OMICS Group · 2014. 9. 4. · •EP9-A2: Method comparison . Assay Validation •Quality of...
Transcript of OMICS Group · 2014. 9. 4. · •EP9-A2: Method comparison . Assay Validation •Quality of...
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and Embryology
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Application of LC-MS/MS in Clinical
Laboratory for Small Molecule Quantification
Sihe Wang, PhD DABCC FACB
Section Head and Medical Director, Clinical
Biochemistry, Cleveland Clinic
JCST July 23, 2014
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Objectives
• To explain the advantages and challenges of LC-
MS/MS in quantifying small molecules in patient
specimens
• To illustrate unique contributions that LCMS/
MS may bring for patient care
25-hydroxyvitamin D
1,25-dihydroxyvitamin D
• To demonstrate how to validate a quantitative
assay by LC-MS/MS
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The Quadrupole Mass Analyzer
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Tandem Mass Spectrometry (MS/MS)
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Specificity through MS/MS
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ElectrosprayIonization (ESI)
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Atmospheric Pressure Chemical
Ionization (APCI)
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Clinical Applications of LC-MS/MS
• Neonatal screening
• Inborn errors of metabolism
• Toxicology and Drugs of abuse
• Pain management drug testing
• Therapeutic drug monitoring
• Endocrine
• Protein identification and quantitation
Vogeser, Clin Chem Lab Med 2003;41:117-126. Dooley, Clin Biochem 2003;36:471-481.
Vogeser, Clin Biochem 2008;41:649-662. Wu, Clin Chimica Acta 2013.
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Advantages of Immunoassays
• Easy to operate
• Low maintenance by laboratory personnel
• Pre-defined performance by manufactures
• Approved by regulatory agencies
• High throughput through automation and parallel reactions
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Limitations of Immunoassay Tests for Small
Molecule Quantification
• Sensitivity—10-100 pg/mL
• Specificity
Structurally similar molecules
Human anti-mouse antibodies
Heterophilic antibodies
• Hard to develop
• Variation between assays using different
antibodies
• Epitopes recognized by different antibodies
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Advantages of LC-MS/MS Methods
• High specificity
• High sensitivity (< 1pg/mL)
• Wide range of applicability
Volatility
Polarity
• Assay flexibility
Relatively easy to establish and change
• Information rich detection
Multiple analytes in one run
Structural information
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Challenges of Using LC-MS/MS
• Instrument complexity
Hard to learn
• Home brewed assays
Lack of regulatory agency cleared assays
• Throughput is limited by
Analytical cycle time
Sample preparation
Interface with lab information system
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Inaccurate Results by LC-MS/MS
• Poor signal stability—appropriate internal
standards
• Potential interference by molecules/metabolites
with identical MW
• Matrix effects
Reduced/enhanced ionization intensity from co-
eluted impurities or matrix
Optimal internal standards
• Cross talk
• Insource transformation
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Operational Challenges
• High initial capital investment
Low reagent cost
Long-term investment
• Challenge to maintain high performance of the
instruments
Hardware
Methodology
• Manual operation
• No commercial interface
• No strong service support
• No consensus on performance characteristics suitable
for clinical use
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Clinical and Laboratory Standards Institute
• C50-A: Mass Spectrometry in Clinical Lab
• EP14-A2: Evaluation of matrix effect
• EP7-A2: Interference testing
• EP6-A: Evaluation of linearity
• EP17-A: Limits of detection and quantitation
• EP10-A3: Preliminary evaluation
• EP9-A2: Method comparison
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Assay Validation
• Quality of chromatograms
• Matrix effects
• Interferences
• Analytical measurable range
• Accuracy (analytical recovery)
• Carry-over
• Precision
• Method comparison
• Robustness
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• Examples—Vitamin D Metabolites
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• In the early 20th century, it was discovered that
cod liver oil and sunshine exposure had antirachitic
effect
• The antirachitic substance was the
4th vitamin discovered and was
called vitamin D
• - Cholecalciferol (D3)
• - Ergocalciferol (D2)
Vitamin D Discovery
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Measure of Vitamin D Nutritional Status
• Lack of data showing free 25(OH)D or
1,25(OH)2D are better indicator of vitamin D
status
• - Vitamin D-DBP can be up-taken by cells
• By consensus, the total 25(OH)D is the accepted
indicator of D status
• - 25(OH)D is not much regulated and primarily
dependent on substrate concentration
- Easily measured (higher concentration)
- Longer half-life (~3 wk) vs 1,25(OH)2D (~4h) and
D (~1 d)
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Vitamin D in Health and Disease
• Bone metabolism
• Muscle strength
• Cancer
• Cardiovascular disease
• Autoimmune disease
• Diabetes (both type I and II)
• Neurological disorders
• Kidney disease
• Bacterial and viral infections
• Pregnancy outcome
• All-cause mortality
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Available Methods for Measuring Serum 25(OH)D
• Immunoassay
Radioimmunoassay (RIA)
Enzyme Immunoassay (EIA)
• Protein binding assay
• HPLC
• LC-MS/MS
25(OH)D2 and 25(OH)D3
Gold standard
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Method Comparison—
IA and LC-MS/MS
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• Healthy volunteers from the laboratory staff
• 100,000 IU of vitamin D3 as four ampoules of Dcure (S.M.B., Brussels, Belgium) (Liege group; n=7). Samples were collected at day 1, 7
• 600,000 IU of vitamin D2 as a single vial of Sterogyl 15 (DB Pharma, La Varenne Saint- Hilaire, France) (Paris group; n=11). Samples were collected at day 0, 7, and 28
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25-Hydroxyvitamin D2 Recovery
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Cleveland Clinic LC-MS/MS Method
• Minimal sample preparation
Protein precipitation with acetonitrile
Online turbulent flow extraction
• Gradient LC method on a polar end-capped C18
column in 5.5 min
• MS in positive APCI mode
401.3→383.2 m/z for 25OHD3, 413.3→395.0 for
25OHD2, and 407.3→389.2 m/z for d6-25OHD3
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Chromatogram
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Analyte Add-In (nmol/L)
Expected (nmol/L)
Mean (nmol/L)
Analytical Recovery
%CV
25OHD2 2.36 2.39 2.98 125.0% 29.2%
4.73 4.78 4.60 96.2% 4.3%
7.10 7.18 7.20 100.3% 7.5%
9.46 9.57 9.45 98.8% 6.9%
18.93 19.13 18.78 98.1% 5.3%
37.86 38.26 36.66 95.8% 5.0%
75.72 76.53 72.57 94.8% 4.8%
151.44 153.06 147.15 96.1% 3.0%
302.88 306.12 277.92 90.7% 1.8%
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25OHD3 2.44 2.49 2.95 118.7% 14.5%
4.87 4.98 5.42 109.0% 10.2%
7.31 7.47 7.81 104.7% 3.1%
9.75 9.96 10.43 104.9% 2.3%
19.50 19.92 18.89 94.7% 3.9%
39.00 39.84 37.94 95.1% 1.4%
78.00 79.68 72.63 91.0% 1.1%
156.00 159.37 144.82 90.7% 2.3%
312.00 318.74 283.55 88.7% 2.3%
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Precision (CLSI EP-10A2)
25OHD3 25OHD2
LOW MID HIGH LOW MID HIGH
Mean (nmol/L) 33.4 61.0 120.5 18.5 70.6 139.9
Total SD 3.0 4.6 11.4 3.1 7.8 15.6
Intra-assay SD 1.6 2.3 7.4 1.7 3.2 9.0
Inter-assay %CV 7.7 6.6 7.1 14.2 10.0 9.2
Intra-assay %CV 4.9 3.8 6.1 9.3 4.6 6.4
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When to Measure 1,25- Dihydroxyvitamin D Clinically?
• Hyper- or hypo-parathyroidism
• Chronic kidney disease
PTH suppression
Compliance with 1,25(OH)D therapy
• Vitamin D-dependent rickets
Types I: low conversion of vitamin D to 1,25(OH)D
Type II: resistance to 1,25(OH)D
• Others
Sarcoidosis
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Radioimmunoassay
for Measuring 1,25-Dihydroxyvitamin D
• Extract sample with acetonitrile, centrifuge and
decant
• Purify pretreatment solution by C18 column
• RIA procedure including pipetting, centrifugation,
decanting and reading on the gamma counter
• Large variation
• Interference
• At least 1-day long procedure
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Cleveland Clinic LC-MS/MS Method
• Triple quadrupole mass spectrometer
• Lithium adduct monitored (0.5 mM lithium
acetate in mobile phase)
• Onyx monolithic C18 columns (100 x 3.0mm)
• Total run time 10 minutes
• Calibrators in charcoal stripped serum down to
LOQ level
• Validation performed in real samples
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Removing Interferences
Example chromatograms
(arrows: interference peaks)
Things we tried and failed • LC gradient • Selection of other MRMs • Two Onyx monolithic columns in tandem • Addition of a SPE • Derivatization with PTAD • Turbulent flow online extraction
What worked robustly • 500 μL serum immunoaffinity extraction of serum samples
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Representative Chromatograms of
Samples with Low 1,25(OH)2D
• Sample 26: 6.1 pg/mL 1,25(OH)2D3
• Sample 29: 12.1 pg/mL 1,25(OH)2D2
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Correlation with an RIA
Slope=0.751 R=0.79 N=40
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Correlation (Total) with a Commercial
LC-MS/MS
Slope=1.02 R=0.98 N=20
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Future of LC-MS/MS in Clinical Lab
• Automate sample preparation
Bar code reader
Hands off extraction
• Reduce solvent consumption, improve throughput
Low-flow LC coupled with MS of high sensitivity
and scan speed
• Reduce transcription errors
Interface between sample preparation, LC-MS/MS,
and LIS
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Conclusion
• LC-MS/MS has high sensitivity and specificity
• LC-MS/MS offers unique contributions to patient care
• There are challenges utilizing LC-MS/MS for clinical
testing
• Vigorous validation of LC-MS/MS methods is
warranted for patient care
• Important to collaborate with manufactures to
improve the current technologies
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Chromatography & Separation Techniques
Related Journals
Journal of Bioanalysis & Biomedicine
Journal of Analytical & Bioanalytical
Techniques
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Chromatography & Separation
Techniques Related Conferences
• 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques
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