Supplier Showcase Seminar QC Enzymatic Analyses l

Supplier Showcase Seminar: QC & Enzymatic Analyses l Thursday, June 19 1: 30 pm - 3: 00 pm l Chair: Patricia Howe, Patricia Howe Wines, California l Theme: Improve and understand your Malic and Glu/Fru analysis l Speakers: Jason Gerard, Tom Eddy Wines, California Patricia Howe, Patricia Howe Wines, California Samantha Kollar, Vinquiry, California Michael Moyer, Walla Community College, Washington Geoffrey Anderson, Unitech Scientific, California

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

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

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

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

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

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

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

ISSUE: Poor ‘splits’ (extreme samples) “only ‘dry’ wines (or only juice samples) affected” Case Study #1(b) RESOLUTION: l check ‘delta ABS’ (raw data) <0. 030 or greater (> high Std ABS) l consider alternate protocols

2 Protocols: Accuracy over a Broad Analytical Range Sample Volumes 1) L-Malic ‘dry’ (for high sensitivity, 0. 02 – 0. 5 G/L) 2 u. L Sample volume 2) L-Malic ‘juice’ (detecting high levels, 0. 2 – 5. 0 G/L) 2 u. L Sample Volume pre-diluted 10 -fold

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)

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 Photometer/Plate Positioning: 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

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

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

Editing a Standard Curve 5 -Point Calibration Curve r 2 = 0. 95972

Editing: Select Calibration Points

Editing a Standard Curve 4 -Point Calibration Curve r 2 = 0. 99997

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

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

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

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

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

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

Quality Control Tracking LIS compatible

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)

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

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