This document describes the development of an automated SPE-LC/MS/MS method for the simultaneous measurement of multiple drugs of abuse in urine and oral fluid samples. Key points: - An online SPE method was optimized on an autosampler to pre-concentrate samples prior to LC/MS/MS analysis, allowing all drugs to be measured in a single run. - Method development focused on optimizing recoveries for the lowest dose and hardest to measure drugs. Automation facilitated rapid testing and transfer of optimized parameters between steps. - The method demonstrated linear response over a 100-500 μL sample loading range and achieved a total analysis time of 4.5 minutes per sample for SPE and LC/MS/

1. Unified Drug Testing by Online SPE-LC/MS/MS
Focus on Productivity Achieved Through Ease of Use:
One totally automated method measures ALL the drugs in urine and/or oral Fluids
Mark Hayward,2 Rick Youngblood,2 Kim Gamble,2
Martin Johnson,1 and Matthew T. Hardison1
1Assurance Scientific Laboratories,
727 Memorial Dr. Suite 103, Bessemer AL 35022
2ITSP Solutions Inc.,
10 South Carolina St., Hartwell GA 30643
SPE cartridge
Syringe
Positive pressure micro scale SPE
Automated like this!

2. Measurement of drugs of abuse in urine
and/or oral fluids (OF) is common
• Pre-employment screening
• DOT / federal mandated testing
• Law enforcement
• Compliance / diagnostic determinations by physicians
– Latter two are growing rapidly!
• Fastest-growing & preferred approach is LC/MS/MS
– Due to high degree of certainty for simultaneous
identification and concentration determination
• Continued growth in the use of LC/MS/MS for the
measurement of drugs of abuse in urine and OF
seems certain

3. Still, there are several technical
challenges that need to be met
• Easily measure low dose drugs at/near 1 ng/g
– For medical purposes [Pesce, et. al. 2012 AACC conference] as
well as for zero tolerance testing
• Simplicity for performing measurements with lab
technicians
– Automation: load samples / run list (prioritized queuing for STAT samples)
– All LC/MS/MS peaks sufficiently intense for automatic
integration
• Ability to achieve high productivity for all work
– Needs apply to ALL testing! [not just high volume tests]
– Investing 90% effort in 10% of tests is not acceptable
• Minimizing the labor and number of workflows required
– Simplicity has always been crucial
– As volume and number of tests continues to grow, simplicity
grows in importance!

4. Pain Management Drug Monitoring
• Required to prevent abuse, addiction, diversion,
mortality and morbidity (urine drug testing)
• Nevertheless, must meet patient needs first (and
not penalize low, irregular dosing)
• Production environment: assembly line
• Needs to be easy, robust, and have low labor
requirements to measure all relevant drugs at all
relevant concentrations
• Needs sufficient capacity relative to capital
investment: ≥100 reimbursable reports per day
per LC/MS/MS
How does this impact one's approach toward the measurement methodology?

5. Measurement range (defining the challenge):
low single digit ng/g for some opioids and benzos
Pesce, et. al.
2012 AACC
conference
Dilute and shoot is insufficient
Measurement at these levels usually requires some
pre-concentration of the sample  SPE, LLE
At the same time, this needs to be easy!  automation

6. Approach chosen: SPE performed
with LC/MS/MS autosampler
• Automated (serial) pre-concentration of
samples, so that all drugs can be measured in
one method
• Remove salts, proteins, and cells for robust
LC/MS/MS operation
– Very little high LogP (greasy) interferences
– Primary role is to filter, pre-concentrate, and remove salts / other H2O
soluble interferences
– Reverse phase should be sufficient
• Modest capital investment: must buy LC
autosampler regardless  choose one that
does more of the work  CTC/PAL  ITSP
ITSP = Instrument Top Sample Prep

7. What is ITSP?
Micro-SPE
performed by a CTC/PAL
ITSP SPE
cartridge
10-45 mg
sorbent
10 mg most
common and
has 32 l
internal
volume
ITSP SPE cartridge
being discarded after use
ITSP SPE: overall system, AKA your autosampler!
Photos: Assurance Scientific Laboratories
Precise syringe
driven flow
ITSP = Instrument Top Sample Prep

8. Technology
Capabilities SPE Technologies
ITSP syringe flow SPE
Parallel tube and plate
based SPE
Pipette based SPE (packed
sorbent)
Pipette based SPE (loose
sorbent, dispersive)
HPLC Pump flow SPE
(2D HPLC)
Total automation Y
Y - but separate workflow flow
from LC/MS and GC/MS [extra
robots = extra $$$]
Y - but separate workflow flow
from LC/MS and GC/MS [extra
robots = extra $$$]
Y - but separate workflow flow
from LC/MS and GC/MS [extra
robots = extra $$$]
Y - requires additional pumps,
valves, and fixed columns [extra LC
hardware = extra $$$]
On-line operation
w/ LC/MS/MS Y N N N Y
On-line operation
w/ GC/MS/MS Y N N N N
Accurate Flow Control Y N N N Y
Single use sorbent Y Y Y Y N
All sorbents available Y Y N N N
Pre-concentrate
w/o dry down Y N N N Y
Fast Y N Y N Y
Method development
automation Y N N N N
Off-line operation to feed
multiple instruments Y Y Y Y N
Full chain of custody Y - bar code reading possible at every
step
Y - with manual labeling and
recording of each step
N N N
Works with any MS
software
Y - SW pre-embedded in all major MS
brands
N - separate work flow N - separate work flow N - separate work flow
Y - each MS manufacturer has
some unique form of 2D HPLC
Ease of use &
maintenance
Y - the PAL is a HPLC/GC autosampler &
requires same skills & maintenance
Conceptually simple, but
laborious and flow control is
challenging. Robots similar to
pipette based SPE.
Easy to do once one has
bought, set up, and learned
how to use separate robotic
pipette systems ($$$)
Easy to do once one has
bought, set up, and learned
how to use separate robotic
pipette systems ($$$)
N
Pros
Automation, accuracy, & multiple
tests on single system set up
Many methods in literature
Easy to do once one has
bought, set up, and learned
how to use separate robotic
pipette systems ($$$)
Easy to do once one has
bought, set up, and learned
how to use separate robotic
pipette systems ($$$)
Single workflow automation
Cons None
Laborious and lack of accurate
flow control
Limited sorbent choices and
bed masses. Flow control not
proven.
Not proven to improve sample
condition and no control of
flow over sorbent
Requires significant expertise
and one never knows
when/how SPE column will fail
(limits applications)
Comparison of SPE technologies

9. Understanding ITSP and how it
differs from other approaches
• ITSP has precise flow control allowing separations to be performed at
their Van Deemter optimum velocity
• This yields separation performance that is difficult (if not impossible) to
achieve with other SPE approaches
• ITSP also is a completely unique form of SPE in that it is a truly low
volume device (32 l)
• This allows elution to performed precisely with l volumes (ca. 50 l
ready for direct injection on LC/MS or GC/MS)
• This allows ITSP to easily pre-concentrate samples by loading 1-10 ml
of sample while eluting with <100 l of solvent
• Other forms of SPE require larger volumes for elution (10 to 50x) and
significant pre-concentration can only be achieved by adding a separate
slow, laborious dry-down step
• No other SPE approach can achieve the precise chromatographic
separations, pre-concentration of sample, robust operation, and total
automation achieved simultaneously by ITSP

10. 80
85
90
95
100
0 1 2 3 4 5 6 7 8
SCX %-Recovery in 20% Water
SCX %-Recovery in 80% Water
Reverse Phase %-Recovery
SPE is Chromatography!
Optimized outcomes require accurate flow
Are you optimized? Or just guessing?
Oxycodone
Van Deemter curves
for SPE by ITSP
SCX optimum velocity = 0.37 mm/s (1.2 l/s by ITSP) with little room for error!
RP optimum velocity
= 1.5 mm/s
(4x higher than SCX!)
SPE flow driven pneumatically or by vacuum cannot achieve and maintain optimal flow!
ITSP=TOTALCONTROLOFYOURSPE!
ITSP=TOTALCONTROLOFYOURSPE!
Flow (l/s) [5 l/s = 1.5 mm/s]
%Recovery

11. SPE-LC/MS/MS method development
helicopter view of strategy
• Focus on simplicity and minimization of steps
• Prioritize hardest to measure drugs (lowest concentration,
low dose opioids and benzos) over the easy to measure
drugs for recovery optimization and pre-concentration
• Focus on relative recoveries for elution  pre-concentrate
• Choose balanced LC/MS conditions that allow separation
and measurement of both acidic and basic drugs as well as
polar and non-polar drugs (1 method, all drugs!)
• Establish linear scalability and stoichiometry in sample
loading as a data driven way to establish the validation
readiness of the method
• Leverage automation to achieve rapid method
development and execution  ITSP with the CTC/PAL
• Develop the method for urine first, then adapt to oral fluid

12. Serially automated SPE method development
Parallel testing of C18 and DVB SPE phases (3x): each step is a sample list!
Hands on view of strategy
• Test SPE cartridge wash with various solvents (3x cartridge volume)
and no wash: rinse cartridge with water, load spiked urine &
measure drug breakthroughs (choose wash solvent)
• Test SPE cartridge conditioning & loaded sample wash with buffers
(at 3x cartridge volume): load spiked urine & measure drug
breakthroughs (choose conditioning/wash buffer)
• Test SPE cartridge elution with various solvents and measure drug
recoveries (choose elution solvent and measure optimal flow)
• Test SPE cartridge elution at multiple volumes with various buffers
in chosen elution solvents and measure drug recoveries (pre-concentrate)
while monitoring LC separation (choose elution buffer based on LC
separation first, then recoveries)
• Vary sample amount over a range of at least 10x and measure drug
recoveries. If linear stoichiometry is not observed, re-optimize
above steps based on data. If linear, re-optimize LC/MS/MS, choose
sample amount, then validate!

13. Automated method development
• Run each of the 5 lists sequentially (described in
previous slide) transferring optima measured into
next list
• Optima results:
– Sorbent: C18 end-capped (gives higher recovery for
benzos / opioids)
– Conditioning: MeOH, then NH4OAc buffer
– Loading: ≥0.5% NH4OAc in sample, Flow 5 l/s
– Wash: aqueous NH4OAc (≥0.5%)
– Elution: 0.2% NH4OAc in MeOH, 75 l at 5 l/s (pre-
concentrates lowest concentration drugs most)
– Sample load range: 100 – 500 l is linear at 0.5%
NH4OAc in sample (can be increased with higher %NH4OAc in sample)
Minimizes
breakthrough

14. Test compatibility of SPE eluent with
LC separation (SPE – LC interfacing)
Codeine
Hydrocodone
80% ACN
Buffered 80% ACN
Of course,
chemical
presentation of
the sample from
SPE to LC is
important
Just like with SPE,
control of the pH
(ionization state)
controls retention
Buffer: NH4OAc
LC column: C18
B = ACN
Elution in 80% ACN limits LC injection volume to 2 l (2.1 x 50 mm
column). Elution in 100% MeOH (buffered) allows 5 l LC injection.
Viscosity has an equally important role in LC injection along with pH.
SPE eluent

15. SPE elution volumeAllows optimization for drug classes
0.4
0.6
0.8
1
40 50 60 70 80 90 100
THCA
6-MAM
Buprenorphine
Codeine
Diazepam
Secobarbital
Phencyclidine
Elution volume (ul)
Normalizedresponse
Favors opiates,
metabolites,
and other
illicits
Favors opioids,
benzos, barbs,
and THCA
Best for PM
C18 SPE data shown
with MeOH elution
DVB with MeOH elution
favors low volume elution
for all drugs
k’ = 1.5 - 2 k’ = 2 - 3
Gains in sensitivity
from lower volume
elution using DVB do
not outweigh the
absolute recoveries
observed with C18 SPE
k’ > 3
Dilutes all
drugs

16. 0
0.5
1
0 200 400 600 800 1000
MDMA
Fentanyl
Amphetamine
Diazepam
Oxymorphone
Pentobarbital
THCA
Sample loading: defining SPE cartridge
capacity and linear working range
Linear range
Current TQs
Older TQs
500 l syringe
loading sample
at 5 l/s
Volume (l) of urine loaded on SPE cartridge
ROI optimum
Pre-concentration 1.5-3x
NormalizedLC/MS/MSresponse
C18 SPE, MeOH elution,
0.5% NH4OAc in sample
Linear response
observed within 100
to 500 l sample load
range for all PM drugs
Current triple quads
(TQs) can measure all
PM drugs in the lower
half of this range
Opiates, metabolites,
and other illicits
saturate cartridge first
at 500 to 1000 l
sample load
Performance below
100 l can be
improved with smaller
syringe and blowing
out cartridge with air
at each step (RTC)
[also dilution to 200 l
with PAL works nicely]
ITSP cartridge volume
is 32 l
Opioids and benzos are
most optimized by design
(recoveries >90%)
Opiates, metabolites, &
illicits fully functional
(recoveries 80-90%) Linear range can be
extended with higher
%NH4OAc in sample

17. Focus on the LC/MS/MS
also can be productive
Original LC/MS/MS
It works
4.5 min
Heat column, increase flow, add
gradient segments
Improve separation where peaks
are crowded
Decrease time between well
separated peaks
4.1 min
Column switching / conditioning
also saves overhead time

18. Acidic drugs can be measured under LC
conditions used for basic drugs
THCA
Pentobarbital
Secobarbital

19. Workflow: minimizing cycle time
PAL operation in the inject ahead mode
SPE 1 SPE 2 SPE 3
LC/MS/MS 1 LC/MS/MS 2
4.5 min 4.5 min
……
……
Total cycle time (SPE + LC/MS/MS) = 4.5 min

20. Summary: Drugs in urine
• An online and automated SPE-LC/MS/MS method has been
developed for pain management (PM) monitoring in urine
samples
• The method is scalable and can be adapted to any LC/MS/MS
simply by adjusting the volume of urine loaded for SPE
• Rather than using multiple panel focused methods, this single
method is used to measure all PM related drugs
• Method development focus has been on the lowest dose,
hardest to measure prescribed drugs (suggests prioritization works!)
• Rework is limited to only the highest dose drugs in the highest
dosed patients (inject less, bring into linear range)
• Robust operation and a cycle time (SPE + LC/MS/MS) of 4.5
min has been achieved
• The use of ITSP with the CTC/PAL for serial automation is a
very efficient way to perform SPE method development: this
method required 3 lab days using 1 SPE-LC/MS/MS system

21. Adapt method developed for urine to
function equally well for oral fluid (OF)
• First things first: choosing a sampling device
• Many considered / criticized, primary concerns:
• Sample stability
• Volumetric sampling accuracy
• We added need for forensic acceptance and success
when challenged at trial
• Quantisal sampling device chosen for its:
• Highest volumetric accuracy (+/-10%)
• Built in filtering (important: all OF samples require
either filtering or centrifugation)
• >1 week sample stability
• Greatest acceptance in clinical and forensic use

22. Adapt / test method for oral fluid (OF)
• SPE and sample buffering increased to 2% NH4OAc to
overcome built in Quantisal buffering
• High opiate / illicit drug recovery requires formation of
drug-OAc ion pair
• Sample volume increased to 1 ml to achieve 0.1 ng/g cut offs
• Drug concentrations lower in OF  13x pre-concentration
• Linear response for 71 drugs achieved
• Method tested with 20 multi medication / not so good
prognosis patient samples where both urine and OF collected
in parallel and tested with both LC/MS/MS and immunoassay
– LC/MS/MS in complete agreement for urine and OF (no false neg)
and “partying” successfully detected in one sample set
– Included patients dosed 1 mg/day opioids / benzos: easily measured
/ auto-integrated at approx 0.5 ng/g in OF (0.1 ng/g cutoffs)
– Immunoassay showed one of its deficiencies by not being able to
detect / confirm 1 mg/day dosed drugs (urine and OF)

23. Unified urine and oral fluid
method results
• Lab time to adapt urine method to OF: 2 days
• Again, leveraging the automation
• Method (urine & OF) validated to both clinical and
forensic standards in multiple labs (cv 3-5%)
• Urine and OF can be measured in the same run
• Still need to include blanks, calibrators, and QCs
for both
• Method (urine & OF) in use for production work and
delivering 100-200 results overnight for each
LC/MS/MS
• High ROI readily achieved for top end LC/MS/MSs

24. What’s next? Larger range of tests
ready to run with a single LC/MS/MS
• We have successfully added Vit D (blood) measurement to
individual PAL systems already measuring drugs in urine / OF
– These 3 tests are currently considered highest ROI in clinical lab
– 2 cartridge types, 2 LC column types, all on-board, all the time
• We have drug methods for blood/serum/plasma/DPS/DBS
samples to add to these systems (ion exchange SPE)
– Includes DPS/DBS disk extraction and IS addition
• We are implementing SPE solvent sourcing to Fast Wash
stations (up to 4) as an alternative to ink wells
– Up to 8 solvents total using minimal PAL rail
– Allows 2-4 weeks of solvent capacity (0.5-2 liter bottles)
• We are implementing 4 way valve cleaning solvent sourcing
for DLW (multiple cleaning chemistries for multiple methods)
• This should allow 4 tests to be setup/ready to run on single
PAL-LC/MS/MS systems requiring only method selection in
MS software and perhaps loading color coded SPE cartridges