Presentation Basic Introduction to Instrumentation Matrix Effects Challenges

Transcription

1 Keys to Implementing Mass Spectrometry in the Clinical Laboratory Paul J. Taylor Chromatography Mass Spectrometry Satellite Meeting, AACB 20th September, 2013 Dept of Clinical Pharmacology, Princess Alexandra Hospital, Brisbane, Australia

2 Presentation Basic Introduction to Instrumentation Matrix Effects Challenges

3 Section 1: Instrumentation

4 Why use HPLC-Tandem Mass Spectrometry? HPLC is a fundamental separation technique that is amenable to most pharmaceuticals and biological molecules from small molecules to peptides to proteins Samples do not require volatility or thermal stability as for GC No need for sample derivatisation

5 Why use HPLC-Tandem Mass Spectrometry? A combination of hydrophobic separation and mass selectivity Tandem mass spectrometry is more sensitive and far more specific than other HPLC detectors Can provide improved selectivity over immunoassays

6 The 3 S Advantages of Mass Spectrometry to Solve Analytical Specificity it Sensitivity Speed Problems (McLafferty FW. Science 1981;214:280-7.)

7 What is Mass Spectrometry?

8 What is Mass Spectrometry? Mass spectrometry is the production and differentiation of ions in the gas phase

9 Keys to Success Ionization Ion transmission

10 Components of a Mass Spectrometer Ionization/ desorption Mass Sorting Detection ti + Source Analyzer Ion Detection Inlet Sample Introduction Solid Liquid Vapour Mass Spectrum Data Analysis

11 Types of Ionization Atmospheric Pressure (API) Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Electron Impact (EI) Chemical Ionization (CI) Fast Atom Bombardment (FAB) Matrix Assisted Laser Desorption (MALDI)

12 Electrospray Ion Source Curtain Gas Electrospray Probe Atmospheric pressure Vacuum HPLC Eluant 3-6 kv N 2 for Nebulization Charged Droplets Ions Orifice Source: PE/Sciex

13 Theory of Electrospray (Ion Evaporation) Liquid Phase Ionization Liquid 5 kv Droplet Droplet Coulomb + Formation Evaporation Explosion Ion Evaporation + CH CH 3 N CH CH 3 + Molecular Ion

14 APCI Interface Liquid from HPLC Heat M H 2 O H 2 O H O 2 M H O 3 + Molecular Ions (M+H) + Air for Nebulization Heat Vaporization Ion molecule Reactions Corona Discharge (5KV)

15 Electrospray Molecular Weigh ht APCI 10 Non-polar Polarity High Polarity

16 Magnetic Sector Time-of-flightflight Ion Trap Quadrupole Types of Analyzers All separate ions which have different charge-to- mass ratios, m/z in magnetic field or electric field

17 Mass Spectrum Flumazenil [M+H] + O N O F N O N [M+Na] + [M+K] +

18 Mass Spectrum of Haemoglobin α (15126) and β (15867) Chains

19 Chromatography

20 Chromatography To obtain maximum signal the column selected should provide adequate retention with highest possible organic solvent content t To obtain retention with polar compounds the use normal phase chromatography may be required* *Naidong W, et al. J Chromatogr B 2001;754:

21 Example Determination of Cocaine in Urine by HPLC-ESI ESI-MS/MS O Basic compound pk a = 5.6 Protonated species Mobile phase at ph 3.0 N O O O Needham SR, et al. J Chromatogr B 2000;748:

22 Example Determination of Cocaine in Urine by HPLC-ESI ESI-MS/MS What column would you start your method development with? A silica based C18 column To obtain adequate retention < 15% organic solvent composition

23 New Stationary Phase - Pentafluorophenylpropyl Bonded to Silica CH 3 F Si O Si F CH 3 F F F

24 PFPP vs C18 cetonitrile:5 il mm Ammonium Formate (ph 3.0) 4.5 fold lower LOD 90% ACN 15% ACN

25 Section 2: Matrix Effects

26 Matrix Effects A major problem when generating ions using atmospheric pressure ionization techniques Must be appreciated when developing a method

27 Matrix Effects Matrix effects are the alteration in ionization efficiency of an interface due to the presence of co- eluting ions The exact mechanism is currently unknown Co-eluting ions alter droplet formation and droplet evaporation leading to variable generation of the ions of interest in the gas phase

28 Matrix Effects Depending on the environment in which ionization and ion evaporation takes place, this competition may: 1. Decrease analyte signal ion suppression 2. Increase analyte signal ion enhancement Greatest influence on electrospray ionization

29 Matrix Effects Compound dependant A study of 4 compounds of different polarities, under the same mass spectrometric conditions: 1. Most polar largest ion suppression 2. Least polar less affected by ion suppression Bonfiglio R, et al. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:

30 How to Assess Matrix Effects Post-extraction addition Post-column infusion

31 Post-Extraction Addition Supplement the compound of interest post- extraction Compare the response with a mobile phase standard

32 Post-Extraction Addition Evaluation (Buhrman et al, J Am Soc Mass Spectrom., 1996.) Method Post- extraction area Mobile phase area Hexane liq-liq Ion suppression (%) Back-extraction C18 SPE

33 Post-extraction Addition Is considered a static method that provides information about matrix effects at the point of elution of the analyte. A more dynamic method of assessing matrix effects is post-column infusion

34 Post-Column Infusion HPLC Analytical Column Mass Spectrometer Infusion Pump Flow Splitter Electrospray Interface

35 Post-Column Infusion Test Mobile Phase 2000 Intensity (cps) Time (min)

36 Protein Precipitation 2000 Inte ensity (cp ps) Time (min)

37 Solid Phase Extraction 2000 ps) Inte ensity (c Time (min)

38 Where Does it Come From? Additives in the mobile phase

39 Acetate Vs Trifluoroacetic acid for Gentamycin Analysis TFA decreases ionization efficiency Lagerwerf FM et al., TrAC 2000;19:

40 Where Does it Come From? Additives in the mobile phase Sample extract

41 Sample Extract Salts retention of compounds Phospholipids???

42 Phospholipids and Matrix Effects Monitor specific transitions In-source multiple reaction monitoring glycerophosphocholines (GPCho) major constituent of phospholipids p p in plasma (Little JL et al. J Chrom B 2006;833:219-30) 30)

43 High Cone Voltage Low Collision Energy m/z

44 Protein Precipitation

45 Where Does it Come From? Additives in the mobile phase Sample extract Previous samples or build-up up on column

46 Endogenous Ion Suppression Protein precipitation of plasma Lagerwerf FM et al., TrAC 2000;19:

47 What Can Be Done to Minimize Matrix Sample preparation Chromatography Different ion source Effects? Internal standard selection compensates

48 Matrix Effects and the Clinical Laboratory Inter-patient variability of the matrix Biological samples from a patient with renal failure, aids patient, burns patient compared to a healthy volunteer

49 Patient to Patient Variability Matuszewski BK, et al., Anal Chem 2003;75:

50 Case Study

51

52 Methods Development for Electrospray Chromatography (k ) differences 50:50 ACN:buffer vs 90:10 ACN:buffer Extraction differences Ether extraction (non-buffered) vs Ether extraction (buffered to ph 9.8)

53 Extraction vs Chromatography Non-selective extraction Selective extraction Plasma # Little separation k =1.75 Good separation k =13.25 Little separation k =1.75 Good separation k = Matuszewski BK, et al. Anal Chem 1998;70:882-9.

54 Extraction vs Chromatography Non-selective extraction Selective extraction Plasma # Little separation k =1.75 Good separation k =13.25 Little separation k =1.75 Good separation k = Matuszewski BK, et al. Anal Chem 1998;70:882-9.

55 Extraction vs Chromatography Non-selective extraction Selective extraction Plasma # Little separation k =1.75 Good separation k =13.25 Little separation k =1.75 Good separation k = Matuszewski BK, et al. Anal Chem 1998;70:882-9.

56 Extraction vs Chromatography Non-selective extraction Selective extraction Plasma # Little separation k =1.75 Good separation k =13.25 Little separation k =1.75 Good separation k = Matuszewski BK, et al. Anal Chem 1998;70:882-9.

57 Comments A combination of effective sample preparation and chromatography is required for successful a quantification method using HPLC-MS

58 Further Reading Annesley TM. Clin Chem 2003;49:1041. Matuszewski BK, et al. Anal Chem 1998;70:822. Matuszewski BK, et al. Anal Chem 2003;75:3019. Taylor PJ. Clin Biochem 2005;38:328. Mallet CR, et al. 2004;18:49. Rogatsky E, et al. 2005;16:1757.

59 Section 3: Challenges

60 Economic Barrier There is a perceived cost barrier A triple quadrupole instrument requires an initial capital investment of greater than $US This compares poorly with immunoassays that are supplied by the manufacturer on a reagent- rental basis

61 Economic Barrier Redundancy d means at least 2 instruments t are required Although the long-term cost per assay may be higher for the immunoassay, it is the lump sum investment for the mass spectrometer that prevents many laboratories from entry into this technology

62 Cost Comparison of Tacrolimus Measurement (Prof M Oellerich, Germany) LC-MS/MS MEIA Number of samples Direct costs ( ) Technician time ( ) Total costs ( ) n=3 n=15 n=3 n=

63 Personnel There is limited availability of suitable trained staff to run and trouble-shoot this equipment Clinical Scientist Mass spectrometrist This point may not be as important t as technology improvements in instrumentation occur

64 To Provide a Viable Alternative ti to Immunoassays

65 Selectivity Automation Accuracy Ease of Use Sensitivity Throughputh

66 Selectivity and Accuracy HPLC-MS has excellent selectivity and accuracy compared with immunoassays that are prone to non-specific anti-body cross-reactivityreactivity

67 Selectivity Automation Accuracy Ease of Use Sensitivity Throughputh

68 Sensitivity and Throughput Differ depending on the analyte under investigation

69 Selectivity Automation Accuracy Ease of Use 1/2 1/2 Sensitivity Throughputh

70 Ease of Use and Automation Immunoassays are superior to HPLC-MS

71 Selectivity X Automation Accuracy 1/2 1/2 Ease of Use Sensitivity Throughputh X

72 Discussion (1) For HPLC-MS technology to compete directly with immunoassays an acceptable increase in sensitivity and throughput are necessary This imbalance may change as demand for this type of instrumentation grows and manufacturers improve systems

73 Discussion (2) HPLC-MS requires significant improvement in ease of use and automation Approaches such as two-dimensional chromatography minimise sample handling and preparation provide some automation

74 Discussion (3) What is required Walk-up instrument 1. Sample is placed directly into the instrumentation 2. The assay requested 3. Sample analyzed 4. Result is reported in a suitable format for clinical interpretation

75 Availability of suitable internal standards

76 Internal Standards Compensate for changes in ionisation efficiency Simultaneous methods may require multiple internal standards depending on the chemical differences of the analytes Stokvis Eetal al. Rapid Commun Mass Spectrom 2005; 19:

77 Isotope Labeled Internal Standards Provide the highest order of reliability Incomplete recovery does not effect results Co-elution any variation in ion formation should alter equally for analyte and IS Matrix effects do not influence IS/analyte ratio

78 Isotope Labeled Internal Standards Labelled internal standards can be expensive and not always available Labelled internal standards must be used for methods in the clinical setting

79 Harmonization

80 Harmonization With the development of slightly different variations of a particular test used by various laboratories there is a greater likelihood of different results Thus harmonization of mass spectrometric methods is required to establish normal ranges, decision points, etc.

81 Discussion HPLC-MS is an important technology that is and will complement and extend the capabilities of the clinical i l laboratory HPLC-MS clearly offers much more versatility and scope than GC-MS and provides improved selectivity over immunoassays

82 Keys to Success Ionization Ion transmission

83 Discussion Matrix effects must be understood and carefully evaluated There are some challenges that need to be resolved This is due in part to mass spectrometry not being a turn-key solution to an analytical problem

84 Final Thoughts Dr Michael Kinter stated in a 1995 review: The expectation of mass spectrometry is limited to developing non-routine information about specialized analytes Anal Chem 1995;67 67: R-7R.

85 Final Thoughts Mass spectrometry is widely appreciated as a powerful analytical method, that can be used for both the qualitative ti and quantitative analysis of nearly all types of molecules. In light of this power, I would propose that mass spectrometry is ready for a substantially larger role in routine clinical analysis.

86 Final Thoughts Mass spectrometry is widely appreciated as a powerful analytical method, that can be used for both the qualitative and quantitative analysis of nearly all types of molecules. In light of this power, I would propose p that mass spectrometry is ready for a substantially larger role in routine clinical analysis. Kinter M. Clin Chem 2004;50: