topics in sample preparation a 5 part series…€¦ · topics in sample preparation a 5 part...
TRANSCRIPT
Topics in Sample Preparation A 5 Part Series…
Part 2 - An Introduction to Pass-Through Sample Preparation
©2015 Waters Corporation 1
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� Recorded version of today’s presentation
©2015 Waters Corporation 3
� PDF Copies of today’s slides
� Discount Offers on Sample Preparation Products (Oasis, Ostro etc…)
� Product specific information
� Reference materials
Today’s Presenter – Xin Zhang
� Xin Zhang, Ph.D - Senior Research Chemist, Waters Corporation
� Today’s Webinar will be presented by Xin Zhang, Senior Research Chemist at Waters Corporation. She received her Ph.D in analytical chemistry from SUNY, Buffalo in 2007. At Waters Corporation, she has worked in Consumables R&D and the Technology
©2015 Waters Corporation 4
worked in Consumables R&D and the Technology Advancement Department.
� She has been focused on evaluating different sample prep techniques such as solid phase extraction(SPE), protein precipitation(PPT), solid supported liquid extraction (SSLE) as well as the Ostro Pass-through sample prepare technique. Recently, she has been working on simplified SPE procedures for the Oasis family. In addition, she also helps customers understand sample preparation by delivering educational seminars and trouble shooting.
Agenda
� Industry overview
– Business & scientific challenges
– Sample preparation techniques
� Ostro Pass-through sample preparation
– What is it?
– How does it work?
©2015 Waters Corporation 5
– How does it work?
– Comparison to traditional techniques (PPT, LLE & SSLE)
� Practical Applications of Ostro
� Summary
� Appendix
– Applications examples
Today’s Industry Challenges
Business Challenges– Increasing revenue
– Decreasing costs
Scientific Challenges– Doing more with less
– Diverse samples/analytes
Meeting the needs of the modern laboratory…
vs.
©2015 Waters Corporation 6
– Decreasing costs
– Maximizing resources– Diverse samples/analytes
– Speed
How can we address these needs?
vs.
Sample Preparation Techniques
� Examples of various sample preparation techniques
– Dilution followed by injection
– Protein precipitation (PPT)
– Filtration
– Ultrafiltration
– Liquid-liquid extraction (LLE)
©2015 Waters Corporation 7
– Liquid-liquid extraction (LLE)
– Solid-supported liquid-liquid extraction (SSLE)
– Solid-phase extraction (SPE)
– Immunoaffinity extraction (IA)
– QUECHERS
– ELISA
Selection: Which One?
� How do you chose a technique to remove phospholipids &
proteins from complex sample matrices?
– Protein precipitation (PPT)
– Ultrafiltration
– Liquid-liquid extraction (LLE)
– Solid-supported liquid-liquid extraction (SSLE)
©2015 Waters Corporation 8
– Solid-phase extraction (SPE)
Objectives:Simple technique
Fast preparation procedureClean, protein-free extracts
Agenda
� Industry overview
– Business & scientific challenges
– Sample preparation techniques
� Ostro Pass-through sample preparation
– What is it?
– How does it work?
©2015 Waters Corporation 9
– How does it work?
– Comparison to traditional techniques (PPT, LLE & SSLE)
� Practical Applications of Ostro
– Drug screening
– Direct analysis of opiates
� Conclusions
� Appendix
– Applications examples
Sample Preparation Techniques
PPT
Add sample
Ostro
Load sample
LLE
Add sample
Add extract
SLE
Add Sample
SPE
Condition
SPE
(Mixed Mode)
Condition
©2015 Waters Corporation 10
Add ACN
Mix/vortex
Filter / centrifuge
Add ACN
Mix/vortex
pass-through
Add extract
Mix
Wait
Separate
Evaporate
Reconstitute
Add extraction solvent
Wait 5 -10 mins
Extract
Evaporate
Reconstitute
Equilibrate
Load sample
Wash
Elute
Equilibrate
Load sample
Wash 1
Wash 2
Elute
Fastest Cleanest
Lowest HighestSensitivity
Sample Preparation
A Simple Approach: What is Pass-Through Sample Preparation?
Apply Sample
Retention of Proteins
©2015 Waters Corporation 11
Analytes of interest
Retention of Phospholipids
OstroSorbent
Proteins
Analytes pass-through free of proteins and phospholipids
What is Ostro?
� The Ostro Pass-through sample preparation plate removes
proteins and phospholipids using a combination of filtration and
sorbent interaction
� Benefits include:
– Cleaner samples in less time
– Minimal-to-no method development
©2015 Waters Corporation 12
– Minimal-to-no method development
– Simple, generic protocol
� Ostro provides consistent, robust methods and increased
throughput using a simple, easy-to-implement protocol
What is Ostro (cont.)?
� 96-well plate for pass-through sample preparation
– Simply and efficiently removes proteins & phospholipids
– Typical applications areas:
o Works with plasma, serum, whole blood, milk, meat, tissue and
other protein/lipid-containing samples
o Bioanalysis, forensics, clinical research, food, environmental, etc.
©2015 Waters Corporation 13
Ostro: Typical Application Areas and Sample Pre-Treatment
Clinical Research (Forensic)
Whole Blood/Plasma
Bioanalysis
Plasma/Serum
Food
Milk/Tissue
©2015 Waters Corporation 14
• 1:3 w/Zinc Sulphate – Whole Blood
• Dilute sample
• If required, acidify or basify samples
• Sample is treated with ACN externally
Ostro: How to Use
Clinical Research (Forensics)
Whole Blood/Plasma
Bioanalysis
Plasma/Serum
Food
Milk/Tissue
©2015 Waters Corporation 15
• 1:3 w/Zinc Sulphate – Whole Blood • Dilute sample
• If required acidify or basify samples
• Sample is treated with ACN externally
Load sample
Add solvent
In-well mixing
Analytes pass-through
Collect & Inject
Analytes pass-through free of proteins, phospholipids, and particulates
Example: Ostro in Bioanalysis
Place Ostro onto collection plate
Pipette 50-200 µL of plasma into wells
Forcefully add 2% formic acid in acetonitrile (3:1 solvent:plasma)
It is possible to work with lower sample volumes (such as 25 µL). When doing so, a higher organic solvent to sample ratio (e.g.,
©2015 Waters Corporation 16
acetonitrile (3:1 solvent:plasma) (methanol not recommended)
Mix thoroughly by aspirating 3x with pipette
Filter samples using vacuum manifold or positive-pressure manifold
Analyze samples
to sample ratio (e.g., 10:1,20:1) is required.
The well volume is 1.9 mL, however in order to mix by aspiration, the maximum volume is 1.4 mL. This translates to a maximum sample size of 350 µL.
The well volume is 1.9 mL, however in order to pass high sample volumes, multiple
Sample is treated with acetonitrile externally
Place Ostro onto collection plate
Example: Ostro in Food Analysis
©2015 Waters Corporation 17
pass high sample volumes, multiple loading need to be done in the same well.Pass acetonitrile-treated
sample through Ostro
Filter samples using vacuum manifold or
positive pressure manifold
Analyze samples
Example: Forensics (Whole Blood)
Take whole blood and add 1:3 w/zinc
Place Ostro onto collection plate
Pipette 50-200 µL of supernant into wells
Forcefully add 2% formic acid in acetonitrile (3:1
solvent:plasma) (methanol
©2015 Waters Corporation 18
add 1:3 w/zinc sulphate, centrifuge, remove supernatant, and use as sample with Ostro
solvent:plasma) (methanol not recommended)
Mix thoroughly by aspirating 3x with pipette
Filter samples (pass through) using vacuum manifold or positive pressure manifold
Analyze samples
Ostro vs. Other Sample Preparation Techniques
PPT OSTRO LLE SSLE
Method Development NO NO YES YES
Highest Selectivity
Direct Inject √ √
Simple /Ease of Use √ √ √* √*
High Throughput √ √ √* √
Reproducibility √ √ √ √
©2015 Waters Corporation 19
Reproducibility √ √ √ √
Reduction of Matrix Effects √ √ √
Highest Sensitivity
Sample Concentration √* √
“Clean” Extracts √ √ √
Recovery √ √ √ √
Generic √ √
Protocol Time √ √
Environmentally Friendly √ √
* Perception of being simple
Use of PPT for Bioanalysis
� If PPT works, it is generally the first choice for those developing
sample preparation methods
– Samples can be diluted with water and directly injected to speed up
workflow (“dilute and shoot”)
– Inexpensive to perform in most cases
– Very simple protocol
©2015 Waters Corporation 20
o Easily transferred from lab to lab
o Inexperienced personnel can perform successfully
� Disadvantages include:
– Variable recoveries
– Not effective for phospholipids
o Doesn’t remove matrix effects
Ostro versus Protein Precipitation
PPT OSTRO LLE SSLE
Method Development NO NO YES YES
Highest Selectivity
Direct Inject √ √
Simple /Ease of Use √ √ √* √*
High Throughput √ √ √ √
©2015 Waters Corporation 21
High Throughput √ √ √ √
Phospholipid Removal √ √
Highest Sensitivity
“Clean” Extracts √ √ √
Recovery √ √ √ √
Generic √ √
Protocol Time √ √
Environmentally Friendly √ √
* Perception of being simple
Comparison of Ostro versus PPT Protocols
Ostro (Total processing time: 11 min)
Load sample (3 min)
PPT(Total processing time: 13 min)
Load sample, (3 min)
©2015 Waters Corporation 22
Add acetonitrile (2 min)
Mix (3 min)
Pass Through (3 min)
Add extraction solvent
(2 min)
Mix (Vortex) (3 min)
Centrifuge (2 min)
Transfer (3 min)
Sample processing time with Ostro is slightly less than PPT
Remaining Phospholipids
2.00 4.00 6.00 8.00 10.00 12.00
%
0
100184.4 > 184.4 (Lipid 184)
1.31e8
7.50
7.43
7.31
7.69
7.76
9.18
8.80
8.319.88
10.08
Traditional PPT
©2015 Waters Corporation 23
Time
2.00 4.00 6.00 8.00 10.00 12.00
%
0
100
2.00 4.00 6.00 8.00 10.00 12.00
184.4 > 184.4 (Lipid 184)
1.31e8
8.30
Ostro
MRM transition 184>184 to demonstrate total remaining PLs
Phospholipid Build-Up: 250 Injections
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
PPT last
injection
PPT 1st
injection
gradient
end
end
organic hold
©2015 Waters Corporation 24
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
Ostro last
injection
Ostro 1st
injection
0.5 min hold at high % organic does not fully elute phospholipids resulting in phospholipid build-up on column and unpredictable
elution in subsequent injections
Advantages of Ostro versus Protein Precipitation
� Simple, universal protocol
– Little-to-no method development
� Removes proteins and phospholipids
– Improved instrument uptime and more robust methods
– PPT requires centrifugation to remove proteins (which takes time)
� Decreased matrix effects
©2015 Waters Corporation 25
� Decreased matrix effects
� Lower sample variability
� Increased recovery
� Increased method robustness
� “The Ostro plate performed significantly better than protein
precipitation and the marketed plates for removal of all classes
of phospholipids*”
*Brian Hoffman, Advion BiosciencesBioanalysis Journal (2010), 2 (12), 1932-1933
Ostro versus Liquid-Liquid Extraction (LLE) in Bioanalysis
PPT OSTRO LLE SSLE
Method Development NO NO YES YES
Highest Selectivity
Direct Inject √ √
Simple /Ease of Use √ √ √* √*
High Throughput √ √ √ √
©2015 Waters Corporation 26
High Throughput √ √ √ √
Phospholipid Removal √ √
Highest Sensitivity
“Clean” Extracts √ √ √
Recovery √ √ √ √
Generic √ √
Protocol Time √ √
Environmental Friendly √ √
* Perception of being simple
Using Liquid-Liquid Extraction (LLE)
� LLE: separation of compounds based on solubility in two
immiscible liquids (e.g., water and an organic solvent)
� Advantages:
– Uses existing glassware; no on-going costs other than labor and
solvents
©2015 Waters Corporation 27
solvents
– Can be more compound specific
� Disadvantages:
– Very time and labor intensive
– Large solvent consumption (purchase and disposal costs)
– Emulsions are possible
– Not readily automatable (poor sample throughput)
Comparison of Ostro versus LLE Protocols
Ostro (Total processing time : 11 min)
Load sample (3 min)
LLE (Total processing time : 20 min)
Load sample (2 min)
Add extraction solvent (2 min)
©2015 Waters Corporation 28
Add acetonitrile (2 min)
Mix (3 min)
Pass Through (3 min)
Add extraction solvent (2 min)
Mixing (1 min)
Centrifuge (5 min)
Transfer (2 min)
Evaporate (5 min)
Reconstitute (2 min )
Vortex (1 min)
Sample processing time for Ostro is less than LLE
Comparison of Analyte Recovery: Ostro versus LLE
Average Analyte Recovery
80
100
120
Average Analyte Recovery
©2015 Waters Corporation 29
0
20
40
60
Oxycodone Valethamate Niflumic Acid Ketoprofen Progesterone Hydrocortisone
Ostro
LLE
weak, polarbase
strong base zwitterion acid hydrophobicneutral
hydrophobicneutral
Comparison of Total Remaining Phospholipids: Ostro versus LLE
%
100 184.4 > 184.4 (Lipid 184)1.34e8
1.79 1.45
1.28 1.59 1.85
Ostro Ostro
©2015 Waters Corporation 30
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
%
0
100 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 0
184.4 > 184.4 (Lipid 184)1.34e8
2.13 1.88 1.79
1.40
1.95
2.00
2.08
2.21
2.26 2.31
2.70
LLE using
100%
MTBE
LLE using 100% MTBE
Ostro eliminates build-up of PLs on column
Comparison of Total Remaining Phospholipids: Ostro versus LLE
Sum of Remaining Residual Phospholipids
2500000
3000000
3500000
4000000
©2015 Waters Corporation 31
0
500000
1000000
1500000
2000000
Ostro LLE MTBE - Ostro LLE MTBE
For even cleaner extracts: combination of the Ostro plate and LLE removes>99.9% of residual phospholipids
Advantages of Ostro versus LLE
� Faster run times
� Simple, easy to implement generic method (little-to-no method
development required)
� Superior removal of proteins and phospholipids
� No emulsions with Ostro
� Can be automated to further increase sample throughput
©2015 Waters Corporation 32
� Can be automated to further increase sample throughput
For additional information, see application note:
720004051EN - Providing a Universal, One-Step Alternative to Liquid-Liquid Extraction in Bioanalysis
Ostro versus SSLE
PPT OSTRO LLE SSLE
Method Development NO NO YES YES
Highest Selectivity
Direct Inject √ √
Simple / Ease of Use √ √ √* √*
High Throughput √ √ √ √
©2015 Waters Corporation 33
High Throughput √ √ √ √
Phospholipid removal √ √
Highest Sensitivity
“Clean” Extracts √ √ √
Recovery √ √ √ √
Generic √ √
Protocol Time √ √
Environmental Friendly √ √
* Perception of being simple
Using Solid-Supported Liquid Extraction (SSLE)
� SSLE = SSLE is partition chromatography using buffered water
immobilized on a solid support (diatomaceous earth) and elution
by a water immiscible solvent
� Advantages:
– More compound specific
©2015 Waters Corporation 34
– More compound specific
� Disadvantages:
– Large solvent consumption (purchase and disposal costs)
– Requires method development
– Multiple dilution buffers required
– Evaporation & reconstitution required
– Processing times
Comparison of Ostro versus SSLE Protocols
Ostro (Total processing time: 11 min)
Load sample (3 min)
SSLE (Total processing time: 28 – 33 min)
Load sample, initiate (3 min)
©2015 Waters Corporation 35
Add acetonitrile (2 min)
Mix (3 min)
Pass Through (3 min)
Wait 5 – 10 min
Add extraction solvent (2 min)
Wait 5 – 10 min
Extract (1 min)
Evaporate (10 – 15 min)
Reconstitute (2 min )
Sample processing time for Ostro is 2-3 times less than SSLE
Comparison of Analyte Recovery and Matrix Effects from Plasma: Ostro versus SSLE
0
20
40
60
80
100
120
Ostro SSLE
0.60
0.80
1.00
1.20
1.40
Ostro SSLE
©2015 Waters Corporation 36
0
0.20
0.40
Recovery for Ostro & SSLE Matrix factors for Ostro & SSLE
Ostro showed very good and consistent recoveries across all the tested
analytes with an average recovery of 95±4%
SSLE showed variability in recovery, since the extraction method was selected
for neutral and basic analytes
Comparable matrix factors for Ostro and SSLE
Comparison of Batch to Batch Reproducibility: Ostro versus SSLE
0
20
40
60
80
100
120
Ostro batch 1 Ostro batch 2 Ostro batch 3
Ostro batch to batch recovery &
reproducibility
©2015 Waters Corporation 37
0
20
40
60
80
100
120
SSLE batch1 SSLE batch2
SSLE batch to batch recovery &
reproducibility
Ostro offers superior recoveries and batch-to-batch reproducibility
Advantages of Ostro versus SSLE
� Simple, easy to implement generic method (little-to-no method
development required)
� 60% in time savings
� Consistently higher and more reproducible recovery across wide
range of analytes
� More environmental friendly methodology
©2015 Waters Corporation 38
� More environmental friendly methodology
� More consistent batch to batch results
For more information, see application note:
720005199EN Advantages of Ostro Pass-through Sample Preparation Versus Solid Supported Liquid Extraction (SSLE)
Agenda
� Industry overview
– Business & scientific challenges
– Sample preparation techniques
� Ostro pass-through sample preparation
– What is it?
– How does it work?
©2015 Waters Corporation 39
– How does it work?
– Comparison to traditional techniques (PPT, LLE & SSLE)
� Practical Applications of Ostro
� Summary
� Appendix: Additional Applications
– Enhanced dried blood spots analysis
– Veterinary drugs from Milk
Ostro in Drug Screening
� Optimizing sample and laboratory workflow is critical in drug
discovery
– Diverse sets of compounds
– Need to achieve acceptable sensitivity
– Clean extracts
– Generic methods
©2015 Waters Corporation 40
– Generic methods
� Goal: develop a method with high recovery that produces
cleaner extracts and facilitates more robust, shorter run times
– 100 µL of plasma extracted using the generic protocol
Ostro in Drug Screening: Chromatographic Separation
1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha-Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam10. Nitrazepam11. Clonazepam d4
110, 11, 12, 13
14, 15, 16, 17, 18, 19, 20, 21
26
100
0.80
1. 7-aminonitrazepam2. 7-aminoclonazepam3. 7-aminoflunitrazepam4. Clozapine5. Midazolam6. Chlordiazepoxide7. Alpha-Hydroxymidazolam8. Bromazepam9. n-Desmethylflunitrazepam10. Nitrazepam11. Clonazepam d4
110, 11, 12, 13
14, 15, 16, 17, 18, 19, 20, 21
26
100
0.80
100100
0.80
©2015 Waters Corporation 41
11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha-Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam26. Prazepam
2
3
4 6
5
7
8
9 22, 23, 24
25
26
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
11. Clonazepam d412. Clonazepam13. Flunitrazepam14. Triazolam15. 2-Hydroxyethylflurazepam16. Hydroxyalprazolam d517. Alpha-Hydroxyalprazolam18. Alprazolam19. Alprazolam d520. Oxazepam21. Clobazam22. Estazolam23. Desalkylflurazepam24. Temazepam25. Nordiazepam26. Prazepam
2
3
4 6
5
7
8
9 22, 23, 24
25
26
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
%
0
Ostro in Drug Screening:Average Analyte Recovery
60
80
100
Average Recovery ~86%
©2015 Waters Corporation 42
0
20
40
Triazo
lam
(T-9
10)
alph
a-Hyd
roxy
mid
azola
m (H
-902)
2-hyd
roxy
ethylf
lura
zepam
(F-9
01)
Hyd
roxy
alpr
azol
am d
5 (A
-904)
Clo
zapin
e (C-0
59)
Mid
azol
am (M
-908)
Praze
pam
(P-9
06)
alph
a-Hyd
roxy
alpr
azola
m (A
-905)
Clo
nazep
am d
4 (C
-905
)
Brom
azep
am (B
-903
)
Clo
nazep
am (C
-907)
Fluni
traze
pam
(F-9
07)
Alpra
zola
m d
5 (A-9
02)
Alpra
zola
m (A
-903)
Temaz
epam
(T-9
07)
Clo
bazam
(C-9
09)
n-D
esm
ethyl
fluni
traze
pam
(D-9
19)
Chl
ordia
zepo
xide
(C-0
22)
Estaz
olam
(E-9
01)
Des
alky
lflur
azepa
m (D
-915
)
Oxa
zepam
(O-9
02)
7-am
inoc
lonaz
epam
(A-9
15)
7-am
inof
luni
traze
pam
(A-9
12)
Nitr
azepam
(N-9
06)
Nor
diaz
epam
(N-9
05)
7-am
inon
itraz
epam
(A-9
14)
Ostro in Drug Screening: Sum of Phospholipids
5000000
6000000
7000000
8000000
9000000
sum of 8 phospholipids
©2015 Waters Corporation 43
0
1000000
2000000
3000000
4000000
Ostro PPT
sum of 8 phospholipids
Ostro removes >99% of phospholipids relative to traditional PPT
Ostro in Drug Screening: Remaining Phospholipids
2.00 4.00 6.00 8.00 10.00 12.00
%
0
100184.4 > 184.4 (Lipid 184)
1.31e8
7.50
7.43
7.31
7.69
7.76
9.18
8.80
8.319.88
10.08
Traditional PPT
©2015 Waters Corporation 44
Time
2.00 4.00 6.00 8.00 10.00 12.00
%
0
100
2.00 4.00 6.00 8.00 10.00 12.00
184.4 > 184.4 (Lipid 184)
1.31e8
8.30
Ostro
MRM transition 184>184 to demonstrate total remaining PLs
Ostro in Drug Screening: Phospholipid Build-up
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100 758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
2.282.131.99
758.4 > 184.4 (Lipid 758)3.19e6
PPT last
injection
PPT 1st
injection
gradient
end
end
organic hold
©2015 Waters Corporation 45
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.750
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
758.4 > 184.4 (Lipid 758)3.19e6
Ostro last
injection
Ostro 1st
injection
0.5 min hold at high % organic does not fully elute phospholipids resulting in phospholipid build-up on column and unpredictable
elution in subsequent injections
Ostro in Drug Screening: Phospholipid Elution in Shortened Gradient
%
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100 184.4 > 184.4 (Lipid 184)1.34e8
0.55
0.02
0.65 0.73 0.87
184.4 > 184.4 (Lipid 184)1.34e8
%
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100 184.4 > 184.4 (Lipid 184)1.34e8
%
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100 184.4 > 184.4 (Lipid 184)1.34e8
0.55
0.02
0.65 0.73 0.87
184.4 > 184.4 (Lipid 184)1.34e8
0.55
0.02
0.65 0.73 0.87
184.4 > 184.4 (Lipid 184)1.34e8
PPT
200 injections
PPT
1 injection
end
gradient
©2015 Waters Corporation 46
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
00.73
184.4 > 184.4 (Lipid 184)1.34e8
0.33
184.4 > 184.4 (Lipid 184)1.34e8
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
Time0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
0
100
0.20 0.40 0.60 0.80 1.00 1.20 1.40
%
00.20 0.40 0.60 0.80 1.00 1.20 1.40
%
00.73
184.4 > 184.4 (Lipid 184)1.34e8
0.73
184.4 > 184.4 (Lipid 184)1.34e8
0.33
184.4 > 184.4 (Lipid 184)1.34e8
Ostro
200 injections
Ostro
1 injection
Gradient cannot be shortened using PPT since PLs continue to elute after gradient has ended
Ostro in Drug Screening:Summary
� Ostro generates high recoveries for a discovery screen with no
method development
� Simple, universal protocol
� Direct injection of Ostro-processed samples
� Removes >99% of PLs
– Improved instrument uptime and more robust methods
©2015 Waters Corporation 47
– Improved instrument uptime and more robust methods
� Reduces sample variability
� Facilitates use of shorter runtimes, improving throughput
Ostro in Forensics:Direct Analysis of Opiates
� Forensic laboratories often need to analyze whole blood
specimens for the presence of different drugs to determine the
precise cause of death, in cases of driving under the influence of
drugs, or other criminal or research purposes
� Many sample preparation strategies have been used for whole
©2015 Waters Corporation 48
� Many sample preparation strategies have been used for whole
blood analysis
� With Ostro the complete sample preparation workflow for whole
blood is done within the wells, including sample pre-treatment
Ostro in Forensics:Sample Preparation Workflow
Add 150 µL of aqueous 0.1 M ZnSO4 /0.1 M NH4CH3COOH to the wells
Add 50 µL of Whole blood
Mix (to Lyse the cells)
Add 600 µL of ACN
©2015 Waters Corporation 49
Vortex
Pass through (vacuum or positive pressure)
Evaporate (N2)
Reconstitute with 50 µL of 0.1% formic acid in 2% acetonitrile
Analyze
Ostro in Forensics: Chromatographic Separation of Opiates and Synthetic Analgesic Compounds
©2015 Waters Corporation 50
Ostro in Forensics: Recoveries of Opiates from Whole Blood
Mean recovery from whole blood samples (n=4)
©2015 Waters Corporation 51
Ostro provides reproducible recoveries
Direct Analysis of Opiates:Summary
� The Ostro pass-through sample preparation plate allows for
rapid, in-well cell lysis and protein precipitation
� Provides additional benefit of phospholipid removal
� Ostro simplifies the forensic laboratory’s workflow by
eliminating multiple transfers & lab wares
� Cost savings & throughput achieved
©2015 Waters Corporation 52
� Cost savings & throughput achieved
Ostro With Dried Blood Spots (DBS)
�Dried blood spot (DBS) analysis is being evaluated in
the pharmaceutical industry because it facilitates the
use of smaller sample sizes and provides easier
handling and long term storage of samples
©2015 Waters Corporation 53
� The goal was to improve current DBS methods and
provide cleaner DBS extracts, as well as offer a
single-step DBS method with no extract transfer or
centrifugation
Ostro With Dried Blood Spots
O N
F N
N
N
O
CH3
Risperidone
MW 410.48
pKa 8.24
API
©2015 Waters Corporation 54
O N
NH
N
N
N
CH3
O N
F N
N
N
O
CH3
OH
Clozapine
MW 326.82
pKa 7.6
9-OH Risperidone
MW 426.48
pKa 7.86
Internal Standard
Metabolite
�Ostro in-well extraction
� 3.0 mm DBS punch
extracted in-well using
250 µL of 95/5
� Traditional DBS
extraction
� 3.0 mm DBS punch
extracted in centrifuge
tube using 250 µL of
Ostro With Dried Blood Spots
©2015 Waters Corporation 55
250 µL of 95/5
methanol/water 95/5 methanol/water
20 µL whole blood was spotted onto Whatman DMPK-C cards and dried for 2 hrs
Ostro With Dried Blood Spots:Sum of Phospholipids
Sum of Phospholipids
30000
35000
40000
45000
50000
Sum of Phospholipids
©2015 Waters Corporation 56
0
5000
10000
15000
20000
25000
30000
Ostro in-well Traditional In-tube
Ostro removes 99.9% of residual PLs relative to the traditional DBS extraction
Ostro With Dried Blood Spots:Phospholipids Remaining
0.50 1.00 1.50 2.00 2.50 3.00
%
0
100
TIC1.36e8
2.84
2.19
Traditional DBS
©2015 Waters Corporation 57
Time0.50 1.00 1.50 2.00 2.50 3.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.000
TIC
1.36e8
DBS in-well using Ostro
TIC of 5 individual PLsNo PL build-up on column with Ostro
Ostro With Dried Blood Spots:Phospholipid Build-up
DBS in-well Ostro1st injection
DBS in-well Ostro
Last injection
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100758.4 > 184.4 (Lipid 758)
4.88e6
758.4 > 184.4 (Lipid 758)4.88e6
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100758.4 > 184.4 (Lipid 758)
4.88e6
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100758.4 > 184.4 (Lipid 758)
4.88e6
758.4 > 184.4 (Lipid 758)4.88e6
758.4 > 184.4 (Lipid 758)4.88e6
©2015 Waters Corporation 58
Traditional DBS
1st injection
Traditional DBSLast injection
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
758.4 > 184.4 (Lipid 758)4.88e6
2.20
758.4 > 184.4 (Lipid 758)4.88e6
2.212.16
3.37Time
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
758.4 > 184.4 (Lipid 758)4.88e6
758.4 > 184.4 (Lipid 758)4.88e6
2.20
758.4 > 184.4 (Lipid 758)4.88e6
2.20
758.4 > 184.4 (Lipid 758)4.88e6
2.212.16
3.37
No PL build-up on column with Ostro
Ostro With Dried Blood Spots:High Sensitivity
%
0
100 427.1 > 109.8 (9-OH Risperidone)
4.71e4
2.232.041.871.29
0.54
0.300.030.78 0.81 1.16
1.851.68 1.58
2.30
3.442.39
2.522.63 3.292.95 3.14
Blank Whole Blood Spot
©2015 Waters Corporation 59
Time0.50 1.00 1.50 2.00 2.50 3.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 0
427.1 > 109.85 (9-OH Risperidone) 4.71e4
1.30984
Whole Blood Spiked at 50 pg/mL with 9-OH
risperidone
Excellent sensitivity for dilute DBS extracts using Xevo TQ-S
Ostro With Dried Blood Spots:Summary
� Simple, single step method for DBS samples
� Removes 99.9% of PLs relative to tradition DBS
extraction
– More robust LC methods
� Achieve lower LODs with high sensitivity analyses
©2015 Waters Corporation 60
� Achieve lower LODs with high sensitivity analyses
� Eliminates build-up of PLs on columns allowing for
the use of shorter gradients
– Improved laboratory throughput
Ostro for Veterinary Drug Analyses in Milk
�Multiple sample preparation methods are available
today for this application
– PPT, LLE, SPE, etc.
�Goal is to compare Ostro performance with protein
precipitation
©2015 Waters Corporation 61
precipitation
Ostro for Veterinary Drug Analyses in Milk: Chromatographic Separation
©2015 Waters Corporation 62
Removal of phospholipids from whole milk using Ostro
Ostro for Veterinary Drug Analyses in Milk: Summary
– Rapid & Simple
– Clean extracts
– Removal of potentially interfering phospholipids from samples
– High recovery for analyte of interest
– Increased throughput
©2015 Waters Corporation 63
Agenda
� Industry overview
– Business & scientific challenges
– Sample preparation techniques
� Ostro pass-through sample preparation
– What is it?
– How does it work?
©2015 Waters Corporation 64
– How does it work?
– Comparison to traditional techniques (PPT, LLE & SSLE)
� Practical Applications of Ostro
� Summary
� Appendix
– Applications examples
Summary
� Ostro pass-through sample preparation device offers far
superior performance when compared to competitive techniques
� Filters samples while removing proteins & phospholipids
� Lower matrix effects, high sensitivity and increased instrument
uptime
� Generic method
©2015 Waters Corporation 65
� Generic method
– Little-to-no method development; rapid implementation
– Simple workflow
� 96-well format
– Automatable for increased sample throughput
� Reproducible, robust methods
Simple protocol Cleaner extracts Consistent results
Thank You for Attending!
� Post-Event Home Page: http://www.waters.com/Feb26
� 30% Product Specific Offer on Ostro Plates
� 25% Offer On Oasis, SepPak Products
– Full Webinar Recording of Today’s Session w/PDF Slide
Deck
– Compilation of TODAY’S KEY Literature, Brochures etc…
©2015 Waters Corporation 66
– Compilation of TODAY’S KEY Literature, Brochures etc…
� For Questions and to Submit your Ideas for our Next Topic
– Please eMail - [email protected]
©2015 Waters Corporation 67
Appendix
©2015 Waters Corporation 68
Appendix
Ostro in Clinical Study
� Antipsychotic drug
marketed as Risperdal®
� 9-OH Risperidone is the
primary circulating species N
F
N
O
N CH3
OH9-Hydroxyrisperidone
M.W. 426.2
Risperidone and 9-OH Metabolite in Human Plasma
©2015 Waters Corporation 69
NO
F
N
NH
N
N
Cl
CH3
NO
N
F
N
O
N CH3
Risperidone
M.W. 410.2
pKa = 8.24
Clozapine, ISTD
M.W. 326.1
pKa’s = 7.1, 5.3
Method Requirements
� Wide linear dynamic range
– 0.025 – 500 ng/mL
� Meets accuracy and precision regulatory requirements for
standards and QC samples
� Reproducible
� Achieve required detection limit
©2015 Waters Corporation 70
� Achieve required detection limit
� Easy
� Fast
� No method development
Wide Linear Dynamic Range:Standard Curve for Risperidone
Compound name: Risperidone
Correlation coefficient: r = 0.997938, r 2̂ = 0.995879
Calibration curve: 0.466273 * x + 0.00860374
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None
Conc
Re
sid
ua
l
-10.0
-5.0
0.0
5.0
10.0
Compound Name: RisperidoneCorrelation coefficient r = 0.997938, r^2 = 0.995879Calibration curve: 0.466273*x + 0.00860374Response type: Internal Std (Ref1), Area*(IS Conc./IS Area)Curve Type: Linear, Origin:Exclude, Weighting 1/x^2, Axis trans: None
©2015 Waters Corporation 71
Conc0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Re
sp
on
se
0
50
100
150
200
Calibration curve is linear over 4.5 orders of magnitude
Calibration curve range: 0.025 (25 pg/mL) to 500 ng/mL in human plasma
Wide Linear Dynamic Range: Standard Curve for 9-OH Risperidone
Compound name: 9-OH Risperidone
Correlation coefficient: r = 0.998454, r^2 = 0.996910
Calibration curve: 0.321752 * x + 0.00172858
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None
Conc
Re
sid
ua
l
-5.0
0.0
5.0
10.0
15.0
20.0
Compound Name: 9-OH RisperidoneCorrelation coefficient r = 0.998454, r^2 = 0.996910Calibration curve: 0.321752*x + 0.00172858Response type: Internal Std (Ref1), Area*(IS Conc./IS Area)Curve Type: Linear, Origin:Exclude, Weighting 1/x^2, Axis trans: None
©2015 Waters Corporation 72
Conc0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500
Re
sp
on
se
0
25
50
75
100
125
150
Curve is linear over 4.5 orders of magnitude
Curve range is 0.025 (25 pg/mL) to 500 ng/mL in human plasma
Method Meets Regulatory Criteria for Accuracy and Precision
Human Plasma Type Area IS Area Conc. %Dev
25 pg/mL Standard 401.8 19390.7 0.026 3.9
50 pg/mL Standard 579.5 18436.2 0.049 -2.1
100 pg/mL Standard 909.5 18751.3 0.086 -14.4
250 pg/mL Standard 2304.7 17667.0 0.261 4.5
500 pg/mL Standard 3135.0 12261.9 0.530 6
2.5 ng/mL Standard 20772.9 18459.5 2.395 -4.2
12.5 ng/mL Standard 112761.8 19344.0 12.483 -0.1
25 ng/mL Standard 218078.2 18115.3 25.800 3.2
©2015 Waters Corporation 73
Meets Regulatory Criteria: all standards within 15% deviation of expected
25 ng/mL Standard 218078.2 18115.3 25.800 3.2
50 ng/mL Standard 428895.1 17760.3 51.773 3.5
250 ng/mL Standard 2180640.3 19283.9 242.503 -3
500 ng/mL Standard 3702304.5 15468.5 513.297 2.7
75 pg/mL QC 847.8 17836.0 0.083 11.3
750 pg/mL QC 5868.9 16136.6 0.762 1.5
7.5 ng/mL QC 48311.8 13565.3 7.620 1.6
75 ng/mL QC 558263.4 15764.1 75.932 1.2
Std Conc(ng/mL)
Average Calculated
Concentration (ng/mL) %RSD
0.075 0.083 11.3
Std Conc(ng/mL)
Average Calculated
Concentration (ng/mL) %RSD
0.075 0.09 20.3
Meets Regulatory Criteria: Accuracy and Precision
Risperidone 9-OH Risperidone
©2015 Waters Corporation 74
0.75 0.762 1.5
7.5 7.62 1.6
75 75.932 1.2
0.75 0.787 5
7.5 8.262 10.2
75 74.686 -0.4
All QC samples meet accuracy and precision regulatory criteria