LC-MS/MS Challenges in a High-Throughput Clinical Chemistry Laboratory Michael Wright

Transcription

1 LC-MS/MS Challenges in a High-Throughput Clinical Chemistry Laboratory Michael Wright SEALS Prince of Wales Hospital Randwick, NSW

2 Clinical Chemistry From an Analytical Chemist Perspective

3 How do you test for interferences? Endogenous compounds Low level (pmol/l) Part of large families of closely related compounds No such thing as a blank sample When you start measuring patients, how do you know that it is only your analyte in the peak you see?

4 Why LC-MS/MS? Triple quadrupole tandem mass spectrometers in MRM mode are seen as a sensitive and specific way of measuring target analytes Replacement/Confirmation test for expensive, imprecise or inaccurate Immunoassays (IA) Replacement for traditional UV, fluorescent, electrochemical detection for HPLC Replacement for lengthy GC-MS methods that have extensive sample clean-up

5 Multiple Reaction Monitoring (MRM) Fixed to one m/z Fragmenting and transferring fragments Fixed to one m/z

6 Isobaric interferences Ion Ratios Quantifier Ion Qualifier Ion 20% acceptance range

7 Fragments of Fragments A * e * Intensity, cps m/z, Da 175

8 Points to cover 1) Specificity of Mass Spectrometry - Selecting the correct stationary phase 2) UHPLC Keeping our eye on the prize 3) What do you want from your LC-MS? 4) Emerging technologies

9 1a Mass Spectrometry s Specificity Due to the superior specificity of Mass Spectrometry extensive chromatography was not required Same paper. A number of false positives were reported that were attributed to the patient being bled in a sitting rather than a supine position

10 Isobaric interferences -Sugars

11 Isobaric interferences Bile Acids Deoxycholic Acid (DC) Cheno-deoxycholic Acid (CDC) Urso-deoxycholic acid (UDC) All have m/z ratios of 391 plus none of them fragment so their transitions are 391/391 GDC fragmentation TDC fragmentation

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13 1. Choosing the correct chromatography When we switched to using a mass spectrometer as a detector we lost most of the mobile phase tools we had LC-MS has made us much more reliant on the stationary phase for our separation

14 Isobaric interferences & the C18 mentality Urinary Free Cortisol Hypersil Gold 50x2.1mm 3µm C18 Patient A Quantifier Ion Qualifier Ion Ion Ratio Failed!!!

15 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...? 2.8e4 2.6e4 Patient A 2.4e4 Max. 2.8e4 cps Cortisol e4 2.0e4? 1.8e4 In te n s ity, c p s 1.6e4 1.4e e4??? 1.0e Time, min

16 Take home message Run at least 1 orthogonal chromatographic run on a few patient samples early in your validation Check the Ion Ratios are clear Check that you get the same results Be careful when setting up steroid panels!

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

18 Take home messages Validate the peak purity at the LLOQ Check you dead volume (t0) to check how much chromatography you actually have (k >1) Start with a form of chromatography that is designed with your analytes in mind (not necessarily what is in the literature!)

19 Unusual patient samples Serum testosterone - paediatrics Serum 25OH Vitamin D3

20 25OH Vitamin D analysis in infants A e5 Intensity, cps 25OHD3 & 3epi- 25OHD3 1.0 B Ascentis Express 50 x 2.1mm 2.7µm C8 5.0 Time, min 3epi- 25OHD3 2.3e4 F 7.24 F epi 25OHD 3 Intensity, cps 25OHD3 Si O F Si F 25OHD3 F Ascentis Express 100 x 2.1mm 2.7µm F5 1.0 Time, min 8.0 Wright MJ, Halsall DJH and Keevil BG. Clin Chem 2012; v. 58, p

21 Take Home Messages Nothing wrong with having back-up confirmation methods Validating a second method for unusual patients can be easier than altering and re-validating your current method When looking at co-eluting interferences remember that, of the resolution equation, selectivity is the most powerful determinant

22 Wow so many interferences. Surely Immunoassay is safer? Urinary Free Cortisol Sample Immulite LCMSMS Urine blank Urine + 2µg/ml THE Urine + 2µg/ml THF 5α Urine + 2µg/ml THF 5β > Urine + 2µg/ml α-cortolone

23 2. Ultra High Pressure Liquid Chromatography For 40 or so years 6000psi was the back-pressure limit on even the most advanced HPLC systems (despite a preliminary study in 1969 demonstrating the benefits of higher pressure systems) In 2004 the first UHPLC systems first came onto the market (Acquity) Resulted in an entirely new design focusing on minimising dispersion

24 Why use UHPLC? Performance (Plates) P= = 1000F L P r2dp2 1100psi 5µm µm psi 1.7µm psi

25 2: Is ultra high-pressure chromatography always the best option? So you can either increase efficiency OR cut the column down and increase the speed Trade off versus increase in number of blockages (or sample clean-up) Patients often waiting in hospital beds for results = turn-around time AND system robustness of high importance Alternative? Fused core, core shell, solid core, superficially poros

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28 Advantages higher performance:pressure Ascentis Express C18 Dim: 50x4.6mm 2.7µm Urine 5HIAA 5HIA A 50% IPA STD QC CHECK 1_2ML #8 STD2 Fused Core 1.5ml 1µl inj 1-5HIA A MinHgt = 0.010, Inhbit = On 1.10 ECD_1 2 - Internal Std Inhbit = Off 1.26 na Internal Std BP= 2070 psi HIA A min min run time

29 Take Home Messages Definitely go for a UHPLC machine if you can afford it! Check your efficiency gains on <2µm particle columns are they worth it versus your system robustness?

30 3: What do you want from your LCMS? Faster or multi-tasking? 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 Weekly workload at Addenbrooke s Studies Newborn Screening MCADD/PKU (580) TDM Tacrolimus, Cyclosporin, Sirolimus (460) 25OH Vitamin D2/3 (400) Serum Testo/17OHP/Andro (80) Plasma and Urine Metanephrines (80) B/S Carnitines (20) B/S Testosterones Skin steroid panel Cell culture steroid panel 3-epi-25OH Vitamin D2/3 Evaluation/validation Urine, Serum and Salivary Cortisol (30) Estradiol Urine and Plasma Creatine/Guanidinoacetate/Creatinine (15) Aldosterone Urine 5HIAA (15) Insulin Homocysteines (10) ALL REQUIRE A BACK-UP DHEAS Thyroxine

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

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

34 Pump(s) 1 Pump(s) 2 WASTE

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36 Multiplexing Run first analysis General Conditioning step (20min) Specific Conditioning step (20min) Start second batch of analysis. Example from Addenbrookes 1) 2) 3) 4) 5) Serum testosterones 48 samples by Online SPE (3½ hours) Conditioning (40min) Urine Cortisols 36 samples by Online SPE (4 hours) Conditioning (40min) Serum 25OH Vit D3/2 96 samples by Online SPE (8 hours) Run put on at 4pm has finished at 9am the following morning

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

38 Confirmation Analysis A e5 Intensity, cps 25OHD3 & 3epi- 25OHD3 1.0 B 5.0 Time, min 3epi- 25OHD3 2.3e epi 25OHD 3 Intensity, cps 25OHD3 25OHD3 1.0 Time, min 8.0

39 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

40 Benefits of multiplexing Cheaper way of obtaining much larger capacity Multiple methods can be run back to back without an analyst being present Value for money from our MS system (easy to upgrade) Systems are available during the day for application development Research studies can be run after all the routine work has been completed

41 4: Emerging Technologies Ion Mobility The ability to separate and identify ionized molecules in the gas phase based on their mobility in a carrier buffer gas The collision cross section of the molecule (the size and shape) determines the mobility? Could you possibly use ion mobility as a form of separation technique in addition to/or replacing HPLC for clinical analysis

42 Differential Mobility

43 Dynamic Cluster/Decluster Model Separations are Chemical in Nature + High field Declustering Mobility Increases Low Field ClusteringMobility Decreases High field Declustering Low Field ClusteringMobility Decreases Mobility Increases Krylov et al., Int. J. Mass Spectrom., 2009, 285,

44 Examples of LC-IMS-MS/MS MRM Separation of Isobaric Compounds (m/z 309) Quinoxyfen Warfarin Phenylbutazone Bestatin Benoxinate Normalized Signal 1.0 MRM with Ion Mobility CV (V) Time, min 3.0

45 Mass Accuracy Carbon Hydrogen Oxygen Nitrogen has has has has CO N2 C2H4 a a a a mass mass mass mass of of of of = 28 = = 28 = = 28 =

46 Unit Resolution m/z = 1432 Unit Resolution

47 High Resolution Transfer Quad, Bovine W, neg ion, 6GHz UCA121RES01 2 (0.051) Cm (2:28) Resolution > 100,000 FWHM TOF MS ES5.33e % m/z 1434

48 Accurate Mass/High Resolution instruments for quantitative work

49 Acknowledgements Addenbrooke s Hospital David Halsall Kevin Taylor Lisa Tanner AB Sciex Steven Ayris Chris Hodgkins Sigma/Supelco Dave Bell Craig Aurand Denise Walworth Kings College Hospital Colin Stone Clare Glicksman Evelina Children s Hospital Neil Dalton Charles Turner Waters Mike Morris Shimadzu Earl McCoy