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

mwright_11@yahoo.co.uk

Questions

Additional Information

Contact email for Michael Wright

mwright_11@yahoo.co.uk

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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