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
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
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)
More
Advantages of Automation
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
Reagent Issues
Spectrophotometers vs. Analyzers
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
& Samples
Fluid Handling, Pipetting
Photometric Analysis
& cleanliness
ISSUE: Poor ‘splits’
“many samples
affected, results were good in
the morning run”
Case Study #1(a)
RESOLUTION:
 check for air bubbles
 check for reagent spills
(photometry issues)
ISSUE: Poor ‘splits’
(extreme samples)
“only ‘dry’ wines (or
only juice samples) affected”
Case Study #1(b)
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 - OK
Blank - OK
Small Sample
Sample
Pipetting Accuracy:
OK
Photometer/Plate Positioning: OK
Medium Sample
OK
OK
Large
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
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 r2 value”
RESOLUTION:
 Verify delta A values (replace G/F reagents)
 Consider editing G/F Standard Curve
 Run Self Test
Editing a Standard Curve
5-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.
Check & clean lamp(s)
Clean Photometer(s) lense(s)
Clean any reagent spills
Check cuvettes for cleanliness &
scratches
Optical Design
4 optical channels
align with 4 microwells
and read each well at
all 8 wavelengths
(340-750nm)
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



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