Laboratory Experiences with
Oral Fluid Testing
Lou Jambor, Ph.D.
Rody Predescu, M.D.
Quest Diagnostics - Los Angeles
Concerns About Drug
Detection Times in Oral Fluid
Why Oral Fluid Testing?
• Ease of Specimen Collection
• Collection easily be performed at the
Workplace
• Clients saves money on collection fees.
• Client saves time and problems with urine
collection.
• Dilution and Adulteration Minimized.
Initial Screen
• 24 - 48 hours is expected
• OraSure Reagents/Intercept Collection
Device.
• Positive rate very similar to Urine
• 96 Well Micro Titer Plates
• Tecan Genesis Instrument with
Automated Plate Washers and Readers.
Tecan Screening Instrument
• We are currently running about 1000
specimens/day
• Instrument Appears to be very rugged
and has had only minimal downtime.
• Relatively easy to train staff on
instrumentation.
Screening Instrumentation
• In 8 hours, we can run 632 donor
specimens on 2 Systems with 1.5
Technicians.
• We would like it to be faster.
1
Screening Cut-Offs
(In Buffered Specimen)
•
•
•
•
•
•
•
Amphetamine
Methamphetamine
Parent THC
Opiates
Cocaine
PCP
Human IgG
100 ng/mL
40 ng/mL
1.0 ng/mL
10 ng/mL
5.0 ng/mL
1.0 ng/mL
0.5 mcg/mL
Screening Controls
• Controls at 50% and 150% of Cut-Off
• Excellent Precision of Controls
Screening Cross Reactivity
• MDMA: 14 ng/mL on Methamphetamine
Assay.
Human IgG
• Test for Human IgG to verify adequate
specimen and human specimen.
Screening Cross Reactivity
• Excellent Sensitivity for Hydrocodone
(13 ng/mL)
• Excellent Sensitivity for 6-Acetyl-Morphine
(16 ng/mL)
Screening Issues
• 300 mcL of Specimen
• About 700 mcL for Confirmation
• MDA: 204 ng/mL on Amphetamine Assay
2
Screening Issues
• Overall 81% Confirmation Rate
• PCP Confirmation Rate at 40%.
Unknown reasons, but appear to be
false positives.
• Occasional Methamphetamine positive
due to Ephedrine. (0.77% Cross
Reactivity)
Screening Summary
• OraSure reagents on Tecan system
works very well.
• No significant issues.
Adventures in GC/MS
Confirmations
• Quest Diagnostics in Atlanta
developed screening and confirmation
methods.
Instrumentation
Summer 2004
• One Varian 1200 Triple Quad for PCP
and parent THC.
• All other tests on Agilent 5973
• Our task was just to duplicate those
methods in Van Nuys.
Amphetamines
Methamp
Methamp
30
30ng/mL
ng/mL
Agilent
Agilent5973
5973
• Amphetamine,Methamphetamine,
MDMA and MDA at 40 ng/mL.
• Liquid/Liquid Extraction derivatized
with PFPA.
• Using 200 mcL of specimen, this is a
straight forward assay on Agilent 5973
GC/MS.
3
Opiates
• Morphine, Codeine, Hydrocodone,
Hydromorphone, and 6-Acetyl Morphine at
10 ng/mL with 300 mcL of specimen.
• This had to be done on the 5973 and it is
clearly a challenge.
• UTC extraction columns are the standard
method.
Opiates
• Extraction and GC/MS separations look
ugly off the start.
• We call Phil Dimson at SPE Ware and the
staff at Biochemical Diagnostics to see if
they have a better method.
• Phil suggests Varian Factor 4 Analytical
column.
Morphine
Morphine10
10
ng/mL
ng/mL5973
5973
Opiates
• Varian Factor Four Column works great.
• Original UTC extraction procedure still the
cleanest extract.
• 6-Acetyl Morphine is easily detected at 10
ng/mL.
Hydrocodone
Hydrocodone
10
10ng/mL
ng/mL5973
5973
6-MAM
6-MAM
10
10ng/mL
ng/mL
Agilent
Agilent
4
Benzoylecgonine
• 2 ng/mL cut-off
• Original Procedure called for extraction
using SPE Ware Trace B columns.
• Agilent 5973 GC/MS with HP-1 analytical
column.
• We had problems.
Benzoylecgonine
• We call Phil Dimson at SPE Ware.
• He fine tunes things a little.
• The real problem was that our Sigma
powdered BE-D3 was contaminated with
Benzoylecgonine.
• Cerilliant Internal Standards are fine.
BE
BE33ng/mL
ng/mL
Agilent
Agilent5973
5973
Benzoylecgonine
• Many other extraction columns were
tried, but the SPE Ware column was
still the cleanest.
• Varian Factor 4 column also very nice
for benzoylecgonine analysis in urine.
• Resolves a co-eluting peak.
PCP and THC
• Both analytes at 0.5 ng/mL
• We can use the Varian 1200 Triple
Quad
Varian 1200
PCP and THC
• Excellent instrument sensitivity.
• We are a factory. We need a standard
data handling method. We would like
to use Agilent Software.
• ProLab conversion between Varian
Instrument and Agilent Software is
available.
5
ProLab
• After much discussion between Varian,
ProLab and Agilent we were able to
run the Varian with Agilent Software.
• This makes life easier.
Varian 1200
• Overall an excellent instrument.
• Needs to be a little more rugged, but
Varian has an excellent applications
chemist to trouble shoot instrument
and methodologies.
Dean Switch
• First column isolates the analyte
• Narrow time (~ 0.8 min ) around
analyte is directed to second column
• Resulting in significant improvement
in signal to noise.
• ~$7,000 Upgrade to 5973.
22ng/mL
ng/mLStd
Std
Dean
DeanSwitch
Switch
No
Cryo
No Cryo
Dean Switch
• All analytes can be performed on the Dean
Switch.
• We routinely run Opiates and PCP on the
Dean Switch.
• We routinely run THC on the Varian 1200
• Benzoylecgonine and Amphetamines
routinely run on conventional 5973
0.5
0.5THC
THCStd.
Std.
Dean
DeanSwitch
Switch
with
Cryo
with Cryo
6
0.5
0.5THC
THCStd
Std
Varian
Varian1200
1200
Positive Rates
Confirmed Positive Rate:
THC
44%
BE:
22%
Opiates:
18%
Amphetamines:
15%
PCP:
.05%
Positive Rates
Oral Fluid
Overall:
5.8%
THC
44%
Opiates
18%
Cocaine
22%
Amphetamines 15%
5.8%
Summary
Urine
4.9%
64%
7%
16%
12%
Future
• I wish the reagent manufactures would
develop screening assays for the high
through-put instruments.
• The Dean Switch holds tremendous
potential for all analytical toxicology
labs.
• Oral Fluid testing will become more
prevalent.
• The Dean Switch allows low level
confirmation with conventional
GC/MS instruments in Oral Fluid.
Positive Rates
Confirmed Positive Rate:
THC
44%
BE:
22%
Opiates:
18%
Amphetamines:
15%
PCP:
.05%
5.8%
7
Positive Rates
Oral Fluid
Overall:
5.8%
THC
44%
Opiates
18%
Cocaine
22%
Amphetamines 15%
Summary
Urine
4.9%
64%
7%
16%
12%
• Oral Fluid testing will become more
prevalent.
• The Dean Switch allows low level
confirmation with conventional
GC/MS instruments in Oral Fluid.
Future
• I wish the reagent manufactures would
develop screening assays for the high
through-put instruments.
• The Dean Switch holds tremendous
potential for all analytical toxicology
labs.
8