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Overcoming the Challenges of Sample Preparation and LC/MS/MS Method
Development for Clinical Applications
Speaker: Sky Countryman, Manager of PhenoLogix and Applied Technologies, Phenomenex Webinar Host: Sonia Nicholas, Clinical Diagnostics Editor of SelectScience
Overcoming the Challenges of Sample
Preparation and LC/MS/MS Method
Development for Clinical Applications
Presented by Sky Countryman
Learning Objectives
• How to determine the best sample
preparation technique
• Method development tips for achieving
separation of target compounds via
LC/MS/MS
• How to increase throughput without
sacrificing results
Myths of LC/MS/MS
1. I don’t need good resolution
2. I don’t need to do any sample clean up
Codeine
Interferences
Endogenous
Interferences
Urine
Contaminant
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Min
8.0e4
1.6e5
2.4e5
3.2e5
Inte
ns
ity
EtG
EtS
XIC of +MRM (3 pairs): 172.2/154.2 Da ID: Gabapentin-1 from Sample 4 (Blank matrix004) of Supression.wiff (Turbo Spray) Max. 1.7e5 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.596 191 287 382 477 572 667 762 858 953 1048
Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
Inte
nsity
, cps
1
The Matrix Effect
Suppression
Enhancement
Normal
Matrix Effects
• What contributes to the matrix effect?
– Disease state
– Endogenous compounds
– Exogenous compounds
– MS source design
– Sample preparation
Poll 1: Which of the following sample
preparation techniques do you perform in
your lab?
• Solid Phase Extraction (SPE)
• Liquid-Liquid Extraction (LLE)
• Phospholipid Removal
• Protein Crash/Precipitation
• Filtration
• Dilution (Dilute and Shoot)
Solid Phase Extraction (SPE) Liquid / Liquid Extraction
Phospholipid Removal
Protein Crash / Precipitation Filtration
Centrifugation
Settling and Decanting
Dilution
Highly Selective
Techniques
Non-Selective
Techniques
Methods Of
Sample Preparation
SPE Clean Up
Relatively
Clean Urine
SPE Extracted
Urine
What Dies First?
$2 $600 $350,000 $80,000
Today’s Agenda
Discuss three case studies where sample prep
played an important role in method stability
1. Vitamin D
2. Aldosterone
3. Pain Panel
Poll 2: Which of the following
applications do you run most often?
• Vitamin D Analysis
• Pain Panel (pain medications)
• Steroids
• Immunosuppressants
• Other
Case Study 1
25-OH D2 & D3 from Plasma
Method Background
• Matrix: plasma
– Vitamin D is protein bound
– High phospholipid content in plasma
• APCI source
– Reduces ion suppression (phospholipids)
• Fast separation
– Kinetex 2.6µm C18 30 x 3.0 mm
– Two minute ballistic gradient
Sample Prep Strategies
Protein
Precipitation • Crash using organic
solution
• Disrupts protein-analyte
binding
• Limited clean up
Phospholipid
Removal • Crash using organic
solution
• Selectively remove
phospholipids using
special designed phase
• Limited interaction with
target compounds
XIC of +MRM (5 pairs): 395.300/209.300 Da ID: D2/1 from Sample 16 (QC2(75ng)-Phree) of P-A batch Phree012313.wiff (Heated Nebu... Max. 5555.6 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
In
te
ns
it
y,
c
ps
XIC of +MRM (5 pairs): 395.300/209.300 Da ID: D2/1 from Sample 16 (QC2(75ng)-Phree) of P-A batch Phree012313.wiff (Heated Nebu... Max. 5555.6 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
5000.0
1.0e4
1.5e4
1.7e4
In
te
ns
it
y,
c
ps
1.58
1.411.18 1.28
25-OH-Vit D2
XIC of +MRM (5 pairs): 383.200/211.100 Da ID: D3/2 from Sample 16 (QC2(75ng)-Phree) of P-A batch Phree012313.wiff (Heated Nebuli... Max. 1.3e4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
In
te
ns
it
y,
c
ps
1.55
25-OH-Vit D3
LC/MS/MS Data
Phospholipid Removal
XIC of +MRM (5 pairs): 395.300/209.300 Da ID: D2/1 from Sample 21 (QC2(75ng)-PPT) of P-A batch Phree012313.wiff (Heated Nebuliz... Max. 1.4e4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
5.0e4
1.0e5
1.4e5
In
te
ns
it
y,
c
ps
XIC of +MRM (5 pairs): 395.300/209.300 Da ID: D2/1 from Sample 21 (QC2(75ng)-PPT) of P-A batch Phree012313.wiff (Heated Nebuliz... Max. 1.4e4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
4.8e4
In
te
ns
it
y,
c
ps
1.59
25-OH-Vit D2
XIC of +MRM (5 pairs): 383.200/211.100 Da ID: D3/2 from Sample 21 (QC2(75ng)-PPT) of P-A batch Phree012313.wiff (Heated Nebuliz... Max. 2.9e4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
2.0e4
4.0e4
6.0e4
6.7e4
In
te
ns
it
y,
c
ps
1.55
25-OH-Vit D3
Protein Precipitation
• No major difference – slightly higher signal from PPT
• Choose the cheapest / easiest sample prep method
What are Phospholipids?
Double Chain = Phosphatidyl choline
Single Chain = Lysophosphatidyl choline
Negative Effects
of Phospholipids
• Signal suppression in positive ESI mode
– Loss in sensitivity for ESI+
– Much less pronounced APCI
– Dependent on source design (older = more suppression)
• Retention time shifts as phospholipids build up
– Tandem Labs reported retention shifts of >1 minute
• Reduce column life
– Build up on column reaches critical concentrations
– Phospholipids crash out of solution and back pressure spikes
LLE:
Depletion of Phospholipids
“Errors in Bioanalysis Due to Phospholipids – Definitive Measurement, Mechanism and Management”; ASMS 2011 Poster by LabCorp, Russel Grant, Matthew Crawford, Brian Rappold and Stacy Dee.
Less solubility
in ACN
Important Classes
of Phospholipids
Phosphatidyl Cholines
are the major
component of lecithin
Detection
of Phospholipids
• Look for precursors:
– Lysos m/z: 496, 522
– m/z: 760, 784, 786
– Product Ion m/z: 184
• The 184184 transition provides signal for all
phosphatidyl cholines and lysophosphatidyl
cholines
Polar head group fragment
Mass ~184
PPT vs. Phree in ESI+
184184 Phospholipid APCI
(Extraction from PPT)
XIC of +MRM (7 pairs): 395.300/209.300 Da ID: D2/1 from Sample 11 (Samp1-PPT(ACN-300uL)) of Phree VS PPT(012513)Scan VitD+... Max. 3644.4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e58.2e5
Inte
ns
ity
, c
ps
1.801.411.21 2.211.52
Phospholipids?
25-OH-Vit D2/D3
Phospholipids?
Phospholipid Elution XIC of +MRM (7 pairs): 184.0/184.0 Da ID: PL(Isource) from Sample 11 (30 ug/mL Amoxapine - PPT Infusion) of PostColumn_MeOH_1... Max. 4.9e6 cps.
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
4.0e6
4.2e6
4.4e6
4.6e6
4.8e6
Inte
ns
ity
, c
ps
7.59
9.81
9.00
7.89
6.71
3.87
8.5610.09
6.49
4.04 10.728.664.955.522.09 10.171.58
5.70 10.91
10.3113.20
10.4013.49
3.47 12.921.483.76 13.733.04
12.62
16.1914.58 14.85 16.80
Lyso
s
PC
-1,4
PC
-1
PC
-2
It takes >10 min
to elute all Phosphatidyl cholines
at 95% MeOH
Final Sample Prep Method
• Dispense: – 300 µL of ACN/MeOH (85/15) into each well
– 100 µL of spiked plasma
• Aspirate: – Manually aspirate or vortex to achieve crash
– Wait 30 seconds
• Vacuum: – Apply vacuum for 1-2 mins at 10-15” Hg
• Collect & inject: – Make direct injection on the LC column
– No need to go through the time consuming dry down step
– Total time for sample prep is ≤4 minutes
Final Method
Table 1. Precision and Accuracy Data for 25 OH Vitamin D2
Expected Conc. (ng/mL) %CV Accuracy
25.0 4.766484 103.742303
75.0 4.609796 89.780394
Table 2. Precision and Accuracy Data for 25 OH Vitamin D3
Expected Conc. (ng/mL) %CV Accuracy
25.0 3.316110 105.772878
75.0 3.469131 97.816781
Accuracy and precision based on quantitation against an internal standard
Conclusions
• Removing phospholipids was determined
to be critical to long term method stability
• Methods were adapted to 96-well plate for
high throughput analysis
• Final methods showed high recoveries for
Vit D
Case Study 2
Aldosterone in Plasma
Method Background
• Required detection limit – 10 pg/mL – API 5000
– Requires sample pre-concentration/clean up
• Ionizable in both APCI and ESI – APCI showed lowest background
– Negative mode detection
Aldosterone Ionization
• Capable of forming [M+H]+, [M-H]-, [M+Na]+,
[M+OCOCH3]-
• [M-H] gives the most intense signal
• It exists as three possible tautomers:
Source: Yamashita et al, Chem Pharma Bull, 56(6), 873-877 (2008)
Chromatography
• Initial chemistries used:
– Kinetex C18, 50x2.1 mm, 2.6 µm
– Kinetex XB-C18, 50x2.1 mm, 2.6 µm and 30x2.1 mm
– Kinetex PFP, 50x2.1 mm, 2.6 µm
– Gemini NX C18, 50x2.0 mm, 3 µm
• The goal was to separate the Aldosterone from its
known isomers: Cortisone & Prednisone
• XB-C18 provided the best separation between the
Aldosterone and its possible interferences
Chromatogram XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 from Sample 4 (5000 pg/mL) of Cal-12-22-2010.wiff (Heated ... Max. 1.3e5 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.081 161 241 321 401 481 561 641 721 800 880 960 1040 1120 1200 1280
Time, min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
1.10e5
1.15e5
1.20e5
1.25e5
Inte
ns
ity
, c
ps
2.77
Notice the
presence of
impurities or
tautomers prior
to Aldosterone
elution
SPE Method
• SPE sorbents tested
– Strata-X
– Strata-X-A
– Strata C18-E
• Strata-X-A provided the
cleanest extract
• Next step: Optimization
Strata-X-A
Optimization: Acidic Load XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 from Sample 16 (0.1 ng/mL Plasma Ext-pH 5, #3) of Ext pH-0... Max. 1.5e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.581 161 241 321 401 481 561 641 721 800 880 960 1040
Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
Inte
ns
ity
, c
ps
4.31
4.66
4.882.825.06
4.22
High level of matrix
background
Optimization: Sample Load
at pH 7 XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 from Sample 17 (0.1 ng/mL Plasma Ext-pH 7, #1) of Ext pH-... Max. 1216.7 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.581 161 241 321 401 481 561 641 721 800 880 960 1040
Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1683
Inte
ns
ity
, c
ps
4.67
2.80
4.88
4.13
3.16
XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 from Sample 21 (0.1 ng/mL Plasma Ext-pH 9, #2) of Ext pH-... Max. 3841.7 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.581 161 241 321 401 481 561 641 721 800 880 960 1040
Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
Inte
ns
ity
, c
ps
2.81
4.88
4.67
5.07
4.36
0.332.17
3.182.580.45
3.47 5.372.28 4.02
Optimization: Sample Load
at pH 9
SPE Optimization
• Increase in pH during the sample load reduced
the amount of the late-eluting matrix components
Final SPE Method
Strata-X-A 60 mg /3 mL
• Load: 0.5 mL plasma diluted 1:2 with 25 mM NH4HCO3, pH 8.8-9
• Wash: – 1mL of 25 mM buffer
– 1mL 25% MeOH in Water
• Dry the SPE bed
• Elute: 2 mL of 1.5% NH4OH in MeOH (2x 1mL elution)
• Post SPE : – Dry the residue @ 50-55°C
– Reconstitute with 100 µL of 30:70 MeOH:H2O containing ~1 ppm Estriol
• Recovery @ 100 pg/mL plasma spike is 85% (n=3)
Problem: Columns died after as little as
<50 injections…rapid increase in back
pressure / split peaks
Pressure Trace
0
100
200
300
400
500
600
0 50 100 150 200 250 300
Injections
Pre
ss
ure
(b
ar)
37950 37951 37952 37953 37954
Contaminant &
particle build up
35000 times magnification
15000 times magnification
SEM images of column frits show build up of proteinaceous
material
Investigation
Solution
• Proteinaceous material was highly methanol /
water soluble
• Very low solubility in organic solvents such as
hexane, ethyl acetate, MeCl2
• Changing elution solvent to EA/IPA/NH4OH
provided elution of Aldosterone but not “junk”
10 pg/mL Aldosterone
in Plasma
Case Study 3
Pain Panel in Urine
Hydrolysis
• Hydrochloric acid
– Very efficient & cheap
– Destroys suboxone and 6-MAM
– Corrosive to metal system components
• Beta-glucuronidase / sulfatase
– Inefficient & costly
– High specificity
– Good for all drug classes
Sample Prep Strategies
• Dilute & shoot
– Cheap & easy
– Very hard on system components
• Solid Phase Extraction
– More costly
– Requires special equipment
– Decreases system maintenance
– Increase column lifetime / method stability
– Provides sample concentration
SPE Extraction
PCP
Morphine Codeine Diazepam Amphetamine
Norbuprenorphine Benzoylecgonine 6-MAM
Developing a single SPE method can be a challenge!
Optimized Method for
41 Pain Panel Compounds
• 18 Opiates
• 12 Benzodiazepines
• 5 Amphetamines
• 4 Analgesics
• 2 Drugs of Abuse
• Method readily adaptable to automated formats
• Good for acid or enzymatic hydrolysis
For complete method details
visit
www.phenomenex.com/clinical
Optimizing Wash Strength
Optimized Wash/Elution
Wash1: 0.1N HCl
Wash2: 100% MeOH
Elution: 5% MeOH in NH4OH
Wash1: 0.1N HCl
Wash2: 100% MeOH
Elution: NH4OH:IPA:Ethyl Acetate
Wash1: Buffer
Wash2: 100% MeOH
Elution: NH4OH:IPA:Ethyl Acetate
Problems with Enzyme
• Hydrolyzed samples contain solubilized
enzyme that must be removed
• Centrifugation works well in test tubes
• Rotor arms that adapt to 96-well plates
reduce maximum spin speed
• Resulting samples can rapidly decrease
column lifetime
For more details visit
www.phenomenex.com/clinical
Hydrolyzed Samples
Processed in 96-Well Plates
Number of injection VS. Increase in Back Pressure for Samples
Without PPT
100
150
200
250
300
350
400
0 2 4 6 8 10 12 14
# Of Injections
Back P
ressu
re
Back pressure (bar)
Beta Glucuronidase Removal
• Protein Precipitation (PPT) Using Impact Precipitation Plates after centrifugation
• SPE procedures also remove enzyme
PPT Samples:
No Increase in Back Pressure
# of injection VS. Increase in Back Pressure for Samples
With PPT (Using Impact-U)
100
150
200
250
300
350
400
0 50 100 150 200 250 300 350 400 450 500
# Of Injections
Back P
ressu
re
Back pressure (bar)
# of injections vs. increase in back pressure for samples
with PPT (using Impact)
Conclusion
• SPE provides the highest level of clean up
– Long term = Lowest amount of system maintenance
• Enzymes must be removed before HPLC analysis
– Centrifugation in 96-well plates is not effective in
removing the solubilized enzyme
– PPT using impact removes the enzyme and is suitable
for high throughput environments
– Once the enzyme is removed, acceptable column
lifetime is observed
In Summary
• LC/MS/MS reduces many of the
challenges to chromatographic analysis
• MS technology continues to improve
• There is still a need for chromatography to
separate isobaric interferences
• Sample prep can significantly improve
method stability
Poll Results
Which of the following sample preparation techniques do you perform in your lab?
• Solid Phase Extraction (SPE) 28%
• Liquid-Liquid Extraction (LLE) 20%
• Phospholipid Removal 8%
• Protein Crash/Precipitation 16%
• Filtration 16%
• Dilution (Dilute and Shoot) 12%
Which of the following applications do you run most often?
• Vitamin D Analysis 22%
• Pain Panel (pain medications) 14%
• Steroids 14%
• Immunosuppressants 17%
• Other 33%
Poll 2: Which of the following applications do you run most often?
• Vitamin D Analysis
• Pain Panel (pain medications)
• Steroids
• Immunosuppressants
• Other
Q & A
Thank you for attending
We hope you found the webinar useful and informative.
If you have any further questions please email [email protected].
