Tandem Mass Spectrometry QA/QC for Newborn Screening: Routine Operations.

1
Tandem Mass Spectrometry QA/QC for Newborn
Screening Routine Operations.

• Mark A. Morrissey, PhD
• Wadsworth Center
• Department of Health
• State of New York

2
Outline

• Routine, but not daily considerations
• Daily analysis of samples
• i.    Sample Preparation
• 1.      Derivatized
• 2.      Un-derivatized
•   ii.    Documentation
• Daily Data Evaluation
•    i.     Data Reduction
• ii.     Evaluation of QC results
• iii. Evaluation of abnormal samples
• d. Summary

3
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

4
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 (C141, 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.

5
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.

6
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

7
Internal Standard Preparation

• In-house Prepared Internal Standards
• Available from Dr. H.J. ten Brink
  (acylcarnitines) and Cambridge Isotope Labs
  (amino acids and acylcarnitines).
• 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

8
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

9
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)

10
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)

11
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.

12
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

13
Derivatized Method

• Add Internal Standard (critical step) we use
  12-channel pipetter.
• Extract 20 minutes on a shaker (room
  temperature).
• Transfer extract to a fresh plate. (We keep
  original plate).
• Evaporate Solvent using warm air and gentle
  warming.
• Add Butanolic HCl, cover (we use Teflon plates)
  and heat to approximately 60 C for 20 minutes
  (ovens corrode over time).
• Evaporate Butanolic HCl using warm air.
• Add reconstitution solvent.
• Cover with Aluminum foil and analyze by MS/MS.

14
Documentation -- Preparation
15
Documentation -- MS/MS Analysis
16
Derivatized Method, Challenges or Hints

• 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.

17
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

18
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.

19
Evaluation of Quality Control Samples.
20
Evaluation of Abnormal Results
21
Evaluation of Results
22
Analysis of Trends
23
Summary

• Start with well defined rules and written
  Standard Operating Procedures
• Be consistent
• Follow long-term trends as well as the daily
  results.