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

ASEV Supplier’s Showcase, Portland OR

June 19, 2008

Improve and understand your Malic & Glu/Fru analyses

Issues in Automated Analyses

Geoffrey Anderson Geoffrey Anderson Unitech Scientific LLC Unitech Scientific LLC

Lakewood, CA Lakewood, CA

Visit us: Visit us: www.unitechscientific.com

Advantages of Automation

Efficiency: more data . . . Faster

Better Accuracy & ReliabilityMulti-point Standard Curves (improved

accuracy over analytical range)Verified Calibrations (Check Wines as Verified Calibrations (Check Wines as

controls, Levy Jennings Charts)controls, Levy Jennings Charts)

Systematic (fewer random errors)

MoreMore Advantages of Automation

EasierRandom Access - Random Access - multiple tests run

togetherEasy assay set-up Easy assay set-up Easy accurateEasy accurate reportingreportingCurve Editing Curve Editing Store Calibration Curves Store Calibration Curves

Managing the Issues

Troubleshooting – Common Themes

Reagent IssuesSpectrophotometers vs. AnalyzersDiscrete & Other Analyzers

Analyzer Components & Functions

Operating software 2 syringe pumps wash headwash headOptical Optical

systemsystem

samples reagents ReactionsDI water

Controlling Potential Sources of Error

Reagents, Standards Fluid Handling, Pipetting Photometric Analysis & Samples & cleanliness

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)

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

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

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 - OKBlank - 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 °CBlank 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 3.0091Medium 1.3440 1.4202 0.0082 0.5756 5.6687Small 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 r2 value”

RESOLUTION:Verify delta A values (replace G/F reagents)Consider editing G/F Standard CurveRun Self Test

Editing a Standard Curve5-Point Calibration Curve

r2 = 0.95972

Editing: Select Calibration Points

Editing a Standard Curve

4-Point Calibration Curve

r2 = 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”

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.

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.Photometer P.M.Check & clean lamp(s)Check & clean lamp(s)Clean Photometer(s) lense(s)Clean Photometer(s) lense(s)Clean any reagent spillsClean any reagent spillsCheck cuvettes for cleanliness & Check cuvettes for cleanliness &

scratchesscratches

Optical DesignOptical Design

4 optical channels

align with 4 microwells

and read each well at

all 8 wavelengths

(340-750nm)

Quality ControlQuality ControlTrackingTracking

LIS compatibleLIS compatible

Preventive Maintenance for Accurate Automated Results

A. Fluid Handling System A. Fluid Handling System (e.g. bubbles)(e.g. bubbles)

B. Photometry System B. Photometry System (lamp voltage, (lamp voltage, spills etc.)spills etc.)

C. System Diagnostics C. System Diagnostics (self test)(self test)

D.D. Daily/WeeklyDaily/Weekly (prime, bleach or change (prime, bleach or change tubing, water supply, confirm performance)tubing, water supply, confirm performance)

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)

Issues in Automated Analyses

Geoffrey Anderson Geoffrey Anderson Unitech Scientific LLC Unitech Scientific LLC

Lakewood, CA Lakewood, CA

Visit us: Visit us: www.unitechscientific.com