1. health and fitness
2. disease
3. cholesterol

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
EtS
3.2e5
Intensity
EtG
2.4e5
1.6e5
Urine
Contaminant
8.0e4
0.2
0.4
0.6
0.8
1.0
Min
1.2
1.4
1.6
1.8
1.6e5
1.5e5
1.4e5
The Matrix Effect
1.3e5
1.2e5
1.1e5
Intensity, cps
1.0e5
Enhancement
9.0e4
8.0e4
7.0e4
Suppression
Normal
6.0e4
5.0e4
4.0e4
1
3.0e4
2.0e4
1.0e4
0.0
0.5
96
1.0
191
1.5
287
2.0
382
2.5
477
3.0
572
Time, min
3.
66
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)
Methods Of
Sample Preparation
Solid Phase Extraction (SPE)
Liquid / Liquid Extraction
Phospholipid Removal
Protein Crash / Precipitation
Filtration
Centrifugation
Settling and Decanting
Dilution
Highly Selective
Techniques
Non-Selective
Techniques
SPE Clean Up
Relatively
Clean Urine
SPE Extracted
Urine
What Dies First?
$2
$600
$80,000
$350,000
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
Phospholipid
Removal
• Crash using organic
solution
• Disrupts protein-analyte
binding
• Limited clean up
• Crash using organic
solution
• Selectively remove
phospholipids using
special designed phase
• Limited interaction with
target compounds
s
p
c
i t
y
,
LC/MS/MS Data
Max. 1.4e4 cps.
s
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...
sn
1.4e5
I,c n pc t pse
1.0e5
Protein Precipitation
yi t ,y
5.0e4
0.0
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Time, min
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...
2.6
2.8
3.0
3.2
3.4
Max. 1.4e4 cps.
is t
0.2
Max. 5555.6 cps.
sns
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...
4.8e4
yi t I,y n ,Ic nt pce t psen
4.0e4
5.0e4
3.0e4
4.0e4
2.0e4
3.0e4
1.0e4
2.0e4
Phospholipid Removal
25-OH-Vit D2
1.59
0.0
1.0e4
2.6
2.8
3.0
3.2
3.4
2.6
2.8
3.0
3.2 Max. 2.9e4
3.4 cps.
Max. 5555.6 cps.
ns
is t
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
Time, min
0.0
XIC of +MRM (5
pairs): 383.200/211.100
D3/2 from1.0
Sample 21
of
(Heated
Nebuliz...
0.2
0.4
0.6 Da ID:0.8
1.2(QC2(75ng)-PPT)
1.4
1.6P-A batch
1.8Phree012313.wiff
2.0
2.2
2.4
Time, min
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...
6.7e4
6.0e4
1.7e4
1.5e4
4.0e4
1.55
1.0e4
25-OH-Vit
D3
2.0e4
n cI nt pe t sen
• No major difference – slightly higher signal from PPT
• Choose the cheapest
/ easiest sample prep method
25-OH-Vit D2
1.58
,I
5000.0
0.0
0.2
0.4
0.6
0.8
1.0
0.2
0.4
0.6
0.8
1.0
y
i t
0.0
1.2
1.18 1.28
1.2
1.4
1.41
1.4
1.6
1.8
Time, min
1.6
1.8
Time, min
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
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
Polar head group fragment
Mass ~184
• Look for precursors:
– Lysos m/z: 496, 522
– m/z: 760, 784, 786
– Product Ion m/z: 184
• The 184184 transition provides signal for all
phosphatidyl cholines and lysophosphatidyl
cholines
PPT vs. Phree in ESI+
184184 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.
8.2e5
8.0e5
7.5e5
Phospholipids?
7.0e5
6.5e5
6.0e5
In te n s ity , c p s
5.5e5
5.0e5
Phospholipids?
4.5e5
4.0e5
3.5e5
3.0e5
25-OH-Vit D2/D3
2.5e5
2.0e5
1.5e5
1.0e5
5.0e4
0.0
0.2
0.4
0.6
0.8
1.0
1.21
1.2
1.41 1.52
1.4
1.6
1.80
1.8
Time, min
2.0
2.21
2.2
2.4
2.6
2.8
3.0
3.2
3.4
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.
7.59
PC-1
PC-2
4.8e6
PC-1,4
4.6e6
4.4e6
4.0e6
3.8e6
3.6e6
3.4e6
3.2e6
Lysos
4.2e6
It takes >10 min
to elute all
9.81
9.00
7.89
Phosphatidyl cholines
In te n s ity , c p s
3.0e6
at 95% MeOH
2.8e6
2.6e6
6.71
2.4e6
3.87
2.2e6
8.56
2.0e6
1.8e6
1.6e6
10.09
6.49
4.04
1.58
2.09
4.95
10.72
8.66
5.52
10.17
5.70
1.4e6
10.91
1.2e6
10.31
13.20
1.0e6
10.40
8.0e5
6.0e5
13.49
3.47
1.48
3.04
4.0e5
12.92
3.76
13.73
12.62
14.58 14.85 16.1916.80
2.0e5
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
Time, min
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
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 fromSample 4 (5000 pg/mL) of Cal-12-22-2010.wiff (Heated ...
Max. 1.3e5 cps.
2.77
1.25e5
1.20e5
1.15e5
1.10e5
1.05e5
1.00e5
9.50e4
9.00e4
8.50e4
In te n s ity , c p s
8.00e4
7.50e4
7.00e4
6.50e4
Notice the
presence of
impurities or
tautomers prior
to Aldosterone
elution
6.00e4
5.50e4
5.00e4
4.50e4
4.00e4
3.50e4
3.00e4
2.50e4
2.00e4
1.50e4
1.00e4
5000.00
0.00
0.5
81
1.0
161
1.5
241
2.0
321
2.5
401
3.0
481
3.5
561
4.0
4.5
641
721
Time, min
5.0
800
5.5
880
6.0
960
6.5
1040
7.0
1120
7.5
1200
8.0
1280
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 fromSample 16 (0.1 ng/mL Plasma Ext-pH 5, #3) of Ext pH-0...
Max. 1.5e4 cps.
4.31
1.5e4
1.4e4
1.3e4
High level of matrix
background
1.2e4
1.1e4
In te n s ity , c p s
1.0e4
9000.0
8000.0
7000.0
6000.0
5000.0
4000.0
3000.0
4.66
4.88
2.82
2000.0
5.06
1000.0
0.0
4.22
0.5
81
1.0
161
1.5
241
2.0
321
2.5
401
3.0
481
3.5
561
Time, min
4.0
641
4.5
721
5.0
800
5.5
880
6.0
960
6.5
1040
Optimization: Sample Load
at pH 7
XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 fromSample 17 (0.1 ng/mL Plasma Ext-pH 7, #1) of Ext pH-...
Max. 1216.7 cps.
1683
1600
1500
1400
1300
4.67
1200
2.80
4.88
In te n s ity , c p s
1100
1000
900
800
700
4.13
600
500
400
300
3.16
200
100
0
0.5
81
1.0
161
1.5
241
2.0
321
2.5
401
3.0
481
3.5
561
4.0
641
4.5
721
5.0
800
5.5
880
6.0
960
6.5
1040
Optimization: Sample Load
at pH 9
XIC of -MRM (3 pairs): 359.100/189.000 Da ID: Aldo 1 fromSample 21 (0.1 ng/mL Plasma Ext-pH 9, #2) of Ext pH-...
Max. 3841.7 cps.
2.81
3800
3600
3400
3200
3000
2800
2600
In te n s ity , c p s
2400
4.88
2200
4.67
2000
1800
5.07
1600
1400
1200
1000
800
600
400
4.36
0.33
2.17
3.47
2.28
200
0
3.18
2.58
0.45
0.5
81
1.0
161
1.5
241
2.0
321
2.5
401
3.0
481
3.5
561
Time, min
5.37
4.02
4.0
641
4.5
721
5.0
800
5.5
880
6.0
960
6.5
1040
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
37950
37951
37952
37953
37954
600
500
Pressure (bar)
400
300
200
100
0
0
50
100
150
Injections
200
250
300
Investigation
SEM images of column frits show build up of proteinaceous
material
35000 times magnification
15000 times magnification
Contaminant &
particle build up
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
Amphetamine
Morphine
Norbuprenorphine
Codeine
Diazepam
PCP
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
For complete method details
visit
www.phenomenex.com/clinical
• Method readily adaptable to automated formats
• Good for acid or enzymatic hydrolysis
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
Wash1: Buffer
Wash2: 100% MeOH
Elution: NH4OH:IPA:Ethyl Acetate
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
400
350
Back Pressure
300
250
Back pressure (bar)
200
150
100
0
2
4
6
8
# Of Injections
10
12
14
Beta Glucuronidase Removal
• Protein Precipitation (PPT) Using Impact
Precipitation Plates after centrifugation
• SPE procedures also remove enzyme
PPT Samples:
No Increase in Back Pressure
# #ofofinjections
injection vs.
VS. increase
Increase in
in back
Back pressure
Pressure for
for samples
Samples
with
With PPT
PPT (using
(Using Impact)
Impact-U)
400
350
Back Pressure
300
250
Back pressure (bar)
200
150
100
0
50
100
150
200
250
300
# Of Injections
350
400
450
500
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
Thank You!
Questions?
SkyC@Phenomenex.com
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
•
Protein Crash/Precipitation
16%
•
Filtration
16%
•
Dilution (Dilute and Shoot)
12%
8%
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
editor@selectscience.net.