane Methadone Prazepam Page 23

24 Contact for applications Page 24

LCMS-8050 Drugs of Abuse: 113 Analytes with Polarity Switching

LCMS-8050 Drugs of Abuse: 113 Analytes with Polarity Switching Liquid Chromatography Mass Spectrometry SSI-LCMS-8 LCMS-8 Drugs of Abuse: Analytes with Polarity Switching LCMS-8 Summary Seventy six analytes and their internal standards are described below. Multiple