Professional Panel Screen by LC/MS/MS. Andrew J. Fischinger Ph.D., MT(ASCP) & Lesli Joseph B.S. Norchem Drug Testing, Flagstaff, AZ

Transcription

1 Professional Panel Screen by LC/MS/MS Andrew J. Fischinger Ph.D., MT(ASCP) & Lesli Joseph B.S. Norchem Drug Testing, Flagstaff, AZ

2 LC/MS/MS GC/MS

3 1200L LC/MS & LC/MS/MS ProStar Binary 210 Solvent Delivery System ProStar 430 AutoSampler Module 1200L MS with ESI

4 Analyzer Components Q1 Multiplier +/- 5kV post acceleration Q3 Q2 Curved 186 mm Path

5 MRM Scanning of m/z Parent > Daughter mass fragments Analyte Parent > Daughter RT Collision Energy Meperidine-D4: > min Volts Ketamine: > min Volts Pentazocine: > min Volts Butorphanol: > min Volts Meperidine: > min Volts Tramadol: > min Volts > min Volts

6 Method Performance Analyte Within thin-run Between-run Limit Accuracy (ng/ml) Linearity LOD/LOQ Precision Precision P Carryover % Ketamine / % CV 2.4 % CV Pentazocine / % CV 4.8 % CV Meperidine / % CV 6.3 % CV Butorphanol / % CV 13.8 % CV Tramadol / % CV 6.4 % CV Interferences: None with over 80 high levels of various drugs of abuse and therapeutic drugs tested as documented in our validation study (as recently documented in our CAP FUDT Inspection)

7 Specimen Preparation for LC/MS/MS Run Internal Standard (IS): Meperidine-D4, 2.0 ng/ml QC: -25 and 25% & neg. controls. Calibrator: 25 ng/ml of Butorphanol & Pentazocine, 300 ng/ml for f Meperidine & Tramadol, and 100 ng/ml for Ketamine. Assay calibrated for 1.00 ml Samples 1. Add 100 mcl of IS to each tube. Add 1.00 ml of Calibrator, controls and urine specimens. 2. Add 1.0 ml of ph 8.5, 2.0 M TRIS buffer to each tube, vortex. 3. Pass 1.0 ml methanol through SPE column (BCD GV-65). 4. Pour specimen into column and let drip through by gravity. 5. Wash column with 2.0 ml of 0.1 M ph 8.5, TRIS buffer. 6. Dry columns under full vacuum for 2 minutes. 7. Elute with 0.5 ml acetonitrile (B organic on LC/MS/MS). 8. Add 250 mcl of A (0.1% aq. formic acid, on LC/MS/MS) to eluent. Vortex. 9. Transfer to labeled LC/MS/MS plastic conical 0.5 ml vials. Cap vials v and tap to dislodge any air bubbles at bottom of vial. 10. Run gradient flow on Varian 1200L w. 420 Autosampler & Nitrogen generator. 11. Gradient: %A & %B at 50% at time sec, ramping to 97.5% B for 50 sec, holding 97.5% B for 7 sec, & going back to 50% B during the last second of the 1min run. NOTE: Due to current software/hardware? Constraints it takes another 55 sec to print out results!

8 C o s t e f f e c t i v

9 LC/MS/MS for Screening? Advantages: 1. No need to derivitize,, faster processing for multiple analytes. 2. Can screen more analytes with a single simple, rapid extraction. 3. More specific, even at peaks appearing at same retention times (RT). 4. Higher sensitivity, no need to dry down SPE eluates. Disadvantages: 1. Same MW parents generally need RT separation, e.g., morphine / hydromorphone, and codeine / hydrocodone. 2. Generally limited to a multiple screen 10 analytes for rapid LC/MS/MS screening runs 1min.

10 LC/MS/MS Screening vs. Confirmations Screening: 1. Need only a minimum of Parent > Daughter transitions. NOTE: One Parent > Daughter monitoring generally OK, except if interfering analytes (other drugs or metabolites) appear within the set Q1 amu or RT peak seek window. 2. Rapid multiple screening 1 min/run, if interfering analytes not seen within RT peak seek window,@ +/-.2min NOTE:Can find other daughter ions to remove interferants. Confirmation: 1. Same considerations as above, but probably need 3 daughters with 2 qualifiers,to meet forensic requirements. 2. Need LOQ and ULOL controls as well as +/- 25% Controls.

11 Tramadol with m/z qualifier 246 m/z secondary daughter ion qualifier insures that we can distinguish from Venlafaxine (Effexor)

12 Venlafaxine (Effexor) vs. Tramadol Effexor (Antidepressant) MW = O-Demethylated RT = ESI > % ESI > % Area (246.0)/Area (58.0) = 164.1% Tramadol (Analgesic, Non- narcotic) MW = RT = (+/- 0.2 min search window) ESI > % ESI > % Area (246.0)/Area (58.0) = 2.25 %

13 Other Advantages of LC/MS/MS Rapid analyses, < 2 minutes usually. High linearities, 5 10,000 ng/ml for typical confirmations of urine drugs of abuse, allowing high sample predilutions & faster TAT s. Automation & increased accuracy with rapid (TECAN) 96 microtiter plate sample with reagents dispenser (Flexispense) addition.

14 Mass Analyzer w/ 2 Guides Ion energy 3.0 ev Ion Guide Q1 Ion Guide RF only RF & DC RF only Pre-filter ion guides (rather than lenses) reduce the radial displacement in the mass filter and improve the peak shape and resolution. This improves tuning and ion throughput over a wide mass range and energy level

15 Ion Guides (Q1 and Q3) Pre-ion guide Post-ion guide Pre-ion and post-ion guides A set of 1 cm long quadrupole rods at entrance and exit of main quadrupole assembly RF only waveform is applied to lessen fringe affects at ends of DC/RF quadrupole field On both Q1 and Q3 Using ion guides and the elimination of lenses increases ion transmission efficiency (signal)

16 Collision Cell 180 degree path 40 degree pre-guide evacuation section 100 degree collision cell (186 mm path) 40 degree post-guide evacuation section Curved for low noise and high sensitivity Removes neutrals from the CID process Positions the detector in off-axis from the source Long path for wide CID energy range Efficient dissociation of stable ions Square geometry with flat surfaces Larger cross sectional area for ion transmission Collision cell without lenses Decreased ion losses

17 Collision Cell: Curved Design Ion Source Neutral Noise Positions the ion detector off-axis from the ion source Removes photons, metastables, & high energy charged droplets from ion detector Detector Ions

18 Summary for MS Analyzer MS Analyzer with pre- and post-filter ion guides Maximum ion transmission Optimal tune over entire mass range No lenses in analyzer Collision cell Long path for wide CID energy range Curved for low noise and high sensitivity Pre- and post-guide evacuation section High performance detector for pos. & neg. ions Long life

19 1200L LC/MS Electrospray Ionization

20 State-of of-the-art Design Features with proven value for maximum performance, robust operation and user convenience: 1. Off-axis orientation of the needle and sampling capillary 2. Adjustable nebulization parameters 3. Full range of X-Y needle position 4. Counter-flow of heated drying gas 5. Wide bore sampling capillary 6. Off-axis orientation of the capillary and ion guide 7. High efficiency, hexapole ion guide 8. Heated spray chamber with slight angle to promote drainage 9. Hinged spray chamber with quick release clips 10. Snap-on spray plate 11. Simplified cleaning procedure for all components 12. Design summary Click on desired topic

21 Off-axis Spray Needle Off-axis orientation between the spray needle and sampling capillary eliminates contamination of the interface by large droplets

22 Adjustable Nebulization Inner Needle Adjustment Adjustment of the inner, liquid capillary needle to maximize nebulization efficiency

23 Adjustable Nebulization Liquid Nebulizing Gas Inner to outer needle position is critical to nebulization efficiency Optimum depends on liquid flow

24 Other Nebulization Parameters Gas flow rate Higher liquid flows require higher gas flows Type of nebulization gas Nitrogen for positive ESI application Less than 1% oxygen Air for some negative ESI application Suppresses electrical discharge at high potentials

25 Fully Adjustable Spray Needle X-Adjustment X-Y adjustment of spray needle to optimize sensitivity and maximize robust operation TOP VIEW

26 Counter-flow Drying Gas Gas Heater N 2 N 2 drying gas Droplets Capillary Restriction Drying gas blows large droplets away from capillary inlet

27 Desolvation and Declustering Gas Heater N 2 N 2 drying gas Ions Droplets Capillary Restriction 1 L/min Drying gas also promotes rapid desolvation of droplets and declustering of solvent from ions

28 Electrospray Droplets Distribution of droplet sizes Number Useable Droplets Desolvated and Declustered Higher Probability Of Thermal Degradation Blown Away By Drying Gas Direct Away (Off-axis needle) 1 micron Droplet Diameter 300 micron

29 Wide Bore, Metal Capillary Gas Heater N 2 Droplets Ions Capillary Wide (0.4 mm) internal diameter maximizes ion transmission 1 L/min Stainless steel capillary reduces potential for charge buildup which could impede ions

30 Summary of ESI Design Objectives 1. Maximize Ion Formation & Transmission - High temperature gas phase desolvation of droplets - Large diameter capillary vacuum restriction - reduce space charge effects 2. Minimize Noise - Metal capillary tube - no charging effects like glass - Off axis spray needle - no large droplets entering interface - Patented off axis ion guide - eliminate residual droplet noise 3. Minimize Source contamination - Off axis spray needle - excess spray directed away from interface - No droplet desolvation inside capillary - no contaminants to plug vacuum restriction - Counter flow gas - blow contaminates away from interface - Large diameter vacuum restriction - more tolerant of overloading

31 ELECTROSPRAY IONIZATION (ESI) Mandatory: Compound of interest must be ionized in solution Metallic capilary needle surrounded by N 2 flow to create spray ESI Needle at +3 to 8 kv for +ESI Voltage applied between probe tip and sampling cone(low ev) Heating device to speed up desolvation

32 LC/MS/MS -ESI ACIDIC DRUGS HC, Barbiturates GC/MS Electron Impact LC/MS/MS + ESI BASIC DRUGS Amphetamine Basic (ammonia) Aq. Solvent Phase Volatile buffer 0.1% formic acid Aq. Solvent Phas Volatile buffer

33 For ESI need low ionic strengths of soft ions, i.e., OAc- & NH 4 +

34 ION SUPRESSION

35 Chromatogram Plots

36 INFUSION to obtain Parent > Daughter and CE s

37 - 25 % Control

38 + 25% Control

39 Butorphanol (25 ng/ml cutoff)

40 Calibrator Mix: Professional Panel Ketamine 100 ng/ml Meperidine 300 ng/ml Tramadol 300 ng/ml Pentazocine 25 ng/ml Butorphanol 25 ng/ml Meperidine-D4 IS 300 ng/ml

41 LOQ Control

42 Meperidine + Urine Screen

43 Typical Negative Screen

44 Pentazocine & Butorphanol 25% C

45 Butorphanol +/- 25% Controls LCMSMS BUTORPHANOL /2/2005 5/4/2005 5/6/2005 5/8/2005 5/10/2005 5/12/2005 5/14/2005 5/16/2005 5/18/2005 5/20/2005 5/22/2005 5/24/2005 5/26/2005 5/28/2005 5/30/2005 QC RUN # QC - QC + RATE VALUE (ng/m

46 Ketamine +/- 25% Controls LCMSMS KETAMINE 5/2/2005 5/4/2005 5/6/2005 5/8/2005 5/10/2005 5/12/2005 5/14/2005 5/16/2005 5/18/2005 5/20/2005 5/22/2005 5/24/2005 5/26/2005 5/28/2005 5/30/2005 DATE QC - QC + RATE VALUE (ng/

47 Meperidine +/- 25% Controls LCMSMS MEPERIDINE 5/2/2005 5/4/2005 5/6/2005 5/8/2005 5/10/2005 5/12/2005 5/14/2005 5/16/2005 5/18/2005 5/20/2005 5/22/2005 5/24/2005 5/26/2005 5/28/2005 5/30/2005 DATE QC - QC + RATE VALUE (ng/m

48 DAY-to to-day PRECISION LCMSMS MEPERIDINE VALUES IN ng/ml DAY QC - QC + CAL ZERO QC - QC + 5/ % 127% 5/ % 124% 5/ % 126% 5/ % 124% 5/ % 124% 5/ % 124% 5/ % 124% 5/ % 121% 5/ % 126% 5/ % 123% 5/ % 130% 5/ % 123% 5/ % 125% 5/ % 121% 5/ % 121% 5/ % 123% 5/ % 120% 5/ % 120% 5/ % 123% 5/ % 125% MEAN C.V.%

49 Meperidine Precision Meperidine Precision LCMSMS MEPERIDINE 5/2/2005 5/4/2005 5/6/2005 5/8/2005 5/10/2005 5/12/2005 5/14/2005 5/16/2005 5/18/2005 5/20/2005 5/22/2005 5/24/2005 5/26/2005 5/28/2005 5/30/2005 DATE QC - QC + RATE VALUE (ng/m

50 Day-to to-day Precision: Ketamine LCMSMS KETAMINE ALL VALUES IN ng/ml DAY QC - QC + CAL ZERO QC - QC + 5/ % 128% 5/ % 114% 5/ % 128% 5/ % 120% 5/ % 124% 5/ % 119% 5/ % 130% 5/ % 125% 5/ % 126% 5/ % 120% 5/ % 139% 5/ % 127% 5/ % 127% 5/ % 123% 5/ % 122% 5/ % 119% 5/ % 120% 5/ % 123% 5/ % 120% 5/ % 122% MEAN C.V.%

51 Pentazocine +/- 25% Controls LCMSMS PENTAZOCINE /2/2005 5/3/2005 5/4/2005 5/5/2005 5/6/2005 5/7/2005 5/8/2005 5/9/2005 5/10/2005 5/11/2005 5/12/2005 5/13/2005 5/14/2005 5/15/2005 5/16/2005 5/17/2005 5/18/2005 5/19/2005 5/20/2005 5/21/2005 5/22/2005 5/23/2005 5/24/2005 5/25/2005 5/26/2005 5/27/2005 5/28/2005 5/29/2005 5/30/2005 5/31/2005 DATE QC - QC + rate value (ng/m

52 Day-to to-day Precision: Pentazocine LC/MS/MS PENT AZOCINE all values in ng/ml DAY QC - QC + CAL ZERO QC - QC + 5/ % 124% 5/ % 140% 5/ % 124% 5/ % 116% 5/ % 132% 5/ % 132% 5/ % 136% 5/ % 116% 5/ % 128% 5/ % 136% 5/ % 120% 5/ % 124% 5/ % 124% 5/ % 124% 5/ % 124% 5/ % 120% 5/ % 116% 5/ % 120% 5/ % 120% 5/ % 124% MEAN C.V.%

53 Tramadol +/- 25% Controls LCMSMS TRAMADOL 5/2/2005 5/4/2005 5/6/2005 5/8/2005 5/10/2005 5/12/2005 5/14/2005 5/16/2005 5/18/2005 5/20/2005 5/22/2005 5/24/2005 5/26/2005 5/28/2005 5/30/2005 DATE QC - QC + RATE VALUES (ng/

54 Day-to to-day Precision: Tramadol LCMSMS TRAMADOL ALL VALUES IN ng/ml DAY QC - QC + CAL ZERO QC - QC + 5/ % 117% 5/ % 119% 5/ % 108% 5/ % 115% 5/ % 121% 5/ % 120% 5/ % 120% 5/ % 140% 5/ % 127% 5/ % 121% 5/ % 127% 5/ % 131% 5/ % 126% 5/ % 125% 5/ % 124% 5/ % 118% 5/ % 120% 5/ % 126% 5/ % 127% 5/ % 130% MEAN C.V.%

55 Composite Precision: Professional Panel - 25% Control Precison date sample id Ketamine Meperidine Tramadol PentazocinButorphanol 11/1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% /1/2004 c1-25% mean standard deviation %CV 3.2% 1.7% 3.1% 3.6% 2.1% mean + 2 Stdev mean - 2 Stdev ta rge t + 20% ta rge t - 20% a ccura cy 97.0% 97.5% 100.3% 92.9% 100.0% N =

56 Composite Precision: Professional Panel + 25% Control Precison date sample id Ketamine Meperidine Tramadol PentazocinButorphano 11/1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% /1/2004 c2 +25% mean standard deviation %CV 3.2% 2.1% 3.3% 2.4% 2.1% mean + 2 Stdev mean - 2 Stdev ta rge t + 20% ta rge t - 20% a ccura cy 99.5% 96.7% 98.7% 90.8% 96.0% N =

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