2. Supplier Showcase Seminar: QC & Enzymatic Analyses Thursday, June 19 1:30 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, California Patricia Howe, Patricia Howe Wines, California Samantha Kollar, Vinquiry, California Michael Moyer, Walla Walla Community College, Washington Geoffrey Anderson, Unitech Scientific, California

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

4. 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) Verified Calibrations (Check Wines as controls, Levy Jennings Charts) Systematic (fewer random errors)

5. More More Advantages of Automation Easier Random Access - Random Access - multiple tests run together Easy assay set-up Easy assay set-up Easy accurate reporting Easy accurate reporting Curve Editing Curve Editing Store Calibration Curves Store Calibration Curves

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

7. Analyzer Components & Functions Operating software2 syringe pumps wash head wash head Optical system samplesreagentsReactions DI water

8. Controlling Potential Sources of Error Reagents, Standards Fluid Handling, Pipetting Photometric Analysis & Samples & cleanliness

9. 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)

10. ISSUE: Poor splits (extreme samples) Case Study #1(b) only dry wines (or only juice samples) affected RESOLUTION: check delta ABS (raw data) high Std ABS) consider alternate protocols

11. 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

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

13. 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

14. 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

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

16. Editing a Standard Curve 5-Point Calibration Curve r 2 = 0.95972

17. Editing: Select Calibration Points

18. Editing a Standard Curve 4-Point Calibration Curve r 2 = 0.99997

19. 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

20. Potential ISSUE: 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

21. 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.

22. ISSUE : Check-wine is Out of Range Case Study #3D-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

23. ISSUE: Self Test: Filter Voltage fails Photometer P.M. RESOLUTION: Photometer P.M. Check & clean lamp(s) Check & clean lamp(s) Clean Photometer(s) lense(s) Clean Photometer(s) lense(s) Clean any reagent spills Clean any reagent spills Check cuvettes for cleanliness & scratches Check cuvettes for cleanliness & scratches

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

25. Quality Control Tracking LIS compatible

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

27. 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)

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