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. Page 3
Clinical applications already available PN 5990-5782EE PN 5990-5785EE PN 5990-5784EE Page 4
Clinical applications already available Sales Tools PN 5990-5783EEE PN 5990-5781EE PN 5990-5426EE Page 5
Therapeutic Drug Monitoring 2D LCMS/MS Method for the Analysis of Immunosuppressive Drugs in Blood : Tacrolimus, Sirolimus, Cyclosporin A and Everolimus Page 6
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
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
Method User Manual Page 9
Method User Manual Page 10
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 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 1.65 1 Flow rate timetable Time (min) Pump A Flowrate(ml/min) Pump B Flowrate(ml/min) 0 0.5 0.1 0.01 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 0.1 2 0.5 0.1 NB Diagram describes a 6 rather than a 10-port switching valve. The plumbing is similar with either system Page 13
LC pump and column setup Isocratic pump Binary pump Page 14
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: 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
Quantitative Results Calibration Levels Spiked into Whole Blood Sirolimus Tacrolimus Everolimus Level Conc. (ng/ml) 1 1.38 2 2.71 3 5.41 4 11.3 5 22.3 6 44.3 QC I 3.64 QC II 11.2 QC III 18.9 Level Conc. (ng/ml) 1 1.22 2 2.46 3 5.03 4 10.2 5 20.5 6 39.7 QC I 3.28 QC II 6.67 QC III 13.3 Cyclosporine A Level Conc. (ng/ml) 1 1.2 2 2.62 3 5.13 4 10.9 5 22.7 6 44.9 QC I 3.34 QC II 10.6 QC III 18.2 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 Page 17
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 Page 18
Quantitative Results Cyclosporine A Sirolimus Tacrolimus Everolimus Page 19
Opiates Page 20
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
47 Drugs of Abuse in 10 minutes 5 Page 22
List of 47 DOA Tested Compound Name RT Precursor Ion Morphine 1.60 286 Oxymorphone 1.74 302 Hydromorphone 1.91 286 Codeine 2.51 300 Oxycodone 2.71 316 7-Aminonitrazepam 2.77 252 7-Aminoclonazepam 2.78 286 Noroxycodone 2.84 302 Hydrocodone 2.85 300 6-MAM 2.93 328 Norhydrocodone 2.96 286 Pregabalin 3.16 160 Gabapentin 3.20 172 7-Aminoflunitrazepam 3.27 284 O-Desmathyltremadol 3.27 250 Bromazepam 4.08 316 Norfentanyl 4.09 233 Tramadol 4.28 264 alpha-hydroxytriazolam 4.53 359 Hydroxyalprazolam 4.54 325 Meperidine 4.66 248 Normeperidine 4.79 234 Desmethylflunitrazepa 4.80 300 Compound Name RT Precursor Ion Oxazepam 4.81 287 Estazolam 4.83 295 Nitrazepam 4.94 282 Lorazepam 5.01 321 Alprazolam 5.07 309 2-hydroxyethylflurazepam 5.12 333 Chlordiazepoxide 5.12 300 Clonazepam 5.13 316 Triazolam 5.18 343 Norbuprenorphine 5.28 414 Fentanyl 5.32 337 Desalkylflurazepam 5.49 289 Flunitrazepam 5.61 314 EDDP 5.64 278 Temazepam 5.66 301 Meprobamate 5.68 219 Nordiazepam 5.72 271 Buprenorphine 5.86 468 Midazolam 6.03 326 Propoxyphane 6.49 340 Diazepam 6.60 285 Norpropoxyphane 6.60 326 Methadone 6.67 310 Prazepam 8.19 325 Page 23
Contact for applications ------------------------------ jan.srbek@hpst.cz Page 24