Meeting the LC-MS needs of a highthroughput. laboratory. April 18 th. 10:00 [New York]/ 15:00 [London]

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1 Meeting the LC-MS needs of a highthroughput clinical laboratory April 18 th 10:00 [New York]/ 15:00 [London]

2 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: Isaac Bruce Commissioning Editor Bioanalysis Today s Presenter: Michael J.P. Wright Cambridge University Hospital Trust

3 Our Speaker Michael J.P. Wright Lead Method Developer Cambridge University Hospital Trust

4 Meeting the LC-MS Needs of a High-Throughput Clinical Chemistry Laboratory Michael Wright Biochemistry Department Addenbrooke s Hospital Cambridge University Hospital Trust

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6 What is Clinical Chemistry? The Question What s wrong with this person? What does it look/sound like? The Matrix - Urine Blood Faeces The human body I think it might be disease X, send a sample to the lab Saliva Cerebrospinal Fluid Bile Amniotic Fluid Dried Blood Spot The Target - Biomarkers

7 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

8 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 highthroughput clinical laboratory 4) Where are we now with clinical chemistry LC-MS?

9 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

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11 Isobaric interferences Urinary Free Cortisol Ascentis Express 100x2.1mm 2.7µm Phenyl Hexyl XIC of +MRM (6 pairs): / Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_08 Ascentis HP Cortisol run.wiff (Heat e4 2.6e4 2.4e4 2.2e4 2.0e4? Max. 2.8e4 cps. Cortisol In te n s ity, c p s 1.8e4 1.6e4 1.4e4 1.2e4 1.0e ?? 2.69?? Time, min

12 Intensity, cps Unusual patient samples Serum testosterone A 1.9e5 25OHD 3 & 2.52 Serum 25OH Vitamin D3 3epi- 25OHD 3 B Time, min

13 Intensity, cps Intensity, cps 25OH Vitamin D analysis in infants A 1.9e5 25OHD 3 & 3epi- 25OHD Ascentis Express 50 x 2.1mm 2.7µm C8 B 2.3e Time, min 3epi- 25OHD 3 3-epi 25OHD F F F 25OHD 3 Si O Si F 25OHD 3 F 1.0 Time, min 8.0 Ascentis Express 100 x 2.1mm 2.7µm F5 Wright MJ, Halsall DJH and Keevil BG. Clin Chem 2012; v. 58, p

14 Polar analytes Creatinine Ascentis Express 50x2.1mm 2.7µm HILIC Guanidinoacetate Ion Suppression trace Creatine 150 x 4.6mm 5µm C18

15 Urinary Xanthine, Hyperxanthine and Sulphocysteine S-sulphocysteine Xanthine Hypoxanthine XIC of -MRM (12 pairs): / Da ID: Xanthine 1 from Sample 1 (HX X SSC ) of 2011_07_26 C18 Run.wiff (Turbo Spray) Max. 1.2e4 cps. XIC of -MRM (12 pairs): / Da ID: Xanthine 1 from Sample 4 (HX X SSC ) of 2011_07_26 HILIC Run.wiff (Turbo Spray) Max. 1.5e5 cps. 1.4e5 1.3e5 1.2e5 1.1e5 1.5e5 1.4e5 1.3e5 1.2e e5 1.1e5 9.0e4 1.0e5 In te n s ity, c p s 8.0e4 7.0e4 6.0e4 In te n s ity, c p s 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 5.0e4 4.0e4 4.0e4 3.0e4 3.0e4 2.0e4 1.0e e4 1.0e Time, min Time, min Ascentis Express 100x 2.1mm 2.7µm C18 Ascentis Express 100x 2.1mm 2.7µm HILIC

16 Chiral Columns for Non-Chiral Separations Teicoplanin (Chirobiotic T) ionic site O HNCOCH 3 H N HOOC HO OH HO H CH 2 OH O Cl O N H H A B NHR O O O O H H N N O H H OH O O OH OH C HO H Cl H N CH 2 OH OH -acceptor O H D O HO N H O H ionic site Key interaction sites; A, B,C and D are cavities NH 2

17 Urinary S-sulphocysteine, Xanthine & Hyperxanthine on Chirobiotic-T Astec 100x2.1 5µm Chirobiotic T

18 2: Is ultra high-pressure chromatography always the best option? Performance (Plates) P P = = 1000F L r 2 d 2 p 5µm psi 3µm psi 1.7µm psi

19 Superficially porous particle columns

20 Ascentis Express Fused Core columns Sub-2µm efficiency achieved at near 3µm pressures ca. 1 ml/min 2.7 µm Fused-Core 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 ca. 1 ml/min 1.7 µm 2.7 µm Fused-Core 3 µm Mobile Phase Velocity (mm/sec) Mobile Phase Velocity (mm/sec) *50x4.6 mm columns, 55/45 ACN/Water

21 MinHgt = 0.010, Inhbit = On Inhbit = Off Advantages higher performance:pressure Urine 5HIAA 5HIAA 50% IPA STD QC CHECK 1_2ML #8 STD2 Fused Core 1.5ml 1µl inj ECD_1 na 4 4-5HIAA Ascentis Express C18 Dim: 50x4.6mm 2.7µm 1-5HIAA 2 - Internal Std Internal Std BP= 2070 psi min run time min

22 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 N 2 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?

23 Solid Phase Extraction? Time consuming Expensive to automate Consumable costs

24 Online Solid Phase Extraction 2 position divert valve On-line SPE cartridge Analytical column Pump(s) 1 Pump(s) 2 Auto-sampler Column Oven Mass Spectrometer WASTE

25 Protein Precipitation step 100µL Serum 25µL Internal Standard 25µL 0.2M ZnSO 4 200µL Methanol Process a plate of Calibrators, QC and 84 patient samples in 30min

26 Online Solid Phase Extraction Autosampler Eluting Pumps Waste Analytical Column Strata mm 20μm C8

27 XIC of +MRM (6 pairs): / Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_08 Ascentis HP Cortisol run.wiff (Heat... Urine Cortisol Serum Testosterone Max. 2.8e4 cps. XIC of +MRM (5 pairs): / Da ID: testo 1 from Sample 59 (Sample 15) of 2011_06_10 New Ascentis Express Method.wiff (... Max. 8.5e5 cps. 2.8e e e4 8.0e5 2.4e4 2.2e4 2.0e4 1.8e e5 7.0e5 6.5e5 6.0e5 5.5e5 In te n s ity, c p s 1.6e4 1.4e4 1.2e In te n s ity, c p s 5.0e5 4.5e5 4.0e5 3.5e5 1.0e e5 2.5e e5 1.5e e5 5.0e Time, min Time, min 25OH Vitamin D 2/3 XIC of +MRM (9 pairs): / Da ID: D3 quant from Sample 6 (Std 4) of 2011_11_21.wiff (Heated Nebulizer) Max. 1.6e5 cps. Thyroxine (T4) 1.6e e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 In te n s ity, c p s 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min

28 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

29 LC-MS Workload at Addenbrooke s Newborn Screening MCADD/PKU TDM Tacrolimus, Cyclosporin, Sirolimus 25OH Vitamin D2/3 Serum androgens Urine Metanephrines B/S Carnitines Urine, Serum and Salivary Cortisol Urine & Plasma Creatine/Guanidinoacetate/Creatinine Urine 5HIAA Homocysteines Total thyroxine Studies DBS Testosterone Skin steroid panel Cell culture steroid panels 3-epi-25OH Vitamin D2/3 Evaluation/validation Plasma metanephrines Androstenedione Aldosterone 17OHP/DHEAS Insulin T3, rt3

30 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?

31 Pump(s) 1 Autosampler Solvent selector 6 position selection valve Analytical column Column Oven Mass Spectrometer

32 Adding Online SPE Solvent selector 6 position selection valve 2 position divert valve Pump(s) 1 Pump(s) 2 On-line SPE cartridge Analytical column Autosampler Column Oven Mass Spectrometer

33 Pump(s) 1 Pump(s) 2 WASTE

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35 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

36 XIC of +MRM (5 pairs): / Da ID: testo 1 from Sample 59 (Sample 15) of 2011_06_10 New Ascentis Express Method.wiff (... Max. 8.5e5 cps. XIC of +MRM (6 pairs): / Da ID: Cortisol 1 from Sample 18 (Patient 7) of 2011_06_07.wiff (Heated Nebulizer) Max. 2.2e4 cps. 8.5e e e5 2.1e4 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 2.0e4 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e In te n s ity, c p s 5.0e5 4.5e5 4.0e5 3.5e5 In te n s ity, c p s 1.3e4 1.2e4 1.1e4 1.0e e5 2.5e e e5 1.0e5 5.0e Time, min Serum Testosterone Time, min Urine Cortisol XIC of +MRM (9 pairs): / Da ID: D3 quant from Sample 6 (Std 4) of 2011_11_21.wiff (Heated Nebulizer) Max. 1.6e5 cps. TIC: from Sample 11 (Std 10) of 2011_11_08 skin samples APCI.wiff (Heated Nebulizer) Max. 2.3e5 cps. 1.6e e e5 2.2e5 1.4e5 2.1e5 2.0e5 1.3e5 1.9e5 1.2e5 1.8e5 1.7e5 1.1e5 1.6e5 In te n s ity, c p s 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 In te n s ity, c p s 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e e4 3.0e4 6.0e4 5.0e4 4.0e e4 1.0e4 3.0e4 2.0e4 1.0e Time, min Serum 25OH Vitamin D Time, min Skin Steroids

37 Confirmation Analysis A 1.9e5 Intensity, cps 25OHD 3 & 3epi - 25OHD B 2.3e4 Intensity, cps Time, min epi 25OHD OHD 3 25OHD Time, min 8.0

38 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 (Bterm) 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

39 HybridSPE Simplify the procedures of protein precipitation and phospholipid removal into one step

40 Phospholipid depletion filter plates 100µL Serum 25µL Internal Standard 25µL 0.2M ZnSO 4 200µL Methanol Protein precipitation plate Hybrid SPE plate Collection plate

41 Craig R Aurand, David S Bell & Michael Wright. Bioanalysis 2012; 4(22) p

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43 Topic 4. Where are we now? Certified serum based reference standards Urinary Free Cortisol Reference Range? Sample Immulite LCMSMS Urine blank Urine + 2µg/ml THE Urine + 2µg/ml THF 5α Urine + 2µg/ml THF 5β > Urine + 2µg/ml α-cortolone Serum Testosterone Clinically useful cross reactivity Steroid Panels

44 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

45 Questions

46 Additional Information Contact 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

47 Thank you Today s presentation has been recorded and a copy will shortly be available on Follow-up: info@bioanalysis-zone.com