1. Optimization of Automated Online
SPE-LC-MS/MS Used in Pain
Management Drug Monitoring
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
SPE cartridge
Syringe
Positive pressure micro scale SPE
Automated like this!

2. Pain Management

3. Perhaps the increase in the use of pain
meds is, in part, the price we pay for
increasing longevity through medicine

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 ones 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
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
• Removes salts, proteins, and cells for robust
LC/MS/MS operation (reverse phase should be sufficient)
• Modest capital investment: must buy LC
autosampler regardless  choose one that
does more of the work  CTC/PAL  ITSP

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

8. SPE-LC/MS/MS method development
helicopter view of strategy
• Focus on simplicity and minimization of steps
• Focus on relative (not absolute) recoveries
• Prioritize hardest to measure drugs (low level opioids
and benzos) over the easy to measure drugs for
recovery optimization
• Choose balanced 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

9. 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 (BT, choose wash solvent)
• Test SPE cartridge conditioning & loaded sample wash with
buffers (at 3x cartridge volume): load spiked urine & measure
drug breakthroughs (BT, choose conditioning/wash buffer)
• Test SPE cartridge elution with various solvents and measure
drug recoveries (choose elution solvent)
• Test SPE cartridge elution with various buffers in chosen elution
solvents and measure drug recoveries 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!

10. SPE cartridge conditioning
Solvent choice (list of 18 analyses, triplicate, 1.5 hr)
C18 - SPE DVB - SPE
MeOH, ACN, or THF 100 l MeOH, ACN, or THF
H2O 100 l H2O
Load sample 100 l Load sample
MeOH best ACN best
Measure breakthrough LC/MS/MS Measure breakthrough
Benzos, opioids least BT opiates least BT
mix mix

11. SPE cartridge conditioning
Enhancing with buffer (list of 6 analyses, triplicate, 0.5 hr)
C18 - SPE DVB - SPE
MeOH 100 l ACN
H2O (10% NH4OAc) 100 l H2O (10% NH4OAc)
Load sample 100 l Load sample
BT cut 25+% BT cut in 25+%
Measure breakthrough LC/MS/MS Measure breakthrough
Benzos, opioids least BT opiates least BT

12. SPE cartridge loading
Enhancing with buffer (added to sample)
C18 - SPE DVB - SPE
MeOH 100 l ACN
H2O (10% NH4OAc) 100 l H2O (10% NH4OAc)
Load sample 100 l Load sample
BT cut 25% again BT cut 25% again
Measure breakthrough LC/MS/MS Measure breakthrough
Benzos, opioids least BT opiates least BT
Add H2O (10% NH4OAc) 50 l in 1 ml Add H2O (10% NH4OAc)

13. SPE elution
2 equal outcomes, same on both phases
C18 - SPE DVB - SPE
Elution Elution
MeOH (100-80%) or 100 l MeOH (100-80%)
ACN (100-70%) ACN (100-70%)
Condition cartridge 100 l ea Condition cartridge
Load sample 100 l Load sample
80% ACN / 100% MeOH 80% ACN / 100% MeOH
Measure recoveries LC/MS/MS Measure recoveries
Benzos, opioids best recoveries opiates best recoveries
Meets PM needs best!

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
NormalizedLC/MS/MSresponse
C18 SPE data shown
with MeOH elution
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 70-90%)

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 can save overhead time

18. Yes, 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
• An online and automated SPE-LC/MS/MS method has
been developed for pain management (PM) monitoring
• Rather than using multiple panel focused methods, this
single method is used to measure all PM drugs
• Method development focus has been on the lowest dose,
hardest to measure prescribed drugs
• 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:
the data in this presentation required 3 lab days using 1
SPE-LC/MS/MS system (much of it for DVB not shown)