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, Assurance 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

SyringePositive pressure micro scale SPE

Automated like this!

Pain Management

Perhaps the increase in the use of pain meds is, in part, the price we pay for

increasing longevity through medicine

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 • 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?

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

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 • 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

What is ITSP?Micro-SPE

performed by a CTC/PAL

ITSP SPE cartridgebeing discarded after use

Photos: Assurance Scientific Laboratories

ITSP SPE cartridge

10-45 mg sorbent

10 mg most common and has 32 µµµµl internal volumeITSP SPE: overall system, AKA your autosampler!

SPE-LC/MS/MS method developmenthelicopter view of strategy

• Focus on simplicity and minimization of steps

• Focus on relative (not absolute) recoveries

• Prioritize hardest to measure drugs (low level opioidsand benzos) over the easy to measure drugs for recovery optimization

• Choose balanced conditions that allow separation and • 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

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 • 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!

SPE cartridge conditioningSolvent choice (list of 18 analyses, triplicate, 1.5 hr)

C18 - SPE DVB - SPE

MeOH, ACN, or THF 100 µµµµl MeOH, ACN, or THF

H2O 100 µµµµl H2O

Load sample 100 µµµµl Load sample

MeOH best ACN bestMeasure breakthrough LC/MS/MS Measure breakthrough

Benzos, opioids least BT opiates least BT

mix mix

SPE cartridge conditioningEnhancing with buffer (list of 6 analyses, triplicate, 0.5 hr)

C18 - SPE DVB - SPE

MeOH 100 µµµµl ACN

H2O (10% NH4OAc) 100 µµµµl H2O (10% NH4OAc)

Load sample 100 µµµµl Load sample

BT cut 25+% BT cut in 25+%Measure breakthrough LC/MS/MS Measure breakthrough

Benzos, opioids least BT opiates least BT

SPE cartridge loadingEnhancing with buffer (added to sample)

C18 - SPE DVB - SPE

MeOH 100 µµµµl ACN

H O (10% NH OAc) 100 µµµµl H O (10% NH OAc)H2O (10% NH4OAc) 100 µµµµl H2O (10% NH4OAc)

Load sample 100 µµµµl Load sample

BT cut 25% again BT cut 25% againMeasure breakthrough LC/MS/MS Measure breakthrough

Benzos, opioids least BT opiates least BT

Add H2O (10% NH4OAc) 50 µµµµl in 1 ml Add H2O (10% NH4OAc)

SPE elution2 equal outcomes, same on both phases

C18 - SPE DVB - SPE

Condition cartridge 100 µµµµl ea Condition cartridge

Load sample 100 µµµµl Load sample

Elution Elution

MeOH (100-80%) or 100 µµµµl MeOH (100-80%)

ACN (100-70%) ACN (100-70%)

80% ACN / 100% MeOH 80% ACN / 100% MeOHMeasure recoveries LC/MS/MS Measure recoveries

Benzos, opioids best recoveries opiates best recoveries

Meets PM needs best!

Test compatibility of SPE eluent with LC separation (SPE – LC interfacing)

Hydrocodone

80% ACN

Buffered 80% ACN

Of course, chemical presentation of the sample from SPE to LC is important

SPE eluent

Codeine

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 µµµµl (2.1 x 50 mm

column). Elution in 100% MeOH (buffered) allows 5 µµµµl LC injection.

Viscosity has an equally important role in LC injection along with pH.

SPE elution volumeAllows optimization for drug classes

0.8

1

THCA

6-MAM

No

rma

lize

d r

esp

on

se

C18 SPE data shown with MeOH elution

DVB with MeOH elution favors low volume elution for all drugs

0.4

0.6

0.8

40 50 60 70 80 90 100

Buprenorphine

Codeine

Diazepam

Secobarbital

Phencyclidine

Elution volume (ul)

No

rma

lize

d r

esp

on

se

Favors opiates, metabolites, and other illicits

Favors opioids, benzos, barbs, and THCABest for PM

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

1

MDMA

Sample loading: defining SPE cartridge capacity and linear working range

Current TQs

ROI optimum

No

rma

lize

d L

C/M

S/M

S r

esp

on

se

C18 SPE data shown with MeOH elution

Linear response observed within 100 to 500 µµµµl 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,

Opiates, metabolites, & illicits fully functional (recoveries 70-90%)

0

0.5

0 200 400 600 800 1000

MDMA

Fentanyl

Amphetamine

Diazepam

Oxymorphone

Pentobarbital

THCA

Linear range

Older TQs

500 µµµµl syringe

loading sample

at 5 µµµµl/sVolume (µµµµl) of urine loaded on SPE cartridge

No

rma

lize

d L

C/M

S/M

S r

esp

on

se

Opiates, metabolites, and other illicitssaturate cartridge first at 500 to 1000 µµµµl sample load

Performance below 100 µµµµl can be improved with smaller syringe and blowing out cartridge with air at each step (����RTC) [also dilution to 200 µµµµl with PAL works nicely]

ITSP cartridge volume is 32 µµµµl

Opioids and benzos are most optimized by design (recoveries >90%)

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

Yes, acidic drugs can be measured under LC conditions used for basic drugs

THCA

PentobarbitalSecobarbital

Workflow: minimizing cycle timePAL operation in the inject ahead mode

SPE 1 SPE 2 SPE 3

LC/MS/MS 1 LC/MS/MS 2 ……

……LC/MS/MS 1 LC/MS/MS 2

4.5 min 4.5 min

……

Total cycle time (SPE + LC/MS/MS) = 4.5 min

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 • 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)