Supplier Showcase Seminar: QC & Enzymatic Analyses

1. Supplier Showcase Seminar: QC & Enzymatic Analyses • Thursday, June 191:30 pm - 3:00 pm • Chair: Patricia Howe, Patricia Howe Wines, California • Theme: Improve and understand your Malic and Glu/Fru analysis • Speakers:Jason Gerard, Tom Eddy Wines, CaliforniaPatricia Howe, Patricia Howe Wines, CaliforniaSamantha Kollar, Vinquiry, CaliforniaMichael Moyer, Walla Walla Community College, Washington Geoffrey Anderson, Unitech Scientific, California

2. ASEV Supplier’s Showcase, Portland OR June 19, 2008Improve and understand your Malic & Glu/Fru analysesIssues in Automated Analyses Geoffrey Anderson Unitech Scientific LLC Lakewood, CA Visit us: www.unitechscientific.com

3. Advantages of Automation • Efficiency: more data . . . Faster • Better Accuracy & Reliability • Multi-point Standard Curves (improved accuracy over analytical range) • Verified Calibrations (Check Wines as controls, Levy Jennings Charts) • Systematic (fewer random errors)

4. MoreAdvantages of Automation • Easier • Random Access - multiple tests run together • Easy assay set-up • Easy accuratereporting • Curve Editing • Store Calibration Curves

5. Managing the Issues Troubleshooting – Common Themes • Reagent Issues • Spectrophotometers vs. Analyzers • Discrete & Other Analyzers

6. Analyzer Components & Functions wash head Optical system Operating software 2 syringe pumps DI water reagents samples Reactions

7. Controlling Potential Sources of ErrorReagents, Standards Fluid Handling, Pipetting Photometric Analysis & Samples & cleanliness

8. ISSUE: Poor ‘splits’ Case Study #1(a)“many samples affected, results were good in the morning run” RESOLUTION: • check for air bubbles • check for reagent spills (photometry issues)

9. ISSUE: Poor ‘splits’(extreme samples) Case Study #1(b)“only ‘dry’ wines (or only juice samples) affected” RESOLUTION: • check ‘delta ABS’ (raw data) <0.030 or greater (> high Std ABS) • consider alternate protocols

10. 2 Protocols:Accuracy over a Broad Analytical Range Sample Volumes 1) L-Malic ‘dry’ (for high sensitivity, 0.02 – 0.5 G/L) 2uL Sample volume 2) L-Malic ‘juice’ (detecting high levels, 0.2 – 5.0 G/L) 2uL Sample Volume pre-diluted 10-fold

11. Problem Solving Approach:Isolate instrument issues Instrument Diagnostics(example) • ‘Self Test’Measure Precision & Accuracy (Pipette delivery of dye + diluent to cuvettes) • Blank (measure d.i. water in cuvette) • Photometer Voltage(s) (i.e. response to light at each wavelength)

12. Distinguish instrument vs. chemistry issues Self Test (example) Sunday, June 08, 2008, 11:01 Lamps - OK Blank - OK Small Sample Medium Sample Large Sample Pipetting Accuracy: OK OK OK Photometer/Plate Positioning: OK OK OK PASSED (Blank out of recommended range) DETAILS: (6.3.1.162/1720/0.95/0.92) Temperature at start: 29.3 °C Temperature at end: 24.8 °C Blank Abs: 0.0016

13. Distinguish instrument vs. chemistry issues Self Test(precision detail) Type Target Mean SD %CV %Diff ------------------------------------------------------------------ Large 2.4190 2.4918 0.0147 0.5898 3.0091 Medium 1.3440 1.4202 0.0082 0.5756 5.6687 Small 0.2690 0.3071 0.0034 1.0930 14.1654

14. ISSUE: Standard Curve Case Study #2(a) “LMA curve is OK, G/F curve has poor duplicates & poor r2 value” RESOLUTION: • Verify delta A values (replace G/F reagents) • Consider editing G/F Standard Curve • Run Self Test

15. Editing a Standard Curve5-Point Calibration Curve r2 = 0.95972

16. Editing: Select Calibration Points

17. Editing a Standard Curve4-Point Calibration Curve r2 = 0.99997

18. ISSUE: Standard Curve Case Study #2(b) “Curve ‘falls-off’ at top end” Likely Reagent Cause: Substrate depletion (Working Reagent) or weak enzyme activity RESOLUTION:Replace G/F Reagents

19. PotentialISSUE: Reagent Stability(Reagents may be left ‘on-board’) RESOLUTION: Anticipate Deterioration & Dilution - Schedule - Fresh Working Reagents - Use Enzyme ALIQUOTS & Standards ALIQUOTS - Control Temperature as required

20. ISSUE: Standard Curve Case Study #2(c) “Everything was great Friday, nothing looks good Monday” • Verify change in system RESOLUTION: • Weekly P.M. (e.g. Self Test, check tubing, etc.) Check for air bubbles, clean supply water, clean cuvettes, etc.

21. ISSUE: Check-wine is ‘Out of Range’Case Study #3“D-Fru is low, D-Glu is OK” Note: D-Fru reaction may not be complete (PGI Reaction is the rate-limiting step) RESOLUTION: Load fresh PGI aliquot

22. ISSUE:‘Self Test’: Filter Voltagefails RESOLUTION: Photometer P.M. • Check & clean lamp(s) • Clean Photometer(s) lense(s) • Clean any reagent spills • Check cuvettes for cleanliness & scratches

23. Optical Design 4 optical channels align with 4 microwells and read each well at all 8 wavelengths (340-750nm)

24. Quality Control Tracking LIS compatible

25. Preventive Maintenancefor Accurate Automated Results A. Fluid Handling System (e.g. bubbles) B. Photometry System (lamp voltage, spills etc.) C. System Diagnostics (self test) • Daily/Weekly (prime, bleach or change tubing, water supply, confirm performance)

26. Data Issues • Sample Data base (avoid transcription errors) • Review results on-screen (Accept or Rerun) • Sorting Data, Print & Distribute Reports & export reports (avoid transcription errors)

27. Issues in Automated Analyses Geoffrey Anderson Unitech Scientific LLC Lakewood, CA Visit us: www.unitechscientific.com