Getting a Handle on Your Biggest Volumes Dr Russell Grant

Transcription

Getting a Handle on Your Biggest Volumes
Dr Russell Grant
Laboratory Corporation of America® Holdings
1
The Different Types of Volume
 Common Needs
• Redundant systems (Automation and instruments)
• High-throughput multiplexing LC
 Many Assays – few samples (Dynamic)
• Manual (real time) and automated
• “Generic” sample preparation
• Sporadic calibration
 Same assay – many samples (Batch)
• “Common” technology usage (Platform expertise)
• Process Automation – end to end + electronic
2
Patient Service Network:
Interaction, Phlebotomy and Triage
3
Beacon Touch
Patient Details Entry/Verify
Touch screen/Draw/Print
Barcode/Verify/Scan
Process/Ship
Laboratory: Parent tube and IS addition Automation
TECAN Freedom Evo®
Benefits to Automated Sample Prep





8 opposable thumbs vs 1
Automated batch building - ID retained
Proven accurate and precise
Error monitoring – PMP
Platform expertise – Not assay
Manual v Automated Correlation
Slope (deming) = 1.008
Correlation Coef = 0.999
Mean Bias = 1.8%
4
Freedom Evo® is a registered trademark of TECAN
Intermediate (96-well plate processing (ALD®)
 2 or 4 x 96-well plates (SPE and SLE)
 12 solvents
 Positive pressure
 Manifold heating
FTE Time
81 min to 12 minutes
Total time minus 5 min
IS Variance reduced 18% to 11%
5
ALD is a registered trademark of SPEWare
Islands of Automation
Manual Batch Prep (n=192)
Automated Batch Prep (n=192)
Prep Batch
10 min
Prep Batch
10 min
Pipette Samples
60 min
Tecan EVO
(Span 8 and
96 tip)
Pipette Samples
35 min
Transfer to 96-well
plate 10 min
Transfer to 96-well
plate 3 min
SLE Extraction
30 min
SLE Extraction
30 min
Evap/Recon
25 min + 5 min
SPEWare ALD
– Single Head
Mass Spec Analysis
420 min
125 min Hands on
Evap/Recon
25 min
Mass Spec Analysis
420 min
Manual
15 min Hands on
Automated
6
Evo® is a registered trademark of Tecan, ALD® is a registered trademark of SPEWare
High Throughput Multiplexing Tools
ARIA® Transcend® TLX-4
MS Detects
(2x600bar binary + 2 valves/channel)
2.8e6
1
2
3
4
Max. 2.9e6 cps.
2.4e6
2.0e6
Intensity, cps
1.6e6
1.2e6
8.0e5
4.0e5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Time, min
Tricyclic antidepressants panel
2.17 Min total cycle time
30 seconds diverted to MS
7
API 5000® /5500® Triple
Quadrupole
ARIA and Transcend are registered trademarks of Thermo Corporation,
API5000 and 5500 are registered trademarks of AB SCIEX
1
TFC-LC – Dynamic
TFC
Waste
LC
MS
AS
Sample Injection/Loading
LOADING
PUMP
Chromatofocusing
ELUTING
PUMP
TFC Column Cleaning/Loop refill
Reconditioning
Load Pump – TFC Extraction Column
Cycle Time
Elute Pump – Analytical Column
Column Recondition
(previous injection)
Chromatofocusing
Gradient Elution/Select to MS
Column Cleaning
TFC-LC Method Folding
Assay Cycle times: 2.1 – 2.8 minutes
8
MS/MS Acquisition: 24 – 60 seconds
Method Development – TFC-LC Chromatofocusing
Elute Pump Flow Rate
1.5mL/min
1.0mL/min
0.5mL/min
10%
1%
30%
20%Loop
LoopComposition
Composition
1.6e5
2.0e5
2.8e5
1.0e5
2.6e5
3.0e5
2.0e5
1.2e5
2.2e5
8.0e4
1.5e5
Intensity, cps
Intensity, cps
Intensity, cps
2.0e5
1.5e5
2.0e5
1.4e5
1.0e5
1.0e5
8.0e4
5.0e4
0.0
9
6.0e4
1.6e5
8.0e4
1.0e5
4.0e4
1.0e5
4.0e4
5.0e4
2.0e4
4.0e4
0.10
0.20
0.30
0.40
0.50
Time, min
0.0
0.10
0.20
0.30
0.40 Time, min
0.1-100ng/mL Calibrator.
50% Loop Composition
Load Pump Flow = 0.5mL/min
100ng/mL Calibrator
Load Pump Flow = 0.5mL/min Elute Pump Flow = 0.5mL/min
Benzodiazepine Panel
Nordiazepam, Diazepam, Clonazepam,
Norchlordiazepoxide, Chlordiazepoxide
Tricyclic Antidepressant Panel
Nortriptyline, Desmethyldoxepin, Desipramine, Amitriptyline, Doxepin, Imipramine, Desmethylclomipramine, Clomipramine
Mean Lipid Recovery
TFC-LC and Phospholipid Depletion [1]
100.0
TFC column = Cyclone P, 50 x 0.5mm
Lyso’s and Glycero’s via TFC-LC
90.0
Loop Size = 100 L
80.0
70.0
Loop contents modified from 10-100% for ACN,
1:1 ACN:MeOH or MeOH.
60.0
50.0
40.0
Glycerophosphatidyl cholines recovery < 1%
30.0
20.0
10.0
Lyso_ACN:MeOH 1:1
Lyso_ACN
Lyso_MeOH
Glyero_ACN:MeOH 1:1
Glyero_ACN
Glyero_MeOH
0.0
10
20
30
40
50
60
70
80
90
Lysophosphatidyl cholines recovery >10% for
ACN and 1:1 ACN:MeOH and >30% for MeOH
100
Protein Precipitated Sample (A)
2.0e6
Max. 2.1e6 cps.
Max. 2.7e5 cps.
A
B
2.4e5
1.6e6
2.0e5
Intensity, cps
Intensity, cps
P
1.2e6
B
Phospholipids (P) co-elution = additional
chromatographic resolution required
1.6e5
TFC-LC direct-injection (B)
1.2e5
8.0e5
P
8.0e4
B
4.0e5
0.0
10
0.2
0.4
0.6
0.8
1.0
1.2
Phospholipid recovery reduced to <5% with 50%
1:1 ACN:MeOH in TFC loop
4.0e4
1.4
1.6
1.8
Time, min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.33
1.4
1.6
1.8
Time, min
Minimize ESI Matrix effects = LC Flexibility
[1] Rappold B, Holland, P, Grant, RP. Balancing Sample Preparation Liquid Chromatography to Remove Phospholipid-Based
Matrix Effects in Positive ESI. Conference abstracts and Proceedings of the 55th American Society for Mass Spectrometry,
2008, Denver, Poster Tues 442).
TFC-LC-API5000 Selectivity and Reproducibility
1.15e6 cps.
4.6e5 cps.
1.0e6
A
B
4.0e5
↓ Organic Composition in Loop = ↓ Signal
8.0e5
3.0e5
Intensity, cps
Intensity, cps
6.0e5
Sensitive instrumentation allows for signal reduction
2.0e5
4.0e5
Stable labeled internal standards/Calibration Curves
1.0e5
2.0e5
0.0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Time, min
Loop composition (A) =
50% elutropic content
Calibrators
0.0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Time, min
Loop composition (B) =
5% elutropic content
QC’s and Samples
Calibrators
Calibrators
Coefficient of Variation = 3.5%
QC’s and Samples
Coefficient of Variation = 2.4%
Double Blanks
Double Blanks
Nortriptyline IS Response
Methotrexate IS Response
Goal is Reproducible Recovery not Absolute
11
Dilution Pre or Post IS addition – Linear 1/x Calibration
Basic Hydrolysis for “Total”
LC-MS/MS versus TFC-LC-MS/MS
2.4e5
Total Dabigatran (Including Glucoronide)
pH 11, 2.5 Hr @ 37C = 182.484 ng/mL
1.8e5
Intensity, cps
Free Dabigatran (Unconjugated)
pH 3 = 105.470 ng/mL
1.2e5
6.0e4
0.0
0.10
0.20
0.30
0.40
0.50
0.60
0.70 Time, min
Sample diluted 1:9, then sub-aliquot 50L + 450L
50ng/mL (D3-Dabigatran at 450 x LLOQ)
Sample 50L + 450L 50ng/mL D3-Dabigatran, then
dilute 1:9 with Diluent (D3-Dabigatran at 45 x LLOQ)
7.5e5
7.0e5
Intensity, cps
3.5e5
Samples (n=6) at 5000ng/mL
(5 x ULOQ)
CV = 6.57%
Bias = -2.20%
5.0e5
Intensity, cps
Samples (n=6) at 5000ng/mL
(5 x ULOQ)
CV = 7.52%
Bias = 12.11%
5.5e5
3.0e5
1.5e5
1.0e5
0.20
12
0.40
0.60
Time, min
0.20
0.40
0.60
Time, min
IS selection aids Data Review
7.5e5
Codeine
Intensity, cps
m/z = 300.2→257.2
5.5e5
m/z = 303.2→241.2
3.5e5
Codeine-d6
1.5e5
m/z = 300.2→215.2
0.0
1.2e6
Intensity, cps
Hydrocodone-d3
Hydrocodone
4.0e5
m/z = 306.2→115.2
0.10
0.20
0.30
0.40
0.50 Time, min
0.0
5.0e5
Morphine
Intensity, cps
m/z = 286.2→185.2
m/z = 286.2→201.2
4.0e5
Hydromorphone-d6
3.0e5
m/z = 292.2→152.2
2.0e5
Morphine-d3
1.0e5
m/z = 289.2→185.2
0.0
8.0e5
0.10
0.20
0.30
0.40
Dihydrocodeine
m/z = 302.2→245.2
Intensity, cps
m/z = 302.2→242.2
m/z = 308.2→202.2
4.0e5
Dihydrocodeine-d3
2.0e5
0.0
m/z = 305.2→230.2
0.10
0.20
0.30
Hyc-d6
306.2
Hyc-d3
303.2
0.40
0.50 Time, min
Oxym-d3
305.2
Oxym-d6
308.2
1.2e6
Intensity, cps
8.0e5
Oxym
302.2
Cod-d3
303.2
Cod-d6
306.2
Dhc-d6
308.2
Dhc-d3
305.2
Dhc
302.2
0.0
0.10
0.20
0.30
0.40
0.50
0.40
0.50 Time, min
0.10
0.20
0.30
0.40
0.50 Time, min
0.10
0.20
0.30
0.40
0.50 Time, min
4.0e5
5.0e5
3.0e5
1.0e5
0.0
M+2 isotopic contribution
4.0e5
13
Intensity, cps
Oxymorphone-d6
0.30
6.0e5
0.0
7.0e5
6.0e5
0.20
2.0e5
0.50 Time, min
Oxymorphone
0.10
8.0e5
Intensity, cps
Hydromorphone
8.0e5
Time, min
Open Access LC Configuration – Multichannel usage
Tricyclics LLOQ – 3 Independent Channels
4.0e4
8.0e4
5.0e4
6.0e4
3.0e4
4.0e4
Intensity, cps
Intensity, cps
Intensity, cps
4.0e4
3.0e4
2.0e4
2.0e4
1.0e4
2.0e4
1.0e4
0.0
0.10
0.20
0.30
0.40 Time, min
0.0
0.10
Channel 2
0.20
0.30
0.40 Time, min
Channel 3
0.0
0.10
0.20
0.30
0.40 Time, min
Channel 4
Analyte Reproducibility across 3 channels in Samples and QC’s
Amitriptyline
0.0 – 3.8% CV
Nortriptyline
0.4 – 5.3% CV
Desipramine
1.8 – 5.3% CV
Imipramine
0.7 – 4.1%CV
Doxepin
0.0 – 7.5% CV
Desmethyldoxepin
0.7 – 8.6% CV
Clomipramine
1.0 – 6.5% CV
Desmethylclomipramine
0.0 – 2.3% CV
3.0
Analyte Area / IS Area
2.0
Amitriptyline Calibration Curve
Overlay from LC Channels 2-4
1.0
Calibrate on 1 channel
0.0
14
250
500
Analyte Conc. / IS Conc.
750
1000
Extending Calibration – Validation of Historical Curves
Levetiracetam + 24 hours
Deming Slope = 1.016
Intercept = -0.7749
Corr Coef, r = 0.9955
Oxcarbazepine + 24 hours
Intercept = -0.8327
Lacosamide + 24 hours
Intercept = -0.9056
15
Extending Calibration – Validation of Historical Curves
Levetiracetam + 144 hours
Intercept = -1.1966
Oxcarbazepine + 144 hours
Intercept = -0.7982
Lacosamide + 144 hours
Intercept = -0.8589
16
Clinical Toxicology – Generic Prep/Analytical Platform
for Rapid TAT and STAT Testing - Dynamic
>90 Analytes, multiple sub-panels matrix agnostic
50uL sample + 450uL IS in 0.1-1% Formic Acid,
Seal plate/Mix/Centrifuge/Inject
Calibrate Daily or weekly
QC bracket Release
3.2e6
4.0e6
5.0e6
T
M
Me
F
T
Intensity, cps
C
1.6e6
Fl
0.0
F
0.10
0.20
0.30
0.40
0.0
Time, min
Nf
0.1
0.2
0.4
0.5
0.6
1.0e6
0.7
0.8
0.0
Time, min
C
Intensity, cps
Intensity, cps
1.2e4
3.0e6
I
8000
0.05
0.15
0.25
0.35
0.45
Time, min
Opiates Profile 1
C
7.5e4
6.0e4
NC
Intensity, cps
Dd
2.4e6
1.8e6
CH
D
4.5e4
3.0e4
N
1.2e6
D
1.5e4
6.0e5
4000
0.0
0.10
0.20
0.30
Tricyclics Profile
17
0.3
B
3.6e6
N Dc
1.6e4
0.0
N
4.2e6
A
2.0e4
M
2.0e6
Opiates Profile 2
Clinical Toxicants
Do
3.0e6
Fx
8.0e5
1.0e6
D
C
6A
M
Intensity, cps
Intensity, cps
2.0e6
H
O
4.0e6
H
2.4e6
W
3.0e6
Oc Hc
Nx
0.40
0.10
0.20
0.30
0.40
0.50
Time, min
Time, min
Benzodiazepines Profile
0.0
0.10
0.20
0.30
0.40
0.50
0.60
Barbiturates Profile
Time, min
Clinical Toxicology Analytical Cassetting Throughput
A Analysis of 6 assays on 4 channels is executed in 6
1.6e6
minutes (A),
C
1.2e6
Intensity, cps
Analytical throughput of 1440 samples/system/day
8.0e5
Clinical Tox profile (C) , Opiates Profile 2 (O2),
Cocaine Profile (Co), Benzodiazepine Profile (B),
Tricyclics Profile (T) and Opiates Profile 1 (O1).
T
B
4.0e5
Co
O2
0.0
0.5
1.0
O1
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
2.4e6
B Analysis of a subset of assays 4 samples in 2.5
T
2.0e6
Time, min
minutes (B),
B
Intensity, cps
C
1.6e6
Analytical throughput of 2304 samples/system/day.
Co
1.2e6
Tricyclics Profile (T), Clinical Tox profile (C), Cocaine
Profile (Co), Benzodiazepine Profile (B)
8.0e5
4.0e5
0.0
18
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Time, min
LC optimization for Batch Mode
Screening Gradient:
45 Sec Load
90 Sec Ramp
45 Sec Wash
60 Sec Equilibration
100% B
Analyte
100% A
4 Min
100% B
Final Gradient:
10 Sec Load
40 Sec Ramp
30 Sec Wash
40 Sec Equilibration
0.5 minute data window
Analyte
100% A
2.0 Min
19
CHANGE
Test reduced load times, truncate
ramp, reduce wash and
equilibration times
Maintain Gradient Pitch (%/sec)*
Testosterone UHT - 4 channel LC-MS/MS (25-5000pg/mL)
1.4e4
1.3e4
1.2e4
1.1e4
3C -Testosterone
13
, Precision
1.0e4
9000.0
Intensity, cps
200µL Serum/Plasma plus 200µL
of Tecan® <2%
SLE, 2-fold concentration, inject 35µL LC-MS/MS
8000.0
7000.0
6000.0
5000.0
4000.0
3000.0
2000.0
Testosterone 2.5 – 5000ng/dL (25-5000pg/mL): m/z 289 – 97
(QN), 289 – 109 (QL)
3C -Testosterone
13
1000.0
0.0
0.20
Time, min
0.30
Female: Red top versus Serum Separator Tube
Transition ratio Bias = -3.32% versus 6.57%
Mean SST Bias = -12.75%
Precision (CV, n = 24) = 1.95% versus 3.06%
(~120ng/dL): 292 – 112 (QN), 292 – 100 (QL)
XIC of +MRM (4 pairs): 289.2/109.1 Da
0.10
from Sample 3 (SST) of 1Hr Acquis.wiff (Heated Nebulizer)
Max. 1.6e5 cps.
1.8e5
1.7e5
1.6e5
1.5e5
1.19
3.62
6.04
3.08
17.70 18.26
8.50
13.41
10.96
22.56
27.43
25.00
15.83
32.87
47.47
37.15
32.30
42.60
45.04
49.92
40.16
34.72
1.4e5
52.37
54.82
57.27
1.3e5
1.2e5
Intensity, cps
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
0.0
20
5
10
15
20
25
30
Time, min
35
40
45
50
55
60
99 samples in 60 minutes or 2376 samples/system/day – Cycle time = 2.4 min
CDC Certification
Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum.
Botelho JC, Shacklady C, Cooper HC, Tai SS, Uytfanghe KV, Thienpont LM, Vesper HW. Clin Chem. 2013 Feb;59(2):372-80.
doi: 10.1373/clinchem.2012.190934. Epub 2012 Dec 4.
21
2D-LC to 1D LC – 25-Hydroxyvitamin D2/D3
0.55
0.59
4200
2500
ARIATM TX4-API 4000:
3600
2000
3000
Intensity, cps
Intensity, cps
1500
1000
2400
1800
1200
500
600
0
0.2
0.4
0.6
0
0.8 Time, min
25-Hydroxyvitamin D2
LOQ (1 ng/mL)
0.2
0.4
0.6
0.8 Time, min
LOQ (1 ng/mL)
860
ARIATM TranscendTM TX4-API 5000:
1400
660
1100
Intensity, cps
Intensity, cps
460
260
800
500
200
60
0
0.05
0.15
0.25
0.35
Time, min
0.45
0.55
LOQ (1 ng/mL)
22
PPT with 2x Concentration
2D-LC
5 minute cycle-time – 1 min acquisition
0
0.05
0.15
0.25
0.35
Time, min
0.45
0.55
LOQ (1 ng/mL)
PPT with 3x Dilution – Supernatant injection
1D-LC
2.5 minute cycle-time – 30 sec acquisition
CDC Certified 2014
Dead volume/recycling and Divert Valve – 1D
Ramp to 95% B over 75
seconds at 1.25 mL/min
Mobile phase B forward-washes the
column at 2 mL/min for 20 seconds
Loading pump back-washes the
column at 2.5 mL/min for 20
seconds
Start – 1.25 ml/min,
80% Mobile Phase B
Step to 100% B for 25 seconds at 1.25
ml/min, then 2 mL/min for 5 sec
Inline 48 -78 secs
Column is reconditioned
with 80% Mobile Phase B
at 1.25 mL/min
Eluting pump
Loading pump
23
2 Pump/2 Valve Assay Switching – Speed and Open Access
MS
Column 1
LOADING
PUMP
AS
Column 2
ELUTING
PUMP
(A) Common Solvents Alternate LC column Functionality
Second (Load) Pump primed ready for redundancy
Column 1
LOADING
PUMP
AS
MS
ELUTING
PUMP
Column 2
(B) Unique Solvents and Alternate LC column Functionality
4-Plex combination of solvent systems and LC columns per channel
24
8-Channel/8-Assay Open Access at >2000 samples/24Hr
Analyte
Androstenedione
Aldosterone
Aldosterone
DHEA
17-OHProgesterone
17-OHProgesterone
Cortisol
Cortisone
1
2
A
25
3
4
B
5
Preanalytical Matrix Channels
SLE+
S/P
1,3,5,7
Hydrolysis/SLE U
1,3,5,7
SLE+
S/P
1,3,5,7
LLE/KMnO4
S/P
2,6
Hydrolysis/LLE U
4
SLE+
S/P
4
SLE+
U
8
SLE+
U
8
6
A
7
8
B
CHANNEL
VALVING SETUP
Multiplexed LC-MS/MS Data – Acquisition Window
Sample Name: "10 ng/dL Standard 1" Sample ID: "25 ng/dL Standard 2" File: "4009.wiff"
PeakName: "004706 Androstenedione" Mass(es): "287.2/97.0 Da"
Comment: "N/C" Annotation: ""
Sample Index:
4
Sample Type:
Standard
Concentration:
25.000
ng/dL
Calculated Conc: 24.030
ng/dL
1.8e4
Acq. Date:
1/4/2011
Acq. Time:
3:26:12 PM
1.8e4
Modified:
Yes
Proc. Algorithm: IntelliQuan - MQII
1.7e4
Noise Percentage:
50
Base. Sub. Window:
1.00
min
1.7e4
Peak-Split. Factor: 4
Report Largest Peak: No
Min. Peak Height: 1000.00
cps
1.6e4
Min. Peak Width:
0.00
sec
Smoothing Width:
15
points
1.6e4
RT Window:
30.0
sec
Expected RT:
Use Relative RT:
0.170
Yes
Sample Name: "10 ng/dL Standard 1" Sample ID: "25 ng/dL Standard 2" File: "4009.wiff"
Peak Name: "D7 Androstenedione(IS)" Mass(es): "294.2/113.1 Da"
Sample Index:
4
Sample Type:
Standard
Concentration:
1.00
ng/dL
Calculated Conc:
N/A
1.5e5
Acq. Date:
1/4/2011
Acq. Time:
3:26:12 PM
0.18
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height: 1000.00 cps
Min. Peak Width:
0.00 sec
Smoothing Width:
9
points
RT Window:
30.0
sec
Expected RT:
0.172
min
Use Relative RT:
No
min
1.5e4
Int. Type:
Base To Base
Retention Time:
0.182 min
Area:
53003.350 counts
Height:
17937.641 cps
Start Time:
0.126 min
End Time:
0.272 min
Int. Type:
Base To Base
Retention Time:
0.172
min
Area:
434964.647
counts
Height:
147296.095 cps
Start Time:
0.114
min
End Time:
0.330
min
1.5e4
1.4e4
1.4e4
Sample Name: "Cal001-1 ng/dL" Sample ID: "Cal1- 1 ng/dL" File: "10033.wiff"
PeakName: "004371 Aldosterone" Mass(es): "359.2/189.0 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.000
ng/mL
1900
ng/mL
Acq. Date:
1/11/2011
Acq. Time:
3:30:23 PM
1850
0.17
1.4e5
Modified:
No
Proc. Algorithm: Specify Parameters - MQ III
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.298 min
Use Relative RT:
Yes
1.4e5
1.3e5
1.3e5
1.2e5
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00 cps
Min. Peak Width:
0.00 sec
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.293
min
Use Relative RT:
No
1750
1700
1650
1600
1550
Int. Type:
Base To Base
Retention Time:
0.282 min
Area:
2898.407 counts
Height:
1490. cps
Start Time:
0.250 min
End Time:
0.333 min
1.2e5
1.1e5
Int. Type:
Valley
Retention Time:
0.278
Area:
354020.738
Height:
1.76e+005
Start Time:
0.225
End Time:
0.377
1500
1450
1.0e5
3300
3200
3100
2800
1.0e4
1.0e5
2200
9.5e4
950
900
850
5.0e4
6000.0
5500.0
4.5e4
5000.0
4.0e4
8.5e4
1.2e5
1100
800
7.0e4
1000
1.0e4
500.0
0.0
0.05
0.10
0.15
0.20
Time, min
0.25
0.30
0.35
0.0
0.40
0.10
0.15
0.20
Time, min
0.25
0.30
0.35
Sample Name: "S2_10ng/dL__" Sample ID: "S2_50ng/dL__" File: "SLE Cross Val Batch 11.wiff"
Peak Name: "IS(IS)" Mass(es): "339.3/113.1 Da"
Sample Index:
4
Sample Type:
Standard
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
Acq. Date:
8/6/2010
1.6e5
Acq. Time:
3:59:29 PM
0.34
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00 cps
Min. Peak Width:
0.00 sec
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.317
min
Use Relative RT:
No
3400
3300
3200
Int. Type:
Base To Base
Retention Time:
0.309
min
Area:
804207.995
counts
Height:
1.59e+005 cps
Start Time:
0.159
min
End Time:
0.488
min
3100
3000
1.5e5
1.4e5
1.4e5
1.3e5
0.20
0.25
Time, min
0.30
0.35
0.40
0.0
0.45
900
880
860
840
820
800
0.01
740
720
1.1e5
660
2700
1.0e5
9.0e4
5.0e4
3.0e4
3.8e4
2.0e4
400
1.0e4
60
0.05
0.10
0.15
0.20
0.25
Time, min
0.30
0.35
0.40
0.45
0.0
0.66
0.67 0.76
0.10
0.15
0.20
0.25
Time, min
0.30
0.35
0.40
0.45
17OHProgesterone (S/P)
SLE+, 10ng/dL, 30s
0
0.2
0.3
1.2e5
0.05
0.10
0.15
Sample Name: "Standard 1ng/mL" Sample ID: "Standard 1 0.10ug/dL" File: "14010.wiff"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
1.6e5
Acq. Date:
1/14/2011
Acq. Time:
3:50:16 PM
0.32
Modified:
No
Noise Percentage:
55
Base. Sub. Window:
1.00
min
Min. Peak Height:
500.00 cps
Min. Peak Width:
0.00 sec
Smoothing Width:
5
points
RT Window:
30.0
sec
Expected RT:
0.331
min
Use Relative RT:
No
6600
6400
6200
6000
5800
0.20
Time, min
0.25
0.30
0.35
5200
0.33
1.6e5
1.5e5
1.5e5
1.4e5
1.4e5
1.3e5
Int. Type:
Base To Base
Retention Time:
0.326
min
Area:
954053.646
counts
Height:
1.61e+005 cps
Start Time:
0.137
min
End Time:
0.541
min
5600
5400
1.3e5
1.2e5
1.2e5
4800
1.1e5
1.1e5
4400
1.0e5
4200
9.5e4
9.0e4
8.0e4
7.5e4
3600
3400
8.5e4
8.0e4
7.5e4
3200
7.0e4
7.0e4
3000
6.5e4
2800
6.5e4
6.0e4
2600
6.0e4
5.5e4
2400
5.5e4
2200
5.0e4
5.0e4
2000
4.5e4
4.5e4
1800
4.0e4
1600
3.5e4
1400
3.0e4
3.0e4
1200
700
2.5e4
2.5e4
600
1000
2.0e4
2.0e4
800
1.5e4
300 0.01
6000.0
0.0
0.35
4600
8.5e4
3.5e4
500
0.30
5000
900
600
1.0e4
0.52
0.64
1.5e4
0.55
400
1.0e4
200
5000.0
200
0.4
Time, min
0.5
0.6
0.7
0.0
5000.0
100
2000.0
0.1
Int. Type:
Base To Base
Retention Time:
0.320 min
Area:
36814.226 counts
Height:
6.76e+003 cps
Start Time:
0.200 min
End Time:
0.494 min
1.3e5
1000
4000.0
20
0.05
1.4e5
4.0e4
0.35
40
5000.0
100
0.64
1.4e5
1200
400
0.25
3800
800
0.32
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
5
points
RT Window:
30.0
sec
Expected RT:
0.326 min
Use Relative RT:
No
1.5e5
1100
1.0e4
0.20
Time, min
9.0e4
2300
8000.0
0.15
PeakName: "120518 Cortisone" Mass(es): "361.3/163.0 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.000
ng/mL
ng/mL
7000
Acq. Date:
1/14/2011
Acq. Time:
3:50:16 PM
6800
0.33
1.5e5
2.2e4
1.2e4
0.10
DHEA (S/P)
LLE/Ox+, 20ng/dL, 20s
1.6e5
2.0e4
1.6e4
2000.0
0.05
4000
1400
1.4e4
0.27
0.45
9.5e4
1300
1.8e4
0.10 0.15
0.40
1600
2.4e4
80
300
200
0.35
1.0e5
1500
0.78
0.30
1.1e5
1700
2.8e4
120
0.25
Time, min
1.1e5
2100
2.6e4
100
1.5e4
0.20
1.2e5
2000
140
0.01
0.15
1.3e5
Int. Type:
Base To Base
Retention Time:
0.326
min
Area:
954053.646
counts
Height:
1.61e+005 cps
Start Time:
0.137
min
End Time:
0.541
min
2200
3.2e4
160
2.5e4
600
Modified:
No
Noise Percentage:
55
Base. Sub. Window:
1.00
min
Min. Peak Height:
500.00 cps
Min. Peak Width:
0.00 sec
Smoothing Width:
5
points
RT Window:
30.0
sec
Expected RT:
0.331
min
Use Relative RT:
No
2700
3.0e4
180
0.10
2600
400
200
800
0.05
Sample Index:
3
Sample Type:
Standard
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
1.6e5
Acq. Date:
1/14/2011
Acq. Time:
3:50:16 PM
0.34
2900
240
700
0.45
2800
380
220
3.5e4
0.40
3000
1900
280
4.0e4
900
5.4e4
1800
260
1000
0.35
3400
3.4e4
300
4.5e4
1100
0.30
3300
3.6e4
320
1300
0.25
Time, min
3600
4.0e4
360
0.20
3500
500
340
5.5e4
0.15
3800
3200
4000.0
Intensity, cps
6.5e4
6.0e4
0.10
3700
2500
420
0.05
Intensity, cps
7.0e4
0
4000
2400
480
100
0.0
3900
4.4e4
460
1.0e4
4200
3100
8000.0
6000.0
0
4100
5.6e4
1.0e4
500
400
100
4300
4.2e4
440
1800
1700
1200
Int. Type:
Base To Base
Retention Time:
0.336 min
Area:
25188.906 counts
Height:
4.44e+003 cps
Start Time:
0.215 min
End Time:
0.510 min
0.33
0.30
0.03
200
Intensity, cps
7.5e4
1400
Modified:
No
Noise Percentage:
55
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
5
points
RT Window:
30.0
sec
Expected RT:
0.341 min
Use Relative RT:
No
1.2e4
0.26
300
2.0e4
PeakName: "004433 Cortisol" Mass(es): "363.3/121.1 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.000
ng/mL
4500
ng/mL
Acq. Date:
1/14/2011
4400
Acq. Time:
3:50:16 PM
0.25
520
Intensity, cps
Intensity, cps
Intensity, cps
Intensity, cps
8.0e4
3.0e4
Aldosterone (U)
SLE+, 20ng/dL, 30s
Intensity, cps
8.5e4
1600
0.45
540
2200
1500
0.40
4.6e4
560
1900
0.35
5.0e4
580
9.5e4
2100
0.30
4.8e4
600
2300
2000
0.25
Time, min
5.2e4
640
620
2400
0.20
6.0e4
680
2600
0.15
5.8e4
700
1.2e5
2500
0.10
6.2e4
Int. Type:
Base To Base
Retention Time:
0.246
min
Area:
299368.948
counts
Height:
7.73e+004 cps
Start Time:
0.129
min
End Time:
0.542
min
760
2800
1.1e5
0.05
Sample Name: "Cal1__" Sample ID: "Cal1__" File: "ValRun23_09232010.wiff"
Peak Name: "TESTO C13(IS)" Mass(es): "292.2/100.1 Da"
Sample Index:
7
Sample Type:
Standard
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
7.6e4
Acq. Date:
9/23/2010
Acq. Time:
3:12:38 PM
7.4e4
Modified:
No
7.2e4
Noise Percentage:
50
Base. Sub. Window:
1.00
min
7.0e4
6.8e4
Min. Peak Height:
0.00 cps
Min. Peak Width:
0.00 sec
6.6e4
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.250
min
6.4e4
Use Relative RT:
No
0.50
920
780
Int. Type:
Base To Base
Retention Time:
0.496 min
Area:
3743.434 counts
Height:
9.36e+002 cps
Start Time:
0.388 min
End Time:
0.588 min
1.2e5
0.44
0.39
0.17
0.21
700
600
4.0e4
200
Aldosterone (S/P)
SLE+, 1ng/dL, 30s
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.496 min
Use Relative RT:
No
1.5e5
1.3e5
2900
0.15
Sample Name: "Cal1__" Sample ID: "Cal1__" File: "ValRun23_09232010.wiff"
PeakName: "331.3 / 109.1" Mass(es): "331.3/109.1 Da"
Sample Index:
7
Sample Type:
Standard
Concentration:
20.000
ng/mL
960
ng/mL
Acq. Date:
9/23/2010
940
Acq. Time:
3:12:38 PM
0.31
0.47
400
1.0e4
1.6e4
1.4e4
800
5.0e4
5000.0
1.8e4
900
300
0.10
2.0e4
1300
6.0e4
1.5e4
0.05
2.2e4
1400
0.01
500
0
0.40
Androstenedione (S/P)
SLE+, 10ng/dL, 20s
Sample Name: "S2_10ng/dL__" Sample ID: "S2_50ng/dL__" File: "SLE Cross Val Batch 11.wiff"
PeakName: "17-Hydroxyprogesterone" Mass(es): "331.3/109.1 Da"
Sample Index:
4
Sample Type:
Standard
Concentration:
50.000
ng/mL
ng/mL
3900
Acq. Date:
8/6/2010
Acq. Time:
3:59:29 PM
3800
Modified:
No
3700
Noise Percentage:
50
Base. Sub. Window:
1.00
min
3600
3500
Min. Peak Height:
200.00
cps
600
2.0e4
50
0.05
1500
1.0e5
700
3.0e4
100
5000.0
1.1e5
1200
0.11
150
1000.0
2.4e4
4.0e4
200
1.5e4
2.6e4
9.0e4
250
1500.0
2.8e4
1700
1600
8.0e4
1300
2.5e4
2.0e4
0.33
3.0e4
3.4e4
1900
1800
900
350
2500.0
3.2e4
2000
1.3e5
3.5e4
3.6e4
2100
1.4e5
2200
1000
0.11
4.0e4
3.8e4
1.6e5
1.5e5
4.5e4
400
4.2e4
1800
1200
0.22
4.6e4
4.4e4
2400
2300
1700
1400
300
2000.0
1.7e5
1100
2.5e4
3000.0
1900
5.0e4
0.17
4.8e4
2600
2500
600
3.0e4
4000.0
3500.0
5.2e4
6.0e4
450
5.4e4
5.0e4
5.5e4
3.5e4
5.8e4
5.6e4
2800
650
500
4500.0
6.0e4
3400
2700
1500
6.5e4
0.24
6.2e4
3300
3000
1.8e5
1600
550
0.16
3100
1.9e5
7.5e4
700
points
sec
min
2900
7.0e4
5.5e4
3
30.0
0.125
No
Int. Type:
Base To Base
Retention Time:
0.155
min
Area:
123737.326
counts
Height:
7.31e+004 cps
Start Time:
0.111
min
End Time:
0.238
min
3200
2100
750
7000.0
2.3e5
2000
800
6500.0
Smoothing Width:
RT Window:
Expected RT:
Use Relative RT:
4000
3600
2.2e5
8.0e4
0.41
4200
4100
Intensity, cps
6.0e4
2400
2300
9.0e4
4400
4300
3800
2.4e5
2500
1.1e5
1050
Sample Name: "Std 1 20 ng/dL" Sample ID: "Std 1 20NG/DL" File: "12004.wiff"
Peak Name: "DHEA IS(IS)" Mass(es): "277.2/97.1 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.00
ng/dL
Calculated Conc:
N/A
7.2e4
Acq. Date:
1/12/2011
Acq. Time:
12:12:51 PM
7.0e4
Modified:
No
6.8e4
Noise Percentage:
70
Base. Sub. Window:
1.00
min
6.6e4
Report Largest Peak: Yes
6.4e4
Min. Peak Width:
0.00 sec
0.16
4600
4500
3700
Intensity, cps
7500.0
2.5e5
Intensity, cps
6.5e4
Int. Type:
Base To Base
Retention Time:
0.162 min
Area:
7617.252 counts
Height:
4450. cps
Start Time:
0.124 min
End Time:
0.206 min
2.7e5
2700
1.1e5
1000
0.39
8000.0
2.8e5
2.6e5
2600
Intensity, cps
7.5e4
7.0e4
3.0e5
2.9e5
2.0e5
0.47
Intensity, cps
8500.0
Intensity, cps
Intensity, cps
Intensity, cps
9000.0
3.1e5
2.1e5
0.49
1100
8.0e4
9500.0
Modified:
No
Noise Percentage:
70
Base. Sub. Window:
1.00
min
Min. Peak Height:
150.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
3
points
RT Window:
30.0
sec
Expected RT:
0.138 min
Use Relative RT:
Yes
3500
1.2e5
1150
8.5e4
Sample Name: "Std 1 20 ng/dL" Sample ID: "Std 1 20NG/DL" File: "12004.wiff"
PeakName: "004102 DHEA" Mass(es): "271.2/213.1 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
20.000
ng/mL
4800
ng/mL
Acq. Date:
1/12/2011
4700
Acq. Time:
12:12:51 PM
0.26
3.2e5
3900
Int. Type:
Valley
Retention Time:
0.264
min
Area:
635630.07
counts
Height:
3.40e+005 cps
Start Time:
0.192
min
End Time:
0.364
min
3000
2900
1.2e5
1200
9.0e4
1.1e4
26
3500
Int. Type:
Base To Base
Retention Time:
0.269 min
Area:
7140.14 counts
Height:
3748.45 cps
Start Time:
0.234 min
End Time:
0.338 min
1.3e5
1300
1.2e4
1.1e4
0
1.5e5
1.4e5
1250
9.5e4
500
1.5e5
1.4e5
min
counts
cps
min
min
Modified:
No
Noise Percentage:
40
Base. Sub. Window:
1.00
min
Report Largest Peak: Yes
Min. Peak Width:
0.00 sec
Smoothing Width:
7
points
RT Window:
30.0
sec
Expected RT:
0.260
min
Use Relative RT:
No
3400
1350
1.2e4
0.00
sec
3
points
30.0
sec
0.330 min
No
1.6e5
1.6e5
Sample Name: "Cal1- .20 ug/L" Sample ID: "Cal001" File: "11032.wiff"
Peak Name: "Aldosterone IS(IS)" Mass(es): "366.2/303.0 Da"
Sample Index:
3
Sample Type:
Standard
3.4e5
Concentration:
1.00
ng/dL
Calculated Conc:
N/A
Acq. Date:
1/12/2011
3.3e5
Acq. Time:
11:28:55 PM
0.27
3600
Modified:
No
Noise Percentage:
50
Base. Sub. Window:
1.00
min
Min. Peak Height:
0.00
cps
Min. Peak Width:
0.00
sec
Smoothing Width:
7
points
RT Window:
30.0
sec
Expected RT:
0.270 min
Use Relative RT:
No
1.3e5
1.1e5
1.3e4
Int. Type:
Valley
Retention Time:
0.342 min
Area:
17737.700 counts
Height:
3.75e+003 cps
Start Time:
0.250 min
End Time:
0.455 min
Sample Name: "Cal1- .20 ug/L" Sample ID: "Cal001" File: "11032.wiff"
Peak Name: "289117 Aldosterone" Mass(es): "359.2/189.0 Da,359.2/188.9 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
0.200
ng/mL
3800
ng/mL
Acq. Date:
1/12/2011
3700
Acq. Time:
11:28:55 PM
0.28
1.7e5
1400
1.3e4
Min. Peak Width:
Smoothing Width:
RT Window:
Expected RT:
Use Relative RT:
Sample Name: "Cal001-1 ng/dL" Sample ID: "Cal1- 1 ng/dL" File: "10033.wiff"
Peak Name: "D7-Aldosterone(IS)" Mass(es): "366.2/194.0 Da"
Sample Index:
3
Sample Type:
Standard
Concentration:
1.00
ng/mL
Calculated Conc:
N/A
Acq. Date:
1/11/2011
1.7e5
Acq. Time:
3:30:23 PM
0.28
1800
0
0.1
0.2
0.3
0.4
Time, min
0.5
0.6
0.7
17OHProgesterone (U)
Hydrolysis/LLE,
20ng/dL, 45s
0.1
0.2
0.3
Time, min
0.4
0.5
0.6
0.0
0.1
0.2
0.3
Time, min
0.4
0.5
0.6
Cortisol (U)
SLE+, 1ng/mL, 40s
0
0.1
0.2
0.3
Time, min
0.4
0.5
0.6
0.0
0.1
0.2
0.3
Time, min
0.4
0.5
Cortisone (U)
SLE, 20ng/mL, 40s
0.6
MedWatch: 184 Drugs/Metabolites in Urine
Urine + IS/Acid, Seal Plate, Mix, Hydrolysis, Centrifuge, Inject
UPLC-API5500 ESI Scheduled MRM, 354 monitored transitions
XIC of +MRM (379 pairs): 304.200/230.100 amu Expected RT: 3.4 ID: ezogabine 2 from Sample 25 (MIX 2 INJ STD) of 08-29-13 X QC.w...
Max. 2.7e6 cps.
4.2e6
4.0e6
3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
2.8e6
2.6e6
Intensity, cps
2.4e6
2.2e6
2.0e6
1.8e6
1.6e6
Opiates
Opioids
Benzodiazepines
Drugs of abuse
Antiepileptics
Antipsychotics
Antidepressants
Analgesics
3.40
1.4e6
1.2e6
1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.0
0.0
27
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
Time, min
2.8
3.0
3.2
3.4
3.63
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
Immunoassay and LC-MS/MS Concordance
28
Pre ASCENT™ Implementation Analyst/Reporter
LIS +
Accession
Receipt
Print
annotate
+ve’s
Reporter
(word)
Review 2
Data sets
29
QC Review
Immuno
Screen
LIS
Upload
LCMS/MS
analysis
Analyst
Batch
Load
Extraction
Analyst
Peak
correct
LIS Entry
Certify
Records
Post ASCENT™ Implementation Ascent
LIS +
Accession
Receipt
Immuno
Screen
LIS
Upload
LCMS/MS
analysis
Web
Review
30
QC Flag +
Screen
Consistent
Ascent
Peak
Correct
LIS
Batch Load
Extraction
Certify
QC File +
LIS Entry
Records
Automated Data review
If [examined value] exceeds [a threshold], then flag with [text string].
40 QA rules (18 custom)
20 “Batch rules”
20 “Chromatogram rules”
31
Automated Data review Impact
Absolute time
Tech Time
200% Manual review
Ascent + 100% Manual
Ascent + outlier review
Requires a LOT of well designed and validated QA rules in Ascent®
Identical to Autoanalyzer Autoverification
32
Ascent is a registered product of Indigo Biosystems
Isotope Dilution versus Reference Method
Towards “Internal Calibration + Random Access”
Isotope Dilution: Range Analyte concentrations (Ac), one IS concentration (Ic),
29006.rdb (070036 Testosterone, To): "Quadratic" Regression ("1 / x" weighting): y = -5.39e-008 x^2 + 0.00588 x + 0.0043 (r = 0.9999)
29
28
26
24
22
Analyte peak area (Ar)
IS peak Area (Ir)
20
A n a ly te A re a / IS A re a
18
16
14
12
10
8
6
4
2
0
0
500
1000
1500
2000
2500
Analyte Conc. / IS Conc.
3000
3500
4000
4500
5000
Analyte concentration (Ac) / Internal standard concentration (Ic)
Reference method: Spike IS at concentration close to Analyte Concentration from
Isotope Dilution
XIC of +MRM (4 pairs): 292.200/100.100 Da ID: 13C3 Testo Quant from Sample 6 (1000ng/dL) of MLC 020413.wiff (Heated Nebulizer), ...
Max. 3.4e4 cps.
0.18
3.4e4
3.2e4
Ac = (Ar * Ic )/Ir
3.0e4
2.8e4
2.6e4
2.4e4
Ac = 93474 * 1000 ng/dL / 108564 =
861.034 ng/dL
2.2e4
In te n s ity , c p s
2.0e4
1.8e4
1.6e4
1.4e4
1.2e4
1.0e4
8000.0
6000.0
4000.0
2000.0
0.0
0.00
33
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
Time, min
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
Internal standard and analyte MUST be
analytically equivalent
Use of IS near Medical Decision Points
XIC of -MRM (6 pairs): 120.100/76.000 Da from Sample 93 (032-304-7358-0) of 35011.wiff (Turbo Spray), Smoothed, Smoothed
Max. 4.8e4 cps.
2.0e5
1.9e5
MMA = 373 nM
1.8e5
1.7e5
1.6e5
1.5e5
IS: (m/z) 120 – 76 1000nM (Y-axis ratio max 15)
1.4e5
1.3e5
Intens ity, c ps
1.2e5
1.1e5
1.0e5
IS: (m/z) 120 – 76* 300nM (Y-axis ratio max 58)
9.0e4
8.0e4
7.0e4
6.0e4
IS: (m/z) 120 – 58 90nM (Y-axis ratio max 190)
0.22
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
0.0
0.05
0.10
0.15
0.20
0.25
Time, min
0.30
0.35
0.40
0.45
Peak Area Ratio
Low MDP IS/ High MDP IS
IS Transition ratio Low MDP/High MDP
34
MMA Concentration (nM)
No “Calibration” – IS at MDP Pseudo Reference Method
Reference method IS at 300 nM
Slope [Deming] = 0.961
Correlation Coefficient = 0.9946
Mean Bias = -1.55%
Patient mean = 271nM
Reference method IS at 80 nM
Mean Bias = 4.45%
Reference method Mean of 300 and 80nM
Mean Bias = 0.97%
35
Medical Decision Points and IS position
Testosterone Medical Decision Points (MDP, ng/dL)
Age (y)
Low
High
1-10
<3
10
11-18 (F)
<3
38
11-18 (M)
<3
970
20 - 50 (F Pre)
10
55
20 - 50 (M)
350
1030
60 - 80 (F Post)
7
10
60 - 80 (M)
7
40
2.5e6
2.4e6
2.3e6
2.2e6
2.1e6
2.0e6
1.9e6
IS: (m/z) 292 – 112 (4000ng/dL - 2.6e6)
IS: (m/z) 292 – 112 (1500ng/dL - 9.6e5)
Testosterone (m/z) 289 – 97 = 517ng/dL (3.4e5)
IS: (m/z) 292 – 112 100ng/dL - 6.8e4
IS: (m/z) 292 – 112 (50ng/dL - 3.7e4)
IS: (m/z) 292 – 112 (10ng/dL - 5187)
IS: (m/z) 292 – 112 (5ng/dL - 2950)
1.8e6
1.7e6
1.6e6
1.5e6
Intensity, cps
1.4e6
1.3e6
1.2e6
1.1e6
1.0e6
9.0e5
8.0e5
7.0e5
6.0e5
5.0e5
4.0e5
3.0e5
2.0e5
1.0e5
0.0
0.00
36
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18 0.20 0.22
Time, min
0.24
0.26
0.28
0.30
0.32
0.34
0.36
0.38
Determining IS Concentration - Gravimetry
Internal Standard Concentration (Ic) = (Ac * Ir) / Ar
Analyte
Analyte Peak
Concentration (Ac) Area (Ar)
IS Peak Area (Ir)
2.5
7054.517
10532.26
5
11290.82
8825.85
10
23821.803
11314.437
50
95506.79
9710.595
200
456837.937
10603.594
1000
1970307.633
9122.446
2500
5253780.503
9596.876
5000
10390778.7
10347.679
Mean IS Concentration equivalent (ng/dL)
Standard Deviation (% CV)
IS #1
3.732
3.908
4.750
5.084
4.642
4.630
4.567
4.979
4.537
10.548
IS #2
5.747
6.495
7.138
7.067
7.484
6.954
6.997
7.042
6.866
7.671
IS #3
45.956
49.458
55.229
55.869
54.440
56.312
55.655
58.074
53.874
7.526
IS #4
80.630
88.675
96.047
98.328
97.518
99.417
100.661
104.044
95.665
7.851
IS #5
1266.153
1403.244
1547.910
1587.144
1543.240
1583.272
1595.970
1607.158
1516.761
7.927
IS #6
3224.713
3622.708
4016.756
4103.246
3901.718
4171.816
4049.695
4205.207
3911.982
8.512
Normalized Ic
1.2
1.1
IS 1
1
IS 2
IS 3
0.9
IS 4
0.8
IS 5
IS 6
0.7
0.6
0
37
1
2
3
4
5
6
Calibrator level
7
8
9
Mean of 6 IS results (Cals) vs CDC Phase 1
Slope [Deming] = 0.980 (0.972 – 0.988)
Intercept = 0.880 (-1.899 – 3.658)
Mean Bias = -1.662%
38
Average IS versus Isotope Dilution
Slope [Deming] = 0.945 (0.941 – 0.949)
Intercept = 1.962 (0.332 – 3.597)
Mean Bias = -4.899%
“Approximate Matching” IS versus Isotope Dilution
Slope [Deming] = 1.002 (0.998 – 1.006)
Intercept = 0.080 (-1.546 – 1.707)
Mean Bias = 0.251%
39
Population Pooling Testosterone Men (N=400)
Pool
25
Mean
CV (%)
Target
50
Mean
CV (%)
Target
100
Mean
CV (%)
Target
200
Mean
CV (%)
Target
400
40
Mean
CV (%)
1
372
376
356
365
2
530
550
520
555
367
539
2.39
3
362
393
362
384
375
4.19
3.07
453
440
465
435
451
448
2.97
4
433
465
439
449
447
3.13
5
434
433
414
441
431
2.69
411
403
426
396
416
410
3.26
6
523
562
518
527
533
3.76
7
507
526
495
534
516
3.44
482
409
424
397
416
412
2.78
429
420
420
421
405
417
1.84
8
465
494
475
507
485
3.88
9
407
427
408
436
420
3.42
500
514
540
513
534
525
2.63
10
398
409
382
407
399
3.08
11
395
402
388
416
400
2.99
409
394
409
389
416
402
3.14
468
469
484
468
478
475
1.61
12
483
499
456
502
485
4.34
13
487
499
475
501
491
2.45
443
444
472
455
458
457
2.52
451
3.27
16
440
465
435
451
448
2.97
422
412
449
433
441
434
3.67
462
497
495
488
480
490
1.57
460
445
473
469
484
468
3.51
460
449
470
437
448
15
392
417
375
404
397
4.50
479
479
498
490
497
491
1.78
430
425
444
413
435
429
3.10
446
443
470
437
457
452
3.27
14
445
479
472
472
467
3.22
Population Pools as Calibrators
Testosterone (MDP, ng/dL)
Age (y)
Low
High
1-10
<3
10
11-18 (F)
<3
38
11-18 (M)
<3
970
20 - 50 (F Pre)
10
55
20 - 50 (M)
350
1030
60 - 80 (F Post)
7
10
60 - 80 (M)
7
40
28000 Males 20 – 50 All
1 - 10 MF
41
11 – 18 F
N
10782
19453
25434
6551
28000
5830
21452
Minus Outliers
11 – 18 M
Testosterone Population (ng/dL)
Mean
Median Measured (ID) Measured (CDC)
5.43
4.70
2.75
3.42
30.68
28.70
27.56
28.35
303.00
285.45
261.81
261.17
24.78
23.00
30.07
29.99
382.69
365.83
450.38
450.25
18.62
17.05
16.98
17.48
371.63
354.63
NA
NA
Boxcox transformation
20 – 50 F
Cumulative Frequency and
Regression Line 20 – 50 M
60 – 80 F
60 – 80 M
Population Pools as Calibrators vs CDC Phase 1
Population Median
Intercept = 1.057
Mean Bias = -9.902%
Pool Targets from ID
Intercept = -0.528
Mean Bias = -0.799%
Pool Targets CDC Assigned
Intercept = 0.101
Mean Bias = -0.821%
42
The Litmus Test
43
A: Standard Isotope Dilution
B: IS Mean (Cals)
C: IS Approximatly Matched (Cals)
D: IS Mean (CDC)
E: IS Approximately Matched (CDC)
F: Population Pool (Median)
G: Population Pool (Assigned by ID)
H: Population Pool (Assigned by CDC)
Conclusions
Dynamic volume:
Bracketing QC’s
Generic sample preparation (good QQQ)
Generic Chromatographic platform
Longitudinal Calibration
Automated data release
Batch volume:
Contiguous Automation
Very high throughput multiplexing
Advanced LC configuration
Robust LC methods
Automated data release
Acknowledgements
LabCorp: Dr Christopher Shuford, Patricia Holland, Matt Crawford, Stacy Dee,
Yvonne Wright, Martin Green, Dr Marcia Eisenberg, Gregory Janis, Dr Karla Walker
Essential Testing: Brian Rappold
44

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