Optimization of Automated Online SPE-LC-MS

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

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

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 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 cartridge10-45 mg sorbent

10 mg most common and has 32 l internal volume

ITSP SPE cartridgebeing discarded after use

ITSP SPE: overall system, AKA your autosampler!

Photos: Assurance Scientific Laboratories

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 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

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!

SPE cartridge conditioningSolvent 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 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 l ACN

H2O (10% NH4OAc) 100 l H2O (10% NH4OAc)


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


SPE cartridge loadingEnhancing with buffer (added to sample)

C18 - SPE DVB - SPE

MeOH 100 l ACN

H2O (10% NH4OAc) 100 l H2O (10% NH4OAc)


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


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

SPE elution2 equal outcomes, same on both phases

C18 - SPE DVB - SPE

Elution ElutionMeOH (100-80%) or 100 l MeOH (100-80%) ACN (100-70%) ACN (100-70%)

Condition cartridge 100 l ea Condition cartridge


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

Benzos, opioids best recoveries opiates best recoveriesMeets PM needs best!

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

5x10

00.025

0.050.075

0.10.125

0.150.175

0.20.225

0.250.275

0.30.325

0.350.375

0.40.425

0.450.475

0.50.525

0.550.575

0.60.625

0.650.675

0.70.725

0.750.775

0.80.825

0.850.875

0.90.925

0.950.975

11.025

1.051.075

1.11.125

1.151.175

1.21.225

1.251.275

1.31.325

1.35

Cpd 9: Codeine: +ESI MRM Frag=140.0V [email protected] (300.0000 -> 199.0000) C18 80% ACN elution 0.175% NH4 Acetate 2ul Loop.d

1.395

1.179

Counts vs. Acquisition Time (min)0.88 0.9 0.92 0.94 0.96 0.98 1 1.02 1.04 1.06 1.08 1.1 1.12 1.14 1.16 1.18 1.2 1.22 1.24 1.26 1.28 1.3 1.32 1.34 1.36 1.38 1.4 1.42 1.44 1.46 1.48 1.5 1.52 1.54 1.56 1.58 1.6 1.62 1.64 1.66 1.68 1.7 1.72 1.74 1.76 1.78

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: NH4OAcLC 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

SPE elution volumeAllows optimization for drug classes

40 50 60 70 80 90 1000.4

0.6

0.8

1

THCA6-MAMBuprenorphineCodeineDiazepamSecobarbitalPhencyclidine

Elution volume (ul)

Nor

mal

ized

resp

onse

Favors opiates, metabolites, and other illicits

Favors opioids, benzos, barbs, and THCABest for PM

C18 SPE data shown with MeOH elutionDVB 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

0 100 200 300 400 500 600 700 800 900 10000

0.5

1

MDMAFentanylAmphetamineDiazepamOxymorphonePentobarbitalTHCA

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

Nor

mal

ized

LC/M

S/M

S re

spon

se

C18 SPE data shown with MeOH elution

Linear response observed within 100 to 500 l sample load range for all PM drugsCurrent triple quads (TQs) can measure all PM drugs in the lower half of this rangeOpiates, metabolites, and other illicits saturate cartridge first at 500 to 1000 l sample loadPerformance 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%)

Focus on the LC/MS/MS also can be productive

6x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

Cpd 99: 11-nor-9-Carboxyl-THC: +ESI MRM Frag=120.0V [email protected] (345.0000 -> 327.2000) C1850 ul Elution.d

4.40

2

Counts vs. Acquisition Time (min)0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4

6x10

00.10.20.30.40.50.60.70.80.9

11.11.21.31.41.51.61.71.81.9

22.12.22.32.42.52.62.72.82.9

33.13.23.33.43.53.63.73.83.9

44.14.24.34.44.54.64.74.84.9

55.15.25.35.45.55.65.7

Cpd 96: 11-nor-9-Carboxyl-THC: +ESI MRM Frag=120.0V CF=0.000 DF=0.000 [email protected] (345.0000 -> 327.2000) 2000B.d

4.067

Counts vs. Acquisition Time (min)0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1

Original LC/MS/MSIt works4.5 min

Heat column, increase flow, add gradient segmentsImprove separation where peaks are crowdedDecrease time between well separated peaks4.1 minColumn switching / conditioning also can save overhead time

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

4x10

00.10.20.30.40.50.60.7

0.80.9

11.11.21.31.41.51.61.71.81.9

22.1

2.22.32.42.52.62.72.82.9

33.13.23.33.43.53.6

3.73.83.9

44.14.24.34.44.54.6

Cpd 96: 11-nor-9-Carboxyl-THC: +ESI MRM Frag=120.0V CF=0.000 DF=0.000 [email protected] (345.0000 -> 327.2000) 2000B.d 1

Counts vs. Acquisition Time (min)2.4 2.45 2.5 2.55 2.6 2.65 2.7 2.75 2.8 2.85 2.9 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 3.45 3.5 3.55 3.6 3.65 3.7 3.75 3.8 3.85 3.9 3.95 4 4.05 4.1 4.15 4.2 4.25 4.3 4.35

THCA

Pentobarbital Secobarbital

Workflow: minimizing cycle timePAL operation in the inject ahead mode

SPE 1 SPE 2 SPE 3LC/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 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)

Documents

Optimization of Automated Online SPE-LC-MS