Meeting the LC-MS Needs of a High-Throughput Clinical Lab
Transcript of Meeting the LC-MS Needs of a High-Throughput Clinical Lab
Meeting the LC-MS needs of a high-throughput clinical laboratory
April 18th 10:00 [New York]/ 15:00 [London]
Meeting the LC-MS Needs of a High-Throughput Clinical Chemistry Laboratory
With examples from endocrinology and biochemical genetics unit disciplines within healthcare laboratories, this presentation will cover alternative sample extraction and liquid chromatography solutions developed to meet the challenges of a busy hospital laboratory.
Today’s Hosts: Today’s Presenter: Isaac Bruce Michael J.P. Wright
Commissioning Editor Cambridge University
Bioanalysis Hospital Trust
Michael J.P. Wright Lead Method Developer
Cambridge University Hospital Trust
Our Speaker
Meeting the LC-MS Needs of a High-Throughput Clinical
Chemistry Laboratory
Michael Wright
Biochemistry Department
Addenbrooke’s Hospital
Cambridge University Hospital Trust
The Question “What’s wrong with this person?”
“I think it might be disease X,
send a sample to the lab”
The Matrix -
Saliva Urine
Blood
Faeces Dried Blood Spot
Amniotic Fluid
Cerebrospinal Fluid
Bile
The human body
The Target - Biomarkers
What is Clinical Chemistry?
What does it look/sound like?
Why LC-MS/MS?
Seen as a sensitive and “specific”/selective way of measuring target analytes
Replacement/Confirmation test for expensive, imprecise or inaccurate Immunoassays (IA)
Replacement for traditional UV, flourescent, electrochemical detection for HPLC
Replacement for lengthy GC-MS methods that have extensive sample clean-up
Topics to cover
1) Selecting the correct stationary phase
2) Is ultra high-pressure chromatography always the best option?
3) Sample preparation options for a high-throughput clinical laboratory
4) Where are we now with clinical chemistry LC-MS?
1: Choosing the appropriate chromatography
Target analytes often differ greatly to those found in TDM or Toxicology Endogenous, Low concentration (pg/mL) often part of large, closely related families of endogenous
compounds
In many cases “dilute and shoot, trap & elute” methods are not suitable for accurate analysis
Use of MS detection limits the buffers/ ion pair reagents that we have available – more reliant on the stationary phase
Isobaric interferences
XIC of +MRM (6 pairs): 363.300/121.200 Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_08 Ascentis HP Cortisol run.wiff (Heat... Max. 2.8e4 cps.
0.0 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 2.8 3.0 3.2 3.4 3.6 3.81 37 74 110 146 183 219 255 292 328 365 401 437 474 510 546 583 619 655 692
Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
Inte
ns
ity,
cp
s
3.01
2.14
2.69
3.71
2.84
3.98
2.541.62 1.891.851.59 2.43 3.18 3.543.280.89
Cortisol
?
?
?
? ?
Urinary Free Cortisol Ascentis Express 100x2.1mm 2.7µm Phenyl Hexyl
25OHD3
3-epi 25OHD3
8.0Time, min
2.3e4
Inte
nsity, c
ps
7.24
6.75
25OHD3
1.0
3epi- 25OHD3
1.0 5.0Time, min
1.9e5
Inte
nsity, c
ps
2.52
25OHD3 &
3epi- 25OHD3
A
B
25OHD3
3-epi 25OHD3
8.0Time, min
2.3e4
Inte
nsity, c
ps
7.24
6.75
25OHD3
1.0
3epi- 25OHD3
1.0 5.0Time, min
1.9e5
Inte
nsity, c
ps
2.52
25OHD3 &
3epi- 25OHD3
A
B
Unusual patient samples
Serum testosterone
Serum 25OH Vitamin D3
25OH Vitamin D analysis in infants
25OHD3
3-epi 25OHD3
8.0Time, min
2.3e4
Inte
ns
ity, c
ps
7.24
6.75
25OHD3
1.0
3epi- 25OHD3
1.0 5.0Time, min
1.9e5
Inte
ns
ity, c
ps
2.52
25OHD3 &
3epi- 25OHD3
A
B
25OHD3
3-epi 25OHD3
8.0Time, min
2.3e4
Inte
ns
ity, c
ps
7.24
6.75
25OHD3
1.0
3epi- 25OHD3
1.0 5.0Time, min
1.9e5
Inte
ns
ity, c
ps
2.52
25OHD3 &
3epi- 25OHD3
A
B
Ascentis Express 100 x 2.1mm 2.7µm F5
Wright MJ, Halsall DJH and Keevil BG. Clin Chem 2012; v. 58, p.1719-1720
F
F
F
F
F
SiOSi
Ascentis Express 50 x 2.1mm 2.7µm C8
Polar analytes
Creatinine
Guanidinoacetate
Creatine
150 x 4.6mm 5µm C18
Ion Suppression trace
Ascentis Express 50x2.1mm 2.7µm HILIC
Urinary Xanthine, Hyperxanthine and Sulphocysteine
XIC of -MRM (12 pairs): 151.100/107.900 Da ID: Xanthine 1 from Sample 1 (HX X SSC ) of 2011_07_26 C18 Run .wiff (Turbo Spray) Max. 1.2e4 cps.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
Inte
ns
ity
, c
ps
0.33
6.08 6.305.22 6.435.15 5.975.75 6.675.334.674.290.21 0.53 4.183.981.99 3.09 3.471.18 1.721.65 2.931.120.79 2.37 2.492.05
XIC of -MRM (12 pairs): 151.100/107.900 Da ID: Xanthine 1 from Sample 4 (HX X SSC ) of 2011_07_26 HILIC Run.wiff (Turbo Spray) Max. 1.5e5 cps.
0.0 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 2.8 3.0 3.2 3.4 3.6 3.8Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
Inte
ns
ity
, c
ps
0.50
0.36
0.97
Ascentis Express 100x 2.1mm 2.7µm C18 Ascentis Express 100x 2.1mm 2.7µm HILIC
S-sulphocysteine Xanthine Hypoxanthine
Chiral Columns for Non-Chiral Separations
ionic site
ionic site
-acceptor
OH
NHR
CH 2 OH
HO
O
HNCOCH 3
HO
O
NH 2
O
HO
Cl
H H O
H
O
H N
N H
O
H
B
A
O Cl
N
O
N
H O
H H O
C
OH O HO
N H H
N H
O
HOOC
H D
OH
CH 2 OH OH
OH
O
Key interaction sites; A, B,C and D are cavities
Teicoplanin (Chirobiotic T)
Urinary S-sulphocysteine, Xanthine & Hyperxanthine on Chirobiotic-T
Astec 100x2.1 5µm Chirobiotic T
P =1000FL
r2dp2
P =
5µm
3µm
1.7µm
1100psi
2900psi
8600psi
12000
22000
32500
Performance
(Plates)
2: Is ultra high-pressure chromatography always the best option?
Superficially porous particle columns
Ascentis Express Fused Core columns
Mobile Phase Velocity (mm/sec)
2 4 6 8 10 12
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,000
3 µm
1.7 µm
2.7 µm Fused-Core
ca. 1 mL/min
Mobile Phase Velocity (mm/sec)
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
2.7 µm Fused-Core
2 4 6 8 10 12
ca. 1 mL/min
Sub-2µm efficiency achieved at near 3µm pressures.
*50x4.6 mm columns, 55/45 ACN/Water
Advantages – higher performance:pressure
1.5min run time 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
-0.02
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
1.26
5HIAA 50% IPA STD QC CHECK 1_2ML #8 STD2 Fused Core 1.5ml 1µl inj ECD_1
nA
min
1 - 0.607
2 - 0.723
3 - 0.823
4 - 5HIAA - 0.933
5 - Internal Std - 1.170
6 - 1.413
1 - 5HIAA 2 - Internal Std
Min
Hgt
= 0
.010
, Inh
bit =
On
Inhb
it =
Off
1
2
3
4
5
6
Urine 5HIAA
BP= 2070 psi
Ascentis® Express C18
Dim: 50x4.6mm 2.7µm
3: Sample preparation options for a high-throughput clinical laboratory
Target analytes present in samples at low concentrations – require sample extraction to remove matrix effects & possibly concentrate sample
Liquid-Liquid Extraction? 1. Aliquot sample + Internal Standard + Solvent 2. Vortex 3. Centrifuge 4. Aspirate the solvent layer (freeze the aq. layer) 5. Dry down under N2 or in rotary evaporator 6. Reconstitute in mobile phase 7. Centrifuge 8. Transfer to auto-sampler vials/plates
Time consuming Not always suitable Expensive to automate SLE?
Solid Phase Extraction?
Time consuming Expensive to automate Consumable costs
Online Solid Phase Extraction
Auto-sampler
Pump(s) 1 Pump(s) 2
Column
Oven Mass
Spectrometer
2 position divert valve
On-line SPE cartridge
Analytical column
WASTE
Protein Precipitation step
Process a plate of Calibrators, QC and 84 patient samples in 30min
100µL Serum 25µL Internal Standard 25µL 0.2M ZnSO4
200µL Methanol
Online Solid Phase Extraction
Autosampler
Waste
Eluting Pumps
Analytical Column
Strata 20×2.0mm 20μm C8
XIC of +MRM (6 pairs): 363.300/121.200 Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_08 Ascentis HP Cortisol run.wiff (Heat... Max. 2.8e4 cps.
0.0 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 2.8 3.0 3.2 3.4 3.6 3.81 37 74 110 146 183 219 255 292 328 365 401 437 474 510 546 583 619 655 692
Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
Inte
ns
ity
, c
ps
3.01
2.14
2.69
3.71
2.84
3.98
2.541.62 1.891.851.59 2.43 3.18 3.543.280.89
Urine Cortisol Serum Testosterone XIC of +MRM (5 pairs): 289.200/97.000 Da ID: testo 1 from Sample 59 (Sample 15) of 2011_06_10 New Ascentis Express Method.wiff (... Max. 8.5e5 cps.
0.0 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 2.8 3.0 3.2 3.41 54 108 161 214 268 321 374 428 481 534 587 641 694 747 801 854 907
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
Inte
ns
ity
, c
ps
2.83
25OH Vitamin D 2/3 Thyroxine (T4) XIC of +MRM (9 pairs): 383.200/257.100 Da ID: D3 quant from Sample 6 (Std 4) of 2011_11_21.wiff (Heated Nebulizer) Max. 1.6e5 cps.
0.0 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 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.81 25 49 74 98 122 146 171 195 219 243 268 292 316 340 365 389 413 437 461 486 510 534 558 583
Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
Inte
ns
ity
, c
ps
2.37
Meeting the needs of the department
Staff Time: LLE > Off-line SPE > On-Line SPE
Consumable Cost: Off-line SPE >On-line SPE > LLE
Automation Cost: LLE >Off-line SPE = On-line SPE
Sample Extraction Time LLE > Off-line SPE > On-line SPE
LC-MS Workload at Addenbrooke’s
• Urine Metanephrines
• Urine 5HIAA
• Urine & Plasma Creatine/Guanidinoacetate/Creatinine
• B/S Carnitines
• Homocysteines
• Urine, Serum and Salivary Cortisol
• 25OH Vitamin D2/3
• Serum androgens
• Newborn Screening MCADD/PKU
• TDM Tacrolimus, Cyclosporin, Sirolimus
• Total thyroxine
• DBS Testosterone
• Skin steroid panel
• Cell culture steroid panels
Studies
• 3-epi-25OH Vitamin D2/3
Evaluation/validation
• Plasma metanephrines
• Insulin
• T3, rT3
• Aldosterone
• 17OHP/DHEAS
• Androstenedione
Multiplexing
“What’s the point of running an overnight batch at 2min/sample when the system then sits unused for 10 hours throughout the rest of the night?”
Autosampler
Pump(s) 1
Column
Oven
Mass
Spectrometer
6 position selection valve
Analytical column
Solvent selector
Adding Online SPE
Autosampler
Pump(s) 1 Pump(s) 2
Column
Oven
Mass
Spectrometer
6 position selection valve
2 position divert valve
On-line SPE cartridge
Analytical column
Solvent selector
Pump(s) 1 Pump(s) 2
WASTE
Multiplexing
Example from Addenbrookes
1) Serum androgens – 48 samples by Online SPE (3½ hours)
2) Conditioning (40min)
3) Urine Cortisols – 36 samples by Online SPE (4 hours)
4) Conditioning (40min)
5) Serum 25OH Vit D3/2 – 96 samples by Online SPE (8 hours)
Run put on at 4pm has finished at 9am the following morning
XIC of +MRM (5 pairs): 289.200/97.000 Da ID: testo 1 from Sample 59 (Sample 15) of 2011_06_10 New Ascentis Express Method.wiff (... Max. 8.5e5 cps.
0.0 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 2.8 3.0 3.2 3.41 54 108 161 214 268 321 374 428 481 534 587 641 694 747 801 854 907
Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5In
ten
sit
y,
cp
s2.83
Serum Testosterone
XIC of +MRM (6 pairs): 363.300/121.200 Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_07.wiff (Heated Nebulizer) Max. 2.2e4 cps.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.51 92 183 274 365 455 546 637 728 819 910 1001 1092 1182
Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e4
1.9e4
2.0e4
2.1e4
2.2e4
Inte
ns
ity
, c
ps
4.80
3.46
4.29
3.654.51
6.09
6.91
6.636.44
5.893.32 3.822.94 4.95 5.202.85 5.682.632.340.11 2.121.710.36 1.630.46
Urine Cortisol
XIC of +MRM (9 pairs): 383.200/257.100 Da ID: D3 quant from Sample 6 (Std 4) of 2011_11_21.wiff (Heated Nebulizer) Max. 1.6e5 cps.
0.0 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 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.81 25 49 74 98 122 146 171 195 219 243 268 292 316 340 365 389 413 437 461 486 510 534 558 583
Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
Inte
ns
ity
, c
ps
2.37
TIC: from Sample 11 (Std 10) of 2011_11_08 skin samples APCI.wiff (Heated Nebulizer) Max. 2.3e5 cps.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.51 49 96 144 191 239 287 336 385 433 470 507 543 580 617 653
Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
2.3e5
Inte
ns
ity
, c
ps
5.46
6.07
7.13
2.47
7.032.17
7.333.64 5.655.30
Serum 25OH Vitamin D Skin Steroids
25OHD 3
3 - epi 25OHD 3
8.0 Time, min
2.3e4
Inte
nsity
, cp
s
7.24
6.75
25OHD 3
1.0
- 3
1.0 5.0 Time, min
1.9e5
Inte
nsity
, cp
s
2.52
25OHD 3 & 3epi - 25OHD 3
A
B
25OHD 3
3 - epi 25OHD 3
8.0 Time, min
2.3e4
Inte
nsity
, cp
s
7.24
6.75
1.0
1.0 5.0 Time, min
1.9e5
Inte
nsity
, cp
s
2.52 A
B
Confirmation Analysis
Multiplexing points to remember
There is a larger amount of tubing - ensure that this is as small ID as possible to reduce extra column volumes and thus longitudinal diffusion (B-term) Using 2.1mm ID columns we found 500µl/min to be the
lowest flow rate we could use whilst not suffering from longitudinal diffusion
Incompatible mobile phases require longer equilibration
Keep Curtain Gas as high as you can on all methods
HybridSPE
•Simplify the procedures of protein precipitation and phospholipid removal into one step
Phospholipid depletion filter plates
Protein precipitation plate
Collection plate
Hybrid SPE plate
100µL Serum 25µL Internal Standard 25µL 0.2M ZnSO4
200µL Methanol
Craig R Aurand, David S Bell & Michael Wright. Bioanalysis 2012; 4(22) p.2681-2691
Topic 4. Where are we now?
Certified serum based reference standards
Urinary Free Cortisol
Reference Range?
Serum Testosterone
Clinically useful cross reactivity
Steroid Panels
Sample Immulite LCMSMS
Urine blank 67 35
Urine + 2µg/ml THE 100 33
Urine + 2µg/ml THF 5α 593 38
Urine + 2µg/ml THF 5β >1300 33
Urine + 2µg/ml α-Cortolone 74 32
Acknowledgements
Addenbrooke’s Hospital David Halsall
Kevin Taylor
Lisa Tanner
AB Sciex Steven Ayris
Sigma/Supelco Dave Bell
Craig Aurand
Denise Walworth
Kings College Hospital
Colin Stone
Clare Glicksman
Evelina Children’s Hospital
Neil Dalton
Charles Turner
Shimadzu
Earl McCoy
Questions
Additional Information
Contact email for Michael Wright
Ascentis Express HPLC Columns
Sigma-aldrich.com/express
HybridSPE-Phospholipid removal
sigma-aldrich.com/hybridspe-pl
Chiral HPLC Columns
sigma-aldrich.com/chiral
Thank you
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