Protonation Sites, Tandem Mass Spectrometry and Computational ...
Implementation of Tandem Mass Spectrometry: Quality ...
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Implementation of Tandem Mass Spectrometry: Quality Control/Quality
Assurance of the New York State Newborn Screening Program.
Mark A. Morrissey, PhDWadsworth CenterDepartment of Health
State of New York
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Brand Names
Important:
The use of brand names in this presentations is for purposes of description only.
No endorsement is implied or intended.
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Definitions
Quality Assurance is a comprehensive set of policies, procedures, and practices necessary to assure that the laboratory’s results are reliable.
Quality Control is a set of laboratory procedures designed to ensure that the test method is working properly.
From NYS DOH, Clinical Laboratory Evaluation Program (CLEP)
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Outline
a. Administrative Preparation
b. Instrument Set-up/Method Validation
c. Routine/Daily Operations
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a. Administrative Preparation
• Appropriate space
• Power and air conditioners
• Delineation of test panel
• Qualified staff
• Assay validation
• Lab certification
• Proficiency testing arrangements
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More…
• Follow-up considerations
• Available medical specialists and diagnostic lab services
• Adequate notification of physician community
• Educational materials: lay and professional
• Advisory committee
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Training Issues
• Instrument vendor site training- Instrument operation- Instrument troubleshooting
• Visit other MS/MS laboratories- 20 + MS/MS testing programs- spit equally between vendors
• APHL and NNSGRC sponsored courses- Baylor Institute of Metabolic Diseases- Duke Medical Center
• Develop training and competency logs
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b. Instrument Set-up/Method Validation
Instrument Performance
Method Validation
Establishment of Cut-off values
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Instrument Performance Issues
• Mass Calibration and Resolution– Establish operating range of instrument
• Probe rinses– Minimize Carryover
• Detection Optimization– Front End (Probe, cone, extractor, etc…) voltages.– Collision chamber, 2nd quadrapole, detector voltages
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Instrument Performance Issues
• State files– Mass calibration
• Method files– Scan Parameters– Analytical run time (HPLC method)
• Data Reduction Software– Calibration Table
• Analyte & internal standard masses• Internal standard concentration• Blood spot volume
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Instrument Performance Issues
• Mass Calibration
– Establish operating range of instrument
• Typical mass range 59amu to 1800 amu• We use 50 to 1100 amu, quarterly
– Materials used
• Polypropylene (PPG)• NaI / RbI solution
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Method Validation Protocol
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Method Validation
Requirements from CLIA or CLEP– Accuracy– Precision– Reportable Range– Specificity and Sensitivity– Reference Intervals– “Any other performance characteristics required for
test performance”
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Method Validation
Establish Accuracy• Calculate recovery from analysis of fortified
samples (6 to 8 levels recommended)– [(S – C)/F] X 100
» Where: S is the observed concentrationC is the background concentrationF is the fortification concentration
• Acceptable recoveries: > 80%• Range is the demonstrated range of acceptable
recoveries (for example: C8 0.06 to 100 µmole/L)
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Recovery (4 instruments, 4 Levels, 4 days)
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Method Validation
• Intra run precision
– extract and prepare a set of blood spots.• Minimum of 10 replicate analyses
– Analyze in the same run on the same day.
– Calculate Coefficient of Variation (CV)
• Expected coefficient of variation: < 10%.
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Method Validation
• Inter run precision
– Multiple day analysis (4 days)
– Prepare 4 extracts for each spiked pool daily
– Calculate Coefficient of Variation (CV)
• Expected coefficient of variation (CV): 5 – 15%
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Method Validation(intra/inter run precision)
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Establishment of Cut-off Values
• Pilot Testing– Do a literature search– Contact existing MS/MS programs– Manufacture of instrument or reagents
• Routine Testing Cutoffs– Analyze several thousand normal specimens– Calculate mean and standard deviation– Recalculate after several thousand samples
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Establishment of Cut-off Values
• Consult metabolic specialist/follow up staff.
• Typical cutoffs: 3 and 10 sd from mean
• Compare cutoffs with other MS/MS programs.
• A balance between false positives/negatives
• Consider separate ranges for age > 7 days.
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Establishment of Cut-off Values
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c. Routine Operations
a. Routine, but not daily considerationsb. Daily analysis of samples
i. Sample Preparation1. Derivatized2. Un-derivatized
ii. Documentationc. Daily Data Evaluation
i. Data Reductionii. Evaluation of QC resultsiii. Evaluation of abnormal samples
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Standard Operating Procedure
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Routine, but not daily considerations
Instrument Maintenance• Follow the manufacturers recommendations.• Most labs use a check-off sheet with signature and date• Activities may include: clean cone, ballast the rough pump
and check oil level, check chiller temp, etc…• Maintained in the instrument logbook• Ultimately up to the area supervisor and laboratory director
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Routine, but not daily considerations
Non-routine maintenance• Documented with nature of the problem, who found the problem, resolution
and the person fixing the problem. • We include preventative maintenance from the manufacturer. They have their
own specification for function checks and mass calibration.
Tuning• Concentrated Standard Solution. May not include all the applicable analytes
(C14:1, C5OH, etc). • Collected material from a used plate (not documented or traceable). We do
this as needed if the QC results are out of range.
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Reagent Preparation
• HPLC grade, or Reagent grade, or better• To filter, or not to filter? • Hazards
– Acetonitrile -- toxic, flammable – Methanol -- toxic, flammable – Butanol -- flammable, irritant– Acetyl Chloride -- corrosive, water reactive. Use a
chilled bath and mix with butanol under argon.
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Internal Standard Preparation
• MMWR recommends a deuterated standard to correspond to each analyte.
• Pre-prepared Internal Standards– Available from Perkin-Elmer or Cambridge Isotope
Laboratories. – Concentration and Expiration are provided by the
supplier.
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Internal Standard Preparation
• In-house Prepared Internal Standards– Available from Dr. H.J. ten Brink, Cambridge
Isotope Labs and CDN Isotopes Inc. – Cheaper, but requires more labor– I estimate that for 250,000 samples it requires:
• Approximately 80 hours to prepare the standard• 40 hours to go through a qualification
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Internal Standard Preparation Procedure
• Weigh individual deuterated stds into a suitable container. • Dissolve in 0.01N HCl (d-C16 in MeOH) for an exact
concentration of 1.0 mg/mL or 0.1 mg/mL.• Add the appropriate amount of each to individual 15-mL
polypropylene centrifuge tubes. (Adds up to about 7.3 mL). We make about 40 tubes.
• Seal with Teflon tape and store at –70 C. Stable for at least 1-year
• Should check concentrations and adjust, if necessary
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Internal Standard Preparation Procedure
• To prepare working internal standard dissolve the entire tube in 2-L of methanol. Store at –20C. Lasts about 10-days, (10,000 samples).
• 2-years worth of standards costs us approximately $10,000 (500,000 samples)
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Preparation of Quality Control Samples
• Standards available from Life Science Resources, H.J. ten Brink (carnitines) and Sigma (amino acids).
• Blood obtained from the Red Cross.• Individual stock standards of amino acids and
acylcarnitines are prepared in saline. • Added individually to 50 mL aliquots of whole blood.
Stored frozen along with a desiccant. Lasts approximately six months. (24/day at each level)
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Preparation of Quality Control Samples
• One level is approximately equal to the request for a repeat specimen level. The other level is approximately 2 to 3 times the concentration of the first.
• Determination of control limits.
• QC Samples available from CDC (bi-annually) for periodic use.
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Sample Preparation and Analysis
• Underivatized - fewer steps, faster– Add Internal Standard (critical step)– Cover plates and extract for 20 –30 minutes– Transfer– Analyze by MS/MS
• Derivatized - published, more sensitive• Both methods require care and consistency in
sample preparation
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Derivatized Method
1. Add Internal Standard (critical step) we use 12-channel pipetter. 2. Extract 20 minutes on a shaker (room temperature). 3. Transfer extract to a fresh plate. (We keep original plate). 4. Evaporate Solvent using warm air and gentle warming. 5. Add Butanolic HCl, cover (we use Teflon plates) and heat to
approximately 60 C for 20 minutes (ovens corrode over time). 6. Evaporate Butanolic HCl using warm air. 7. Add reconstitution solvent. 8. Cover with Aluminum foil and analyze by MS/MS.
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Documentation -- Preparation
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Documentation -- MS/MS Analysis
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Derivatized Method, Challenges or Hints
0102030405060708090
1stQtr
2ndQtr
3rdQtr
4thQtr
EastWestNorth
High Quality Control C4
0.50
1.00
1.50
2.00
2.50
3.00
3.50
0 50 100 150 200
Day
Con
c (µ
mol
e/L)
9413
9412
9405
9406
LCL
UCL
• We have seen high C4 when samples were extracted in polystyrene plates.
• Overheating can cause high leucine results (QC results may appear normal)
• We have observed low response for Methionine internal standard. This is under investigation.
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Data Reduction and Evaluation
• Objectives– Determine Concentration of Analytes– Incorporate Results into a Database of Samples– Compare Values to a set of Rules– Produce Reports of Normal and Abnormal
Samples
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Data Reduction and Evaluation
• First Step –review all the “chromatograms.”– Missed injections– Failure of the pump program
• Data Conversion – Neolynx generates a tab-delimited file that can
be read by Excel or a database program. – Ratios can be calculated by the MS software,
Excel, or the database.
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Evaluation of Quality Control Samples.
• Quality Control Plan• Documents quality control decisions
– Acceptable plate quality control• Control results within ± 3 sd• Allow some number outside ± 3 sd
• Latitude in decision making• Document decisions• Checked and Reviewed Daily• Reviewed for trends
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Evaluation of Quality Control Samples.
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Analysis of TrendsSample Average Phenylalanine
0.25
0.45
0.65
0.85
1.05
1.25
1.45
1.65
1.85
0 50 100 150 200 250 300 350
Day
Co
nc (
mg
/dL
)
Average High Quality Control Phenylalanine
2
3
4
5
6
7
8
9
10
0 50 100 150 200 250 300 350
Day
Co
nc (
mg
/dL
)
9413
9412
9405
9406
LCL
UCL
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Evaluation of Abnormal Results
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Evaluation of Results
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References
• Morbidity and Mortality Weekly Report, April 13, 2001
• Clinical Laboratory Improvement Amendments• Clinical Laboratory Evaluation Program• Basic QC Practices, 2nd Edition, James O.
Westgard, Ph.D.
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Summary
• Plan Ahead• Train & Educate• Rigorous Method Validation is Required• Start with well defined rules and written Standard
Operating Procedures• Be consistent• Follow long-term trends as well as the daily
results.